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Sample records for plasmid purification process

  1. Plasmid pVAX1-NH36 purification by membrane and bead perfusion chromatography.

    Science.gov (United States)

    Franco-Medrano, Diana Ivonne; Guerrero-Germán, Patricia; Montesinos-Cisneros, Rosa María; Ortega-López, Jaime; Tejeda-Mansir, Armando

    2017-03-01

    The demand for plasmid DNA (pDNA) has increased in response to the rapid advances in vaccines applications to prevent and treat infectious diseases caused by virus, bacteria or parasites, such as Leishmania species. The immunization protocols require large amounts of supercoiled plasmid DNA (sc-pDNA) challenging the development of efficient and profitable processes for capturing and purified pDNA molecules from large volumes of lysates. A typical bioprocess involves four steps: fermentation, primary recovery, intermediate recovery and final purification. Ion-exchange chromatography is one of the key operations in the purification schemes of pDNA owing the chemical structure of these macromolecules. The goal of this research was to compare the performance of the final purification step of pDNA using ion-exchange chromatography on columns packed with Mustang Q membranes or perfusive beads POROS 50 HQ. The experimental results showed that both matrixes could separate the plasmid pVAX1-NH36 (3936 bp) from impurities in clarified Escherichia coli lysates with an adequate resolution. In addition, a 24- and 21-fold global purification factor was obtained. An 88 and 63% plasmid recuperation was achieved with ion-exchange membranes and perfusion beads, respectively. A better understanding of perfusion-based matrices for the purification of pDNA was developed in this research.

  2. The technology of large-scale pharmaceutical plasmid purification ...

    African Journals Online (AJOL)

    Further test demonstrated that the pcDNAlacZ purified with CTAB and authoritative endotoxin-free plasmid Kit had the similar transfection efficiency in vivo and in vitro. CTAB can be used for plasmid purification; the main advantages of the DNAs purified with CTAB include the avoidance of animal-derived enzymes, toxic ...

  3. Plasmid fermentation process for DNA immunization applications.

    Science.gov (United States)

    Carnes, Aaron E; Williams, James A

    2014-01-01

    Plasmid DNA for immunization applications must be of the highest purity and quality. The ability of downstream purification to efficiently produce a pure final product is directly influenced by the performance of the upstream fermentation process. While several clinical manufacturing facilities already have validated fermentation processes in place to manufacture plasmid DNA for use in humans, a simple and inexpensive laboratory-scale fermentation process can be valuable for in-house production of plasmid DNA for use in animal efficacy studies. This chapter describes a simple fed-batch fermentation process for producing bacterial cell paste enriched with high-quality plasmid DNA. A constant feeding strategy results in a medium cell density culture with continuously increasing plasmid amplification towards the end of the process. Cell banking and seed culture preparation protocols, which can dramatically influence final product yield and quality, are also described. These protocols are suitable for production of research-grade plasmid DNA at the 100 mg-to-1.5 g scale from a typical 10 L laboratory benchtop fermentor.

  4. The technology of large-scale pharmaceutical plasmid purification ...

    African Journals Online (AJOL)

    STORAGESEVER

    2010-01-04

    Jan 4, 2010 ... DNA vaccine, the cost of purification must be decreased. Although commonly .... Three mice were killed every 4 days interval. Tissues of heart, liver, .... Now, methods such as chromatography had good prospects in plasmid ...

  5. Functionalized tetrapod-like ZnO nanostructures for plasmid DNA purification, polymerase chain reaction and delivery

    International Nuclear Information System (INIS)

    Nie Leng; Gao Lizeng; Yan Xiyun; Wang Taihong

    2007-01-01

    Functionalized tetrapodal ZnO nanostructures are tested in plasmid DNA experiments (1) as a solid-phase adsorbent for plasmid DNA purification (2) as improving reagents in a polymerase chain reaction (PCR) and (3) as novel carriers for gene delivery. The amino-modification, the tetrapod-like shape of the nanostructure and its high biocompatibility all contribute to measurements showing promise for applications. A sol-gel method is used for silica coating and amino-modification. Plasmid DNA is purified through reversible conjugations of amino-modified ZnO tetrapods with DNA. Also, as additional reagents, functionalized tetrapods are shown to improve the amount of PCR product. For transfection, ZnO tetrapods provide some protection against deoxyribonuclease cleavage of plasmid DNA and deliver plasmid DNA into cells with little cytotoxicity

  6. Fiscal 1999 achievement report on the venture business assisting type regional consortium - Minor business creation base type. Development of simplified extraction/purification method for highly purified large-size plasmids; 1999 nendo chiiki consortium kenkyu kaihatsu jigyo seika hokokusho. Kanbenka kojundo ogata plasmid chushutsu seiseiho no kaihatsu

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    2001-03-01

    The project aims to develop biotechnological industries in Okinawa Prefecture by embodying a method for obtaining large-size plasmids in a short time at low cost without a special apparatus, for which efforts were made to develop and commercialize a kit comprising an eluate from plasmid cells and a set of columns and eluate for purification. Under this project, University of the Ryukyus well experienced in the handling of plasmid DNAs and Advanced Medical Biological Science Institute jointly developed a 'simplified extraction/purification method for highly purified large-size plasmids.' The developed techniques or methods are described below. A concentration method was developed not using centrifugal operation for escherichia coli cultured in a closed mass culture device. A buffer exchange method in a closed system was developed, continuous from escherichia coli concentration to elution. A column assisted DNA purification method consuming but a short time was established. With these combined, the development is now complete of the technology and device for plasmid DNA elution from escherichia coli by an uninterrupted operation in a completely closed system. (NEDO)

  7. Fiscal 1999 achievement report on the venture business assisting type regional consortium - Minor business creation base type. Development of simplified extraction/purification method for highly purified large-size plasmids; 1999 nendo chiiki consortium kenkyu kaihatsu jigyo seika hokokusho. Kanbenka kojundo ogata plasmid chushutsu seiseiho no kaihatsu

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    2001-03-01

    The project aims to develop biotechnological industries in Okinawa Prefecture by embodying a method for obtaining large-size plasmids in a short time at low cost without a special apparatus, for which efforts were made to develop and commercialize a kit comprising an eluate from plasmid cells and a set of columns and eluate for purification. Under this project, University of the Ryukyus well experienced in the handling of plasmid DNAs and Advanced Medical Biological Science Institute jointly developed a 'simplified extraction/purification method for highly purified large-size plasmids.' The developed techniques or methods are described below. A concentration method was developed not using centrifugal operation for escherichia coli cultured in a closed mass culture device. A buffer exchange method in a closed system was developed, continuous from escherichia coli concentration to elution. A column assisted DNA purification method consuming but a short time was established. With these combined, the development is now complete of the technology and device for plasmid DNA elution from escherichia coli by an uninterrupted operation in a completely closed system. (NEDO)

  8. Binding mechanisms for histamine and agmatine ligands in plasmid deoxyribonucleic acid purifications.

    Science.gov (United States)

    Sousa, Ângela; Pereira, Patrícia; Sousa, Fani; Queiroz, João A

    2014-10-31

    Histamine and agmatine amino acid derivatives were immobilized into monolithic disks, in order to combine the specificity and selectivity of the ligand with the high mass transfer and binding capacity offered by monolithic supports, to purify potential plasmid DNA biopharmaceuticals. Different elution strategies were explored by changing the type and salt concentration, as well as the pH, in order to understand the retention pattern of different plasmids isoforms The pVAX1-LacZ supercoiled isoform was isolated from a mixture of pDNA isoforms by using NaCl increasing stepwise gradient and also by ammonium sulfate decreasing stepwise gradient, in both histamine and agmatine monoliths. Acidic pH in the binding buffer mainly strengthened ionic interactions with both ligands in the presence of sodium chloride. Otherwise, for histamine ligand, pH values higher than 7 intensified hydrophobic interactions in the presence of ammonium sulfate. In addition, circular dichroism spectroscopy studies revealed that the binding and elution chromatographic conditions, such as the combination of high ionic strength with extreme pH values can reversibly influence the structural stability of the target nucleic acid. Therefore, ascending sodium chloride gradients with pH manipulation can be preferable chromatographic conditions to be explored in the purification of plasmid DNA biopharmaceuticals, in order to avoid the environmental impact of ammonium sulfate. Copyright © 2014. Published by Elsevier B.V.

  9. Enhanced extraction and purification of plasmid DNA from escherichia coli by applying a hybrid magnetic nanocomposite

    Energy Technology Data Exchange (ETDEWEB)

    Silva, R.J. da; Chavez-Guajardo, A.E.; Medina-llamas, J.C.; Alcaraz-Espinoza, J.J.; Melo, C.P. de [Universidade Federal de Pernambuco (UFPE), PE (Brazil)

    2016-07-01

    Full text: Plasmid DNA (pDNA), a special kind of nucleic acid usually found in bacteria, is a small molecule physically distinct from chromosomal DNA that can replicate independently. This genetic material has been used in a wide set of biotechnological methodologies, such as genetic engineering, production of recombinant drugs and gene therapy, among others. In all these applications, the extraction and purification of pDNA appears as a crucial step. In this work, we describe the synthesis of a polyaniline and maghemite (PANI/?-Fe2O3) magnetic nanocomposite (MNC) and its use in a new Escherichia coli (E. coli) pDNA extraction and purification protocol. We have used transmission electron microscopy (TEM), UV-Vis spectroscopy, infrared spectroscopy (FTIR), X-ray diffraction (XRD), dynamic light scattering (DLS) and magnetic measurements to characterize the MNC, which was synthesized through an emulsion polymerization method. The yield, purity and quality of the pDNA extracted by using our proposed MNC protocol were evaluated through UV-Vis, agarose gel electrophoreses and PCR techniques, respectively. After comparing our results to those obtained by use of a commercial kit (Promega Wizard Plus SV Minipreps), we suggest that the novel protocol here proposed appears as a competitive alternative methodology. Not only the purification step can be completed within only 10 min, but the high adsorption capacity of the MNC results in pDNA yields that are almost twice the best values obtained by using the commercial kit. Hence, this new MNC methodology can be of general interest and find widespread use in different types of biomedical applications. (author)

  10. A rapid and economic in-house DNA purification method using glass syringe filters.

    Directory of Open Access Journals (Sweden)

    Yun-Cheol Kim

    Full Text Available BACKGROUND: Purity, yield, speed and cost are important considerations in plasmid purification, but it is difficult to achieve all of these at the same time. Currently, there are many protocols and kits for DNA purification, however none maximize all four considerations. METHODOLOGY/PRINCIPAL FINDINGS: We now describe a fast, efficient and economic in-house protocol for plasmid preparation using glass syringe filters. Plasmid yield and quality as determined by enzyme digestion and transfection efficiency were equivalent to the expensive commercial kits. Importantly, the time required for purification was much less than that required using a commercial kit. CONCLUSIONS/SIGNIFICANCE: This method provides DNA yield and quality similar to that obtained with commercial kits, but is more rapid and less costly.

  11. Process optimisation for anion exchange monolithic chromatography of 4.2kbp plasmid vaccine (pcDNA3F).

    Science.gov (United States)

    Ongkudon, Clarence M; Danquah, Michael K

    2010-10-15

    Anion exchange monolithic chromatography is increasingly becoming a prominent tool for plasmid DNA purification but no generic protocol is available to purify all types of plasmid DNA. In this work, we established a simple framework and used it to specifically purify a plasmid DNA model from a clarified alkaline-lysed plasmid-containing cell lysate. The framework involved optimising ligand functionalisation temperature (30-80°C), mobile phase flow rate (0.1-1.8mL/min), monolith pore size (done by changing the porogen content in the polymerisation reaction by 50-80%), buffer pH (6-10), ionic strength of binding buffer (0.3-0.7M) and buffer gradient elution slope (1-10% buffer B/min). We concluded that preferential pcDNA3F adsorption and optimum resolution could be achieved within the tested conditions by loading the clarified cell lysate into 400nm pore size of monolith in 0.7M NaCl (pH 6) of binding buffer followed by increasing the NaCl concentration to 1.0M at 3%B/min. Copyright © 2010 Elsevier B.V. All rights reserved.

  12. Nanospines incorporation into the structure of the hydrophobic cryogels via novel cryogelation method: an alternative sorbent for plasmid DNA purification.

    Science.gov (United States)

    Üzek, Recep; Uzun, Lokman; Şenel, Serap; Denizli, Adil

    2013-02-01

    In this study, it was aimed to prepare hydrophobic cryogels for plasmid DNA (pDNA) purification from Escherichia coli lysate. The hydrophobicity was achieved by incorporating a hydrophobic ligand, N-methacryloyl-(L)-phenylalanine (MAPA), into the cryogel backbone. In addition to the conventional cryogelation process, freeze-drying step was included to create nanospines. Three different cryogels {poly(2-hydoxyethyl methacrylate-N-methacryloyl-L-phenylalanine)-freeze dried, [P(HEMA-MAPA)-FD]; poly(2-hydoxyethyl methacrylate-N-methacryloyl-L-phenylalanine, [P(HEMA-MAPA)] and poly(2-hydoxyethyl methacrylate)-freeze dried, [P(HEMA)-FD]} were prepared, characterized, and used for DNA (salmon sperm DNA) adsorption studies from aqueous solution. The specific surface areas of cryogels were determined to be 21.4 m(2)/g for P(HEMA)-FD, 17.65 m(2)/g for P(HEMA-MAPA) and 36.0 m(2)/g for P(HEMA-MAPA)-FD. The parameters affecting adsorption such as temperature, initial DNA concentration, salt type and concentration were examined in continuous mode. The maximum adsorption capacities were observed as 45.31 mg DNA/g, 27.08 mg DNA/g and 1.81 mg DNA/g for P(HEMA-MAPA)-FD, P(HEMA-MAPA) and P(HEMA)-FD, respectively. Desorption process was performed using acetate buffer (pH 5.50) without salt. First, pDNA was isolated from E. coli lysate and the purity of pDNA was then determined by agarose gel electrophoresis. Finally, the chromatographic performance of P(HEMA-MAPA)-FD cryogel for pDNA purification was tested in FPLC. The resolution (R(s)) was 2.84, and the specific selectivity for pDNA was 237.5-folds greater than all impurities. Copyright © 2012 Elsevier B.V. All rights reserved.

  13. Surface processes during purification of InP quantum dots

    Directory of Open Access Journals (Sweden)

    Natalia Mordvinova

    2014-08-01

    Full Text Available Recently, a new simple and fast method for the synthesis of InP quantum dots by using phosphine as phosphorous precursor and myristic acid as surface stabilizer was reported. Purification after synthesis is necessary to obtain samples with good optical properties. Two methods of purification were compared and the surface processes which occur during purification were studied. Traditional precipitation with acetone is accompanied by a small increase in photoluminescence. It occurs that during the purification the hydrolysis of the indium precursor takes place, which leads to a better surface passivation. The electrophoretic purification technique does not increase luminescence efficiency but yields very pure quantum dots in only a few minutes. Additionally, the formation of In(OH3 during the low temperature synthesis was explained. Purification of quantum dots is a very significant part of postsynthetical treatment that determines the properties of the material. But this subject is not sufficiently discussed in the literature. The paper is devoted to the processes that occur at the surface of quantum dots during purification. A new method of purification, electrophoresis, is investigated and described in particular.

  14. Purification of influenza deoxyribonucleic acid-based vaccine using agmatine monolith.

    Science.gov (United States)

    Bicho, D; Caramelo-Nunes, C; Sousa, A; Sousa, F; Queiroz, J A; Tomaz, C T

    2016-02-15

    Lately, researchers have made several efforts to improve vaccine production to fight highly contagious respiratory diseases like influenza. One of the most promising options for reducing the impact of this virus is DNA vaccination. However, a large quantity of highly pure plasmid DNA (pDNA) is necessary to attain this goal. The present work describes the production and purification of the plasmid NTC7482-41H-VA2HA expressing influenza virus hemagglutinin using an agmatine monolith. This ligand was chosen to purify supercoiled (sc) pDNA from complex lysates because of its versatile multimodal character. Its natural intervention in several biological systems together with its similarity with the highly studied arginine ligand allowed the development of a simpler and more specific purification process. Agmatine works under two strategies: descending ammonium sulfate gradient and ascending sodium chloride gradient. Furthermore, pH manipulation revealed an important role in pDNA isoforms selectivity. Dynamic binding capacity (DBC) experiments were performed varying different parameters and showed an increase with pDNA concentration, while high flow rate and high pH had the opposite effect. Sc pDNA was purified with high yield and was efficient with respect to cell transfection and cell viability. This monolith showed to be appropriate to purify the plasmid NTC7482-41H-VA2HA, providing a valuable tool for pDNA influenza vaccines preparation. Copyright © 2016. Published by Elsevier B.V.

  15. Processing of Nonconjugative Resistance Plasmids by Conjugation Nicking Enzyme of Staphylococci

    Energy Technology Data Exchange (ETDEWEB)

    Pollet, Rebecca M.; Ingle, James D.; Hymes, Jeff P.; Eakes, Thomas C.; Eto, Karina Yui; Kwong, Stephen M.; Ramsay, Joshua P.; Firth, Neville; Redinbo, Matthew R. (Curtin U.); (Sydney); (UNC)

    2016-01-04

    mechanism of antimicrobial resistance transfer in bacteria such asStaphylococcus aureusis an important step toward potentially slowing the spread of antimicrobial-resistant infections. This work establishes protein-DNA interactions essential for the transfer of theStaphylococcus aureusmultiresistance plasmid pSK41 by its relaxase, NES. This enzyme also processed variantoriT-like sequences found on numerous plasmids previously considered nontransmissible, suggesting that in conjunction with an uncharacterized accessory protein, these plasmids may be transferred horizontally via a relaxase intransmechanism. These findings have important implications for our understanding of staphylococcal resistance plasmid evolution.

  16. Rational and systematic protein purification process development: the next generation.

    Science.gov (United States)

    Nfor, Beckley K; Verhaert, Peter D E M; van der Wielen, Luuk A M; Hubbuch, Jürgen; Ottens, Marcel

    2009-12-01

    Current biopharmaceutical manufacturing strongly relies on using purification platform processes, offering harmonization of practices and speed-to-market. However, the ability of such processes to respond quickly to anticipated higher quality and capacity demands is under question. Here, we describe novel approaches for purification process development that incorporate biothermodynamics, modern high throughput experimentation and simulation tools. Such development leads to production platform-specific databases containing thermodynamic protein descriptors of major host cell proteins over a range of experimental conditions. This will pave the way for in silico purification process development, providing better process understanding and the potential to respond quickly to product quality and market demands. Future efforts will focus on improving this field further and enabling more rationale in process development.

  17. Quantification bias caused by plasmid DNA conformation in quantitative real-time PCR assay.

    Science.gov (United States)

    Lin, Chih-Hui; Chen, Yu-Chieh; Pan, Tzu-Ming

    2011-01-01

    Quantitative real-time PCR (qPCR) is the gold standard for the quantification of specific nucleic acid sequences. However, a serious concern has been revealed in a recent report: supercoiled plasmid standards cause significant over-estimation in qPCR quantification. In this study, we investigated the effect of plasmid DNA conformation on the quantification of DNA and the efficiency of qPCR. Our results suggest that plasmid DNA conformation has significant impact on the accuracy of absolute quantification by qPCR. DNA standard curves shifted significantly among plasmid standards with different DNA conformations. Moreover, the choice of DNA measurement method and plasmid DNA conformation may also contribute to the measurement error of DNA standard curves. Due to the multiple effects of plasmid DNA conformation on the accuracy of qPCR, efforts should be made to assure the highest consistency of plasmid standards for qPCR. Thus, we suggest that the conformation, preparation, quantification, purification, handling, and storage of standard plasmid DNA should be described and defined in the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) to assure the reproducibility and accuracy of qPCR absolute quantification.

  18. Plasmid segregation mechanisms

    DEFF Research Database (Denmark)

    Ebersbach, G.; Gerdes, Kenn

    2005-01-01

    Bacterial plasmids encode partitioning (par) loci that ensure ordered plasmid segregation prior to cell division. par loci come in two types: those that encode actin-like ATPases and those that encode deviant Walker-type ATPases. ParM, the actin-like ATPase of plasmid R1, forms dynamic filaments...... that segregate plasmids paired at mid-cell to daughter cells. Like microtubules, ParM filaments exhibit dynamic instability (i.e., catastrophic decay) whose regulation is an important component of the DNA segregation process. The Walker box ParA ATPases are related to MinD and form highly dynamic, oscillating...... filaments that are required for the subcellular movement and positioning of plasmids. The role of the observed ATPase oscillation is not yet understood. However, we propose a simple model that couples plasmid segregation to ParA oscillation. The model is consistent with the observed movement...

  19. Effect of water purification process in radioactive content: analysis on small scale purification plants

    International Nuclear Information System (INIS)

    Lopez del Rio, H.; Quiroga S, J. C.; Davila R, J. I.; Mireles G, F.

    2009-10-01

    Water from small scale purification plants is a low cost alternative for consumers in comparison to the bottled commercial presentations. Because of its low cost per liter, the consumption of this product has increased in recent years, stimulating in turn the installation of purification systems for these small businesses. The purpose of this study was to estimate the efficiency of small scale purification systems located in the cities of Zacatecas and Guadalupe, Zacatecas, to reduce the radioactive content of water. It was measured the total alpha and beta activity in water samples of entry and exit to process, through the liquid scintillation technique. In general it was observed that the process is more efficient in removing alpha that beta activity. The fraction of total alpha activity removed varied between 27 and 100%, while between 0 and 77% of the total beta activity was removed by the analyzed plants. In all cases, the total radioactivity level was lower than the maximum permissible value settled by the official mexican standard for drinking water. (Author)

  20. Molecular processes as basis for plasmid-mediated bacterial UV-light resistance and mutagenesis

    International Nuclear Information System (INIS)

    Aleshkin, G.I.; Brukhanskij, G.V.; Skavronskaya, A.G.

    1985-01-01

    The increase of UV-resistance and UV-induced mutagenesis by lambda 1 pint intmid as well as molecular-genetic mechanisms of plasmid participation in reparation and DNA replication and its degradation after UV-irradiation in plasmid cells on pKM101 plasmid model have been investigated. Data testifying to the necessity of intmid integration in chromosome as obligatory stage of intmid participation in increasing UV-resistance of bacterial cells are obtained. It has been found that intmid raises UV-resistance of cells and increases respectively the UV-induced reverants efficiency. On the basis of the experiment data the conclusion is drawn that the intmid capacity to raise UV-resistance and, possibly, mutagenesis is bound not only with its integration into chromosome but also with pol A + chromosome replication by dependendent imtmid replication complex. It is shown that pKM101 plasmid ensures functioning in E coli cells of inducible, chloroamphenicol-resistant DNA replication, highly resistant to UV-light harmful effect and that the volume of excision reparation in E. coli cells carrying pKM101 plasmid is increased as compared with the volume of reparation in plasmid legs cells. The combination of the data obtained gives grounds to the authors to assume that inducible replication, inducible reparation of DNA and inducible decrease of DNA degradation determined by pKM101 plasmid may serve as recA + lexA + basis dependent increase of UV-resistance and mutagenesis and that these processes provide the possibility of functioning of integrative replication mechanism of plasmid participation in ensuring UV-resistance and mutagenesis of plants

  1. Molecular Cloning Expression And Purification Studies With An ORF Of Mycobacterium Tuberculosis

    Directory of Open Access Journals (Sweden)

    Chiranjibi Chaudhary

    2017-08-01

    Full Text Available The study was initiated to develop a recombinant strain for expression and production of large scale protein and to develop its purification protocol. The MRAORF-X was amplified from the genomic DNA of M. tuberculosis H37Ra. The amplicon was successfully cloned in a cloning vector pGEM-T Easy and transformed in cloning host DH5amp945. Recombinant clones were identified by blue-white screening and insert presence was confirmed by restriction digestion of plasmid isolated from white colonies. Expression vector pET32a was used for protein expression. The recombinant plasmid was transformed into expression host BL21 and protein expression was checked by SDS-PAGE. The desired protein was approximately 60 kDa in size including tags. The purification protocol was established for purification from inclusion bodies. The purity of purified protein was assessed by SDS-PAGE gel run and presence of a single band at 60 kDa suggested that the inclusion bodies were a good source of purified protein.

  2. Resident enhanced repair: novel repair process action on plasmid DNA transformed into Escherichia coli K-12

    International Nuclear Information System (INIS)

    Strike, P.; Roberts, R.J.

    1982-01-01

    The survival of UV-irradiated DNA of plasmid NTP16 was monitored after its transformation into recipient cells containing an essentially homologous undamaged plasmid, pLV9. The presence of pLV9 resulted in a substantial increase in the fraction of damaged NTP16 molecules which survived in the recipient cells. This enhanced survival requires the host uvrA + and uvrB + gene products, but not the host recA + gene product. The requirement for both homologous DNA and the uvrA + gene products suggests that a novel repair process may act on plasmid DNA. Possible mechanisms for this process are considered

  3. A one-step miniprep for the isolation of plasmid DNA and lambda phage particles.

    Directory of Open Access Journals (Sweden)

    George Lezin

    Full Text Available Plasmid DNA minipreps are fundamental techniques in molecular biology. Current plasmid DNA minipreps use alkali and the anionic detergent SDS in a three-solution format. In addition, alkali minipreps usually require additional column-based purification steps and cannot isolate other extra-chromosomal elements, such as bacteriophages. Non-ionic detergents (NIDs have been used occasionally as components of multiple-solution plasmid DNA minipreps, but a one-step approach has not been developed. Here, we have established a one-tube, one-solution NID plasmid DNA miniprep, and we show that this approach also isolates bacteriophage lambda particles. NID minipreps are more time-efficient than alkali minipreps, and NID plasmid DNA performs better than alkali DNA in many downstream applications. In fact, NID crude lysate DNA is sufficiently pure to be used in digestion and sequencing reactions. Microscopic analysis showed that the NID procedure fragments E. coli cells into small protoplast-like components, which may, at least in part, explain the effectiveness of this approach. This work demonstrates that one-step NID minipreps are a robust method to generate high quality plasmid DNA, and NID approaches can also isolate bacteriophage lambda particles, outperforming current standard alkali-based minipreps.

  4. A low inventory adsorptive process for tritium extraction and purification

    International Nuclear Information System (INIS)

    Keefer, B.; Bora, B.; Chew, M.; Rump, M.; Kveton, O.K.

    1990-08-01

    The fuel cycles of future fusion power systems present a diverse spectrum of challenges to gas separation technology, for extraction, concentration, purification and confinement of tritium in fusion fuel cycles. Economic and safety factors motivate process design for minimum tritium inventory, functional simplicity, and overall reliability. A new gas separation process with some features of interest to fusion has been demonstrated under the auspices of the Canadian Fusion Fuels Technology Project. This process (Thermally Coupled Pressure Swing Adsorption or 'TCPSA') is potentially applicable to several fusion applications for separation purification of hydrogen, notably for tritium extraction from breeder blanket purge helium. Recent experimental tests have been directed toward fusion applications, primarily extraction and concentration of tritium-rich hydrogen from the blanket purge helium stream, and also considering purification of this and other hydrogen isotope streams such as the plasma exhaust. For example, hydrogen at 0.1% concentration in helium has been extracted in a TCPSA module operating at 195 K, with the process performed in a single working space to achieve simultaneous high extraction and concentration of the hydrogen. With methane or carbon oxides as the impurities, substantially complete separation is achieved by the same apparatus at ambient temperature. Engineering projections for scale-up to ITER blanket purge extraction and purification applications indicate a low working inventory of tritium

  5. Poly(hydroxyethyl methacrylate) based magnetic nanoparticles for plasmid DNA purification from Escherichia coli lysate

    International Nuclear Information System (INIS)

    Perçin, Işık; Karakoç, Veyis; Akgöl, Sinan; Aksöz, Erol; Denizli, Adil

    2012-01-01

    The aim of this study is to prepare poly(hydroxyethyl methacrylate-N-methacryloyl-(L)-histidine) [PHEMAH] magnetic nanoparticles for plasmid DNA (pDNA) purification from Escherichia coli (E. coli) cell lysate. Magnetic nanoparticles were produced by surfactant free emulsion polymerization. mPHEMAH nanoparticles were characterized by elemental analysis, Fourier transform infrared spectroscopy (FTIR), atomic force microscopy (AFM), vibrating sample magnetometer (VSM), electron spin resonance (ESR), thermogravimetric analyses (TGA) and transmission electron microscopy (TEM). Surface area, average particle size and size distribution were also performed. Specific surface area of the mPHEMAH nanoparticles was found to be 1180 m 2 /g. Elemental analysis of MAH for nitrogen was estimated as 0.18 mmol/g polymer. The amount of pDNA adsorbed onto the mPHEMAH nanoparticles first increased and then reached a saturation value at around 1.0 mg/mL of pDNA concentration. Compared with the mPHEMA nanoparticles (50 μg/g polymer), the pDNA adsorption capacity of the mPHEMAH nanoparticles (154 mg/g polymer) was improved significantly due to the MAH incorporation into the polymeric matrix. The maximum pDNA adsorption was achieved at 25 °C. The overall recovery of pDNA was calculated as 92%. The mPHEMAH nanoparticles could be used six times without decreasing the pDNA adsorption capacity significantly. The results indicate that the PHEMAH nanoparticles promise high selectivity for pDNA. - Highlights: ► Magnetic nanoparticles have several advantages over conventional adsorbents. ► MAH acted as the pseudospecific ligand, ligand immobilization step was eliminated. ► pDNA adsorption amount was 154 mg/g. ► Fifty-fold capacity increase was obtained when compared to conventional matrices.

  6. Expression and purification of recombinant Shiga toxin 2B from ...

    African Journals Online (AJOL)

    sunny t

    2016-05-25

    May 25, 2016 ... MATERIALS AND METHODS. Bacterial strains, plasmid and media ... toxin 2B gene after purified by wizard genomic DNA purification kit. (Promega, USA) ..... This result was approximately two times higher compared to Halo .... manual, 3rd Eds. New York: Cold Spring Harbor Laboratory Press,. Cold Spring ...

  7. Application of hydrometallurgy techniques in quartz processing and purification: a review

    Science.gov (United States)

    Lin, Min; Lei, Shaomin; Pei, Zhenyu; Liu, Yuanyuan; Xia, Zhangjie; Xie, Feixiang

    2018-04-01

    Although there have been numerous studies on separation and purification of metallic minerals by hydrometallurgy techniques, applications of the chemical techniques in separation and purification of non-metallic minerals are rarely reported. This paper reviews disparate areas of study into processing and purification of quartz (typical non-metallic ore) in an attempt to summarize current work, as well as to suggest potential for future consolidation in the field. The review encompasses chemical techniques of the quartz processing including situations, progresses, leaching mechanism, scopes of application, advantages and drawbacks of micro-bioleaching, high temperature leaching, high temperature pressure leaching and catalyzed high temperature pressure leaching. Traditional leaching techniques including micro-bioleaching and high temperature leaching are unequal to demand of modern glass industry for quality of quartz concentrate because the quartz products has to be further processed. High temperature pressure leaching and catalyzed high temperature pressure leaching provide new ways to produce high-grade quartz sand with only one process and lower acid consumption. Furthermore, the catalyzed high temperature pressure leaching realizes effective purification of quartz with extremely low acid consumption (no using HF or any fluoride). It is proposed that, by integrating the different chemical processes of quartz processing and expounding leaching mechanisms and scopes of application, the research field as a monopolized industry would benefit.

  8. Origin and Evolution of Rickettsial Plasmids.

    Directory of Open Access Journals (Sweden)

    Khalid El Karkouri

    Full Text Available Rickettsia species are strictly intracellular bacteria that have undergone a reductive genomic evolution. Despite their allopatric lifestyle, almost half of the 26 currently validated Rickettsia species have plasmids. In order to study the origin, evolutionary history and putative roles of rickettsial plasmids, we investigated the evolutionary processes that have shaped 20 plasmids belonging to 11 species, using comparative genomics and phylogenetic analysis between rickettsial, microbial and non-microbial genomes.Plasmids were differentially present among Rickettsia species. The 11 species had 1 to 4 plasmid (s with a size ranging from 12 kb to 83 kb. We reconstructed pRICO, the last common ancestor of the current rickettsial plasmids. pRICO was vertically inherited mainly from Rickettsia/Orientia chromosomes and diverged vertically into a single or multiple plasmid(s in each species. These plasmids also underwent a reductive evolution by progressive gene loss, similar to that observed in rickettsial chromosomes, possibly leading to cryptic plasmids or complete plasmid loss. Moreover, rickettsial plasmids exhibited ORFans, recent gene duplications and evidence of horizontal gene transfer events with rickettsial and non-rickettsial genomes mainly from the α/γ-proteobacteria lineages. Genes related to maintenance and plasticity of plasmids, and to adaptation and resistance to stress mostly evolved under vertical and/or horizontal processes. Those involved in nucleotide/carbohydrate transport and metabolism were under the influence of vertical evolution only, whereas genes involved in cell wall/membrane/envelope biogenesis, cycle control, amino acid/lipid/coenzyme and secondary metabolites biosynthesis, transport and metabolism underwent mainly horizontal transfer events.Rickettsial plasmids had a complex evolution, starting with a vertical inheritance followed by a reductive evolution associated with increased complexity via horizontal gene

  9. Research progress of novel adsorption processes in water purification:A review

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    As an effective, efficient, and economic approach for water purification, adsorbents and adsorption processes have been widely studied and applied in different aspects for a long time. In the recent years, a lot of novel adsorption processes have been developed for enhancing the efficiency of removing the organic and inorganic contaminants from water. This article reviews some new adsorbents and advanced adsorption methods that specialize in their compositions, structures, functions, and characteristics used in water treatment. The review emphasizes adsorption/catalytic oxidation process, adsorption/catalytic reduction process, adsorption coupled with redox process, biomimetic sorbent and its sorption behaviors of POPs, and modified adsorbents and their water purification efficiency.

  10. Poly(hydroxyethyl methacrylate) based magnetic nanoparticles for plasmid DNA purification from Escherichia coli lysate

    Energy Technology Data Exchange (ETDEWEB)

    Percin, Is Latin-Small-Letter-Dotless-I k [Department of Biology, Hacettepe University, Ankara (Turkey); Karakoc, Veyis [Department of Chemistry, Biochemistry Division, Hacettepe University, Ankara (Turkey); Akgoel, Sinan [Department of Biochemistry, Ege University, Izmir (Turkey); Aksoez, Erol [Department of Biology, Hacettepe University, Ankara (Turkey); Denizli, Adil, E-mail: denizli@hacettepe.edu.tr [Department of Chemistry, Biochemistry Division, Hacettepe University, Ankara (Turkey)

    2012-07-01

    The aim of this study is to prepare poly(hydroxyethyl methacrylate-N-methacryloyl-(L)-histidine) [PHEMAH] magnetic nanoparticles for plasmid DNA (pDNA) purification from Escherichia coli (E. coli) cell lysate. Magnetic nanoparticles were produced by surfactant free emulsion polymerization. mPHEMAH nanoparticles were characterized by elemental analysis, Fourier transform infrared spectroscopy (FTIR), atomic force microscopy (AFM), vibrating sample magnetometer (VSM), electron spin resonance (ESR), thermogravimetric analyses (TGA) and transmission electron microscopy (TEM). Surface area, average particle size and size distribution were also performed. Specific surface area of the mPHEMAH nanoparticles was found to be 1180 m{sup 2}/g. Elemental analysis of MAH for nitrogen was estimated as 0.18 mmol/g polymer. The amount of pDNA adsorbed onto the mPHEMAH nanoparticles first increased and then reached a saturation value at around 1.0 mg/mL of pDNA concentration. Compared with the mPHEMA nanoparticles (50 {mu}g/g polymer), the pDNA adsorption capacity of the mPHEMAH nanoparticles (154 mg/g polymer) was improved significantly due to the MAH incorporation into the polymeric matrix. The maximum pDNA adsorption was achieved at 25 Degree-Sign C. The overall recovery of pDNA was calculated as 92%. The mPHEMAH nanoparticles could be used six times without decreasing the pDNA adsorption capacity significantly. The results indicate that the PHEMAH nanoparticles promise high selectivity for pDNA. - Highlights: Black-Right-Pointing-Pointer Magnetic nanoparticles have several advantages over conventional adsorbents. Black-Right-Pointing-Pointer MAH acted as the pseudospecific ligand, ligand immobilization step was eliminated. Black-Right-Pointing-Pointer pDNA adsorption amount was 154 mg/g. Black-Right-Pointing-Pointer Fifty-fold capacity increase was obtained when compared to conventional matrices.

  11. Comparative symbiotic plasmid analysis indicates that symbiosis gene ancestor type affects plasmid genetic evolution.

    Science.gov (United States)

    Wang, X; Zhao, L; Zhang, L; Wu, Y; Chou, M; Wei, G

    2018-07-01

    Rhizobial symbiotic plasmids play vital roles in mutualistic symbiosis with legume plants by executing the functions of nodulation and nitrogen fixation. To explore the gene composition and genetic constitution of rhizobial symbiotic plasmids, comparison analyses of 24 rhizobial symbiotic plasmids derived from four rhizobial genera was carried out. Results illustrated that rhizobial symbiotic plasmids had higher proportion of functional genes participating in amino acid transport and metabolism, replication; recombination and repair; carbohydrate transport and metabolism; energy production and conversion and transcription. Mesorhizobium amorphae CCNWGS0123 symbiotic plasmid - pM0123d had similar gene composition with pR899b and pSNGR234a. All symbiotic plasmids shared 13 orthologous genes, including five nod and eight nif/fix genes which participate in the rhizobia-legume symbiosis process. These plasmids contained nod genes from four ancestors and fix genes from six ancestors. The ancestral type of pM0123d nod genes was similar with that of Rhizobium etli plasmids, while the ancestral type of pM0123d fix genes was same as that of pM7653Rb. The phylogenetic trees constructed based on nodCIJ and fixABC displayed different topological structures mainly due to nodCIJ and fixABC ancestral type discordance. The study presents valuable insights into mosaic structures and the evolution of rhizobial symbiotic plasmids. This study compared 24 rhizobial symbiotic plasmids that included four genera and 11 species, illuminating the functional gene composition and symbiosis gene ancestor types of symbiotic plasmids from higher taxonomy. It provides valuable insights into mosaic structures and the evolution of symbiotic plasmids. © 2018 The Society for Applied Microbiology.

  12. Development of partitioning process: purification of DIDPA

    Energy Technology Data Exchange (ETDEWEB)

    Watanabe, Masayuki; Morita, Yasuji; Kubota, Masumitsu [Japan Atomic Energy Research Inst., Tokai, Ibaraki (Japan). Tokai Research Establishment

    1998-04-01

    The partitioning process has developed and demonstrated that the solvent extraction with diisodecylphosphoric acid (DIDPA) can successfully separate transuranium elements from a high-level liquid waste. In the solvent extraction, DIDPA is decomposed by radiolysis and hydrolysis. The main degradation product is monoisodecyl phosphoric acid (MIDPA). Ethylene glycol has been used for removing the product by a solvent extraction method. However this method has two drawbacks that two phases separate slowly and the used ethylene glycol is not regeneratable. First it was found that the addition of acetone or methanol with 20 volume % improved the phase separation. Then a new purification method was developed by using an aqueous solution of methanol or acetone. The new purification method is as excellent as the ethylene glycol method for the removal of MIDPA. (author)

  13. [Alcohol-purification technology and its particle sedimentation process in manufactory of Fufang Kushen injection].

    Science.gov (United States)

    Liu, Xiaoqian; Tong, Yan; Wang, Jinyu; Wang, Ruizhen; Zhang, Yanxia; Wang, Zhimin

    2011-11-01

    Fufang Kushen injection was selected as the model drug, to optimize its alcohol-purification process and understand the characteristics of particle sedimentation process, and to investigate the feasibility of using process analytical technology (PAT) on traditional Chinese medicine (TCM) manufacturing. Total alkaloids (calculated by matrine, oxymatrine, sophoridine and oxysophoridine) and macrozamin were selected as quality evaluation markers to optimize the process of Fufang Kushen injection purification with alcohol. Process parameters of particulate formed in the alcohol-purification, such as the number, density and sedimentation velocity, were also determined to define the sedimentation time and well understand the process. The purification process was optimized as that alcohol is added to the concentrated extract solution (drug material) to certain concentration for 2 times and deposited the alcohol-solution containing drug-material to sediment for some time, i.e. 60% alcohol deposited for 36 hours, filter and then 80% -90% alcohol deposited for 6 hours in turn. The content of total alkaloids was decreased a little during the depositing process. The average settling time of particles with the diameters of 10, 25 microm were 157.7, 25.2 h in the first alcohol-purified process, and 84.2, 13.5 h in the second alcohol-purified process, respectively. The optimized alcohol-purification process remains the marker compositions better and compared with the initial process, it's time saving and much economy. The manufacturing quality of TCM-injection can be controlled by process. PAT pattern must be designed under the well understanding of process of TCM production.

  14. Purification process of recombinant monoclonal antibodies with mixed mode chromatography.

    Science.gov (United States)

    Maria, Sophie; Joucla, Gilles; Garbay, Bertrand; Dieryck, Wilfrid; Lomenech, Anne-Marie; Santarelli, Xavier; Cabanne, Charlotte

    2015-05-08

    An innovative process to purify mAb from CHO cell culture supernatant was developed. This three-step process involved two mixed mode resins and an anion exchange membrane. We used a human IgG mixture to determine the optimal conditions for each purification step. Thereafter, the whole process was evaluated and improved for the purification of a recombinant mAb produced in the supernatant of CHO cells. Once optimized, yield and purity of 88% and 99.9%, respectively were comparable to those obtained in a conventional process based on a capture step using protein A. In addition, aggregates, HCPs and DNA levels in the purified fraction were below regulatory specifications. Then we used mass spectrometry to identify contaminating proteins in the antibody fraction in order to highlight the behavior of HCPs. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. Rapid and inexpensive method for isolating plasmid DNA

    International Nuclear Information System (INIS)

    Aljanabi, S. M.; Al-Awadi, S. J.; Al-Kazaz, A. A.; Baghdad Univ.

    1997-01-01

    A small-scale and economical method for isolating plasmid DNA from bacteria is described. The method provides DNA of suitable quality for most DNA manipulation techniques. This DNA can be used for restriction endonuclease digestion, southern blot hybridization, nick translation and end labeling of DNA probes, Polymerase Chain Reaction (PCR) -based techniques, transformation, DNA cycle-sequencing, and Chain-termination method for DNA sequencing. The entire procedure is adapted to 1.5 ml microfuge tubes and takes approximately 30 mins. The DNA isolated by this method has the same purity produced by CTAB and cesium chloride precipitation and purification procedures respectively. The two previous methods require many hours to obtain the final product and require the use of very expensive equipment as ultracentrifuge. This method is well suited for the isolation of plasmid DNA from a large number of bacterial samples and in a very short time and low cost in laboratories where chemicals, expensive equipment and finance are limited factors in conducting molecular research. (authors). 11refs. 11refs

  16. Optimization of mass flow rate in RGTT200K coolant purification for Carbon Monoxide conversion process

    International Nuclear Information System (INIS)

    Sumijanto; Sriyono

    2016-01-01

    Carbon monoxide is a species that is difficult to be separated from the reactor coolant helium because it has a relatively small molecular size. So it needs a process of conversion from carbon monoxide to carbondioxide. The rate of conversion of carbon monoxide in the purification system is influenced by several parameters including concentration, temperature and mass flow rate. In this research, optimization of the mass flow rate in coolant purification of RGTT200K for carbon monoxide conversion process was done. Optimization is carried out by using software Super Pro Designer. The rate of reduction of reactant species, the growth rate between the species and the species products in the conversion reactions equilibrium were analyzed to derive the mass flow rate optimization of purification for carbon monoxide conversion process. The purpose of this study is to find the mass flow rate of purification for the preparation of the basic design of the RGTT200K coolant helium purification system. The analysis showed that the helium mass flow rate of 0.6 kg/second resulted in an un optimal conversion process. The optimal conversion process was reached at a mass flow rate of 1.2 kg/second. A flow rate of 3.6 kg/second – 12 kg/second resulted in an ineffective process. For supporting the basic design of the RGTT200K helium purification system, the mass flow rate for carbon monoxide conversion process is suggested to be 1.2 kg/second. (author)

  17. Decisional tool to assess current and future process robustness in an antibody purification facility.

    Science.gov (United States)

    Stonier, Adam; Simaria, Ana Sofia; Smith, Martin; Farid, Suzanne S

    2012-07-01

    Increases in cell culture titers in existing facilities have prompted efforts to identify strategies that alleviate purification bottlenecks while controlling costs. This article describes the application of a database-driven dynamic simulation tool to identify optimal purification sizing strategies and visualize their robustness to future titer increases. The tool harnessed the benefits of MySQL to capture the process, business, and risk features of multiple purification options and better manage the large datasets required for uncertainty analysis and optimization. The database was linked to a discrete-event simulation engine so as to model the dynamic features of biopharmaceutical manufacture and impact of resource constraints. For a given titer, the tool performed brute force optimization so as to identify optimal purification sizing strategies that minimized the batch material cost while maintaining the schedule. The tool was applied to industrial case studies based on a platform monoclonal antibody purification process in a multisuite clinical scale manufacturing facility. The case studies assessed the robustness of optimal strategies to batch-to-batch titer variability and extended this to assess the long-term fit of the platform process as titers increase from 1 to 10 g/L, given a range of equipment sizes available to enable scale intensification efforts. Novel visualization plots consisting of multiple Pareto frontiers with tie-lines connecting the position of optimal configurations over a given titer range were constructed. These enabled rapid identification of robust purification configurations given titer fluctuations and the facility limit that the purification suites could handle in terms of the maximum titer and hence harvest load. Copyright © 2012 American Institute of Chemical Engineers (AIChE).

  18. Contribution to the study of new hydrogen production, purification and storage processes

    International Nuclear Information System (INIS)

    Manaud, Jean-Pierre

    1984-01-01

    This research thesis addresses the various aspects of hydrogen production, purification and process within the scope of hydrogen-based energy production. Hydrogen production is achieved by water decomposition through a thermo-chemical process. The author reports the thermodynamic assessment of a water decomposition thermo-chemical cycle for chlorine and sulphur-related cycles. He reports the experimental investigation of hydrogen purification by selective diffusion, the study of contamination of a CeMg12 alloy by nitrogen, oxygen and water vapour with application to hydrogen storage under the form of hydrides [fr

  19. Reversible entrapment of plasmid deoxyribonucleic acid on different chromatographic supports.

    Science.gov (United States)

    Gabor, Boštjan; Černigoj, Urh; Barut, Miloš; Štrancar, Aleš

    2013-10-11

    HPLC based analytical assay is a powerful technique that can be used to efficiently monitor plasmid DNA (pDNA) purity and quantity throughout the entire purification process. Anion exchange monolithic and non-porous particle based stationary phases were used to study the recovery of the different pDNA isoforms from the analytical column. Three differently sized pDNA molecules of 3.0kbp, 5.2kbp and 14.0kbp were used. Plasmid DNA was injected onto columns under the binding conditions and the separation of the isoforms took place by increasing the ionic strength of the elution buffer. While there was no substantial decrease of the recovered supercoiled and linear isoforms of the pDNA with the increase of the plasmid size and with the increase of the flow rate (recoveries in all cases larger than 75%), a pronounced decrease of the oc isoform recovery was observed. The entrapment of the oc pDNA isoform occurred under non-binding conditions as well. The partial oc isoform elution from the column could be achieved by decreasing the flow rate of the elution mobile phase. The results suggested a reversible entrapment of the oc isoform in the restrictions within the pores of the monolithic material as well as within the intra-particle space of the non-porous particles. This phenomenon was observed on both types of the stationary phase morphologies and could only be connected to the size of a void space through which the pDNA needs to migrate. A prediction of reversible pDNA entrapment was successfully estimated with the calculation of Peclet numbers, Pe, which defines the ratio between a convective and diffusive mass transport. Copyright © 2013 Elsevier B.V. All rights reserved.

  20. Plasmids in Gram negatives: molecular typing of resistance plasmids.

    Science.gov (United States)

    Carattoli, Alessandra

    2011-12-01

    A plasmid is defined as a double stranded, circular DNA molecule capable of autonomous replication. By definition, plasmids do not carry genes essential for the growth of host cells under non-stressed conditions but they have systems which guarantee their autonomous replication also controlling the copy number and ensuring stable inheritance during cell division. Most of the plasmids confer positively selectable phenotypes by the presence of antimicrobial resistance genes. Plasmids evolve as an integral part of the bacterial genome, providing resistance genes that can be easily exchanged among bacteria of different origin and source by conjugation. A multidisciplinary approach is currently applied to study the acquisition and spread of antimicrobial resistance in clinically relevant bacterial pathogens and the established surveillance can be implemented by replicon typing of plasmids. Particular plasmid families are more frequently detected among Enterobacteriaceae and play a major role in the diffusion of specific resistance genes. For instance, IncFII, IncA/C, IncL/M, IncN and IncI1 plasmids carrying extended-spectrum beta-lactamase genes and acquired AmpC genes are currently considered to be "epidemic resistance plasmids", being worldwide detected in Enterobacteriaceae of different origin and sources. The recognition of successful plasmids is an essential first step to design intervention strategies preventing their spread. Copyright © 2011 Elsevier GmbH. All rights reserved.

  1. In Silico Detection and Typing of Plasmids using PlasmidFinder and Plasmid Multilocus Sequence Typing

    DEFF Research Database (Denmark)

    Carattoli, Alessandra; Zankari, Ea; García-Fernández, Aurora

    2014-01-01

    In the work presented here, we designed and developed two easy-to-use Web tools for in silico detection and characterization of whole-genome sequence (WGS) and whole-plasmid sequence data from members of the family Enterobacteriaceae. These tools will facilitate bacterial typing based on draft...... genomes of multidrug-resistant Enterobacteriaceae species by the rapid detection of known plasmid types. Replicon sequences from 559 fully sequenced plasmids associated with the family Enterobacteriaceae in the NCBI nucleotide database were collected to build a consensus database for integration...... sequences identified in the 559 fully sequenced plasmids. For plasmid multilocus sequence typing (pMLST) analysis, a database that is updated weekly was generated from www.pubmlst.org and integrated into a Web tool called pMLST. Both databases were evaluated using draft genomes from a collection...

  2. The importance of the ammonia purification process in ammonia-water absorption systems

    International Nuclear Information System (INIS)

    Fernandez-Seara, Jose; Sieres, Jaime

    2006-01-01

    Practical experience in working with ammonia-water absorption systems shows that the ammonia purification process is a crucial issue in order to obtain an efficient and reliable system. In this paper, the detrimental effects of the residual water content in the vapour refrigerant are described and quantified based on the system design variables that determine the effectiveness of the purification process. The study has been performed considering a single stage system with a distillation column with complete condensation. The ammonia purification effectiveness of the column is analysed in terms of the efficiencies in the stripping and rectifying sections and the reflux ratio. By varying the efficiencies from 0 to 1, systems with neither the rectifying nor stripping section, with either the rectifying or stripping section, or with both sections can be considered. The impact of the ammonia purification process on the absorption system performance is studied based on the column efficiencies and reflux ratio; and its effects on refrigerant concentration, system COP, system pressures and main system mass flow rates and concentrations are analysed. When the highest efficiency rectifying sections are used a combination of generation temperature and reflux ratio which leads to optimum COP values is found. The analysis covers different operating conditions with air and water cooled systems from refrigeration to air conditioning applications by changing the evaporation temperature. The importance of rectification in each kind of application is evaluated

  3. Magnetic bead purification of labeled DNA fragments forhigh-throughput capillary electrophoresis sequencing

    Energy Technology Data Exchange (ETDEWEB)

    Elkin, Christopher; Kapur, Hitesh; Smith, Troy; Humphries, David; Pollard, Martin; Hammon, Nancy; Hawkins, Trevor

    2001-09-15

    We have developed an automated purification method for terminator sequencing products based on a magnetic bead technology. This 384-well protocol generates labeled DNA fragments that are essentially free of contaminates for less than $0.005 per reaction. In comparison to laborious ethanol precipitation protocols, this method increases the phred20 read length by forty bases with various DNA templates such as PCR fragments, Plasmids, Cosmids and RCA products. Our method eliminates centrifugation and is compatible with both the MegaBACE 1000 and ABIPrism 3700 capillary instruments. As of September 2001, this method has produced over 1.6 million samples with 93 percent averaging 620 phred20 bases as part of Joint Genome Institutes Production Process.

  4. Process for purification of gas mixtures

    Energy Technology Data Exchange (ETDEWEB)

    Vasiliev, S Z; Letitschevskij, V I; Maergojz, I I; Michailov, L A; Puschkarev, L I

    1977-06-23

    The process relates to the purification of gas mixtures of N, H, and Ar, or N and H, or N and O which contain CO, CO/sub 2/ and water vapour. Single-stage adsorption occurs under standard pressure at temperatures from -40 to +4/sup 0/C up to the point of CO penetration through the zeolite layer. Zeolite is of type A or X combined with Ca, Na, Ag, Cd, Co, Ni, Mn or a natural zeolite of the type klinoptilolite. Regeneration is achieved at constant temperature and pressure of 1-5x10/sup -1/ Torr or by heating to 120-600/sup 0/C.

  5. Purification of supercoiled DNA of plasmid Col E1 by RPC-5 chromatography

    Energy Technology Data Exchange (ETDEWEB)

    Best, A.N.; Allison, D.P.; Novelli, G.D.

    1981-07-01

    Col E1 DNA can be purified to a high degree by RPC-5 chromatography of a partially purified cell lysate with a very shallow linear NaC1 gradient at pH 7.8. Electron micrographs demonstrated that the purest fractions were composed of 93% supercoiled (form I) DNA and 7% open circular (form II) DNA. The actual chromatography can be accomplished in 13 to 14 h and is designed for the production of several milligrams of plasmid DNA.

  6. Production and pharmaceutical formulation of plasmid DNA vaccines

    NARCIS (Netherlands)

    van der Heijden, I.

    2013-01-01

    Research leading to the thesis ‘Production and pharmaceutical formulation of plasmid DNA vaccines‘ can be divided into two parts. The first part describes the development of a Good Manufacturing Practice (GMP) compliant plasmid DNA production process of pDNA vaccines for the treatment of Human

  7. The application of modified montmorillonite in the processes of baromembrane purification of water from U (VI)

    International Nuclear Information System (INIS)

    Yurlova, L.Yu.; Krivoruchko, A.P.

    2010-01-01

    The processes of uranium-containing water purification by ultra- and nanofiltration methods combined with the use of montmorillonite modified by polyethyleneimine are studied. It is shown that the application of montmorillonite allows one to obtain the high indices of the uranium-containing water purification by baromembrane methods.

  8. A Simulation of Pre-Arcing Plasma Discharge Processes in Water Purification

    International Nuclear Information System (INIS)

    Rodriguez-Mendez, B. G.; Piedad-Beneitez, A. de la; Lopez-Callejas, R.; Godoy-Cabrera, O. G.; Benitez-Read, J. S.; Pacheco-Sotelo, J. O.; Pena-Eguiluz, R.; Mercado-Cabrera, A.; Valencia-A, R.; Barocio, S. R.

    2006-01-01

    The simulation of a water purification system within a coaxial cylinder reactor operated by 1 kHz frequency plasma discharges in pre-arcing regimes is presented. In contrast with precedent works, this computational model considers three mechanisms of the system operation: (a) the relevant physical characteristics of water (b) the ionisation and expansion processes in the spark channel including the near-breakdown electric current generated by the rate of change of the effective capacitance and resistance in the discharge, and (c) the energy associated with this initial spark in the water. The outcome of the model seems to meet all main requirements to allow the design and construction of specific water purification technology devices

  9. Process and economic evaluation of the extraction and purification of recombinant beta-glucuronidase from transgenic corn

    Science.gov (United States)

    Evangelista; Kusnadi; Howard; Nikolov

    1998-07-01

    A process model for the recovery and purification of recombinant beta-glucuronidase (rGUS) from transgenic corn was developed, and the process economics were estimated. The base-case bioprocessing plant operates 7500 h/year processing 1.74 million (MM) kg of transgenic corn containing 0.015% (db) rGUS. The process consists of milling the corn into flour, extraction of protein by using 50 mM sodium phosphate buffer, and rGUS purification by ion exchange and hydrophobic interaction chromatography. About 137 kg of rGUS of 83% (db) purity can be produced annually. The production cost amounted to $43 000/kg of rGUS. The cost of milling, protein extraction, and rGUS purification accounted for 6, 40, and 48% of annual operating cost, respectively. The cost of transgenic corn was 31% of the raw material costs or 6% of the annual operating cost. About 78% of the cost of buffer and water were incurred in the protein extraction section, while 88% of other consumables were from the purification section. The sensitivity analysis indicated that rGUS can be produced profitably from corn even at the 0.015% (db) expression level, assuming a selling price of $100 000/kg GUS. An increase in rGUS expression levels up to 0.08% significantly improves the process economics.

  10. Industrial compatible re-growth of vertically aligned multiwall carbon nanotubes by ultrafast pure oxygen purification process

    DEFF Research Database (Denmark)

    Bu, Ian Y.Y.; Hou, Kai; Engstrøm, Daniel Southcott

    2011-01-01

    amorphous carbon and reactivate nickel catalyst. Controlling of the purification temperature is important for high yield CNTs, as excessive high annealing temperature results in deformation of the CNTs. Unlike hazardous wet purification treatments, purified CNTs remained vertically aligned and offer......Reproducible high-yield purification process of multiwalled carbon nanotubes (CNTs) was developed by thermal annealing in ultrapure oxygen. The optimized condition involves thermal annealing via a PID controlled heater in high purity oxygen at temperature of 450°C for 180s, which burns out...

  11. Materials measurement and accounting in an operating plutonium conversion and purification process. Phase I. Process modeling and simulation

    International Nuclear Information System (INIS)

    Thomas, C.C. Jr.; Ostenak, C.A.; Gutmacher, R.G.; Dayem, H.A.; Kern, E.A.

    1981-04-01

    A model of an operating conversion and purification process for the production of reactor-grade plutonium dioxide was developed as the first component in the design and evaluation of a nuclear materials measurement and accountability system. The model accurately simulates process operation and can be used to identify process problems and to predict the effect of process modifications

  12. Application of dielectric constant measurement in microwave sludge disintegration and wastewater purification processes.

    Science.gov (United States)

    Kovács, Petra Veszelovszki; Lemmer, Balázs; Keszthelyi-Szabó, Gábor; Hodúr, Cecilia; Beszédes, Sándor

    2018-05-01

    It has been numerously verified that microwave radiation could be advantageous as a pre-treatment for enhanced disintegration of sludge. Very few data related to the dielectric parameters of wastewater of different origins are available; therefore, the objective of our work was to measure the dielectric constant of municipal and meat industrial wastewater during a continuous flow operating microwave process. Determination of the dielectric constant and its change during wastewater and sludge processing make it possible to decide on the applicability of dielectric measurements for detecting the organic matter removal efficiency of wastewater purification process or disintegration degree of sludge. With the measurement of dielectric constant as a function of temperature, total solids (TS) content and microwave specific process parameters regression models were developed. Our results verified that in the case of municipal wastewater sludge, the TS content has a significant effect on the dielectric constant and disintegration degree (DD), as does the temperature. The dielectric constant has a decreasing tendency with increasing temperature for wastewater sludge of low TS content, but an adverse effect was found for samples with high TS and organic matter contents. DD of meat processing wastewater sludge was influenced significantly by the volumetric flow rate and power level, as process parameters of continuously flow microwave pre-treatments. It can be concluded that the disintegration process of food industry sludge can be detected by dielectric constant measurements. From technical purposes the applicability of dielectric measurements was tested in the purification process of municipal wastewater, as well. Determination of dielectric behaviour was a sensitive method to detect the purification degree of municipal wastewater.

  13. Process for the biological purification of waste water

    DEFF Research Database (Denmark)

    1992-01-01

    Process for the biological purification of waste water by the activated sludge method, the waste water being mixed with recirculated sludge and being subjected to an anaerobic treatment, before the waste water thus treated is alternately subjected to anoxic and aerobic treatments and the waste...... water thus treated is led into a clarification zone for settling sludge, which sludge is recirculated in order to be mixed with the crude waste water. As a result, a simultaneous reduction of the content both of nitrogen and phosphorus of the waste water is achieved....

  14. The bubble method of water purification

    Science.gov (United States)

    Smirnov, B. M.; Babaeva, N. Yu.; Naidis, G. V.; Panov, V. A.; Saveliev, A. S.; Son, E. E.; Tereshonok, D. V.

    2018-02-01

    The processes of water purification from admixture molecules are analyzed. The purification rate is limited due to a low diffusion coefficient of the admixture molecules in water. At non-small concentrations of the admixture molecules, the water purication can proceed through association of molecules in condensed nanoparticles which fall on the bottom of the water volume. The rate of association may be increased in an external electric field, but in reality this cannot change significantly the rate of the purification process. The bubble method of water purification is considered, where air bubbles formed at the bottom of the water volume, transfer admixture molecules to the interface. This method allows one to clean small water volumes fast. This mechanism of water purification is realized experimentally and exhibits the promises of the bubble purification method.

  15. Plasmid flux in Escherichia coli ST131 sublineages, analyzed by plasmid constellation network (PLACNET), a new method for plasmid reconstruction from whole genome sequences.

    Science.gov (United States)

    Lanza, Val F; de Toro, María; Garcillán-Barcia, M Pilar; Mora, Azucena; Blanco, Jorge; Coque, Teresa M; de la Cruz, Fernando

    2014-12-01

    Bacterial whole genome sequence (WGS) methods are rapidly overtaking classical sequence analysis. Many bacterial sequencing projects focus on mobilome changes, since macroevolutionary events, such as the acquisition or loss of mobile genetic elements, mainly plasmids, play essential roles in adaptive evolution. Existing WGS analysis protocols do not assort contigs between plasmids and the main chromosome, thus hampering full analysis of plasmid sequences. We developed a method (called plasmid constellation networks or PLACNET) that identifies, visualizes and analyzes plasmids in WGS projects by creating a network of contig interactions, thus allowing comprehensive plasmid analysis within WGS datasets. The workflow of the method is based on three types of data: assembly information (including scaffold links and coverage), comparison to reference sequences and plasmid-diagnostic sequence features. The resulting network is pruned by expert analysis, to eliminate confounding data, and implemented in a Cytoscape-based graphic representation. To demonstrate PLACNET sensitivity and efficacy, the plasmidome of the Escherichia coli lineage ST131 was analyzed. ST131 is a globally spread clonal group of extraintestinal pathogenic E. coli (ExPEC), comprising different sublineages with ability to acquire and spread antibiotic resistance and virulence genes via plasmids. Results show that plasmids flux in the evolution of this lineage, which is wide open for plasmid exchange. MOBF12/IncF plasmids were pervasive, adding just by themselves more than 350 protein families to the ST131 pangenome. Nearly 50% of the most frequent γ-proteobacterial plasmid groups were found to be present in our limited sample of ten analyzed ST131 genomes, which represent the main ST131 sublineages.

  16. [Replication of Streptomyces plasmids: the DNA nucleotide sequence of plasmid pSB 24.2].

    Science.gov (United States)

    Bolotin, A P; Sorokin, A V; Aleksandrov, N N; Danilenko, V N; Kozlov, Iu I

    1985-11-01

    The nucleotide sequence of DNA in plasmid pSB 24.2, a natural deletion derivative of plasmid pSB 24.1 isolated from S. cyanogenus was studied. The plasmid amounted by its size to 3706 nucleotide pairs. The G-C composition was equal to 73 per cent. The analysis of the DNA structure in plasmid pSB 24.2 revealed the protein-encoding sequence of DNA, the continuity of which was significant for replication of the plasmid containing more than 1300 nucleotide pairs. The analysis also revealed two A-T-rich areas of DNA, the G-C composition of which was less than 55 per cent and a DNA area with a branched pin structure. The results may be of value in investigation of plasmid replication in actinomycetes and experimental cloning of DNA with this plasmid as a vector.

  17. Plasmid Flux in Escherichia coli ST131 Sublineages, Analyzed by Plasmid Constellation Network (PLACNET), a New Method for Plasmid Reconstruction from Whole Genome Sequences

    Science.gov (United States)

    Garcillán-Barcia, M. Pilar; Mora, Azucena; Blanco, Jorge; Coque, Teresa M.; de la Cruz, Fernando

    2014-01-01

    Bacterial whole genome sequence (WGS) methods are rapidly overtaking classical sequence analysis. Many bacterial sequencing projects focus on mobilome changes, since macroevolutionary events, such as the acquisition or loss of mobile genetic elements, mainly plasmids, play essential roles in adaptive evolution. Existing WGS analysis protocols do not assort contigs between plasmids and the main chromosome, thus hampering full analysis of plasmid sequences. We developed a method (called plasmid constellation networks or PLACNET) that identifies, visualizes and analyzes plasmids in WGS projects by creating a network of contig interactions, thus allowing comprehensive plasmid analysis within WGS datasets. The workflow of the method is based on three types of data: assembly information (including scaffold links and coverage), comparison to reference sequences and plasmid-diagnostic sequence features. The resulting network is pruned by expert analysis, to eliminate confounding data, and implemented in a Cytoscape-based graphic representation. To demonstrate PLACNET sensitivity and efficacy, the plasmidome of the Escherichia coli lineage ST131 was analyzed. ST131 is a globally spread clonal group of extraintestinal pathogenic E. coli (ExPEC), comprising different sublineages with ability to acquire and spread antibiotic resistance and virulence genes via plasmids. Results show that plasmids flux in the evolution of this lineage, which is wide open for plasmid exchange. MOBF12/IncF plasmids were pervasive, adding just by themselves more than 350 protein families to the ST131 pangenome. Nearly 50% of the most frequent γ–proteobacterial plasmid groups were found to be present in our limited sample of ten analyzed ST131 genomes, which represent the main ST131 sublineages. PMID:25522143

  18. Plasmid flux in Escherichia coli ST131 sublineages, analyzed by plasmid constellation network (PLACNET, a new method for plasmid reconstruction from whole genome sequences.

    Directory of Open Access Journals (Sweden)

    Val F Lanza

    2014-12-01

    Full Text Available Bacterial whole genome sequence (WGS methods are rapidly overtaking classical sequence analysis. Many bacterial sequencing projects focus on mobilome changes, since macroevolutionary events, such as the acquisition or loss of mobile genetic elements, mainly plasmids, play essential roles in adaptive evolution. Existing WGS analysis protocols do not assort contigs between plasmids and the main chromosome, thus hampering full analysis of plasmid sequences. We developed a method (called plasmid constellation networks or PLACNET that identifies, visualizes and analyzes plasmids in WGS projects by creating a network of contig interactions, thus allowing comprehensive plasmid analysis within WGS datasets. The workflow of the method is based on three types of data: assembly information (including scaffold links and coverage, comparison to reference sequences and plasmid-diagnostic sequence features. The resulting network is pruned by expert analysis, to eliminate confounding data, and implemented in a Cytoscape-based graphic representation. To demonstrate PLACNET sensitivity and efficacy, the plasmidome of the Escherichia coli lineage ST131 was analyzed. ST131 is a globally spread clonal group of extraintestinal pathogenic E. coli (ExPEC, comprising different sublineages with ability to acquire and spread antibiotic resistance and virulence genes via plasmids. Results show that plasmids flux in the evolution of this lineage, which is wide open for plasmid exchange. MOBF12/IncF plasmids were pervasive, adding just by themselves more than 350 protein families to the ST131 pangenome. Nearly 50% of the most frequent γ-proteobacterial plasmid groups were found to be present in our limited sample of ten analyzed ST131 genomes, which represent the main ST131 sublineages.

  19. Correction in the efficiency of uranium purification process by solvent extraction

    International Nuclear Information System (INIS)

    Franca Junior, J.M.

    1981-01-01

    An uranium solvent extraction, of high purification, with full advantage of absorbed uranium in the begining of process, is described. Including a pulsed column, called correction column, the efficiency of whole process is increased, dispensing the recycling of uranium losses from leaching column. With the correction column the uranium losses go in continuity, for reextraction column, increasing the efficiency of process. The purified uranium is removed in the reextraction column in aqueous phase. The correction process can be carried out with full efficiency using pulsed columns or chemical mixer-settlers. (M.C.K.) [pt

  20. Statistical and Judgmental Criteria for Scale Purification

    DEFF Research Database (Denmark)

    Wieland, Andreas; Durach, Christian F.; Kembro, Joakim

    2017-01-01

    of scale purification, to critically analyze the current state of scale purification in supply chain management (SCM) research and to provide suggestions for advancing the scale-purification process. Design/methodology/approach A framework for making scale-purification decisions is developed and used...

  1. Plasmid profiles as indicators of the source of contamination of Staphylococcus aureus endemic within poultry processing plants.

    Science.gov (United States)

    Dodd, C E; Chaffey, B J; Waites, W M

    1988-06-01

    A total of 530 strains of Staphylococcus aureus were isolated from the defeathering machinery of a chicken processing plant and from neck skin samples of carcasses at different stages of processing in two visits 4 weeks apart. Eleven different plasmid profiles were detected in the isolates, eight being common to both visits. The plasmid profiles of the strains forming the majority of the population on the freshly slaughtered birds were rarely present in the strains isolated from the pluckers (except at the entry to the first plucker) and were present in only a small proportion of the strains isolated from carcasses after plucking. However, the profiles from the strains isolated from the pluckers on both visits were different from those forming the majority of the population on the incoming birds but formed the major part of the carcass flora after plucking, suggesting that such strains were endemic. These strains were found as a small proportion of the isolates made from the incoming birds, suggesting that this was the route by which the endemic strains were introduced into the plant. Such endemic strains exhibited a clumping growth, even in liquid shake culture, which may have made it easier for them to become established on the pluckers and to resist cleaning and disinfection. This clumping phenotype was correlated with the presence of a 7.5-megadalton plasmid.

  2. Characterization of pLAC1, a cryptic plasmid isolated from Lactobacillus acidipiscis and comparative analysis with its related plasmids.

    Science.gov (United States)

    Asteri, Ioanna-Areti; Papadimitriou, Konstantinos; Boutou, Effrossyni; Anastasiou, Rania; Pot, Bruno; Vorgias, Constantinos E; Tsakalidou, Effie

    2010-07-15

    The pLAC1 plasmid of Lactobacillus acidipiscis ACA-DC 1533, a strain isolated from traditional Kopanisti cheese, was characterised. Nucleotide sequence analysis revealed a circular molecule of 3478bp with a G+C content of 37.2%. Ab initio annotation indicated four putative open reading frames (orfs). orf1 and orf4 were found to encode a replication initiation protein (Rep) and a mobilization protein (Mob), respectively. The deduced products of orf2 and orf3 revealed no significant homology to other known proteins. However, in silico examination of the plasmid sequence supported the existence of a novel operon that includes rep, orf2 and orf3 in pLAC1 and that this operon is highly conserved also in plasmids pLB925A02, pSMA23, pLC88 and pC7. RT-PCR experiments allowed us to verify that these three genes are co-transcribed as a single polycistronic mRNA species. Furthermore, phylogenetic analysis of pLAC1 Rep and Mob proteins demonstrated that they may have derived from different plasmid origins, suggesting that pLAC1 is a product of a modular evolution process. Comparative analysis of full length nucleotide sequences of pLAC1 and related Lactobacillus plasmids showed that pLAC1 shares a very similar replication backbone with pLB925A02, pSMA23 and pLC88. In contrast, mob of pLAC1 was almost identical with the respective gene of plasmids pLAB1000, pLB4 and pPB1. These findings lead to the conclusion that pLAC1 acquired mob probably via an ancestral recombination event. Our overall work highlights the importance of characterizing plasmids deriving from non-starter 'wild' isolates in order to better appreciate plasmid divergence and evolution of lactic acid bacteria. 2010 Elsevier B.V. All rights reserved.

  3. Evaluation of the efficiency of the processes of purification of antimony to semiconductor grade purity

    International Nuclear Information System (INIS)

    Walis, L.; Rowinska, L.; Panczyk, E.

    1992-01-01

    A complex of techniques for purification of antimony from arsenic has been examined with the aid of radiotracer 76 As. The investigated processes comprised vacuum distillation, zone melting and remelting of the metal under artificial slags. The purification efficiencies for the above processes were high and amounted to 94% (for 30% of the charge), 50% (for 50% of the charge) and 99.5% (for 60% of the charge), respectively. Attempts were made to determine the kinetics of the separation of arsenic from antimony by distillation. The application of the radioactive tracer made it possible to determine rapidly the distribution of impurities after each stage of the process within a wide concentration range (10 -2 -10 -7 g/g). (author). 7 refs, 4 figs, 6 tabs

  4. A flow-through chromatography process for influenza A and B virus purification.

    Science.gov (United States)

    Weigel, Thomas; Solomaier, Thomas; Peuker, Alessa; Pathapati, Trinath; Wolff, Michael W; Reichl, Udo

    2014-10-01

    Vaccination is still the most efficient measure to protect against influenza virus infections. Besides the seasonal wave of influenza, pandemic outbreaks of bird or swine flu represent a high threat to human population. With the establishment of cell culture-based processes, there is a growing demand for robust, economic and efficient downstream processes for influenza virus purification. This study focused on the development of an economic flow-through chromatographic process avoiding virus strain sensitive capture steps. Therefore, a three-step process consisting of anion exchange chromatography (AEC), Benzonase(®) treatment, and size exclusion chromatography with a ligand-activated core (LCC) was established, and tested for purification of two influenza A virus strains and one influenza B virus strain. The process resulted in high virus yields (≥68%) with protein contamination levels fulfilling requirements of the European Pharmacopeia for production of influenza vaccines for human use. DNA was depleted by ≥98.7% for all strains. The measured DNA concentrations per dose were close to the required limits of 10ng DNA per dose set by the European Pharmacopeia. In addition, the added Benzonase(®) could be successfully removed from the product fraction. Overall, the presented downstream process could potentially represent a simple, robust and economic platform technology for production of cell culture-derived influenza vaccines. Copyright © 2014 Elsevier B.V. All rights reserved.

  5. Theoretical analysis of a biogas-fed PEMFC system with different hydrogen purifications: Conventional and membrane-based water gas shift processes

    International Nuclear Information System (INIS)

    Authayanun, Suthida; Aunsup, Pounyaporn; Patcharavorachot, Yaneeporn; Arpornwichanop, Amornchai

    2014-01-01

    Highlights: • Thermodynamic analysis of the biogas-fed PEMFC system is performed. • Conventional and membrane-based WGS processes for H 2 purification are studied. • A flowsheet model of the PEMFC system is developed. • Effect of key parameters on yields of H 2 and carbon in the biogas reformer is shown. • Performance of PEMFC systems with different H 2 purification processes is analyzed. - Abstract: This study presents a thermodynamic analysis of biogas reforming and proton electrolyte membrane fuel cell (PEMFC) integrated process with different hydrogen purifications: conventional and membrane-based water gas shift processes. The aim is to determine the optimal reforming process for hydrogen production from biogas in the PEMFC system. The formation of carbon is concerned in the hydrogen production. The simulation results show that increases in the steam-to-methane ratio and reformer temperature can improve the hydrogen yield and reduce the carbon formation. From the performance analysis, it is found that when the PEMFC is operated at high temperature and fuel utilization, the overall system efficiency enhances. The performance of the PEMFC system with the installation of a water gas shift membrane unit in the hydrogen purification step is slightly increased, compared with a conventional process

  6. Electron beam silicon purification

    Energy Technology Data Exchange (ETDEWEB)

    Kravtsov, Anatoly [SIA ' ' KEPP EU' ' , Riga (Latvia); Kravtsov, Alexey [' ' KEPP-service' ' Ltd., Moscow (Russian Federation)

    2014-11-15

    Purification of heavily doped electronic grade silicon by evaporation of N-type impurities with electron beam heating was investigated in process with a batch weight up to 50 kilos. Effective temperature of the melt, an indicative parameter suitable for purification process characterization was calculated and appeared to be stable for different load weight processes. Purified material was successfully approbated in standard CZ processes of three different companies. Each company used its standard process and obtained CZ monocrystals applicable for photovoltaic application. These facts enable process to be successfully scaled up to commercial volumes (150-300 kg) and yield solar grade silicon. (copyright 2014 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim) (orig.)

  7. Antibiotic resistance plasmids of Staphylococcus aureus and their clinical importance

    International Nuclear Information System (INIS)

    Lacey, R.W.

    1975-01-01

    A variety of plasmids were isolated physically, and most antibiotic resistance is thought to be plasmid mediated. A number of characters (e.g., resistance to erythromycin or methicillin, and production of pigment) are determined by genes that do not give clear indications of either plasmid or chromosomal location. Although the formation of a particular plasmid is probably, even in bacterial terms, a very rare event, once formed such an element can spread rapidly among the bacterial population. The spectacular increase in the incidence of penicillinase-producing hospital strains in the late 1940's could have been due in part to this process. Evidence is stronger, however, for the intercell transfer of recently isolated plasmids coding for resistance to fusidic acid (and penicillinase production), or for neomycin, or for tetracycline resistance. Study of bacterial plasmids can resolve fundamental biochemical problems, and give some insight into the life of the cell at the molecular level. But the immediate application of the study of staphylococcal plasmids may be directed towards improving the effectiveness of antibiotic therapy. The most important aspect of future anti-staphylococcal chemotherapy should thus be the limitation of the use of antibiotics, particularly for application to the skin and nose. (U.S.)

  8. Yeast transformation mediated by Agrobacterium strains harboring an Ri plasmid: comparative study between GALLS of an Ri plasmid and virE of a Ti plasmid.

    Science.gov (United States)

    Kiyokawa, Kazuya; Yamamoto, Shinji; Sato, Yukari; Momota, Naoto; Tanaka, Katsuyuki; Moriguchi, Kazuki; Suzuki, Katsunori

    2012-07-01

    Agrobacterium strains containing a Ti plasmid can transfer T-DNA not only to plants but also to fungi, including the yeast Saccharomyces cerevisiae. However, no Agrobacterium strain harboring an Ri plasmid has been evaluated in fungal transformation. Some Ri plasmids have GALLS , instead of virE1 and virE2. GALLS protein can functionally substitute in plant transformation for a structurally different protein VirE2. In this study, we compared the yeast transformation ability among Agrobacterium donors: a strain containing a Ti plasmid, strains harboring either an agropine-type or a mikimopine-type Ri plasmid, and a strain having a modified Ri plasmid supplemented with a Ti plasmid type virE operon. Agrobacterium strains possessing GALLS transformed yeast cells far less efficiently than the strain containing virE operon. Production of GALLS in recipient yeast cells improved the yeast transformation mediated by an Agrobacterium strain lacking neither GALLS nor virE operon. A reporter assay to detect mobilization of the proteins fused with Cre recombinase revealed that VirE2 protein is much more abundant in yeast cells than GALLS. Based on these results, we concluded that the low yeast transformability mediated by Agrobacterium strains having the Ri plasmid is because of low amount of mobilized GALLS in yeast cells. © 2012 The Authors Journal compilation © 2012 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.

  9. Transformation of Saccharomyces cerevisiae with UV-irradiated single-stranded plasmid.

    Science.gov (United States)

    Zgaga, Z

    1991-08-01

    UV-irradiated single-stranded replicative plasmids were used to transform different yeast strains. The low doses of UV used in this study (10-75 J/m2) caused a significant decrease in the transforming efficiency of plasmid DNA in the Rad+ strain, while they had no effect on transformation with double-stranded plasmids of comparable size. Neither the rev3 mutation, nor the rad18 or rad52 mutations influenced the efficiency of transformation with irradiated single-stranded plasmid. However, it was found to be decreased in the double rev3 rad52 mutant. Extracellular irradiation of plasmid that contains both URA3 and LEU2 genes (psLU) gave rise to up to 5% Leu- transformants among selected Ura+ ones in the repair-proficient strain. Induction of Leu- transformants was dose-dependent and only partially depressed in the rev3 mutant. These results suggest that both mutagenic and recombinational repair processes operate on UV-damaged single-stranded DNA in yeast.

  10. The various sodium purification techniques

    International Nuclear Information System (INIS)

    Courouau, J.L.; Masse, F.; Rodriguez, G.; Latge, C.; Redon, B.

    1997-01-01

    In the framework of sodium waste treatment, the sodium purification phase plays an essential role in the chain of operations leading to the transformation of the active sodium, considered as waste, into a stable sodium salt. The objectives of the purification operations are: To keep a low impurity level, particularly a low concentration in oxygen and hydrogen, in order to allow its transfer to a processing plant, and in order to avoid risks of plugging and/or corrosion in sodium facilities; To reduce the sodium activity in order to limit the dose rate close to the facilities, and in order to reduce the activity of the liquid and gaseous effluents. After a recall of the different kind of impurities that can be present in sodium, and of the different purification methods that could be associated with, the following points are highlighted: (i) Oxygen and hydrogen purification needs, and presentation of some selection criteria for a purification unit adapted to a sodium processing plant, as well as 2 cold trap concepts that are in accordance with these criteria: PSICHOS and PIRAMIDE. (ii) Tritium reduction in a bulk of liquid sodium by swamping, isotopic exchange, or permeation throughout a membrane. (iii) Caesium trapping on carbonaceous matrix. The main matrices used at present are R.V.C. (Reticulated Vitreous Carbon) and Actitex/Pica products. Tests in the laboratory and on an experimental device have demonstrated the performances of these materials, which are able to reduce sodium activity in Cs 134 and Cs 137 to very low values. The sodium purification processes as regards to the hydrogen, oxygen and caesium, that are aimed at facilitating the subsequent treatment of sodium, are therefore mastered operations. Regarding the operations associated with the reduction of the tritium activity, the methods are in the process of being qualified, or to be qualified. (author)

  11. CARTOGRAPHIE DU PLASMIDE pSU100, PLASMIDE CRYPTIQUE DE LACTOBACILLUS CASEI

    Directory of Open Access Journals (Sweden)

    F BENSALAH

    2003-06-01

    Ce plasmide appelé pSU100 a été cloné dans le vecteur de transformation pUC18 au site EcoRI chez E. coli JM103. Les profils électrophorétiques de restriction obtenus par des digestions simples, doubles et triples sous l’action de 33 endonucléases, ont contribué à l’élaboration d’une carte de restriction de ce plasmide. Cinq sites uniques ont été identifiés, ainsi que d’autres sites doubles et multiples. Une étude préliminaire du rôle physiologique de ce plasmide a permis de déceler une résistance à la kanamycine.

  12. Structural analysis of the ParR/parC plasmid partition complex

    DEFF Research Database (Denmark)

    Møller-Jensen, Jakob; Ringgaard, Simon; Mercogliano, Christopher P

    2007-01-01

    Accurate DNA partition at cell division is vital to all living organisms. In bacteria, this process can involve partition loci, which are found on both chromosomes and plasmids. The initial step in Escherichia coli plasmid R1 partition involves the formation of a partition complex between the DNA...

  13. Chlamydial plasmids and bacteriophages.

    Science.gov (United States)

    Pawlikowska-Warych, Małgorzata; Śliwa-Dominiak, Joanna; Deptuła, Wiesław

    2015-01-01

    Chlamydia are absolute pathogens of humans and animals; despite being rather well recognised, they are still open for discovery. One such discovery is the occurrence of extrachromosomal carriers of genetic information. In prokaryotes, such carriers include plasmids and bacteriophages, which are present only among some Chlamydia species. Plasmids were found exclusively in Chlamydia (C.) trachomatis, C. psittaci, C. pneumoniae, C. suis, C. felis, C. muridarum and C. caviae. In prokaryotic organisms, plasmids usually code for genes that facilitate survival of the bacteria in the environment (although they are not essential). In chlamydia, their role has not been definitely recognised, apart from the fact that they participate in the synthesis of glycogen and encode proteins responsible for their virulence. Furthermore, in C. suis it was evidenced that the plasmid is integrated in a genomic island and contains the tetracycline-resistance gene. Bacteriophages specific for chlamydia (chlamydiaphages) were detected only in six species: C. psittaci, C. abortus, C. felis, C. caviae C. pecorum and C. pneumoniae. These chlamydiaphages cause inhibition of the developmental cycle, and delay transformation of reticulate bodies (RBs) into elementary bodies (EBs), thus reducing the possibility of infecting other cells in time. Plasmids and bacteriophages can be used in the diagnostics of chlamydioses; although especially in the case of plasmids, they are already used for detection of chlamydial infections. In addition, bacteriophages could be used as therapeutic agents to replace antibiotics, potentially addressing the problem of increasing antibiotic-resistance among chlamydia.

  14. Purification and characterization of the d-xylose isomerase gene from Escherichia coli

    Energy Technology Data Exchange (ETDEWEB)

    Ho, N W.Y.; Rosenfeld, S; Stevis, P; Tsao, G T

    1983-11-01

    A DNA fragment containing both the Escherichia coli D-xylose isomerase (D-xylose ketol-isomerase, EC 5.3.1.5) gene and the D-xylulokinase (ATP: D-xylulose 5-phosphotransferase, EC 2.7.1.17) gene has been cloned on an E. coli plasmid. The D-xylose isomerase gene was separated from the D-xylulokinase gene by the construction of a new deletion plasmid, pLX7. The D-xylose isomerase gene cloned on pLX7 was found still to be an intact gene. The precise location of the D-xylose isomerase gene on the plasmid pLX7 was further determined by the construction of two more plasmids, pLX8 and pLX9. This is believed to be the first D-xylose isomerase gene that has been isolated and extensively purified from any organism. D-Xylose isomerase, the enzyme product of the D-xylose isomerase gene, is responsible for the conversion of D-xylose to D-xylulose, as well as D-glucose to D-fructose. It is widely believed that yeast cannot ferment D-xylose to ethanol primarily because of the lack of D-xylose isomerase in yeast. D-Xylose isomerase (also known as D-glucose isomerase) is also used for the commercial production of high-fructose syrups. The purification of the D-xylose isomerase gene may lead to the following industrial applications: (1) cloning and expression of the gene in yeast to make the latter organism capable of directly fermenting D-xylose to ethanol, and (2) cloning of the gene on a high-copy-number plasmid in a proper host to overproduce the enzyme, which should have a profound impact on the high-fructose syrup technology. 14 references.

  15. Mechanisms of Evolution in High-Consequence Drug Resistance Plasmids

    Directory of Open Access Journals (Sweden)

    Susu He

    2016-12-01

    Full Text Available The dissemination of resistance among bacteria has been facilitated by the fact that resistance genes are usually located on a diverse and evolving set of transmissible plasmids. However, the mechanisms generating diversity and enabling adaptation within highly successful resistance plasmids have remained obscure, despite their profound clinical significance. To understand these mechanisms, we have performed a detailed analysis of the mobilome (the entire mobile genetic element content of a set of previously sequenced carbapenemase-producing Enterobacteriaceae (CPE from the National Institutes of Health Clinical Center. This analysis revealed that plasmid reorganizations occurring in the natural context of colonization of human hosts were overwhelmingly driven by genetic rearrangements carried out by replicative transposons working in concert with the process of homologous recombination. A more complete understanding of the molecular mechanisms and evolutionary forces driving rearrangements in resistance plasmids may lead to fundamentally new strategies to address the problem of antibiotic resistance.

  16. Purification of crude biodiesel using dry washing and membrane technologies

    OpenAIRE

    Atadashi, I.M.

    2015-01-01

    Purification of crude biodiesel is mandatory for the fuel to meet the strict international standard specifications for biodiesel. Therefore, this paper carefully analyzed recently published literatures which deal with the purification of biodiesel. As such, dry washing technologies and the most recent membrane biodiesel purification process have been thoroughly examined. Although purification of biodiesel using dry washing process involving magnesol and ion exchange resins provides high-quali...

  17. Application and improvement of reciprocating-sieve plate extraction column in natural uranium extraction and purification process

    International Nuclear Information System (INIS)

    Wang Xuejun; Li Linyan; Liu Jing; Liu Xin; Yang Lifeng; Xiao Shaohua; Liu Hao

    2013-01-01

    Reciprocating-sieve plate extraction column is commonly used in the extraction process. Optimization and application were conducted successfully via production practice in some chemical and pharmaceutical plants, and good results are obtained while it is applied in the natural uranium extraction and purification process. The key component of reciprocating-sieve plate extraction column is gear-drive equipment in which drive motor serves as its core. Hence, it is important to select appropriate mode of speed regulation. In this paper, the principle and performance of several mode of speed regulation are compared. Both electromagnetic slip and frequency speed-regulation can be applied in general industrial process, but frequency speed-regulation with low energy cost can be used in wider operating range. The application of frequency speed-regulation mode used in reciprocating-sieve plate extraction column will increase the convenience and stability of natural uranium extraction and purification process. (authors)

  18. Relationship between the losses of furfural in the oil purification process and the furfural quality

    Energy Technology Data Exchange (ETDEWEB)

    Shiriaeva, G.P.; Baiburskaia, E.L.; Kalenik, G.S.; Martynenko, A.G.; Okhrimenko, N.V.

    1981-01-01

    It is demonstrated that the use of low quality furfural in the purification process of oil results in a situation where approximately 10% of the solvent delivered to these enterprises make up the avoidable losses.

  19. Large-scale preparation of plasmid DNA.

    Science.gov (United States)

    Heilig, J S; Elbing, K L; Brent, R

    2001-05-01

    Although the need for large quantities of plasmid DNA has diminished as techniques for manipulating small quantities of DNA have improved, occasionally large amounts of high-quality plasmid DNA are desired. This unit describes the preparation of milligram quantities of highly purified plasmid DNA. The first part of the unit describes three methods for preparing crude lysates enriched in plasmid DNA from bacterial cells grown in liquid culture: alkaline lysis, boiling, and Triton lysis. The second part describes four methods for purifying plasmid DNA in such lysates away from contaminating RNA and protein: CsCl/ethidium bromide density gradient centrifugation, polyethylene glycol (PEG) precipitation, anion-exchange chromatography, and size-exclusion chromatography.

  20. Purification process of natural graphite as anode for Li-ion batteries: chemical versus thermal

    Science.gov (United States)

    Zaghib, K.; Song, X.; Guerfi, A.; Rioux, R.; Kinoshita, K.

    The intercalation of Li ions in natural graphite that was purified by chemical and thermal processes was investigated. A new chemical process was developed that involved a mixed aqueous solution containing 30% H 2SO 4 and 30% NH xF y heated to 90 °C. The results of this process are compared to those obtained by heating the natural graphite from 1500 to 2400 °C in an inert environment (thermal process). The first-cycle coulombic efficiency of the purified natural graphite obtained by the chemical process is 91 and 84% after the thermal process at 2400 °C. Grinding the natural graphite before or after purification had no significant effect on electrochemical performance at low currents. However, grinding to a very small particle size before purification permitted optimization of the size distribution of the particles, which gives rise to a more homogenous electrode. The impurities in the graphite play a role as microabrasion agents during grinding which enhances its hardness and improves its mechanical properties. Grinding also modifies the particle morphology from a 2- to a 3-D structure (similar in shape to a potato). This potato-shaped natural graphite shows high reversible capacity at high current densities (about 90% at 1 C rate). Our analysis suggests that thermal processing is considerably more expensive than the chemical process to obtain purified natural graphite.

  1. Plasmid and chromosome partitioning: surprises from phylogeny

    DEFF Research Database (Denmark)

    Gerdes, Kenn; Møller-Jensen, Jakob; Bugge Jensen, Rasmus

    2000-01-01

    Plasmids encode partitioning genes (par) that are required for faithful plasmid segregation at cell division. Initially, par loci were identified on plasmids, but more recently they were also found on bacterial chromosomes. We present here a phylogenetic analysis of par loci from plasmids and chr...

  2. PLUTONIUM PURIFICATION PROCESS EMPLOYING THORIUM PYROPHOSPHATE CARRIER

    Science.gov (United States)

    King, E.L.

    1959-04-28

    The separation and purification of plutonium from the radioactive elements of lower atomic weight is described. The process of this invention comprises forming a 0.5 to 2 M aqueous acidffc solution containing plutonium fons in the tetravalent state and elements with which it is normally contaminated in neutron irradiated uranium, treating the solution with a double thorium compound and a soluble pyrophosphate compound (Na/sub 4/P/sub 2/O/sub 7/) whereby a carrier precipitate of thorium A method is presented of reducing neptunium and - trite is advantageous since it destroys any hydrazine f so that they can be removed from solutions in which they are contained is described. In the carrier precipitation process for the separation of plutonium from uranium and fission products including zirconium and columbium, the precipitated blsmuth phosphate carries some zirconium, columbium, and uranium impurities. According to the invention such impurities can be complexed and removed by dissolving the contaminated carrier precipitate in 10M nitric acid, followed by addition of fluosilicic acid to about 1M, diluting the solution to about 1M in nitric acid, and then adding phosphoric acid to re-precipitate bismuth phosphate carrying plutonium.

  3. Conjugal properties of the Sinorhizobium meliloti plasmid mobilome.

    Science.gov (United States)

    Pistorio, Mariano; Giusti, María A; Del Papa, María F; Draghi, Walter O; Lozano, Mauricio J; Tejerizo, Gonzalo Torres; Lagares, Antonio

    2008-09-01

    The biology and biochemistry of plasmid transfer in soil bacteria is currently under active investigation because of its central role in prokaryote adaptation and evolution. In this work, we examined the conjugal properties of the cryptic plasmids present in a collection of the N(2)-fixing legume-symbiont Sinorhizobium meliloti. The study was performed on 65 S. meliloti isolates recovered from 25 humic soils of Argentina, which were grouped into 22 plasmid-profile types [i.e. plasmid operational taxonomic units (OTUs)]. The cumulative Shannon index calculated for the observed plasmid profiles showed a clear saturation plateau, thus indicating an adequate representation of the S. meliloti plasmid-profile types in the isolates studied. The results show that isolates of nearly 14% of the plasmid OTUs hosted transmissible plasmids and that isolates of 29% of the plasmid OTUs were able to retransfer the previously characterized mobilizable-cryptic plasmid pSmeLPU88b to a third recipient strain. It is noteworthy that isolates belonging to 14% of the plasmid OTUs proved to be refractory to the entrance of the model plasmid pSmeLPU88b, suggesting either the presence of surface exclusion phenomena or the occurrence of restriction incompatibility with the incoming replicon. Incompatibility for replication between resident plasmids and plasmid pSmeLPU88b was observed in c. 20% of the OTUs. The results reported here reveal a widespread compatibility among the conjugal functions of the cryptic plasmids in S. meliloti, and this fact, together with the observed high proportion of existing donor genotypes, points to the extrachromosomal compartment of the species as being an extremely active plasmid mobilome.

  4. Construction and confirmation of the plasmid of human mitochondrial DNA 4977 bp deletion induced by ionizing radiation

    International Nuclear Information System (INIS)

    Chen Xiaosui; Zhou Lijun; Wang Yuxiao; Qu Jia; Feng Jiangbing; Lu Xue; Chen Deqing; Liu Qingjie

    2006-01-01

    Objective: To construct a stable plasmid that spanning deleted human mitochondrial DNA (mtDNA) 4977 bp induced by ionizing radiation and another one for control DNA fragment, in order to use in the human mitochondrial genome study in the future. Methods: The peripheral blood, which had no mtDNA 4977 bp deletion found in previous study, was exposed to 10 Gy 60 Co γ-rays in vitro. The total cell DNA was extracted and PCR was carried out: a nest-PCR of three-round PCR was used for the mtDNA 4977 bp deletion and one- round regular PCR was used for the control ND1 gene. The PCR products were used for transfection by electroporation and the positive clones were obtained after screening. The plasmid DNA was isolated and sequenced after enzymatic digestion and purification. The sequence result was BLASTed with the human mitochondrial genome. Results: The sizes of PCR products for the flanked 4977 bp deletion and the ND1 gene were similar with those predicted according to GeneBank. The sequences for the positive clones were above 99 per cent homologous with the human mitochondrial genome after BLASTed. Conclusion: The plasmids for deleted human mtDNA 4977 bp and control DNA fragment have been constructed successfully, and they could be used in the quality and quantity studies on human mtDNA 4977 bp deletion. (authors)

  5. Protein Structure Initiative Material Repository: an open shared public resource of structural genomics plasmids for the biological community

    Science.gov (United States)

    Cormier, Catherine Y.; Mohr, Stephanie E.; Zuo, Dongmei; Hu, Yanhui; Rolfs, Andreas; Kramer, Jason; Taycher, Elena; Kelley, Fontina; Fiacco, Michael; Turnbull, Greggory; LaBaer, Joshua

    2010-01-01

    The Protein Structure Initiative Material Repository (PSI-MR; http://psimr.asu.edu) provides centralized storage and distribution for the protein expression plasmids created by PSI researchers. These plasmids are a resource that allows the research community to dissect the biological function of proteins whose structures have been identified by the PSI. The plasmid annotation, which includes the full length sequence, vector information and associated publications, is stored in a freely available, searchable database called DNASU (http://dnasu.asu.edu). Each PSI plasmid is also linked to a variety of additional resources, which facilitates cross-referencing of a particular plasmid to protein annotations and experimental data. Plasmid samples can be requested directly through the website. We have also developed a novel strategy to avoid the most common concern encountered when distributing plasmids namely, the complexity of material transfer agreement (MTA) processing and the resulting delays this causes. The Expedited Process MTA, in which we created a network of institutions that agree to the terms of transfer in advance of a material request, eliminates these delays. Our hope is that by creating a repository of expression-ready plasmids and expediting the process for receiving these plasmids, we will help accelerate the accessibility and pace of scientific discovery. PMID:19906724

  6. Expansion of the IncX plasmid family for improved identification and typing of novel plasmids in drug-resistant Enterobacteriaceae

    DEFF Research Database (Denmark)

    Johnson, Timothy J.; Bielak, Eliza Maria; Fortini, Daniela

    2012-01-01

    and biofilm formation. Previous plasmid-based replicon typing procedures have indicated that the prevalence of IncX plasmids is low among members of the Enterobacteriaceae. However, examination of a number of IncX-like plasmid sequences and their occurrence in various organisms suggests that IncX plasmid...

  7. Giant linear plasmids in Streptomyces: a treasure trove of antibiotic biosynthetic clusters.

    Science.gov (United States)

    Kinashi, Haruyasu

    2011-01-01

    Many giant linear plasmids have been isolated from Streptomyces by using pulsed-field gel electrophoresis and some of them were found to carry an antibiotic biosynthetic cluster(s); SCP1 carries biosynthetic genes for methylenomycin, pSLA2-L for lankacidin and lankamycin, and pKSL for lasalocid and echinomycin. Accumulated data suggest that giant linear plasmids have played critical roles in genome evolution and horizontal transfer of secondary metabolism. In this review, I summarize typical examples of giant linear plasmids whose involvement in antibiotic production has been studied in some detail, emphasizing their finding processes and interaction with the host chromosomes. A hypothesis on horizontal transfer of secondary metabolism involving giant linear plasmids is proposed at the end.

  8. Denitrification of acid wastes from uranium purification processes

    International Nuclear Information System (INIS)

    Clark, F.E.; Francis, C.W.; Francke, H.C.; Strohecker, J.W.

    1975-11-01

    Laboratory and pilot-plant investigations have shown the technical feasibility of removing nitrates from neutralized acid wastes from uranium purification processes by biological denitrification, a dissimilatory process in which the nitrate ion is reduced to nitrogen gas by specific bacteria. The process requires anaerobic conditions and an organic carbon source, as well as other life-sustaining constituents. These denitrification studies produced process design information on a columnar denitrification plant and on continuous-flow, stirred-bed reactors. Denitrification, using packed columns, was found to be desirable for soluble salts, such as those of sodium and ammonium; denitrification, using stirred reactors, was found to be desirable for mixtures containing insoluble salts, such as those of calcium and aluminum. Packed columns were found to have denitrification rates ranging up to 122 grams of nitrate per day per cubic decimeter of column volume; stirred-bed reactors have been shown to have reaction rates near 10 grams of nitrate per day per cubic decimeter of reactor volume. The continuous-flow, stirred-bed reactors were selected for scaleup studies because of the solids-removal problems associated with packed columns when operating on feeds containing high concentrations of insoluble salts or ions which form insoluble salts with the products of the denitrification reaction

  9. Hydrogen purification by periodic adsorption

    Energy Technology Data Exchange (ETDEWEB)

    Barg, Christian; Secchi, Argimiro R.; Trierweiler, Jorge O. [Rio Grande do Sul Univ., Porto Alegre, RS (Brazil). Dept. de Engenharia Quimica]. E-mail: cbarg@enq.ufrgs.br; arge@enq.ufrgs.br; jorge@enq.ufrgs.br

    2000-07-01

    The periodic adsorption processes have been widely used for industrial applications, mainly because it spends less energy than the usual gas separation processes, like the cryogenic distillation. The largest commercial application of periodic adsorption processes is the pressure swing adsorption (PSA) applied to hydrogen purification. Although its wide use in the chemical and petrochemical industry, there are no reports in the open literature about complete modeling studies of a complex commercial unit, with multiple adsorbents and multiple beds and several feed components. This study has as objective the modeling, optimization and dynamical analysis of an industrial PSA unit for hydrogen purification. (author)

  10. Impact of co-carriage of IncA/C plasmids with additional plasmids on the transfer of antimicrobial resistance in Salmonella enterica isolates.

    Science.gov (United States)

    Han, Jing; Pendleton, Sean J; Deck, Joanna; Singh, Ruby; Gilbert, Jeffrey; Johnson, Timothy J; Sanad, Yasser M; Nayak, Rajesh; Foley, Steven L

    2018-04-20

    Antimicrobial resistance in Salmonella enterica is often plasmid encoded. A key resistance plasmid group is the incompatibility group (Inc) A/C plasmids that often carry multiple resistance determinants. Previous studies showed that IncA/C plasmids were often co-located with other plasmids. The current study was undertaken to evaluate the impact of plasmid co-carriage on antimicrobial resistance and plasmid transfer. A total of 1267 Salmonella isolates, representing multiple serotypes and sources were previously subjected to susceptibility testing and 251 isolates with resistance to at least 5 antimicrobial agents were identified for further study. Each isolate was subjected to PCR-based replicon typing, and those with IncA/C plasmids were selected for plasmid isolation, PCR-based mapping of IncA/C plasmid backbone genes, and conjugation assays to evaluate resistance plasmid transferability. Of the 87 identified IncA/C positive isolates, approximately 75% carried a plasmid with another identified replicon type, with the most common being I1 (39%), FIA, FIIA, FIB and HI2 (each 15%). PCR-based mapping indicated significant diversity in IncA/C backbone content, especially in regions encoding transfer-associated and hypothetical proteins. Conjugation experiments showed that nearly 68% of the isolates transferred resistance plasmids, with 90% containing additional identified plasmids or larger (>50 kb) non-typeable plasmids. The majority of IncA/C-positive strains were able to conjugally transfer antimicrobial resistance to the recipient, encoded by IncA/C and/or co-carried plasmids. These findings highlight the importance of co-located plasmids for resistance dissemination either by directly transferring resistance genes or by potentially providing the needed conjugation machinery for IncA/C plasmid transfer. Copyright © 2018. Published by Elsevier B.V.

  11. Venturi purification device and its application in purification of gaseous waste of nuclear facilities

    International Nuclear Information System (INIS)

    Kong Jinsong; Yu Ren; Yang Huanlei

    2013-01-01

    The working principle of Venturi purification device and its purification of aerosol have been described. Then, taking the gaseous iodine as an example, the absorption process of insoluble gas pollutants is discussed, the calculation methods of the gas-liquid contact area, mass transfer rate and efficiency of mass transfer are educed, and the factors that affect the efficiency of mass transfer are analyzed. (authors)

  12. Purification of Contaminated MIL-PRF-83282 Hydraulic Fluid Using the Pall Purifier and Multiple Process Configurations (Preprint)

    National Research Council Canada - National Science Library

    Snyder, Jr., Carl E; Gschwender, Lois J; Gunderson, Stephen L; Fultz, George W

    2006-01-01

    .... This report describes a project that evaluated the effectiveness of various hydraulic fluid purification process configurations on the removal of water and particulate contaminants from MIL-PRF-83282...

  13. Mechanisms of Evolution in High-Consequence Drug Resistance Plasmids.

    Science.gov (United States)

    He, Susu; Chandler, Michael; Varani, Alessandro M; Hickman, Alison B; Dekker, John P; Dyda, Fred

    2016-12-06

    The dissemination of resistance among bacteria has been facilitated by the fact that resistance genes are usually located on a diverse and evolving set of transmissible plasmids. However, the mechanisms generating diversity and enabling adaptation within highly successful resistance plasmids have remained obscure, despite their profound clinical significance. To understand these mechanisms, we have performed a detailed analysis of the mobilome (the entire mobile genetic element content) of a set of previously sequenced carbapenemase-producing Enterobacteriaceae (CPE) from the National Institutes of Health Clinical Center. This analysis revealed that plasmid reorganizations occurring in the natural context of colonization of human hosts were overwhelmingly driven by genetic rearrangements carried out by replicative transposons working in concert with the process of homologous recombination. A more complete understanding of the molecular mechanisms and evolutionary forces driving rearrangements in resistance plasmids may lead to fundamentally new strategies to address the problem of antibiotic resistance. The spread of antibiotic resistance among Gram-negative bacteria is a serious public health threat, as it can critically limit the types of drugs that can be used to treat infected patients. In particular, carbapenem-resistant members of the Enterobacteriaceae family are responsible for a significant and growing burden of morbidity and mortality. Here, we report on the mechanisms underlying the evolution of several plasmids carried by previously sequenced clinical Enterobacteriaceae isolates from the National Institutes of Health Clinical Center (NIH CC). Our ability to track genetic rearrangements that occurred within resistance plasmids was dependent on accurate annotation of the mobile genetic elements within the plasmids, which was greatly aided by access to long-read DNA sequencing data and knowledge of their mechanisms. Mobile genetic elements such as

  14. Characterization of new plasmids from methylotrophic bacteria.

    Science.gov (United States)

    Brenner, V; Holubová, I; Benada, O; Hubácek, J

    1991-07-01

    Several tens of methanol-utilizing bacterial strains isolated from soil were screened for the presence of plasmids. From the obligate methylotroph Methylomonas sp. strain R103a plasmid pIH36 (36 kb) was isolated and its restriction map was constructed. In pink-pigmented facultative methylotrophs (PPFM), belonging to the genus Methylobacterium four plasmids were detected: plasmids pIB200 (200 kb) and pIB14 (14 kb) in the strain R15d and plasmids pWU14 (14 kb) and pWU7 (7.8 kb) in the strain M17. Because of the small size and the presence of several unique REN sites (HindIII, EcoRI, NcoI), plasmid pWU7 was chosen for the construction of a vector for cloning in methylotrophs. Cointegrates pKWU7A and pKWU7B were formed between pWU7 and the E. coli plasmid pK19 Kmr, which were checked for conjugative transfer from E. coli into the methylotrophic host.

  15. Purification of crude biodiesel using dry washing and membrane technologies

    Directory of Open Access Journals (Sweden)

    I.M. Atadashi

    2015-12-01

    Full Text Available Purification of crude biodiesel is mandatory for the fuel to meet the strict international standard specifications for biodiesel. Therefore, this paper carefully analyzed recently published literatures which deal with the purification of biodiesel. As such, dry washing technologies and the most recent membrane biodiesel purification process have been thoroughly examined. Although purification of biodiesel using dry washing process involving magnesol and ion exchange resins provides high-quality biodiesel fuel, considerable amount of spent absorbents is recorded, besides the skeletal knowledge on its operating process. Further, recent findings have shown that biodiesel purification using membrane technique could offer high-quality biodiesel fuel with less wastewater discharges. Thus, both researchers and industries are expected to benefit from the development of membrane technique in purifying crude biodiesel. As well biodiesel purification via membranes has been shown to be environmentally friendly. For these reasons, it is important to explore and exploit membrane technology to purify crude biodiesel.

  16. Explanatory chapter: how plasmid preparation kits work.

    Science.gov (United States)

    Koontz, Laura

    2013-01-01

    To isolate plasmid DNA from bacteria using a commercial plasmid miniprep kit (if interested, compare this protocol with Isolation of plasmid DNA from bacteria). Copyright © 2013 Elsevier Inc. All rights reserved.

  17. An efficient procedure for the expression and purification of HIV-1 protease from inclusion bodies.

    Science.gov (United States)

    Nguyen, Hong-Loan Thi; Nguyen, Thuy Thi; Vu, Quy Thi; Le, Hang Thi; Pham, Yen; Trinh, Phuong Le; Bui, Thuan Phuong; Phan, Tuan-Nghia

    2015-12-01

    Several studies have focused on HIV-1 protease for developing drugs for treating AIDS. Recombinant HIV-1 protease is used to screen new drugs from synthetic compounds or natural substances. However, large-scale expression and purification of this enzyme is difficult mainly because of its low expression and solubility. In this study, we constructed 9 recombinant plasmids containing a sequence encoding HIV-1 protease along with different fusion tags and examined the expression of the enzyme from these plasmids. Of the 9 plasmids, pET32a(+) plasmid containing the HIV-1 protease-encoding sequence along with sequences encoding an autocleavage site GTVSFNF at the N-terminus and TEV plus 6× His tag at the C-terminus showed the highest expression of the enzyme and was selected for further analysis. The recombinant protein was isolated from inclusion bodies by using 2 tandem Q- and Ni-Sepharose columns. SDS-PAGE of the obtained HIV-1 protease produced a single band of approximately 13 kDa. The enzyme was recovered efficiently (4 mg protein/L of cell culture) and had high specific activity of 1190 nmol min(-1) mg(-1) at an optimal pH of 4.7 and optimal temperature of 37 °C. This procedure for expressing and purifying HIV-1 protease is now being scaled up to produce the enzyme on a large scale for its application. Copyright © 2015 Elsevier Inc. All rights reserved.

  18. Plasmid-derived DNA Strand Displacement Gates for Implementing Chemical Reaction Networks.

    Science.gov (United States)

    Chen, Yuan-Jyue; Rao, Sundipta D; Seelig, Georg

    2015-11-25

    DNA nanotechnology requires large amounts of highly pure DNA as an engineering material. Plasmid DNA could meet this need since it is replicated with high fidelity, is readily amplified through bacterial culture and can be stored indefinitely in the form of bacterial glycerol stocks. However, the double-stranded nature of plasmid DNA has so far hindered its efficient use for construction of DNA nanostructures or devices that typically contain single-stranded or branched domains. In recent work, it was found that nicked double stranded DNA (ndsDNA) strand displacement gates could be sourced from plasmid DNA. The following is a protocol that details how these ndsDNA gates can be efficiently encoded in plasmids and can be derived from the plasmids through a small number of enzymatic processing steps. Also given is a protocol for testing ndsDNA gates using fluorescence kinetics measurements. NdsDNA gates can be used to implement arbitrary chemical reaction networks (CRNs) and thus provide a pathway towards the use of the CRN formalism as a prescriptive molecular programming language. To demonstrate this technology, a multi-step reaction cascade with catalytic kinetics is constructed. Further it is shown that plasmid-derived components perform better than identical components assembled from synthetic DNA.

  19. Identification of IncA/C Plasmid Replication and Maintenance Genes and Development of a Plasmid Multilocus Sequence Typing Scheme.

    Science.gov (United States)

    Hancock, Steven J; Phan, Minh-Duy; Peters, Kate M; Forde, Brian M; Chong, Teik Min; Yin, Wai-Fong; Chan, Kok-Gan; Paterson, David L; Walsh, Timothy R; Beatson, Scott A; Schembri, Mark A

    2017-02-01

    Plasmids of incompatibility group A/C (IncA/C) are becoming increasingly prevalent within pathogenic Enterobacteriaceae They are associated with the dissemination of multiple clinically relevant resistance genes, including bla CMY and bla NDM Current typing methods for IncA/C plasmids offer limited resolution. In this study, we present the complete sequence of a bla NDM-1 -positive IncA/C plasmid, pMS6198A, isolated from a multidrug-resistant uropathogenic Escherichia coli strain. Hypersaturated transposon mutagenesis, coupled with transposon-directed insertion site sequencing (TraDIS), was employed to identify conserved genetic elements required for replication and maintenance of pMS6198A. Our analysis of TraDIS data identified roles for the replicon, including repA, a toxin-antitoxin system; two putative partitioning genes, parAB; and a putative gene, 053 Construction of mini-IncA/C plasmids and examination of their stability within E. coli confirmed that the region encompassing 053 contributes to the stable maintenance of IncA/C plasmids. Subsequently, the four major maintenance genes (repA, parAB, and 053) were used to construct a new plasmid multilocus sequence typing (PMLST) scheme for IncA/C plasmids. Application of this scheme to a database of 82 IncA/C plasmids identified 11 unique sequence types (STs), with two dominant STs. The majority of bla NDM -positive plasmids examined (15/17; 88%) fall into ST1, suggesting acquisition and subsequent expansion of this bla NDM -containing plasmid lineage. The IncA/C PMLST scheme represents a standardized tool to identify, track, and analyze the dissemination of important IncA/C plasmid lineages, particularly in the context of epidemiological studies. Copyright © 2017 American Society for Microbiology.

  20. Expression, purification, and activity assay of peptide deformylase from Escherichia coli and Staphylococcus aureus.

    Science.gov (United States)

    Che, Xuchun; Hu, Jinwei; Wang, Lijuan; Zhu, Zhifeng; Xu, Qiong; Lv, Junqiang; Fu, Zheng; Sun, Yajun; Sun, Jia; Lin, Gang; Lu, Rong; Yao, Zhi

    2011-11-01

    Peptide deformylase (PDF) is considered an attractive target for screening novel antibiotics. The PDF from Escherichia coli and Staphylococcus aureus are representative of the gram-negative species type of PDF (type I PDF) and the gram-positive species type of PDF (type II PDF), respectively. They could be used for screening broad-spectrum antibiotics. Herein, we cloned the def gene by PCR, inserted it into plasmid pET-22b-def, and transformed the plasmid into E. coli BL21 (DE3) cells, then the cells were induced by IPTG to express PDF. E. coli Ni(2+)-PDF was extracted and purified by ion-exchange chromatography and gel filtration chromatography. S. aureus PDFs were extracted and purified using the MagExtractor kit. The nickel form of S. aureus PDF was obtained by adding NiCl(2) to all reagents used for purification. Iron-enriched S. aureus PDF was obtained by adding FeCl(3) to the growth medium for E. coli BL21 (DE3) cells and adding FeCl(3) and catalase to all reagents used for purification. The activities of PDFs were analyzed, compared, and grouped according to the experimental conditions that produced optimal activity, and we used actinonin as an inhibitor of PDF and calculated the IC(50) value. We obtained high expression of E. coli and S. aureus PDF with high activity and stability. The function of PDFs was inhibited by actinonin in a dose-dependent manner. Results may be helpful for future mechanistic investigations of PDF as well as high-throughput screening for other PDF inhibitors.

  1. Behaviour analysis of uranium purification pilot plant taking into consideration the incorporation of a second column of extraction in the process flow chart

    International Nuclear Information System (INIS)

    Souza Barbosa, E.M. de.

    1979-01-01

    This work analyses the effect of the incorporation of a second column of extraction by solvents in pulsed plate columns in the Process of Uranium Purification. Operating with the aqueous phase of washing this second column of extraction recovers the dragged Uranium, increasing the efficiency of the process. The experiments were carried out in the display unit of Uranium purification in the Institute of Energetic and Nuclear Researches in Sao Paulo, where the dimension and the performance were studied with the following parameters: real increase in the efficiency and decontamination in the process. An efficiency superior to 98% in mass was obtained against 84,6% in the conventional process. With a performance similar to the first column of extract on the decontamination, about 99%, obtained in this column, maintained the grade of nuclear purity peculiar to the Process of Uranium Purification by solvents in pulsed plate columns. (author) [pt

  2. Plasmid-mediated UV-protection in Streptococcus lactis

    Energy Technology Data Exchange (ETDEWEB)

    Chopin, M.C.; Rouault, A. (Institut National de la Recherche Agronomique, Rennes (France). Lab. de Recherches de Technologie Laitiere); Moillo-Batt, A. (Institut National de la Sante et de la Recherche Medicale (INSERM), Hopital de Pontchaillon, 35 - Rennes (France))

    1985-02-01

    Streptococcus lactis strain IL594 contains 9 plasmids, designated pIL1 to pIL9. On the basis of protoplast-induced curing experiments the authors showed that derivatives containing pIL7 were resistant to UV-irradiation while derivatives lacking pIL7 were sensitive. The pIL7-determined UV-protection was confirmed by co-transfer of the plasmid and of the character into a plasmid-free derivative of S. lactis IL594. Moreover, prophage induction required higher UV-fluence in this derivative carrying pIL7 than in the plasmid-free strain. This is the first report of a plasmid-mediated UV-protection in group N streptococci.

  3. Plasmid-mediated UV-protection in Streptococcus lactis

    International Nuclear Information System (INIS)

    Chopin, M.-C.; Rouault, A.

    1985-01-01

    Streptococcus lactis strain IL594 contains 9 plasmids, designated pIL1 to pIL9. On the basis of protoplast-induced curing experiments the authors showed that derivatives containing pIL7 were resistant to UV-irradiation while derivatives lacking pIL7 were sensitive. The pIL7-determined UV-protection was confirmed by cotransfer of the plasmid and of the character into a plasmid-free derivative of S. lactis IL594. Moreover, prophage induction required higher UV-fluence in this derivative carrying pIL7 than in the plasmid-free strain. This is the first report of a plasmid-mediated UV-protection in group N streptococci. (orig.)

  4. Contribution to the study of the process of purification of plutonium by extraction with trilaurylamine

    International Nuclear Information System (INIS)

    Saey, Jean-Claude

    1966-01-01

    This work addresses the process of plutonium purification which uses trilaurylamine nitrate. In order to use this nitrate in its solid state and at ordinary temperature, a secondary solvent must be added which must have some properties: low volume mass and viscosity, high boiling and ignition temperatures, rather low miscibility with water, high stability in front of joint actions of nitric acid and radiations, and no reaction with the alkylammonium nitrate and the complex. Thus, the author addresses phenomena of immiscibility and identifies some important molecular characteristics which could lead to the selection of another secondary solvent than dodecane. The decalin seem interesting and its behaviour is studied. A mixing of dodecane and decalin is used as extraction mixing. The obtained results are discussed. Finally, the author notices that using this mixing in the plutonium purification process results in a large increase of metal concentrations and a decrease of risks of crystallisation, without any major drawback, in a continuously operating micro-industrial installation

  5. Behavior of IncQ Plasmids in Agrobacterium tumefaciens

    NARCIS (Netherlands)

    Hille, Jacques; Schilperoort, Rob

    1981-01-01

    Inc-Q plasmids were introduced into Agrobacterium tumefuciens, by mobilization from Escherichia coli with an Inc-P plasmid, or by transformation with purified plasmid DNA. It was found that they were stably maintained. The presence of an Inc-Q plasmid did not influence tumorigenicity. These results

  6. Purification process of uranium hexafluoride containing traces of plutonium fluoride and/or neptunium fluoride

    International Nuclear Information System (INIS)

    Aubert, J.; Bethuel, L.; Carles, M.

    1983-01-01

    In this process impure uranium hexafluoride is contacted with a metallic fluoride chosen in the group containing lead fluoride PbF 2 , uranium fluorides UFsub(4+x) (0 3 at a temperature such as plutonium and/or neptunium are reduced and pure uranium hexafluoride is recovered. Application is made to uranium hexafluoride purification in spent fuel reprocessing [fr

  7. Persistence Mechanisms of Conjugative Plasmids

    DEFF Research Database (Denmark)

    Bahl, Martin Iain; Hansen, Lars H.; Sørensen, Søren Johannes

    2009-01-01

    Are plasmids selfish parasitic DNA molecules or an integrated part of the bacterial genome? This chapter reviews the current understanding of the persistence mechanisms of conjugative plasmids harbored by bacterial cells and populations. The diversity and intricacy of mechanisms affecting the suc...

  8. Evaluation of strategies to control Fab light chain dimer during mammalian expression and purification: A universal one-step process for purification of correctly assembled Fab.

    Science.gov (United States)

    Spooner, Jennifer; Keen, Jenny; Nayyar, Kalpana; Birkett, Neil; Bond, Nicholas; Bannister, David; Tigue, Natalie; Higazi, Daniel; Kemp, Benjamin; Vaughan, Tristan; Kippen, Alistair; Buchanan, Andrew

    2015-07-01

    Fabs are an important class of antibody fragment as both research reagents and therapeutic agents. There are a plethora of methods described for their recombinant expression and purification. However, these do not address the issue of excessive light chain production that forms light chain dimers nor do they describe a universal purification strategy. Light chain dimer impurities and the absence of a universal Fab purification strategy present persistent challenges for biotechnology applications using Fabs, particularly around the need for bespoke purification strategies. This study describes methods to address light chain dimer formation during Fab expression and identifies a novel CH 1 affinity resin as a simple and efficient one-step purification for correctly assembled Fab. © 2015 Wiley Periodicals, Inc.

  9. Separation process design for isolation and purification of natural products

    DEFF Research Database (Denmark)

    Malwade, Chandrakant R.

    Natural products are defined as secondary metabolites produced by plants and form a vast pool of compounds with unlimited chemical and functional diversity. Many of these secondary metabolites are high value added chemicals that are frequently used as ingredients in food, cosmetics, pharmaceuticals...... and other consumer products. Therefore, process technology towards industrial scale production of such high value chemicals from plants has significant value. Natural products can be obtained in pure form via synthetic or semi-synthetic route, but due to their complicated nature these methods have not been...... developed to the extent of industrial production for majority of natural products. Thus, isolation and purification of such natural products from plants is the most viable way to obtain natural products in pure form. This PhD project is mainly concerned with the design of separation process to isolate...

  10. Lysine purification with cation exchange resin

    International Nuclear Information System (INIS)

    Khayati, GH.; Mottaghi Talab, M.; Hamooni Hagheeghat, M.; Fatemi, M.

    2003-01-01

    L-lysine is an essential amino acid for the growth most of animal species and the number one limiting amino acid for poultry. After production and biomass removal by filtration and centrifugation, the essential next step is the lysine purification and recovery. There are different methods for lysine purification. The ion exchange process is one of the most commonly used purification methods. Lysine recovery was done from broth by ion exchange resin in three different ways: repeated passing, resin soaking and the usual method. Impurities were isolated from the column by repeated wash with distilled water. Recovery and purification was done with NH 4 OH and different alcohol volumes respectively. The results showed that repeated passing is the best method for lysine absorption (maximum range 86.21 %). Washing with alkali solution revealed that most of lysine is obtained in the first step of washing. The highest degree of lysine purification was achieved with the use of 4 volumes of alcohol

  11. Water purification in Borexino

    Energy Technology Data Exchange (ETDEWEB)

    Giammarchi, M. [Infn Milano (Italy); Balata, M.; Ioannucci, L.; Nisi, S. [Laboratori Nazionali del Gran Sasso (Italy); Goretti, A.; Ianni, A. [Princeton University (United States); Miramonti, L. [Dip. di Fisica dell' Università di Milano e Infn (Italy)

    2013-08-08

    Astroparticle Physics and Underground experiments searching for rare nuclear events, need high purity materials to act as detectors or detector shielding. Water has the advantage of being cheap, dense and easily available. Most of all, water can be purified to the goal of obatining a high level of radiopurity. Water Purification can be achieved by means of a combination of processes, including filtration, reverse osmosis, deionization and gas stripping. The Water Purification System for the Borexino experiment, will be described together with its main performances.

  12. DYNAMIC MODELLING AND ADVANCED PREDICTIVE CONTROL OF A CONTINUOUS PROCESS OF ENZYME PURIFICATION

    Directory of Open Access Journals (Sweden)

    Dechechi E.C.

    1997-01-01

    Full Text Available A dynamic mathematical model, simulation and computer control of a Continuous Affinity Recycle Extraction (CARE process, a protein purification technique based on protein adsorption on solid-phase adsorbents is described in this work. This process, consisting of three reactors, is a multivariable process with considerable time delay in the on-line analyses of the controlled variable. An advanced predictive control configuration, specifically the Dynamic Matrix Control (DMC, was applied. The DMC algorithm was applied in process schemes where the aim was to maintain constant the enzyme concentration in the outlet of the third reactor. The performance of the DMC controller was analyzed in the feed-flow disturbances and the results are presented.

  13. Plasmid-mediated mineralization of 4-chlorobiphenyl

    International Nuclear Information System (INIS)

    Shields, M.S.; Hooper, S.W.; Sayler, G.S.

    1985-01-01

    Strains of Alcaligenes and Acinetobacter spp. were isolated from a mixed culture already proven to be proficient at complete mineralization of monohalogenated biphenyls. These strains were shown to harbor a 35 x 10(6)-dalton plasmid mediating a complete pathway for 4-chlorobiphenyl (4CB) oxidation. Subsequent plasmid curing of these bacteria resulted in the abolishment of the 4CB mineralization phenotype and loss of even early 4CB metabolism by Acinetobacter spp. Reestablishment of the Alcaligenes plasmid, denoted pSS50, in the cured Acinetobacter spp. via filter surface mating resulted in the restoration of 4CB mineralization abilities. 4CB mineralization, however, proved to be an unstable characteristic in some subcultured strains. Such loss was not found to coincide with any detectable alteration in plasmid size. Cultures capable of complete mineralization, as well as those limited to partial metabolism of 4CB, produced 4-chlorobenzoate as a metabolite. Demonstration of mineralization of a purified 14 C-labeled chlorobenzoate showed it to be a true intermediate in 4CB mineralization. Unlike the mineralization capability, the ability to produce a metabolite has proven to be stable on subculture. These results indicate the occurrence of a novel plasmid, or evolved catabolic plasmid, that mediates the complete mineralization of 4CB

  14. Ionic behavior of treated water at a water purification plant

    OpenAIRE

    Yanagida, Kazumi; Kawahigashi, Tatsuo

    2012-01-01

    [Abstract] Water at each processing stage in a water purification plant was extracted and analyzed to investigate changes of water quality. Investigations of water at each processing stage at the water purification plant are discussed herein.

  15. Effect of water purification process in radioactive content: analysis on small scale purification plants; Efecto del proceso de purificacion de agua en el contenido radiactivo: analisis en plantas purificadoras a pequena escala

    Energy Technology Data Exchange (ETDEWEB)

    Lopez del Rio, H.; Quiroga S, J. C.; Davila R, J. I.; Mireles G, F. [Universidad Autonoma de Zacatecas, Unidad Academica de Estudios Nucleares, Calle Cipres No. 10, Fracc. La Penuela, 98000, Zacatecas (Mexico)], e-mail: hlopez@uaz.edu.mx

    2009-10-15

    Water from small scale purification plants is a low cost alternative for consumers in comparison to the bottled commercial presentations. Because of its low cost per liter, the consumption of this product has increased in recent years, stimulating in turn the installation of purification systems for these small businesses. The purpose of this study was to estimate the efficiency of small scale purification systems located in the cities of Zacatecas and Guadalupe, Zacatecas, to reduce the radioactive content of water. It was measured the total alpha and beta activity in water samples of entry and exit to process, through the liquid scintillation technique. In general it was observed that the process is more efficient in removing alpha that beta activity. The fraction of total alpha activity removed varied between 27 and 100%, while between 0 and 77% of the total beta activity was removed by the analyzed plants. In all cases, the total radioactivity level was lower than the maximum permissible value settled by the official mexican standard for drinking water. (Author)

  16. Construction of Biologically Functional Bacterial Plasmids In Vitro

    Science.gov (United States)

    Cohen, Stanley N.; Chang, Annie C. Y.; Boyer, Herbert W.; Helling, Robert B.

    1973-01-01

    The construction of new plasmid DNA species by in vitro joining of restriction endonuclease-generated fragments of separate plasmids is described. Newly constructed plasmids that are inserted into Escherichia coli by transformation are shown to be biologically functional replicons that possess genetic properties and nucleotide base sequences from both of the parent DNA molecules. Functional plasmids can be obtained by reassociation of endonuclease-generated fragments of larger replicons, as well as by joining of plasmid DNA molecules of entirely different origins. Images PMID:4594039

  17. Solvent-extraction purification of neptunium

    International Nuclear Information System (INIS)

    Kyser, E.A.; Hudlow, S.L.

    2008-01-01

    The Savannah River Site (SRS) has recovered 237 Np from reactor fuel that is currently being processed into NpO 2 for future production of 238 Pu. Several purification flowsheets have been utilized. An oxidizing solvent-extraction (SX) flowsheet was used to remove Fe, sulfate ion, and Th while simultaneously 237 Np, 238 Pu, u, and nonradioactive Ce(IV) was extracted into the tributyl phosphate (TBP) based organic solvent. A reducing SX flowsheet (second pass) removed the Ce and Pu and recovered both Np and U. The oxidizing flowsheet was necessary for solutions that contained excessive amounts of sulfate ion. Anion exchange was used to perform final purification of Np from Pu, U, and various non-actinide impurities. The Np(IV) in the purified solution was then oxalate-precipitated and calcined to an oxide for shipment to other facilities for storage and future target fabrication. Performance details of the SX purification and process difficulties are discussed. (authors)

  18. Research and Development of Crystal Purification for Product of Uranium Crystallization Process

    Energy Technology Data Exchange (ETDEWEB)

    Yano, K. [Japan Atomic Energy Agency - JAEA (Japan)

    2009-06-15

    Uranium crystallization has been developed as a part of advanced aqueous reprocessing for FBR spent fuel. Although the purity of uranyl nitrate hexahydrate (UNH) crystal from the crystallization process is supposed to meet a specification of FBR blanket fuel, an improvement of its purity is able to reduce the cost of fuel fabrication and storage (in case interim storage of recovered uranium is required). In this work, UNH crystal purification was developed as additional process after crystallization. Contamination of the crystal is caused by mother solution and solid state impurities. They are inseparable by washing and filtration. Mother solution on the surface of UNH crystals is removable by washing, but it is difficult to remove that in an obstructed part of crystalline aggregate by washing. Major elements of solid state impurities are cesium and barium. Cesium precipitates with tetravalent plutonium as a double nitrate, Cs{sub 2}Pu(NO{sub 3}){sub 6}. Barium crystallizes as Ba(NO{sub 3}){sub 2} because of its low solubility in nitric acid solution. It is difficult to separate their particle from UNH crystal by solid-liquid separation such as simple filtration. As a kind of crystal purification, there are some methods using sweating. Sweating is a phenomenon that a crystal melts partly below its melting point and it is caused by depression of freezing point due to impurity. It is considerably applicable for removal of mother solution. Concerning the solid state impurities, which has higher melting point than that of UNH crystal, it is supposed that they are separable by melting UNH crystal and filtration. The behaviors of impurities and applicability of sweating and melting-filtration operations to the purification for UNH crystal were investigated experimentally on a beaker and an engineering scale. With regard to behaviors of impurities, the conditions of cesium and barium precipitation were surveyed and it was clarified that there were most impurities on the

  19. Modelling a water purification process for quality monitoring

    NARCIS (Netherlands)

    Meulen, van der F.H.; Luca, S.; Overal, G.; Dubbeldam, J.L.A.; Di Bucchianico, A.; Jongbloed, G.; Dubbeldam, J.; Groenevelt, W.; Heemink, A.W.; Lahaye, D.; Meerman, C.; Meulen, van der F.

    2014-01-01

    This paper deals with a quality engineering problem introduced by ‘Waterlaboratorium Noord’ (WLN) situated at the Netherlands. In-terest lies in determining an optimal sampling frequency that provides suÿcient information on the water quality in a drinking water purifica-tion plant. The water

  20. Broad-Host-Range IncP-1 plasmids and their resistance potential

    Directory of Open Access Journals (Sweden)

    Magdalena ePopowska

    2013-03-01

    Full Text Available The plasmids of the incompatibility group IncP-1, also called IncP, as extrachromosomal genetic elements can transfer and replicate virtually in all Gram-negative bacteria. They are composed of backbone genes that encode a variety of essential functions and accessory genes that have implications for human health and environmental bioremediation. Broad-host-range IncP plasmids are known to spread genes between distinct phylogenetic groups of bacteria. These genes often code for resistances to a broad spectrum of antibiotics, heavy metals and quaternary ammonium compounds used as disinfectants. The backbone of these plasmids carries modules that enable them to effectively replicate, move to a new host via conjugative transfer and to be stably maintained in bacterial cells. The adaptive, resistance and virulence genes are mainly located on mobile genetic elements integrated between the functional plasmid backbone modules. Environmental studies have demonstrated the wide distribution of IncP-like replicons in manure, soils and wastewater treatment plants. They also are present in strains of pathogenic or opportunistic bacteria, which can be a cause for concern, because they may encode multiresistance. Their broad distribution suggests that IncP plasmids play a crucial role in bacterial adaptation by utilizing horizontal gene transfer. This review summarizes the variety of genetic information and physiological functions carried by IncP plasmids, which can contribute to the spread of antibiotic and heavy metal resistance while also mediating the process of bioremediation of pollutants. Due to the location of the resistance genes on plasmids with a broad host range and the presence of transposons carrying these genes it seems that the spread of these genes would be possible and quite hazardous in infection control. Future studies are required to determine the level of risk of the spread of resistance genes located on these plasmids.

  1. Prevalence of plasmid-bearing and plasmid-free Chlamydia trachomatis infection among women who visited obstetrics and gynecology clinics in Malaysia.

    Science.gov (United States)

    Yeow, Tee Cian; Wong, Won Fen; Sabet, Negar Shafiei; Sulaiman, Sofiah; Shahhosseini, Fatemeh; Tan, Grace Min Yi; Movahed, Elaheh; Looi, Chung Yeng; Shankar, Esaki M; Gupta, Rishien; Arulanandam, Bernard P; Hassan, Jamiyah; Abu Bakar, Sazaly

    2016-03-18

    The 7.5 kb cryptic plasmid of Chlamydia trachomatis has been shown to be a virulence factor in animal models, but its significance in humans still remains unknown. The aim of this study was to investigate the prevalence and potential involvement of the C. trachomatis cryptic plasmid in causing various clinical manifestations; including infertility, reproductive tract disintegrity, menstrual disorder, and polycystic ovarian syndrome (PCOS) among genital C. trachomatis-infected patients. A total of 180 female patients of child bearing age (mean 30.9 years old, IQR:27-35) with gynecological complications and subfertility issues, who visited Obstetrics and Gynecology clinics in Kuala Lumpur, Malaysia were recruited for the study. Prevalence of genital chlamydial infection among these patients was alarmingly high at 51.1% (92/180). Of the 92 chlamydia-infected patients, 93.5% (86/92) were infected with plasmid-bearing (+) C. trachomatis while the remaining 6.5% (6/92) were caused by the plasmid-free (-) variant. Our data showed that genital C. trachomatis infection was associated with infertility issues, inflammation in the reproductive tract (mucopurulent cervicitis or endometriosis), irregular menstrual cycles and polycystic ovarian syndrome (PCOS). However, no statistical significance was detected among patients with plasmid (+) versus plasmid (-) C. trachomatis infection. Interestingly, plasmid (+) C. trachomatis was detected in all patients with PCOS, and the plasmid copy numbers were significantly higher among PCOS patients, relative to non-PCOS patients. Our findings show a high incidence of C. trachomatis infection among women with infertility or gynecological problems in Malaysia. However, due to the low number of plasmid (-) C. trachomatis cases, a significant role of the plasmid in causing virulence in human requires further investigation of a larger cohort.

  2. Genetic characterization of plasmid pRJ5 of Staphylococcus aureus compared to plasmid pE194

    International Nuclear Information System (INIS)

    Oliveira, S.S. de; Freire Bastos, M.C. de

    1993-01-01

    The pRJ5, a naturally occurring constitutive macrolide, lincosamide and streptogramin B (MLS) resistance plasmid of Staphylococcus aureus, was compared to pE194, a plasmid that confers the inducible phenotype. pRJ5 was stable in all strains of S. aureus tested, even under growth at 43 O C, which distinguished it from pE194 which was shown to be thermo-sensitive for replication. pRJ5, like pE194, was highly unstable in Bacillus subtilis when the cells were grown in nonselective conditions. Multimeric forms of pRJ5 DNA were detected in the few cells of B. subtilis that retained this plasmid. pE194 was transduced by phages φ 11 and φ 443 at frequencies 400 and 20-fold higher, respectively, than pRJ5. Both plasmids were co-transduced with the plasmid pRJ4. pRJ5 was shown to be compatible with pE194. Therefore they belong to distinct Inc groups. Hybridization studies revealed that pRJ5 shares a 1.35 kb region of homology to pE194, which is limited to the erm gene, conferring MLS resistance. (author)

  3. Antimicrobial susceptibility pattern and plasmid-mediated ...

    African Journals Online (AJOL)

    negative Staphylococci (CoNS) were isolated from clinical samples and isolates subjected to antibiotic susceptibility testing, plasmid curing and plasmid DNA isolation. Result: The highest percentages isolates were recovered from urine samples and ...

  4. Entanglement of purification: from spin chains to holography

    Science.gov (United States)

    Nguyen, Phuc; Devakul, Trithep; Halbasch, Matthew G.; Zaletel, Michael P.; Swingle, Brian

    2018-01-01

    Purification is a powerful technique in quantum physics whereby a mixed quantum state is extended to a pure state on a larger system. This process is not unique, and in systems composed of many degrees of freedom, one natural purification is the one with minimal entanglement. Here we study the entropy of the minimally entangled purification, called the entanglement of purification, in three model systems: an Ising spin chain, conformal field theories holographically dual to Einstein gravity, and random stabilizer tensor networks. We conjecture values for the entanglement of purification in all these models, and we support our conjectures with a variety of numerical and analytical results. We find that such minimally entangled purifications have a number of applications, from enhancing entanglement-based tensor network methods for describing mixed states to elucidating novel aspects of the emergence of geometry from entanglement in the AdS/CFT correspondence.

  5. Frequent conjugative transfer accelerates adaptation of a broad-host-range plasmid to an unfavorable Pseudomonas putida host.

    Science.gov (United States)

    Heuer, Holger; Fox, Randal E; Top, Eva M

    2007-03-01

    IncP-1 plasmids are known to be promiscuous, but it is not understood if they are equally well adapted to various species within their host range. Moreover, little is known about their fate in bacterial communities. We determined if the IncP-1beta plasmid pB10 was unstable in some Proteobacteria, and whether plasmid stability was enhanced after long-term carriage in a single host and when regularly switched between isogenic hosts. Plasmid pB10 was found to be very unstable in Pseudomonas putida H2, and conferred a high cost (c. 20% decrease in fitness relative to the plasmid-free host). H2(pB10) was then evolved under conditions that selected for plasmid maintenance, with or without regular plasmid transfer (host-switching). When tested in the ancestral host, the evolved plasmids were more stable and their cost was significantly reduced (9% and 16% for plasmids from host-switched and nonswitched lineages, respectively). Our findings suggest that IncP-1 plasmids can rapidly adapt to an unfavorable host by improving their overall stability, and that regular conjugative transfer accelerates this process.

  6. Radioautographic test for genetic cotton transformation by pCaVItoxneo hybrid plasmid

    International Nuclear Information System (INIS)

    Imamkhodjaeva, A.S.

    2006-01-01

    Full text: Search for novel technologies in biology, application of up-to-date methods in gene engineering, manipulation with the recombinant DNA, in particular, open opportunities for experiments with plants. To identify some DNA fragments in an organism's genome, radioautographic methods, such as dot- and blot-hybridization are frequently used. As a rule, genomic DNA is first isolated from the plant's organ. Its purification and subsequent manipulation is followed by hybridization with a probe labeled with radioactive components. The purified DNA, cDNA of RNA reverse transcription or a DNA fragment cloned in E-coli could serve as the probe. Radioautography shows homologically hybridized fragments. We have performed express dot-hybridization analysis on hybrid plasmid transformation of G.Hirsutum L. (108F) and G. Barbadense L. (C-6037) cotton sorts. pCaVItoxneo plasmid obtained on the basis of independently replicated plasmid-like DNA of the G.Hirsutum L. (pGHm2) cotton mitochondria was used (Yusupov T., 1994). There are hybrid two-domain gene of insectotoxin and enzymatically active kanamycine - phosphotransferase in the plasmid. The whole content is controlled by the plant promoter of cauliflower mosaic virus (19 S SFMV). The plasmid in question was added to the pollen sprouting medium followed by the transfer of the suspension on the pistil stigmas of the pre-prepared cotton flowers. The seed budding as the result of the experiment were analyzed by means of dot-hybridization method. DNA probes used for radioactive hybridization were labeled by method of Fainberg and Vagelstein (1990). To perform that DNA was dissolved in Tris-EDTA (10:1), containing 10mM of Tris HCl and 1mM EDTA, denaturated at 100 d eg C for 2 minutes with subsequent addition of oligonucleotide primers and annealing. DNA synthesis in the presence of 32 P labeled dATP and dCTP (Tashkent) was performed in the reaction mixture of potassium-phosphate buffer containing 67mM of MgCl 2 , 1 mg/ml of

  7. Improving the large scale purification of the HIV microbicide, griffithsin.

    Science.gov (United States)

    Fuqua, Joshua L; Wanga, Valentine; Palmer, Kenneth E

    2015-02-22

    Griffithsin is a broad spectrum antiviral lectin that inhibits viral entry and maturation processes through binding clusters of oligomannose glycans on viral envelope glycoproteins. An efficient, scaleable manufacturing process for griffithsin active pharmaceutical ingredient (API) is essential for particularly cost-sensitive products such as griffithsin -based topical microbicides for HIV-1 prevention in resource poor settings. Our previously published purification method used ceramic filtration followed by two chromatography steps, resulting in a protein recovery of 30%. Our objective was to develop a scalable purification method for griffithsin expressed in Nicotiana benthamiana plants that would increase yield, reduce production costs, and simplify manufacturing techniques. Considering the future need to transfer griffithsin manufacturing technology to resource poor areas, we chose to focus modifying the purification process, paying particular attention to introducing simple, low-cost, and scalable procedures such as use of temperature, pH, ion concentration, and filtration to enhance product recovery. We achieved >99% pure griffithsin API by generating the initial green juice extract in pH 4 buffer, heating the extract to 55°C, incubating overnight with a bentonite MgCl2 mixture, and final purification with Capto™ multimodal chromatography. Griffithsin extracted with this protocol maintains activity comparable to griffithsin purified by the previously published method and we are able to recover a substantially higher yield: 88 ± 5% of griffithsin from the initial extract. The method was scaled to produce gram quantities of griffithsin with high yields, low endotoxin levels, and low purification costs maintained. The methodology developed to purify griffithsin introduces and develops multiple tools for purification of recombinant proteins from plants at an industrial scale. These tools allow for robust cost-effective production and purification of

  8. Novel plasmids and resistance phenotypes in Yersinia pestis: unique plasmid inventory of strain Java 9 mediates high levels of arsenic resistance.

    Science.gov (United States)

    Eppinger, Mark; Radnedge, Lyndsay; Andersen, Gary; Vietri, Nicholas; Severson, Grant; Mou, Sherry; Ravel, Jacques; Worsham, Patricia L

    2012-01-01

    Growing evidence suggests that the plasmid repertoire of Yersinia pestis is not restricted to the three classical virulence plasmids. The Java 9 strain of Y. pestis is a biovar Orientalis isolate obtained from a rat in Indonesia. Although it lacks the Y. pestis-specific plasmid pMT, which encodes the F1 capsule, it retains virulence in mouse and non-human primate animal models. While comparing diverse Y. pestis strains using subtractive hybridization, we identified sequences in Java 9 that were homologous to a Y. enterocolitica strain carrying the transposon Tn2502, which is known to encode arsenic resistance. Here we demonstrate that Java 9 exhibits high levels of arsenic and arsenite resistance mediated by a novel promiscuous class II transposon, named Tn2503. Arsenic resistance was self-transmissible from Java 9 to other Y. pestis strains via conjugation. Genomic analysis of the atypical plasmid inventory of Java 9 identified pCD and pPCP plasmids of atypical size and two previously uncharacterized cryptic plasmids. Unlike the Tn2502-mediated arsenic resistance encoded on the Y. enterocolitica virulence plasmid; the resistance loci in Java 9 are found on all four indigenous plasmids, including the two novel cryptic plasmids. This unique mobilome introduces more than 105 genes into the species gene pool. The majority of these are encoded by the two entirely novel self-transmissible plasmids, which show partial homology and synteny to other enterics. In contrast to the reductive evolution in Y. pestis, this study underlines the major impact of a dynamic mobilome and lateral acquisition in the genome evolution of the plague bacterium.

  9. Novel plasmids and resistance phenotypes in Yersinia pestis: unique plasmid inventory of strain Java 9 mediates high levels of arsenic resistance.

    Directory of Open Access Journals (Sweden)

    Mark Eppinger

    Full Text Available Growing evidence suggests that the plasmid repertoire of Yersinia pestis is not restricted to the three classical virulence plasmids. The Java 9 strain of Y. pestis is a biovar Orientalis isolate obtained from a rat in Indonesia. Although it lacks the Y. pestis-specific plasmid pMT, which encodes the F1 capsule, it retains virulence in mouse and non-human primate animal models. While comparing diverse Y. pestis strains using subtractive hybridization, we identified sequences in Java 9 that were homologous to a Y. enterocolitica strain carrying the transposon Tn2502, which is known to encode arsenic resistance. Here we demonstrate that Java 9 exhibits high levels of arsenic and arsenite resistance mediated by a novel promiscuous class II transposon, named Tn2503. Arsenic resistance was self-transmissible from Java 9 to other Y. pestis strains via conjugation. Genomic analysis of the atypical plasmid inventory of Java 9 identified pCD and pPCP plasmids of atypical size and two previously uncharacterized cryptic plasmids. Unlike the Tn2502-mediated arsenic resistance encoded on the Y. enterocolitica virulence plasmid; the resistance loci in Java 9 are found on all four indigenous plasmids, including the two novel cryptic plasmids. This unique mobilome introduces more than 105 genes into the species gene pool. The majority of these are encoded by the two entirely novel self-transmissible plasmids, which show partial homology and synteny to other enterics. In contrast to the reductive evolution in Y. pestis, this study underlines the major impact of a dynamic mobilome and lateral acquisition in the genome evolution of the plague bacterium.

  10. Self-packed filter plates: a good alternative for pre-packed filter plates for developing purification processes of therapeutic proteins

    NARCIS (Netherlands)

    Li, X.; Roo, de G.; Burgers, K.; Ottens, M.; Eppink, M.H.M.

    2012-01-01

    The use of high throughput screening (HTS) has successfully been applied in the past years in downstream process development of therapeutic proteins. Different HTS applications were introduced to speed up the purification process development of these proteins. In the light of these findings, studies

  11. Technological assumptions for biogas purification.

    Science.gov (United States)

    Makareviciene, Violeta; Sendzikiene, Egle

    2015-01-01

    Biogas can be used in the engines of transport vehicles and blended into natural gas networks, but it also requires the removal of carbon dioxide, hydrogen sulphide, and moisture. Biogas purification process flow diagrams have been developed for a process enabling the use of a dolomite suspension, as well as for solutions obtained by the filtration of the suspension, to obtain biogas free of hydrogen sulphide and with a carbon dioxide content that does not exceed 2%. The cost of biogas purification was evaluated on the basis of data on biogas production capacity and biogas production cost obtained from local water treatment facilities. It has been found that, with the use of dolomite suspension, the cost of biogas purification is approximately six times lower than that in the case of using a chemical sorbent such as monoethanolamine. The results showed travelling costs using biogas purified by dolomite suspension are nearly 1.5 time lower than travelling costs using gasoline and slightly lower than travelling costs using mineral diesel fuel.

  12. Finite Element Modeling of Adsorption Processes for Gas Separation and Purification

    International Nuclear Information System (INIS)

    Humble, Paul H.; Williams, Richard M.; Hayes, James C.

    2009-01-01

    Pacific Northwest National Laboratory (PNNL) has expertise in the design and fabrication of automated radioxenon collection systems for nuclear explosion monitoring. In developing new systems there is an ever present need to reduce size, power consumption and complexity. Most of these systems have used adsorption based techniques for gas collection and/or concentration and purification. These processes include pressure swing adsorption, vacuum swing adsorption, temperature swing adsorption, gas chromatography and hybrid processes that combine elements of these techniques. To better understand these processes, and help with the development of improved hardware, a finite element software package (COMSOL Multiphysics) has been used to develop complex models of these adsorption based operations. The partial differential equations used include a mass balance for each gas species and adsorbed species along with a convection conduction energy balance equation. These equations in conjunction with multicomponent temperature dependent isotherm models are capable of simulating separation processes ranging from complex multibed PSA processes, and multicomponent temperature programmed gas chromatography, to simple two component temperature swing adsorption. These numerical simulations have been a valuable tool for assessing the capability of proposed processes and optimizing hardware and process parameters.

  13. Bioinspired Materials for Water Purification

    Directory of Open Access Journals (Sweden)

    Alfredo Gonzalez-Perez

    2016-06-01

    Full Text Available Water scarcity issues associated with inadequate access to clean water and sanitation is a ubiquitous problem occurring globally. Addressing future challenges will require a combination of new technological development in water purification and environmental remediation technology with suitable conservation policies. In this scenario, new bioinspired materials will play a pivotal role in the development of more efficient and environmentally friendly solutions. The role of amphiphilic self-assembly on the fabrication of new biomimetic membranes for membrane separation like reverse osmosis is emphasized. Mesoporous support materials for semiconductor growth in the photocatalytic degradation of pollutants and new carriers for immobilization of bacteria in bioreactors are used in the removal and processing of different kind of water pollutants like heavy metals. Obstacles to improve and optimize the fabrication as well as a better understanding of their performance in small-scale and pilot purification systems need to be addressed. However, it is expected that these new biomimetic materials will find their way into the current water purification technologies to improve their purification/removal performance in a cost-effective and environmentally friendly way.

  14. ProteinTracker: an application for managing protein production and purification.

    Science.gov (United States)

    Ponko, Stefan C; Bienvenue, David

    2012-05-10

    Laboratories that produce protein reagents for research and development face the challenge of deciding whether to track batch-related data using simple file based storage mechanisms (e.g. spreadsheets and notebooks), or commit the time and effort to install, configure and maintain a more complex laboratory information management system (LIMS). Managing reagent data stored in files is challenging because files are often copied, moved, and reformatted. Furthermore, there is no simple way to query the data if/when questions arise. Commercial LIMS often include additional modules that may be paid for but not actually used, and often require software expertise to truly customize them for a given environment. This web-application allows small to medium-sized protein production groups to track data related to plasmid DNA, conditioned media samples (supes), cell lines used for expression, and purified protein information, including method of purification and quality control results. In addition, a request system was added that includes a means of prioritizing requests to help manage the high demand of protein production resources at most organizations. ProteinTracker makes extensive use of existing open-source libraries and is designed to track essential data related to the production and purification of proteins. ProteinTracker is an open-source web-based application that provides organizations with the ability to track key data involved in the production and purification of proteins and may be modified to meet the specific needs of an organization. The source code and database setup script can be downloaded from http://sourceforge.net/projects/proteintracker. This site also contains installation instructions and a user guide. A demonstration version of the application can be viewed at http://www.proteintracker.org.

  15. Multilocus sequence typing of IncN plasmids

    DEFF Research Database (Denmark)

    García-Fernández, Aurora; Villa, Laura; Moodley, Arshnee

    2011-01-01

    that spread and persistence of this particular IncN-carrying blaVIM-1 lineage in Greece. CONCLUSIONS: This study proposes the use of pMLST as a suitable and rapid method for identification of IncN epidemic plasmid lineages. The recent spread of blaCTX-M-1 among humans and animals seems to be associated......OBJECTIVES: Incompatibility group N (IncN) plasmids have been associated with the dissemination of antimicrobial resistance and are a major vehicle for the spread of blaVIM-1 in humans and blaCTX-M-1 in animals. A plasmid multilocus sequence typing (pMLST) scheme was developed for rapid...... in different countries from both animals and humans belonged to ST1, suggesting dissemination of an epidemic plasmid through the food chain. Fifteen of 17 plasmids carrying blaVIM-1 from Klebsiella pneumoniae and Escherichia coli, isolated during a 5year period in Greece were assigned to ST10, suggesting...

  16. Selfish restriction modification genes: resistance of a resident R/M plasmid to displacement by an incompatible plasmid mediated by host killing.

    Science.gov (United States)

    Naito, Y; Naito, T; Kobayashi, I

    1998-01-01

    Previous work from this laboratory demonstrated that plasmids carrying a type II restriction-modification gene complex are not easily lost from their bacterial host because plasmid-free segregant cells are killed through chromosome cleavage. Here, we have followed the course of events that takes place when an Escherichia coli rec BC sbcA strain carrying a plasmid coding for the PaeR7I restriction-modification (R/M) gene complex is transformed by a plasmid with an identical origin of replication. The number of transformants that appeared was far fewer than with the restriction-minus (r-) control. Most of the transformants were very small. After prolonged incubation, the number and the size of the colonies increased, but this increase never attained the level of the r- control. Most of the transformed colonies retained the drug-resistance of the resident, r+ m+ plasmid. These results indicate that post-segregational host killing occurs when a plasmid bearing an R/M gene complex is displaced by an incompatible plasmid. Such cell killing eliminates the competitor plasmid along with the host and, thus, would allow persistence of the R/M plasmid in the neighboring, clonal host cells in nature. This phenomenon is reminiscent of mammalian apoptosis and other forms of altruistic cell death strategy against infection. This type of resistance to displacement was also studied in a wild type Escherichia coli strain that was normal for homologous recombination (rec+). A number of differences between the recBC sbcA strain and the rec+ strain were observed and these will be discussed.

  17. Development, upscaling and validation of the purification process for human-cl rhFVIII (Nuwiq®), a new generation recombinant factor VIII produced in a human cell-line.

    Science.gov (United States)

    Winge, Stefan; Yderland, Louise; Kannicht, Christoph; Hermans, Pim; Adema, Simon; Schmidt, Torben; Gilljam, Gustav; Linhult, Martin; Tiemeyer, Maya; Belyanskaya, Larisa; Walter, Olaf

    2015-11-01

    Human-cl rhFVIII (Nuwiq®), a new generation recombinant factor VIII (rFVIII), is the first rFVIII produced in a human cell-line approved by the European Medicines Agency. To describe the development, upscaling and process validation for industrial-scale human-cl rhFVIII purification. The purification process involves one centrifugation, two filtration, five chromatography columns and two dedicated pathogen clearance steps (solvent/detergent treatment and 20 nm nanofiltration). The key purification step uses an affinity resin (VIIISelect) with high specificity for FVIII, removing essentially all host-cell proteins with >80% product recovery. The production-scale multi-step purification process efficiently removes process- and product-related impurities and results in a high-purity rhFVIII product, with an overall yield of ∼50%. Specific activity of the final product was >9000 IU/mg, and the ratio between active FVIII and total FVIII protein present was >0.9. The entire production process is free of animal-derived products. Leaching of potential harmful compounds from chromatography resins and all pathogens tested were below the limit of quantification in the final product. Human-cl rhFVIII can be produced at 500 L bioreactor scale, maintaining high purity and recoveries. The innovative purification process ensures a high-purity and high-quality human-cl rhFVIII product with a high pathogen safety margin. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  18. A Hybrid MPC-PID Control System Design for the Continuous Purification and Processing of Active Pharmaceutical Ingredients

    Directory of Open Access Journals (Sweden)

    Maitraye Sen

    2014-05-01

    Full Text Available In this work, a hybrid MPC (model predictive control-PID (proportional-integral-derivative control system has been designed for the continuous purification and processing framework of active pharmaceutical ingredients (APIs. The specific unit operations associated with the purification and processing of API have been developed from first-principles and connected in a continuous framework in the form of a flowsheet model. These integrated unit operations are highly interactive along with the presence of process delays. Therefore, a hybrid MPC-PID is a promising alternative to achieve the desired control loop performance as mandated by the regulatory authorities. The integrated flowsheet model has been simulated in gPROMSTM (Process System Enterprise, London, UK. This flowsheet model has been linearized in order to design the control scheme. The ability to track the set point and reject disturbances has been evaluated. A comparative study between the performance of the hybrid MPC-PID and a PID-only control scheme has been presented. The results show that an enhanced control loop performance can be obtained under the hybrid control scheme and demonstrate that such a scheme has high potential in improving the efficiency of pharmaceutical manufacturing operations.

  19. Uranium hexafluoride purification

    International Nuclear Information System (INIS)

    Araujo, Eneas F. de

    1986-01-01

    Uranium hexafluoride might contain a large amount of impurities after manufacturing or handling. Three usual methods of purification of uranium hexafluoride were presented: selective sorption, sublimation, and distillation. Since uranium hexafluoride usually is contaminated with hydrogen fluoride, a theoretical study of the phase equilibrium properties was performed for the binary system UF 6 -HF. A large deviation from the ideal solution behaviour was observed. A purification unity based on a constant reflux batch distillation process was developed. A procedure was established in order to design the re boiler, condenser and packed columns for the UF 6 -HF mixture separation. A bench scale facility for fractional distillation of uranium hexafluoride was described. Basic operations for that facility and results extracted from several batches were discussed. (author)

  20. Compatibility and entry exclusion of IncA and IncC plasmids revisited: IncA and IncC plasmids are compatible.

    Science.gov (United States)

    Ambrose, Stephanie J; Harmer, Christopher J; Hall, Ruth M

    2018-02-24

    In an early study, IncA and IncC plasmids that were reported to be compatible were grouped as the "A-C complex" based on similarities and on strong entry exclusion. However, recently, the term IncA/C has been used frequently to describe plasmids belonging to both of these two groups. Granted that the supporting data was not included in the original reports and that the consensus iteron sequences have since been shown to be essentially identical, we have addressed the question again. The original IncA plasmid, RA1, and the IncC plasmid pRMH760, were introduced into the same cell by transformation, and were found to be maintained stably for over 100 generations in the absence of selection for either plasmid, i.e. they were compatible. We conclude that use of the term IncA/C for this important plasmid group is indeed incorrect and it causes unnecessary confusion. Granted the importance of IncC plasmids in the spread of antibiotic resistance genes, we recommend that use of the misleading terms IncA/C, IncA/C 1 and IncA/C 2 should cease. In addition, RA1 and pRMH760 were shown to each completely prevent entry of the other via conjugative transfer into the cell they reside in. Copyright © 2018 Elsevier Inc. All rights reserved.

  1. Ecological and genetic determinants of plasmid distribution in Escherichia coli.

    Science.gov (United States)

    Medaney, Frances; Ellis, Richard J; Raymond, Ben

    2016-11-01

    Bacterial plasmids are important carriers of virulence and antibiotic resistance genes. Nevertheless, little is known of the determinants of plasmid distribution in bacterial populations. Here the factors affecting the diversity and distribution of the large plasmids of Escherichia coli were explored in cattle grazing on semi-natural grassland, a set of populations with low frequencies of antibiotic resistance genes. Critically, the population genetic structure of bacterial hosts was chararacterized. This revealed structured E. coli populations with high diversity between sites and individuals but low diversity within cattle hosts. Plasmid profiles, however, varied considerably within the same E. coli genotype. Both ecological and genetic factors affected plasmid distribution: plasmid profiles were affected by site, E. coli diversity, E. coli genotype and the presence of other large plasmids. Notably 3/26 E. coli serotypes accounted for half the observed plasmid-free isolates indicating that within species variation can substantially affect carriage of the major conjugative plasmids. The observed population structure suggest that most of the opportunities for within species plasmid transfer occur between different individuals of the same genotype and support recent experimental work indicating that plasmid-host coevolution, and epistatic interactions on fitness costs are likely to be important in determining occupancy. © 2016 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.

  2. Materials for Molybdenum 99 purification

    International Nuclear Information System (INIS)

    Wilkinson, M. Victoria; Mondino, Angel V.; Manzini, Alberto C.

    2003-01-01

    The National Atomic Energy Commission (CNEA) produces fission Mo 99, an isotope of wide use in nuclear medicine. In order to simplify the current Mo 99 production process, to shorten its duration and reduce impurities in the final product, alternative methods for purification steps were looked for. In this work a variety of new materials for the purification columns were designed, all of them with carbon. These materials were studied and a material which contribute with the best results for molybdenum retention, was selected. The preparation procedure and the working conditions were determined. (author)

  3. The characteristics of micrococcus (deinococcus) radiodurans sark plasmids

    International Nuclear Information System (INIS)

    Sjarief, Sri Hariani; Kikuchi, Masahiro; Watanabe, Hiroshi.

    1994-01-01

    The characterization of micrococcus (deinococcus) radiodurans sark plasmids. This bacterium has been classified as a new genus deinococcus radiodurans which is resistant to gamma-rays. It can repair itself completely almost all of DNA damages including double strand breaks induced by gamma-rays up to about 5 KGy. To reveal the repair mechanism, several investigations had been done to develop a cloning vector available for the genetic analysis. For this purpose D. radiodurans Sark are to be prepared as a vector by studying the characteristics of its plasmid. Plasmids were isolated by electrophoresis using 0.6% low-melting-temperature agarose in TAE and run for 5.5 hours, followed by the identification. An antibiotic marker was also carried out in this experiment to identify its location in the genetic materials of the cell, beside making a restriction map of the plasmid. Results have shown that D. radiodurans Sark has 4 plasmids (P1, P2, P3, and P4) and the refampicin resistant genes were not found in the plasmid. (authors). 14 refs; 4 figs

  4. Affinity chromatography: A versatile technique for antibody purification.

    Science.gov (United States)

    Arora, Sushrut; Saxena, Vikas; Ayyar, B Vijayalakshmi

    2017-03-01

    Antibodies continue to be extremely utilized entities in myriad applications including basic research, imaging, targeted delivery, chromatography, diagnostics, and therapeutics. At production stage, antibodies are generally present in complex matrices and most of their intended applications necessitate purification. Antibody purification has always been a major bottleneck in downstream processing of antibodies, due to the need of high quality products and associated high costs. Over the years, extensive research has focused on finding better purification methodologies to overcome this holdup. Among a plethora of different techniques, affinity chromatography is one of the most selective, rapid and easy method for antibody purification. This review aims to provide a detailed overview on affinity chromatography and the components involved in purification. An array of support matrices along with various classes of affinity ligands detailing their underlying working principles, together with the advantages and limitations of each system in purifying different types of antibodies, accompanying recent developments and important practical methodological considerations to optimize purification procedure are discussed. Copyright © 2016 Elsevier Inc. All rights reserved.

  5. Health physics system scheme for the uranium purification plant

    International Nuclear Information System (INIS)

    Meyer, M.; Oliveira, E.C.; Sordi, G.A.A.; Abrao, A.

    1976-01-01

    After describing the two uranium purification processes used in the Chemical Engineerring Division of the Instituto de Energia Atomica, it is examined the possible hazards and methods to control or eliminate them. Since these purification process present several stages, in each one of them it is evaluated the hazards and tried to give adequate solutions to protect both, personnel and installations, from the potential radiation hazards

  6. Increasing plasmid transformation efficiency of natural spizizen method in Bacillus Subtilis by a cell permeable peptide

    Directory of Open Access Journals (Sweden)

    Mehrdad Moosazadeh Moghaddam

    2013-01-01

    Full Text Available Introduction: Some of bacterial species are able to uptake DNA molecule from environment, the yield of this process depends on some conditions such as plasmid size and host type. In the case of Bacillus subtilis, DNA uptake has low efficacy. Using Spizizen minimal medium is common method in plasmid transformation into B. subtilis, but rate of this process is not suitable and noteworthy. The aim of this study was investigation of novel method for improvement of DNA transformation into B. subtilis based on CM11 cationic peptide as a membrane permeable agent.Materials and methods: In this study, for optimization of pWB980 plasmid transformation into B. subtilis, the CM11 cationic peptide was used. For this purpose, B. subtilis competent cell preparation in the present of different concentration of peptide was implemented by two methods. In the first method, after treatment of bacteria with different amount of peptide for 14h, plasmid was added. In the second method, several concentration of peptide with plasmid was exposed to bacteria simultaneously. Bacteria that uptake DNA were screened on LB agar medium containing kanamycin. The total transformed bacteria per microgram of DNA was calculated and compared with the control.Results: Plasmid transformation in best conditions was 6.5 folds higher than the control. This result was statistically significant (P value <0.001.Discussion and conclusion: This study showed that CM11 cationic peptide as a membrane permeable agent was able to increase plasmid transformation rate into B. subtilis. This property was useful for resolution of low transformation efficacy.

  7. The broad-host-range plasmid pSFA231 isolated from petroleum-contaminated sediment represents a new member of the PromA plasmid family.

    Science.gov (United States)

    Li, Xiaobin; Top, Eva M; Wang, Yafei; Brown, Celeste J; Yao, Fei; Yang, Shan; Jiang, Yong; Li, Hui

    2014-01-01

    A self-transmissible broad-host-range (BHR) plasmid pSFA231 was isolated from petroleum-contaminated sediment in Shen-fu wastewater irrigation zone, China, using the triparental mating exogenous plasmid capture method. Based on its complete sequence the plasmid has a size of 41.5 kb and codes for 50 putative open reading frames (orfs), 29 of which represent genes involved in replication, partitioning and transfer functions of the plasmid. Phylogenetic analysis grouped pSFA231 into the newly defined PromA plasmid family, which currently includes five members. Further comparative genomic analysis shows that pSFA231 shares the common backbone regions with the other PromA plasmids, i.e., genes involved in replication, maintenance and control, and conjugative transfer. Nevertheless, phylogenetic divergence was found in specific gene products. We propose to divide the PromA group into two subgroups, PromA-α (pMRAD02, pSB102) and PromA-β (pMOL98, pIPO2T, pSFA231, pTer331), based on the splits network analysis of the RepA protein. Interestingly, a cluster of hypothetical orfs located between parA and traA of pSFA231 shows high similarity with the corresponding regions on pMOL98, pIPO2T, and pTer331, suggesting these hypothetical orfs may represent "essential" plasmid backbone genes for the PromA-β subgroup. Alternatively, they may also be accessory genes that were first acquired and then stayed as the plasmid diverged. Our study increases the available collection of complete genome sequences of BHR plasmids, and since pSFA231 is the only characterized PromA plasmid from China, our findings also enhance our understanding of the genetic diversity of this plasmid group in different parts of the world.

  8. The broad-host-range plasmid pSFA231 isolated from petroleum-contaminated sediment represents a new member of the PromA plasmid family

    Directory of Open Access Journals (Sweden)

    Xiaobin eLi

    2015-01-01

    Full Text Available A self-transmissible broad-host-range (BHR plasmid pSFA231 was isolated from petroleum-contaminated sediment in Shen-fu wastewater irrigation zone, China, using the triparental mating exogenous plasmid capture method. Based on its complete sequence the plasmid has a size of 41.5 kb and codes for 50 putative open reading frames (orfs, 28 of which represent genes involved in replication, partitioning and transfer functions of the plasmid. Phylogenetic analysis grouped pSFA231 into the newly defined PromA plasmid family, which currently includes five members. Further comparative genomic analysis shows that pSFA231 shares the common backbone regions with the other PromA plasmids, i.e., genes involved in replication, maintenance and control, and conjugative transfer. Nevertheless, phylogenetic divergence was found in specific gene products. We propose to divide the PromA group into two subgroups, PromA-α (pMRAD02, pSB102 and PromA-β (pMOL98, pIPO2T, pSFA231, pTer331, based on the splits network analysis of the RepA protein. Interestingly, a cluster of hypothetical orfs located between parA and traA of pSFA231 shows high similarity with the corresponding regions on pMOL98, pIPO2T and pTer331, suggesting these hypothetical orfs may represent ‘‘essential’’ plasmid backbone genes for the PromA-β subgroup. Alternatively, they may also be accessory genes that were first acquired and then stayed as the plasmid diverged. Our study increases the available collection of complete genome sequences of BHR plasmids, and since pSFA231 is the only characterized PromA plasmid from China, our findings also enhance our understanding of the genetic diversity of this plasmid group in different parts of the world.

  9. Effect of synthetic iron colloids on the microbiological NH(4)(+) removal process during groundwater purification.

    Science.gov (United States)

    Wolthoorn, Anke; Temminghoff, Erwin J M; van Riemsdijk, Willem H

    2004-04-01

    Subsurface aeration is used to oxidise Fe in situ in groundwater that is used to make drinking water potable. In a groundwater system with pH>7 subsurface aeration results in non-mobile Fe precipitate and mobile Fe colloids. Since originally the goal of subsurface aeration is to remove iron in situ, the formation of non-mobile iron precipitate, which facilitates the metal's removal, is the desired result. In addition to this intended effect, subsurface aeration may also strongly enhance the microbiological removal of ammonium (NH(4)(+)) in the purification station. Mobile iron colloids could be the link between subsurface aeration and the positive effect on the NH(4)(+) removal process. Therefore, the objective of this study was to assess whether synthetic iron colloids could improve the NH(4)(+) removal process. The effect of synthetic iron colloids on the NH(4)(+) removal process was studied using an artificial purification set-up on a laboratory scale. Columns that purified groundwater with or without added synthetic iron colloids were set up in duplicate. The results showed that the NH(4)(+) removal was significantly ( alpha = 0.05 ) increased in columns treated with the synthetic iron colloids. Cumulative after 4 months about 10% more NH(4)(+) was nitrified in the columns that was treated with the groundwater containing synthetic iron colloids. The results support the hypothesis that mobile iron colloids could be the link between subsurface aeration and the positive effect on the NH(4)(+) removal process.

  10. Extraction and purification of yellow cake

    International Nuclear Information System (INIS)

    Yousif, E.H.

    2006-01-01

    This dissertation has reviewed current studies on production and purification of yellow cake from uranium ores by both acid and alkaline leaching processes. It comprises three chapters, the first one deal with uranium minerals, uranium deposits, geology of uranium and uranium isotopes. The second chapter covers mining and milling methods, uranium leaching chemistry, precipitation, and purification of uranium concentrate by solvent extraction and possible impurities that commonly interfered with yellow cake. The last chapter presented ongoing literature review.(Author)

  11. [Studies on purification of total glycosides of paeony (TGP) from Paeonia lactiflora by ethanol gradient combined with resin processing].

    Science.gov (United States)

    Shu, Xin-Ying; Meng, Xian-Sheng; Pan, Ying; Han, Ling; Bao, Yong-Rui; Guo, Xiao-Rui

    2010-11-01

    To study the purification technology of TGP from Paeonia lactiflora by ethanol gradient combined with resin processing and determine the optimum technological conditions and parameters. Using orthogonal test design to investigate the effect of ethanol gradient treatment on the content of TGP. Moreover, from the static and dynamic adsorption nine types of macroporous adsorption resin were evaluated to select the best resin type and the optimum separation and purification conditions. The best technology of Paeonia lactiflora ethanol precipitation was concentration of 1 g crude drug/mL precipitated by 95% ethanol to 90% concentration and then frozen for 10 h. HPD300 resin was the optimal model for the separation and purification of TGP from Paeonia lactiflora, with 5BV of 50% ethanol eluenting and the ratio of herb to resin was 2:1 . This technology is suitable and advanced for industry production and it is simple and convenient, rapid, accurate, etc.

  12. Plasmids foster diversification and adaptation of bacterial populations in soil.

    Science.gov (United States)

    Heuer, Holger; Smalla, Kornelia

    2012-11-01

    It is increasingly being recognized that the transfer of conjugative plasmids across species boundaries plays a vital role in the adaptability of bacterial populations in soil. There are specific driving forces and constraints of plasmid transfer within bacterial communities in soils. Plasmid-mediated genetic variation allows bacteria to respond rapidly with adaptive responses to challenges such as irregular antibiotic or metal concentrations, or opportunities such as the utilization of xenobiotic compounds. Cultivation-independent detection and capture of plasmids from soil bacteria, and complete sequencing have provided new insights into the role and ecology of plasmids. Broad host range plasmids such as those belonging to IncP-1 transfer a wealth of accessory functions which are carried by similar plasmid backbones. Plasmids with a narrower host range can be more specifically adapted to particular species and often transfer genes which complement chromosomally encoded functions. Plasmids seem to be an ancient and successful strategy to ensure survival of a soil population in spatial and temporal heterogeneous conditions with various environmental stresses or opportunities that occur irregularly or as a novel challenge in soil. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  13. Performance of photocatalyst based carbon nanodots from waste frying oil in water purification

    International Nuclear Information System (INIS)

    Aji, Mahardika Prasetya; Wiguna, Pradita Ajeng; Susanto,; Rosita, Nita; Suciningtyas, Siti Aisyah; Sulhadi

    2016-01-01

    Carbon Nanodots (C-Dots) from waste frying oil could be used as a photocatalyst in water purification with solar light irradiation. Performance of C-Dots as a photocatalyst was tested in the process of water purification with a given synthetic sewage methylene blue. The tested was also conducted by comparing the performance C-Dots made from frying oil, waste fryng oil as a photocatalyst and solution of methylene blue without photocatalyst C-Dots. Performance of C-Dots from waste frying oil were estimated by the results of absorbance spectrum. The results of measurement absorbance spectrum from the process of water purification with photocatalyst C-Dots showed that the highest intensity at a wavelength 664 nm of methylene blue decreased. The test results showed that the performance of photocatalyst C-Dots from waste frying oil was better in water purification. This estimated that number of particles C-dots is more in waste frying oil because have experieced repeated the heating process so that the higher particles concentration make the photocatalyst process more effective. The observation of the performance C-Dots from waste frying oil as a photocatalyst in the water purification processes become important invention for solving the problems of waste and water purification.

  14. Performance of photocatalyst based carbon nanodots from waste frying oil in water purification

    Energy Technology Data Exchange (ETDEWEB)

    Aji, Mahardika Prasetya, E-mail: mahardika190@gmail.com; Wiguna, Pradita Ajeng; Susanto,; Rosita, Nita; Suciningtyas, Siti Aisyah; Sulhadi [Department of Physics, Faculty of Mathematics and Natural Science Universitas Negeri Semarang, Jalan Raya Sekaran Gunungpati 50229 Indonesia (Indonesia)

    2016-04-19

    Carbon Nanodots (C-Dots) from waste frying oil could be used as a photocatalyst in water purification with solar light irradiation. Performance of C-Dots as a photocatalyst was tested in the process of water purification with a given synthetic sewage methylene blue. The tested was also conducted by comparing the performance C-Dots made from frying oil, waste fryng oil as a photocatalyst and solution of methylene blue without photocatalyst C-Dots. Performance of C-Dots from waste frying oil were estimated by the results of absorbance spectrum. The results of measurement absorbance spectrum from the process of water purification with photocatalyst C-Dots showed that the highest intensity at a wavelength 664 nm of methylene blue decreased. The test results showed that the performance of photocatalyst C-Dots from waste frying oil was better in water purification. This estimated that number of particles C-dots is more in waste frying oil because have experieced repeated the heating process so that the higher particles concentration make the photocatalyst process more effective. The observation of the performance C-Dots from waste frying oil as a photocatalyst in the water purification processes become important invention for solving the problems of waste and water purification.

  15. Customization of copolymers to optimize selectivity and yield in polymer-driven antibody purification processes.

    Science.gov (United States)

    Capito, Florian; Skudas, Romas; Stanislawski, Bernd; Kolmar, Harald

    2013-01-01

    This manuscript describes customization of copolymers to be used for polymer-driven protein purification in bioprocessing. To understand how copolymer customization can be used for fine-tuning, precipitation behavior was analyzed for five target antibodies (mAbs) and BSA as model impurity protein, at ionic strength similar to undiluted cell culture fluid. In contrast to the use of standardized homopolymers, customized copolymers, composed of 2-acrylamido-2-methylpropane sulfonic acid (AMPS) and 4-(acryloylamino)benzoic acid (ABZ), exhibited antibody precipitation yields exceeding 90%. Additionally, copolymer average molecular weight (Mw ) was varied and its influence on precipitation yield and contaminant coprecipitation was investigated. Results revealed copolymer composition as the major driving force for precipitation selectivity, which was also dependent on protein hydrophobicity. By adjusting ABZ content and Mw of the precipitant for each of the mAbs, conditions were found that allowed for high precipitation yield and selectivity. These findings may open up new avenues for using polymers in antibody purification processes. © 2013 American Institute of Chemical Engineers.

  16. Development of solvent extraction process for erbium purification

    International Nuclear Information System (INIS)

    Singh, D.K.; Anitha, M.; Vijayalakshmi, R.; Chakravartty, J.K.

    2016-01-01

    Erbium an important heavy rare earth (HRE) finds valuable application in space and nuclear energy technology. High purity erbium oxide is used as coating material for test blanket module of fusion reactor to prevent the tritium permeation. The total concentration of HRE (including Er) in only proven resource of rare earths in Indian monazite mineral is < 0.05%. Its separation from such a low concentration and also from host of other chemically similar elements like Y, Dy, Ho, Yb, Tm etc is quite difficult and challenging. A solvent extraction process employing PC88A and Aliquat336 has been developed for the purification of erbium oxide from two types of HRE fractions having % composition; (i) Y 2 O 3 : 0.18, Tb 4 O 7 : 0.28, Dy 2 CO 2 : 47.07, Er 2 O 3 : 35.03, HO 2 O 3 : 10.11, Yb 2 O 3 : 5.88, Tm 2 O 3 : 1.43 and (ii) Dy 2 O 3 : 6.39, Er 2 O 3 : 49.43, HO 2 O 3 : 10.43, Tm 2 O 3 : 2.7, Y 2 O 3 : 24.08, Yb 2 O 3 : 6.96. PC88A was used to process low Y content concentrate from chloride medium whereas Aliquat336 was found to be suitable in thiocynate medium to treat the concentrate with high Y content. Effects of process variables such as acidity, extractant concentration, total oxide concentration in feed, number of stages, phase ratio, scrubbing agent were investigated for both the systems

  17. Extraction and partial purification of chitinase from mycosymbiont and its relation with mycorrhiza association process

    International Nuclear Information System (INIS)

    Darusman, L.K.; Purwakusumah, E.D.; Nurlia, N.

    1999-01-01

    Extraction effectivity and partial purification of chitinase extracellular metabolite from Scleroderma columnare, Pisolithus tinctorius, Trichoderma harzianum and the root of Shorea selanica have been searched. S. columnare and P. tinctorius were the mycosymbiont for S. selanica while T. harzianum was not. (NH4)2SO4 was the very effective solvent for crude extracellular enzyme extraction, followed by PEG-6000 and ethanol 50 percent respectively. The activity of P. tinctorius was the lowest among the microbe while S. columnare was the highest. The activity lowered by purification process but specific activity was not. The chitinase activity of inoculated root was higher in the S. columnare association than P. tinctorius's also the percentage of root chitin content and infection rate. It mean that S. columnare more effective as mycosymbiont. The periods of association lowered the activity of root chitinase but this phenomenon did not happen in the root of S. selanica with T. harzianum infection

  18. ProteinTracker: an application for managing protein production and purification

    Directory of Open Access Journals (Sweden)

    Ponko Stefan C

    2012-05-01

    Full Text Available Abstract Background Laboratories that produce protein reagents for research and development face the challenge of deciding whether to track batch-related data using simple file based storage mechanisms (e.g. spreadsheets and notebooks, or commit the time and effort to install, configure and maintain a more complex laboratory information management system (LIMS. Managing reagent data stored in files is challenging because files are often copied, moved, and reformatted. Furthermore, there is no simple way to query the data if/when questions arise. Commercial LIMS often include additional modules that may be paid for but not actually used, and often require software expertise to truly customize them for a given environment. Findings This web-application allows small to medium-sized protein production groups to track data related to plasmid DNA, conditioned media samples (supes, cell lines used for expression, and purified protein information, including method of purification and quality control results. In addition, a request system was added that includes a means of prioritizing requests to help manage the high demand of protein production resources at most organizations. ProteinTracker makes extensive use of existing open-source libraries and is designed to track essential data related to the production and purification of proteins. Conclusions ProteinTracker is an open-source web-based application that provides organizations with the ability to track key data involved in the production and purification of proteins and may be modified to meet the specific needs of an organization. The source code and database setup script can be downloaded from http://sourceforge.net/projects/proteintracker. This site also contains installation instructions and a user guide. A demonstration version of the application can be viewed at http://www.proteintracker.org.

  19. ProteinTracker: an application for managing protein production and purification

    Science.gov (United States)

    2012-01-01

    Background Laboratories that produce protein reagents for research and development face the challenge of deciding whether to track batch-related data using simple file based storage mechanisms (e.g. spreadsheets and notebooks), or commit the time and effort to install, configure and maintain a more complex laboratory information management system (LIMS). Managing reagent data stored in files is challenging because files are often copied, moved, and reformatted. Furthermore, there is no simple way to query the data if/when questions arise. Commercial LIMS often include additional modules that may be paid for but not actually used, and often require software expertise to truly customize them for a given environment. Findings This web-application allows small to medium-sized protein production groups to track data related to plasmid DNA, conditioned media samples (supes), cell lines used for expression, and purified protein information, including method of purification and quality control results. In addition, a request system was added that includes a means of prioritizing requests to help manage the high demand of protein production resources at most organizations. ProteinTracker makes extensive use of existing open-source libraries and is designed to track essential data related to the production and purification of proteins. Conclusions ProteinTracker is an open-source web-based application that provides organizations with the ability to track key data involved in the production and purification of proteins and may be modified to meet the specific needs of an organization. The source code and database setup script can be downloaded from http://sourceforge.net/projects/proteintracker. This site also contains installation instructions and a user guide. A demonstration version of the application can be viewed at http://www.proteintracker.org. PMID:22574679

  20. Mutation in ESBL Plasmid from Escherichia coli O104:H4 Leads Autoagglutination and Enhanced Plasmid Dissemination

    Directory of Open Access Journals (Sweden)

    Mickaël Poidevin

    2018-02-01

    Full Text Available Conjugative plasmids are one of the main driving force of wide-spreading of multidrug resistance (MDR bacteria. They are self-transmittable via conjugation as carrying the required set of genes and cis-acting DNA locus for direct cell-to-cell transfer. IncI incompatibility plasmids are nowadays often associated with extended-spectrum beta-lactamases producing Enterobacteria in clinic and environment. pESBL-EA11 was isolated from Escherichia coli O104:H4 outbreak strain in Germany in 2011. During the previous study identifying transfer genes of pESBL-EA11, it was shown that transposon insertion at certain DNA region of the plasmid, referred to as Hft, resulted in great enhancement of transfer ability. This suggested that genetic modifications can enhance dissemination of MDR plasmids. Such ‘superspreader’ mutations have attracted little attention so far despite their high potential to worsen MDR spreading. Present study aimed to gain our understanding on regulatory elements that involved pESBL transfer. While previous studies of IncI plasmids indicated that immediate downstream gene of Hft, traA, is not essential for conjugative transfer, here we showed that overexpression of TraA in host cell elevated transfer rate of pESBL-EA11. Transposon insertion or certain nucleotide substitutions in Hft led strong TraA overexpression which resulted in activation of essential regulator TraB and likely overexpression of conjugative pili. Atmospheric Scanning Electron Microscopy observation suggested that IncI pili are distinct from other types of conjugative pili (such as long filamentous F-type pili and rather expressed throughout the cell surface. High transfer efficiency in the mutant pESBL-EA11 was involved with hyperpiliation which facilitates cell-to-cell adhesion, including autoagglutination. The capability of plasmids to evolve to highly transmissible mutant is alarming, particularly it might also have adverse effect on host pathogenicity.

  1. Recovery and purification of ethylene

    Science.gov (United States)

    Reyneke, Rian [Katy, TX; Foral, Michael J [Aurora, IL; Lee, Guang-Chung [Houston, TX; Eng, Wayne W. Y. [League City, TX; Sinclair, Iain [Warrington, GB; Lodgson, Jeffery S [Naperville, IL

    2008-10-21

    A process for the recovery and purification of ethylene and optionally propylene from a stream containing lighter and heavier components that employs an ethylene distributor column and a partially thermally coupled distributed distillation system.

  2. Plasmid-associated sensitivity of Bacillus thuringiensis to UV light

    International Nuclear Information System (INIS)

    Benoit, T.G.; Wilson, G.R.; Bull, D.L.; Aronson, A.I.

    1990-01-01

    Spores and vegetative cells of Bacillus thuringiensis were more sensitive to UV light than were spores or cells of plasmid-cured B. thuringiensis strains or of the closely related Bacillus cereus. Introduction of B. thuringiensis plasmids into B. cereus by cell mating increased the UV sensitivity of the cells and spores. Protoxins encoded by one or more B. thuringiensis plasmids were not involved in spore sensitivity, since a B. thuringiensis strain conditional for protoxin accumulation was equally sensitive at the permissive and nonpermissive temperatures. In addition, introduction of either a cloned protoxin gene, the cloning vector, or another plasmid not containing a protoxin gene into a plasmid-cured strain of B. thuringiensis all increased the UV sensitivity of the spores. Although the variety of small, acid-soluble proteins was the same in the spores of all strains examined, the quantity of dipicolinic acid was about twice as high in the plasmid-containing strains, and this may account for the differences in UV sensitivity of the spores. The cells of some strains harboring only B. thuringiensis plasmids were much more sensitive than cells of any of the other strains, and the differences were much greater than observed with spores

  3. RepA and RepB exert plasmid incompatibility repressing the transcription of the repABC operon.

    Science.gov (United States)

    Pérez-Oseguera, Angeles; Cevallos, Miguel A

    2013-11-01

    Rhizobium etli CFN42 has a multipartite genome composed of one chromosome and six large plasmids with low copy numbers, all belonging to the repABC plasmid family. All elements essential for replication and segregation of these plasmids are encoded within the repABC operon. RepA and RepB direct plasmid segregation and are involved in the transcriptional regulation of the operon, and RepC is the initiator protein of the plasmid. Here we show that in addition to RepA (repressor) and RepB (corepressor), full transcriptional repression of the operon located in the symbiotic plasmid (pRetCFN42d) of this strain requires parS, the centromere-like sequence, and the operator sequence. However, the co-expression of RepA and RepB is sufficient to induce the displacement of the parental plasmid. RepA is a Walker-type ATPase that self associates in vivo and in vitro and binds specifically to the operator region in its RepA-ADP form. In contrast, RepA-ATP is capable of binding to non-specific DNA. RepA and RepB form high molecular weight DNA-protein complexes in the presence of ATP and ADP. RepA carrying ATP-pocket motif mutations induce full repression of the repABC operon without the participation of RepB and parS. These mutants specifically bind the operator sequence in their ATP or ADP bound forms. In addition, their expression in trans exerts plasmid incompatibility against the parental plasmid. RepA and RepB expressed in trans induce plasmid incompatibility because of their ability to repress the repABC operon and not only by their capacity to distort the plasmid segregation process. Copyright © 2013 Elsevier Inc. All rights reserved.

  4. CONTINUOUS CHELATION-EXTRACTION PROCESS FOR THE SEPARATION AND PURIFICATION OF METALS

    Science.gov (United States)

    Thomas, J.R.; Hicks, T.E.; Rubin, B.; Crandall, H.W.

    1959-12-01

    A continuous process is presented for separating metal values and groups of metal values from each other. A complex mixture. e.g., neutron-irradiated uranium, can be resolved into component parts. In the present process the values are dissolved in an acidic solution and adjusted to the proper oxidation state. Thenceforth the solution is contacted with an extractant phase comprising a fluorinated beta -diketone in an organic solvent under centain pH conditions whereupon plutonium and zirconium are extracted. Plutonium is extracted from the foregoing extract with reducing aqueous solutions or under specified acidic conditions and can be recovered from the aqueous solution. Zirconium is then removed with an oxalic acid aqueous phase. The uranium is recovered from the residual original solution using hexone and hexone-diketone extractants leaving residual fission products in the original solution. The uranium is extracted from the hexone solution with dilute nitric acid. Improved separations and purifications are achieved using recycled scrub solutions and the "self-salting" effect of uranyl ions.

  5. Emulsion Liquid Membrane Technology in Organic Acid Purification

    International Nuclear Information System (INIS)

    Norela Jusoh; Norasikin Othman; Nur Alina Nasruddin

    2016-01-01

    Emulsion Liquid Membrane (ELM) process have shown a great potential in wide application of industrial separations such as in removal of many chemicals, organic compounds, metal ions, pollutants and biomolecules. This system promote many advantages including simple operation, high selectivity, low energy requirement, and single stage extraction and stripping process. One potential application of ELM is in the purification of succinic acid from fermentation broth. This study outline steps for developing emulsion liquid membrane process in purification of succinic acid. The steps include liquid membrane formulation, ELM stability and ELM extraction of succinic acid. Several carrier, diluent and stripping agent was screened to find appropriate membrane formulation. After that, ELM stability was investigated to enhance the recovery of succinic acid. Finally, the performance of ELM was evaluated in the extraction process. Results show that formulated liquid membrane using Amberlite LA2 as carrier, palm oil as diluent and sodium carbonate, Na_2CO_3 as stripping agent provide good performance in purification. On the other hand, the prepared emulsion was observed to be stable up to 1 hour and sufficient for extraction process. In conclusion, ELM has high potential to purify succinic acid from fermentation broth. (author)

  6. Purification and Genetic Characterization of Enterocin I from Enterococcus faecium 6T1a, a Novel Antilisterial Plasmid-Encoded Bacteriocin Which Does Not Belong to the Pediocin Family of Bacteriocins

    Science.gov (United States)

    Floriano, Belén; Ruiz-Barba, José L.; Jiménez-Díaz, Rufino

    1998-01-01

    Enterocin I (ENTI) is a novel bacteriocin produced by Enterococcus faecium 6T1a, a strain originally isolated from a Spanish-style green olive fermentation. The bacteriocin is active against many olive spoilage and food-borne gram-positive pathogenic bacteria, including clostridia, propionibacteria, and Listeria monocytogenes. ENTI was purified to homogeneity by ammonium sulfate precipitation, binding to an SP-Sepharose fast-flow column, and phenyl-Sepharose CL-4B and C2/C18 reverse-phase chromatography. The purification procedure resulted in a final yield of 954% and a 170,000-fold increase in specific activity. The primary structure of ENTI was determined by amino acid and nucleotide sequencing. ENTI consists of 44 amino acids and does not show significant sequence similarity with any other previously described bacteriocin. Sequencing of the entI structural gene, which is located on the 23-kb plasmid pEF1 of E. faecium 6T1a, revealed the absence of a leader peptide at the N-terminal region of the gene product. A second open reading frame, ORF2, located downstream of entI, encodes a putative protein that is 72.7% identical to ENTI. entI and ORF2 appear to be cotranscribed, yielding an mRNA of ca. 0.35 kb. A gene encoding immunity to ENTI was not identified. However, curing experiments demonstrated that both enterocin production and immunity are conferred by pEF1. PMID:9835578

  7. Unraveling the regulatory network of IncA/C plasmid mobilization: When genomic islands hijack conjugative elements.

    Science.gov (United States)

    Carraro, Nicolas; Matteau, Dominick; Burrus, Vincent; Rodrigue, Sébastien

    2015-01-01

    Conjugative plasmids of the A/C incompatibility group (IncA/C) have become substantial players in the dissemination of multidrug resistance. These large conjugative plasmids are characterized by their broad host-range, extended spectrum of antimicrobials resistance, and prevalence in enteric bacteria recovered from both environmental and clinical settings. Until recently, relatively little was known about the basic biology of IncA/C plasmids, mostly because of the hindrance of multidrug resistance for molecular biology experiments. To circumvent this issue, we previously developed pVCR94ΔX, a convenient prototype that codes for a reduced set of antibiotic resistances. Using pVCR94ΔX, we then characterized the regulatory pathway governing IncA/C plasmid dissemination. We found that the expression of roughly 2 thirds of the genes encoded by this plasmid, including large operons involved in the conjugation process, depends on an FlhCD-like master activator called AcaCD. Beyond the mobility of IncA/C plasmids, AcaCD was also shown to play a key role in the mobilization of different classes of genomic islands (GIs) identified in various pathogenic bacteria. By doing so, IncA/C plasmids can have a considerable impact on bacterial genomes plasticity and evolution.

  8. Plasmid-Mediated Antimicrobial Resistance in Staphylococci and Other Firmicutes.

    Science.gov (United States)

    Schwarz, Stefan; Shen, Jianzhong; Wendlandt, Sarah; Fessler, Andrea T; Wang, Yang; Kadlec, Kristina; Wu, Cong-Ming

    2014-12-01

    In staphylococci and other Firmicutes, resistance to numerous classes of antimicrobial agents, which are commonly used in human and veterinary medicine, is mediated by genes that are associated with mobile genetic elements. The gene products of some of these antimicrobial resistance genes confer resistance to only specific members of a certain class of antimicrobial agents, whereas others confer resistance to the entire class or even to members of different classes of antimicrobial agents. The resistance mechanisms specified by the resistance genes fall into any of three major categories: active efflux, enzymatic inactivation, and modification/replacement/protection of the target sites of the antimicrobial agents. Among the mobile genetic elements that carry such resistance genes, plasmids play an important role as carriers of primarily plasmid-borne resistance genes, but also as vectors for nonconjugative and conjugative transposons that harbor resistance genes. Plasmids can be exchanged by horizontal gene transfer between members of the same species but also between bacteria belonging to different species and genera. Plasmids are highly flexible elements, and various mechanisms exist by which plasmids can recombine, form cointegrates, or become integrated in part or in toto into the chromosomal DNA or into other plasmids. As such, plasmids play a key role in the dissemination of antimicrobial resistance genes within the gene pool to which staphylococci and other Firmicutes have access. This chapter is intended to provide an overview of the current knowledge of plasmid-mediated antimicrobial resistance in staphylococci and other Firmicutes.

  9. Plasmid DNA damage induced by helium atmospheric pressure plasma jet

    Science.gov (United States)

    Han, Xu; Cantrell, William A.; Escobar, Erika E.; Ptasinska, Sylwia

    2014-03-01

    A helium atmospheric pressure plasma jet (APPJ) is applied to induce damage to aqueous plasmid DNA. The resulting fractions of the DNA conformers, which indicate intact molecules or DNA with single- or double-strand breaks, are determined using agarose gel electrophoresis. The DNA strand breaks increase with a decrease in the distance between the APPJ and DNA samples under two working conditions of the plasma source with different parameters of applied electric pulses. The damage level induced in the plasmid DNA is also enhanced with increased plasma irradiation time. The reactive species generated in the APPJ are characterized by optical emission spectra, and their roles in possible DNA damage processes occurring in an aqueous environment are also discussed.

  10. Comparative genomics of the IncA/C multidrug resistance plasmid family.

    Science.gov (United States)

    Fricke, W Florian; Welch, Timothy J; McDermott, Patrick F; Mammel, Mark K; LeClerc, J Eugene; White, David G; Cebula, Thomas A; Ravel, Jacques

    2009-08-01

    Multidrug resistance (MDR) plasmids belonging to the IncA/C plasmid family are widely distributed among Salmonella and other enterobacterial isolates from agricultural sources and have, at least once, also been identified in a drug-resistant Yersinia pestis isolate (IP275) from Madagascar. Here, we present the complete plasmid sequences of the IncA/C reference plasmid pRA1 (143,963 bp), isolated in 1971 from the fish pathogen Aeromonas hydrophila, and of the cryptic IncA/C plasmid pRAx (49,763 bp), isolated from Escherichia coli transconjugant D7-3, which was obtained through pRA1 transfer in 1980. Using comparative sequence analysis of pRA1 and pRAx with recent members of the IncA/C plasmid family, we show that both plasmids provide novel insights into the evolution of the IncA/C MDR plasmid family and the minimal machinery necessary for stable IncA/C plasmid maintenance. Our results indicate that recent members of the IncA/C plasmid family evolved from a common ancestor, similar in composition to pRA1, through stepwise integration of horizontally acquired resistance gene arrays into a conserved plasmid backbone. Phylogenetic comparisons predict type IV secretion-like conjugative transfer operons encoded on the shared plasmid backbones to be closely related to a group of integrating conjugative elements, which use conjugative transfer for horizontal propagation but stably integrate into the host chromosome during vegetative growth. A hipAB toxin-antitoxin gene cluster found on pRA1, which in Escherichia coli is involved in the formation of persister cell subpopulations, suggests persistence as an early broad-spectrum antimicrobial resistance mechanism in the evolution of IncA/C resistance plasmids.

  11. Generation, concentration and purification for ionic entangled states

    International Nuclear Information System (INIS)

    Yang Ming; Cao Zhuoliang

    2007-01-01

    In cavity QED, the atoms would be sent through the sequential arrays of cavities for the generation of multi-cavity entanglement, or several atoms would be sent into the same cavity mode one bye one for the generation of multi-atom entanglement. The complexity of these processes will impose limitations on the experimental feasibility of it. So, following our previous publication [International Journal Of Quantum Information 2, 231 (2004)] we will propose an alternative scheme for the preparation of multi-cavity W state via cavity QED, which uses the geometrical method to do what other authors have proposed previously using sequential arrays of cavities. Due to the impossibility that one quantum system can be isolated from the environment absolutely, the entanglement of the entangled objects will decrease exponentially with the propagating distance of the objects, and the practically available quantum entangled states are all non-maximally entangled states or the more general case--mixed states. Following our previous publications [Phys. Rev. A 72, 042307 (2005), ibid. 71, 012308 (2005)], we will propose an entanglement generation, concentration and purification scheme for atomic or ionic system, which is mainly based on Cavity QED and linear optical elements. This purification process avoids the controlled-NOT (C-NOT) operations needed in the original purification protocol, which simplifies the whole purification process

  12. Overview of the recombinant proteins purification by affinity tags and ...

    African Journals Online (AJOL)

    From protein within isolation process which the same matter increases labor costs further and prevents application of these tags in industrial scale. Therefore proper replacement is emphasized for enzymatic removal of purification tags. Keywords: protein purification; recombinant proteins; self-cleavable tags; Intein tags; ...

  13. Purification Simulation With Vapor Permeation and Distillation-Adsorption In Bioethanol Plant

    OpenAIRE

    Misri Gozan; Mia Sari Setiawan; Kenny Lischer

    2017-01-01

    High purity of Bioethanol is required in biofuel mixing with gasoline (EXX). In bioethanol production line, the azeotropic property of ethanol-water becomes the barrier for purification process. This study examined two bioethanol separation processes by support of simulation tools, Superpro Designer 9.0 software. Ethanol purity and a low costeconomical process were the major considerations. Purification method of vapor permeation membrane technology was compared with distillation-adsorption m...

  14. Oil quality of passion fruit seeds subjected to a pulp-waste purification process

    Directory of Open Access Journals (Sweden)

    Suelen Alvarenga Regis

    2015-06-01

    Full Text Available Passion fruit seeds must be clean and dry before the extraction processing to obtain high-quality oil for edible and cosmetic purposes. This research studies the viability of a cleaning process of seeds by evaluating the oil quality. The research examined 2 maturation stages of the fruit and one purification process of the seeds, compared to the control. The oil quality was evaluated by fatty acid composition, acidity, peroxide value and oxidative stability. The pulp waste suffered a thermal treatment in an alkaline water solution at 60°C for 10min and was further purified in an experimental decanter. In the control treatment, the pulp waste was processed using only water at ambient conditions. The passion fruit seeds were totally cleaned by the thermal/chemical treatment, allowing a faster drying (less than 50% of the drying time of the seeds and a bit higher yield of oil extraction (proportionally around 7.7%, without changes in quality of the oil

  15. Microbiological and technical aspects of anaerobic waste water purification

    International Nuclear Information System (INIS)

    Aivasidis, A.

    1994-01-01

    Anaerobic waste water purification is likely to be another example of how innovations can result from the joint use of biological and technical concepts. No matter how far the optimization of oxygen input with aerobic waste water purification advances it will still be the less a real competitor for anaerobic techniques the more polluted the waste water is. The principle of carrier fixation to avoid their washing out, too, has often been observed in nature with sessile microorganisms. With highly polluted water, anaerobic purification does not only work at no expenditure of energy but it can also make excess energy available for use in other processes. Another important argument for anaerobic methods of waste water purification is probably the clearly reduced production of excess sludge. (orig.) [de

  16. Frequency and diversity of small cryptic plasmids in the genus Rahnella

    Directory of Open Access Journals (Sweden)

    Summers David K

    2010-02-01

    Full Text Available Abstract Background Rahnella is a widely distributed genus belonging to the Enterobacteriaceae and frequently present on vegetables. Although Rahnella has interesting agro-economical and industrial properties and several strains possess antibiotic resistances and toxin genes which might spread within microbial communities, little is known about plasmids of this genus. Thus, we isolated a number of Rahnella strains and investigated their complements of small plasmids. Results In total 53 strains were investigated and 11 plasmids observed. Seven belonged to the ColE1 family; one was ColE2-like and three shared homology to rolling circle plasmids. One of them belonged to the pC194/pUB110 family and two showed similarity to poorly characterised plasmid groups. The G+C content of two rolling circle plasmids deviated considerably from that of Rahnella, indicating that their usual hosts might belong to other genera. Most ColE1-like plasmids formed a subgroup within the ColE1 family that seems to be fairly specific for Rahnella. Intriguingly, the multimer resolution sites of all ColE1-like plasmids had the same orientation with respect to the origin of replication. This arrangement might be necessary to prevent inappropriate synthesis of a small regulatory RNA that regulates cell division. Although the ColE1-like plasmids did not possess any mobilisation system, they shared large parts with high sequence identity in coding and non-coding regions. In addition, highly homologous regions of plasmids isolated from Rahnella and the chromosomes of Erwinia tasmaniensis and Photorhabdus luminescens could be identified. Conclusions For the genus Rahnella we observed plasmid-containing isolates at a frequency of 19%, which is in the average range for Enterobacteriaceae. These plasmids belonged to diffent groups with members of the ColE1-family most frequently found. Regions of striking sequence homology of plasmids and bacterial chromosomes highlight the

  17. Plasmid mediated enhancement of uv resistance in Streptococcus faecalis

    International Nuclear Information System (INIS)

    Miehl, R.; Miller, M.; Yasbin, R.E.

    1980-01-01

    A 38.5-Mdal plasmid of Streptococcus faecalis subdp. zymogenes has been shown to enhance survival following uv irradiation. In addition, the presence of this plasmid increases the mutation frequencies following uv irradiation and enhanced W-reactivation. The data presented indicate that S. faecalis has an inducible error-prone repair system and that the plasmid enhances these repair functions

  18. Multiple drug resistant carbapenemases producing Acinetobacter baumannii isolates harbours multiple R-plasmids

    Directory of Open Access Journals (Sweden)

    Rajagopalan Saranathan

    2014-01-01

    Full Text Available Background & objectives: The nosocomial human pathogen Acinetobacter baumannii has high propensity to develop resistance to antimicrobials and to become multidrug resistant (MDR, consequently complicating the treatment. This study was carried out to investigate the presence of resistant plasmids (R-plasmids among the clinical isolates of A. baumannii. In addition, the study was performed to check the presence of common β-lactamases encoding genes on these plasmids. Methods: A total of 55 clinical isolates of A. baumannii were included in the study and all were subjected to plasmid DNA isolation, followed by PCR to check the presence of resistance gene determinants such as blaOXA-23 , blaOXA-51, blaOXA-58 and blaIMP-1 on these plasmids that encode for oxacillinase (OXA and metallo-β-lactamase (MBL type of carbapenemases. Plasmid curing experiments were carried out on selected isolates using ethidium bromide and acridine orange as curing agents and the antibiotic resistance profiles were evaluated before and after curing. Results: All the isolates were identified as A. baumannii by 16SrDNA amplification and sequencing. Plasmid DNA isolated from these isolates showed the occurrence of multiple plasmids with size ranging from 500bp to ≥ 25 kb. The percentage of blaOXA-51 and blaOXA-23 on plasmids were found to be 78 and 42 per cent, respectively and 20 isolates (36% carried blaIMP-1 gene on plasmids. Significant difference was observed in the antibiograms of plasmid cured isolates when compared to their parental ones. The clinical isolates became susceptible to more than two antibiotic classes after curing of plasmids indicating plasmid borne resistance. Interpretation & conclusions: Our study determined the plasmid mediated resistance mechanisms and occurrence of different resistance genes on various plasmids isolated from MDR A. baumannii. The present findings showed the evidence for antibiotic resistance mediated through multiple plasmids in

  19. Movement and equipositioning of plasmids by ParA filament disassembly

    DEFF Research Database (Denmark)

    Ringgaard, Simon; van Zon, Jeroen; Howard, Martin

    2009-01-01

    , plasmids consistently migrate behind disassembling ParA cytoskeletal structures, suggesting that ParA filaments pull plasmids by depolymerization. The perpetual cycles of ParA assembly and disassembly result in continuous relocation of plasmids, which, on time averaging, results in equidistribution...

  20. Purification and detoxification of petroleum refinery wastewater by electrocoagulation process.

    Science.gov (United States)

    Gousmi, N; Sahmi, A; Li, H Z; Poncin, S; Djebbar, R; Bensadok, K

    2016-09-01

    The treatment of synthetic oily wastewater having the characteristics of a typical petroleum refinery wastewater (PRW) by electrocoagulation (EC) using iron and aluminum electrodes was conducted in an electrolytic reactor equipped with fluid recirculation. During the treatment, the emulsion stability was followed by the measurement of Zeta potential and particle sizes. Effects of some operating conditions such as electrodes material, current density and electrolysis time on removal efficiencies of turbidity, and chemical oxygen demand (COD) were investigated in detail. The PRW purification by the EC process was found to be the most effective using aluminum as the anode and cathode, current density of 60 A/m(2) and 30 min of electrolysis time. Under these conditions, the process efficiencies were 83.52% and 99.94%, respectively, for COD and turbidity removals which correspond to final values of 96 mg O2/L and 0.5 NTU. A moderate energy consumption (0.341 kWh) was needed to treat 1 m(3) of PRW. Besides, the ecotoxicity test proved that toxic substances presented in the PRW, and those inhibiting the germination growth of whet, were eliminated by the EC technique.

  1. Purification of crude glycerol from transesterification reaction of palm oil using direct method and multistep method

    Science.gov (United States)

    Nasir, N. F.; Mirus, M. F.; Ismail, M.

    2017-09-01

    Crude glycerol which produced from transesterification reaction has limited usage if it does not undergo purification process. It also contains excess methanol, catalyst and soap. Conventionally, purification method of the crude glycerol involves high cost and complex processes. This study aimed to determine the effects of using different purification methods which are direct method (comprises of ion exchange and methanol removal steps) and multistep method (comprises of neutralization, filtration, ion exchange and methanol removal steps). Two crude glycerol samples were investigated; the self-produced sample through the transesterification process of palm oil and the sample obtained from biodiesel plant. Samples were analysed using Fourier Transform Infrared Spectroscopy, Gas Chromatography and High Performance Liquid Chromatography. The results of this study for both samples after purification have showed that the pure glycerol was successfully produced and fatty acid salts were eliminated. Also, the results indicated the absence of methanol in both samples after purification process. In short, the combination of 4 purification steps has contributed to a higher quality of glycerol. Multistep purification method gave a better result compared to the direct method as neutralization and filtration steps helped in removing most excess salt, fatty acid and catalyst.

  2. Chemical purification of Gunungpati elephant foot yam flour to improve physical and chemical quality on processed food

    Science.gov (United States)

    Paramita, Octavianti; Wahyuningsih, Ansori, Muhammad

    2017-03-01

    This study was aimed at improving the physicochemical quality of elephant foot yam flour in Gunungpati, Semarang by chemical purification. The utilization of elephant foot yam flour in several processed food was also discussed in this study. The flour purification discussed in this study was expected to become a reference for the manufacturers of elephant foot yam flour and its processed food in Gunungpati. This study modified the elephant foot yam flour using pre - gelatinization method. The physical and chemical quality of each elephant foot yam flour purification sample were assessed using proximate analysis. The likability test was conducted for its processed food. 20 grams of elephant foot yam flour was put into a beaker glass, then 60 ml of water was added. The suspension was then heated at a temperature of 60 ° C and 70 ° C while stirred until it was homogeneous and thickened for 10, 30 and 60 minutes. The flour which had been heated was then cooled at room temperature for 1 hour and then at a temperature of 0 ° C until it was frozen. Furthermore, flour was dried in an oven at a temperature of 60 ° C for 9 hours. The dried flour was sifted with a 80 mesh sieve. Chemical test was conducted after elephant foot yam was pre-gelatinized to determine changes in the quality flour: test levels of protein, fat, crude fiber content, moisture content, ash content and starch content. In addition, color tests and granular test on elephant foot yam flour were also conducted. The pre-gelatinization as chemical treatment on elephant foot yam flour in this study was able to change the functional properties of elephant foot yam flour towards a better processing characterized by a brighter color (L = 70, a = 6 and b = 12), the hydrolysis of polysaccharides flour into shorter chain (flour content decreased to 44%), the expansion of granules in elephant foot yam resulting in a process - ready flour, and better monolayer water content of 9%. The content of protein and fiber

  3. Restriction Fragment Length Polymorphisms of Virulence Plasmids in Rhodococcus equi

    Science.gov (United States)

    Takai, Shinji; Shoda, Masato; Sasaki, Yukako; Tsubaki, Shiro; Fortier, Guillaume; Pronost, Stephane; Rahal, Karim; Becu, Teotimo; Begg, Angela; Browning, Glenn; Nicholson, Vivian M.; Prescott, John F.

    1999-01-01

    Virulent Rhodococcus equi, which is a well-known cause of pyogranulomatous pneumonia in foals, possesses a large plasmid encoding virulence-associated 15- to 17-kDa antigens. Foal and soil isolates from five countries—Argentina, Australia, Canada, France, and Japan—were investigated for the presence of 15- to 17-kDa antigens by colony blotting, using the monoclonal antibody 10G5, and the gene coding for 15- to 17-kDa antigens by PCR. Plasmid DNAs extracted from positive isolates were digested with restriction endonucleases BamHI, EcoRI, EcoT22I, and HindIII, and the digestion patterns that resulted divided the plasmids of virulent isolates into five closely related types. Three of the five types had already been reported in Canadian and Japanese isolates, and the two new types had been found in French and Japanese isolates. Therefore, we tentatively designated these five types 85-kb type I (pREAT701), 85-kb type II (a new type), 87-kb type I (EcoRI and BamHI type 2 [V. M. Nicholson and J. F. Prescott, J. Clin. Microbiol. 35:738–740, 1997]), 87-kb type II (a new type), and 90-kb (pREL1) plasmids. The 85-kb type I plasmid was found in isolates from Argentina, Australia, Canada, and France. Plasmid 87-kb type I was isolated in specimens from Argentina, Canada, and France. The 85-kb type II plasmid appeared in isolates from France. On the other hand, plasmids 87-kb type II and 90-kb were found only in isolates from Japan. These results revealed geographic differences in the distribution of the virulence plasmids found in the five countries and suggested that the restriction fragment length polymorphism of virulence plasmids might be useful to elucidate the molecular epidemiology of virulent R. equi in the world. PMID:10488224

  4. Plasmid DNA Delivery: Nanotopography Matters.

    Science.gov (United States)

    Song, Hao; Yu, Meihua; Lu, Yao; Gu, Zhengying; Yang, Yannan; Zhang, Min; Fu, Jianye; Yu, Chengzhong

    2017-12-20

    Plasmid DNA molecules with unique loop structures have widespread bioapplications, in many cases relying heavily on delivery vehicles to introduce them into cells and achieve their functions. Herein, we demonstrate that control over delicate nanotopography of silica nanoparticles as plasmid DNA vectors has significant impact on the transfection efficacy. For silica nanoparticles with rambutan-, raspberry-, and flower-like morphologies composed of spike-, hemisphere-, and bowl-type subunit nanotopographies, respectively, the rambutan-like nanoparticles with spiky surfaces demonstrate the highest plasmid DNA binding capability and transfection efficacy of 88%, higher than those reported for silica-based nanovectors. Moreover, it is shown that the surface spikes of rambutan nanoparticles provide a continuous open space to bind DNA chains via multivalent interactions and protect the gene molecules sheltered in the spiky layer against nuclease degradation, exhibiting no significant transfection decay. This unique protection feature is in great contrast to a commercial transfection agent with similar transfection performance but poor protection capability against enzymatic cleavage. Our study provides new understandings in the rational design of nonviral vectors for efficient gene delivery.

  5. Rotating Reverse-Osmosis for Water Purification

    Science.gov (United States)

    Lueptow, RIchard M.

    2004-01-01

    A new design for a water-filtering device combines rotating filtration with reverse osmosis to create a rotating reverse- osmosis system. Rotating filtration has been used for separating plasma from whole blood, while reverse osmosis has been used in purification of water and in some chemical processes. Reverse- osmosis membranes are vulnerable to concentration polarization a type of fouling in which the chemicals meant not to pass through the reverse-osmosis membranes accumulate very near the surfaces of the membranes. The combination of rotating filtration and reverse osmosis is intended to prevent concentration polarization and thereby increase the desired flux of filtered water while decreasing the likelihood of passage of undesired chemical species through the filter. Devices based on this concept could be useful in a variety of commercial applications, including purification and desalination of drinking water, purification of pharmaceutical process water, treatment of household and industrial wastewater, and treatment of industrial process water. A rotating filter consists of a cylindrical porous microfilter rotating within a stationary concentric cylindrical outer shell (see figure). The aqueous suspension enters one end of the annulus between the inner and outer cylinders. Filtrate passes through the rotating cylindrical microfilter and is removed via a hollow shaft. The concentrated suspension is removed at the end of the annulus opposite the end where the suspension entered.

  6. DNA sequence analysis of plasmids from multidrug resistant Salmonella enterica serotype Heidelberg isolates.

    Directory of Open Access Journals (Sweden)

    Jing Han

    Full Text Available Salmonella enterica serovar Heidelberg is among the most detected serovars in swine and poultry, ranks among the top five serotypes associated with human salmonellosis and is disproportionately associated with invasive infections and mortality in humans. Salmonella are known to carry plasmids associated with antimicrobial resistance and virulence. To identify plasmid-associated genes in multidrug resistant S. enterica serovar Heidelberg, antimicrobial resistance plasmids from five isolates were sequenced using the 454 LifeSciences pyrosequencing technology. Four of the isolates contained incompatibility group (Inc A/C multidrug resistance plasmids harboring at least eight antimicrobial resistance genes. Each of these strains also carried a second resistance plasmid including two IncFIB, an IncHI2 and a plasmid lacking an identified Inc group. The fifth isolate contained an IncI1 plasmid, encoding resistance to gentamicin, streptomycin and sulfonamides. Some of the IncA/C plasmids lacked the full concert of transfer genes and yet were able to be conjugally transferred, likely due to the transfer genes carried on the companion plasmids in the strains. Several non-IncA/C resistance plasmids also carried putative virulence genes. When the sequences were compared to previously sequenced plasmids, it was found that while all plasmids demonstrated some similarity to other plasmids, they were unique, often due to differences in mobile genetic elements in the plasmids. Our study suggests that Salmonella Heidelberg isolates harbor plasmids that co-select for antimicrobial resistance and virulence, along with genes that can mediate the transfer of plasmids within and among other bacterial isolates. Prevalence of such plasmids can complicate efforts to control the spread of S. enterica serovar Heidelberg in food animal and human populations.

  7. Simple method for identification of plasmid-coded proteins

    International Nuclear Information System (INIS)

    Sancar, A.; Hack, A.M.; Rupp, W.D.

    1979-01-01

    Proteins encoded by plasmid DNA are specifically labeled in uv-irradiated cells of Escherichia coli carrying recA and uvrA mutations because extensive degradation of the chromosome DNA occurs concurrently with amplification of plasmid DNA

  8. Presence and analysis of plasmids in human and animal associated arcobacter species.

    Directory of Open Access Journals (Sweden)

    Laid Douidah

    Full Text Available In this study, we report the screening of four Arcobacter species for the presence of small and large plasmids. Plasmids were present in 9.9% of the 273 examined strains. One Arcobacter cryaerophilus and four Arcobacter butzleri plasmids were selected for further sequencing. The size of three small plasmids isolated from A. butzleri and the one from A. cryaerophilus strains ranged between 4.8 and 5.1 kb, and the size of the large plasmid, isolated from A. butzleri, was 27.4 kbp. The G+C content of all plasmids ranged between 25.4% and 26.2%. A total of 95% of the large plasmid sequence represents coding information, which contrasts to the 20 to 30% for the small plasmids. Some of the open reading frames showed a high homology to putative conserved domains found in other related organisms, such as replication, mobilization and genes involved in type IV secretion system. The large plasmid carried 35 coding sequences, including seven genes in a contiguous region of 11.6 kbp that encodes an orthologous type IV secretion system found in the Wolinella succinogenes genome, Helicobacter pylori and Campylobacter jejuni plasmids, which makes this plasmid interesting for further exploration.

  9. A new helium gas recovery and purification system

    International Nuclear Information System (INIS)

    Yamamotot, T.; Suzuki, H.; Ishii, J.; Hamana, I.; Hayashi, S.; Mizutani, S.; Sanjo, S.

    1974-01-01

    A helium gas recovery and purification system, based on the principle of gas permeation through a membrane, is described. The system can be used for the purification of helium gas containing air as a contaminant. The apparatus, operating at ambient temperature does not need constant attention, the recovery ratio of helium gas is satisfactory and running costs are low. Gases other than helium can be processed with the apparatus. (U.K.)

  10. Application of methylation in improving plasmid transformation into Helicobacter pylori.

    Science.gov (United States)

    Zhao, Huilin; Xu, Linlin; Rong, Qianyu; Xu, Zheng; Ding, Yunfei; Zhang, Ying; Wu, Yulong; Li, Boqing; Ji, Xiaofei

    2018-05-23

    Helicobacter pylori is an important gastrointestinal pathogen. Its strains possess different levels of powerful restriction modification systems, which are significant barriers to genetic tools used for studying the role of functional genes in its pathogenesis. Methylating vectors in vitro was reported as an alternative to overcome this barrier in several bacteria. In this study we used two H. pylori-E. coli shuttle plasmids and several single/double-crossover homologous recombination gene-targeting plasmids, to test the role of methylation in H. pylori transformation. According to our results, transformants could be obtained only after shuttle plasmids were methylated before transformation. It is helpful in gene complementation and over-expression although at a low frequency. The frequency of gene-targeting transformation was also increased after methylation, especially for the single-crossover recombination plasmids, the transformants of which could only be obtained after methylation. For the double-crossover recombination targeting plasmids, the initial yield of transformants was 0.3-0.8 × 10 2 CFUs per microgram plasmid DNA. With the help of methylation, the yield was increased to 0.4-1.3 × 10 2 CFUs per microgram plasmid DNA. These results suggest that in vitro methylation can improve H. pylori transformation by different plasmids, which will benefit the pathogenic mechanism research. Copyright © 2018. Published by Elsevier B.V.

  11. Plasmid marker rescue transformation proceeds by breakage-reunion in Bacillus subtilis

    International Nuclear Information System (INIS)

    Weinrauch, Y.; Dubnau, D.

    1987-01-01

    Bacillus subtilis carrying a plasmid which replicates with a copy number of about 1 was transformed with linearized homologous plasmid DNA labeled with the heavy isotopes 2 H and 15 N, in the presence of 32 Pi and 6-(p-hydroxyphenylazo)-uracil to inhibit DNA replication. Plasmid DNA was isolated from the transformed culture and fractionated in cesium chloride density gradients. The distribution of total and donor plasmid DNA was examined, using specific hybridization probes. The synthesis of new DNA, associated with the integration of donor moiety, was also monitored. Donor-specific sequences were present at a density intermediate between that of light and hybrid DNA. This recombinant DNA represented 1.4% of total plasmid DNA. The latter value corresponded well with the transforming activity (1.7%) obtained for the donor marker. Newly synthesized material associated with plasmid DNA at the recombinant density amounted to a minor portion of the recombinant plasmid DNA. These data suggest that, like chromosomal transformation, plasmid marker rescue transformation does not require replication for the integration of donor markers and, also like chromosomal transformation, proceeds by a breakage-reunion mechanism. The extent of donor DNA replacement of recipient DNA per plasmid molecule of 54 kilobases (27 kilobase pairs) was estimated as 16 kilobases

  12. THE ENDOGENOUS BACILLUS-SUBTILIS (NATTO) PLASMIDS PTA1015 AND PTA1040 CONTAIN SIGNAL PEPTIDASE-ENCODING GENES - IDENTIFICATION OF A NEW STRUCTURAL MODULE ON CRYPTIC PLASMIDS

    NARCIS (Netherlands)

    MEIJER, WJJ; DEJONG, A; BEA, G; WISMAN, A; TJALSMA, H; VENEMA, G; BRON, S; MAARTEN, J; VANDIJL, JM

    Various strains of Bacillus subtilis (natto) contain small cryptic plasmids that replicate via the rolling-circle mechanism. Like plasmids from other Gram-positive bacteria, these plasmids are composed of several distinct structural modules. A new structural module was identified on the B. subtilis

  13. Plasma processes including electron beam for off-gases purification

    International Nuclear Information System (INIS)

    Chmielewski, A.G.; Witman, S.; Licki, J.

    2011-01-01

    Complete text of publication follows. Non-thermal plasma technologies based on different methods of plasma generation are being applied for ozone generation for different applications, waste water and off-gases treatment. Plasmas create reactive species, in particular ions, radicals or other reactive compounds, which can decompose pollutant molecules, organic particulate matter or soot. Electron beam flue gas treatment is another plasma-based technology which has been successfully demonstrated on industrial scale coal fired power plants. High efficiency of SO 2 (> 95%) and NO x (> 70%) has been obtained and industrial plant applying this process has been built in Poland. The further investigations carried out all over the world have illustrated that the process can be applied for poly-aromatic hydrocarbons (PAH) destruction as well, and just recently research laboratories in the US and South Korea have reported in the feasibility of the process for mercury removal from the flue gas. The recent studies concern a new type of accelerators implementation in the industrial scale, application of the process in the high sulfur oil fired boilers and Diesel off - gases purification. The treatment of the flue gases with the high NOx concentration is a special challenge for the technology since the main energy consumption (and applied accelerators power) is related to this pollutant content in the processed off gases. The pulse beams and scavenger application can be a solution to reduce investment and operational costs. The further development of the technology is directly connected with high power accelerators development. Acknowledgement: The R and D activities are supported by the European Regional Development Found in the frame of the project PlasTEP 'Dissemination and fostering of plasma based technological innovation for environment protection in the Baltic Sea Region'.

  14. Investigation of diversity of plasmids carrying the blaTEM-52 gene

    DEFF Research Database (Denmark)

    Bielak, Eliza Maria; Bergenholtz, Rikke D.; Jørgensen, Mikael Skaanning

    2011-01-01

    OBJECTIVES: To investigate the diversity of plasmids that carry blaTEM-52 genes among Escherichia coli and Salmonella enterica originating from animals, meat products and humans. METHODS: A collection of 22 blaTEM-52-encoding plasmids was characterized by restriction fragment length polymorphism...... of self-transfer to a plasmid-free E. coli recipient. CONCLUSIONS: The blaTEM-52 gene found in humans could have been transmitted on transferable plasmids originating from animal sources. Some of the blaTEM-52 plasmids carry replicons that differ from the classical ones. Two novel replicons were detected...

  15. Continuous processing of recombinant proteins: integration of refolding and purification using simulated moving bed size-exclusion chromatography with buffer recycling.

    Science.gov (United States)

    Wellhoefer, Martin; Sprinzl, Wolfgang; Hahn, Rainer; Jungbauer, Alois

    2014-04-11

    Continuous processing of recombinant proteins was accomplished by combining continuous matrix-assisted refolding and purification by tandem simulated moving bed (SMB) size-exclusion chromatography (SEC). Recombinant proteins, N(pro) fusion proteins from inclusion bodies were dissolved with NaOH and refolded in the SMB system with a closed-loop set-up with refolding buffer as the desorbent buffer and buffer recycling of the refolding buffer of the raffinate by tangential flow filtration. For further purification of the refolded proteins, a second SMB operation also based on SEC was added. The whole system could be operated isocratically with refolding buffer as the desorbent buffer, and buffer recycling could also be applied in the purification step. Thus, a significant reduction in buffer consumption was achieved. The system was evaluated with two proteins, the N(pro) fusion pep6His and N(pro) fusion MCP-1. Refolding solution, which contained residual N(pro) fusion peptide, the cleaved autoprotease N(pro), and the cleaved target peptide was used as feed solution. Full separation of the cleaved target peptide from residual proteins was achieved at a purity and recovery in the raffinate and extract, respectively, of approximately 100%. In addition, more than 99% of the refolding buffer of the raffinate was recycled. A comparison of throughput, productivity, and buffer consumption of the integrated continuous process with two batch processes demonstrated that up to 60-fold higher throughput, up to 180-fold higher productivity, and at least 28-fold lower buffer consumption can be obtained by the integrated continuous process, which compensates for the higher complexity. Copyright © 2014 Elsevier B.V. All rights reserved.

  16. Impurity impacts on the purification process in oxy-fuel combustion based CO2 capture and storage system

    International Nuclear Information System (INIS)

    Li, H.; Yan, J.; Yan, J.; Anheden, M.

    2009-01-01

    Based on the requirements of CO 2 transportation and storage, non-condensable gases, such as O 2 , N 2 and Ar should be removed from the CO 2 -stream captured from an oxy-fuel combustion process. For a purification process, impurities have great impacts on the design, operation and optimization through their impacts on the thermodynamic properties of CO 2 -streams. Study results show that the increments of impurities will make the energy consumption of purification increase; and make CO 2 purity of separation product and CO 2 recovery rate decrease. In addition, under the same operating conditions, energy consumptions have different sensitivities to the variation of the impurity mole fraction of feed fluids. The isothermal compression work is more sensitive to the variation of SO 2 ; while the isentropic compression work is more sensitive to the variation of Ar. In the flash system, the energy consumption of condensation in is more sensitive to the variation of Ar; but in the distillation system, the energy consumption of condensation is more sensitive to the variation of SO 2 , and CO 2 purity of separation is more sensitive to the variation of SO 2 . (author)

  17. Conjugative plasmid pAW63 brings new insights into the genesis of the Bacillus anthracis virulence plasmid pXO2 and of the Bacillus thuringiensis plasmid pBT9727

    Directory of Open Access Journals (Sweden)

    Mahillon Jacques

    2005-07-01

    Full Text Available Abstract Background Bacillus cereus, Bacillus anthracis and Bacillus thuringiensis belong to the genetically close-knit Bacillus cereus sensu lato group, a family of rod-shaped Gram-positive bacteria. pAW63 is the first conjugative plasmid from the B. cereus group to be completely sequenced. Results The 71,777 bp nucleotide sequence of pAW63 reveals a modular structure, including a 42 kb tra region encoding homologs of the Type IV secretion systems components VirB11, VirB4 and VirD4, as well as homologs of Gram-positive conjugation genes from Enterococcus, Lactococcus, Listeria, Streptococcus and Staphylococcus species. It also firmly establishes the existence of a common backbone between pAW63, pXO2 from Bacillus anthracis and pBT9727 from the pathogenic Bacillus thuringiensis serovar konkukian strain 97-27. The alignment of these three plasmids highlights the presence of well conserved segments, in contrast to distinct regions of high sequence plasticity. The study of their specific differences has provided a three-point reference framework that can be exploited to formulate solid hypotheses concerning the functionalities and the molecular evolution of these three closely related plasmids. This has provided insight into the chronology of their divergence, and led to the discovery of two Type II introns on pAW63, matching copies of the mobile element IS231L in different loci of pXO2 and pBT9727, and the identification on pXO2 of a 37 kb pathogenicity island (PAI containing the anthrax capsule genes. Conclusion The complete sequence determination of pAW63 has led to a functional map of the plasmid yielding insights into its conjugative apparatus, which includes T4SS-like components, as well as its resemblance to other large plasmids of Gram-positive bacteria. Of particular interest is the extensive homology shared between pAW63 and pXO2, the second virulence plasmid of B. anthracis, as well as pBT9727 from the pathogenic strain B. thuringiensis

  18. Plasmid-dependent attachment of Agrobacterium tumefaciens to plant tissue culture cells.

    Science.gov (United States)

    Matthysse, A G; Wyman, P M; Holmes, K V

    1978-11-01

    Kinetic, microscopic, and biochemical studies show that virulent Ti (tumor inducing)-plasmid-containing strains of Agrobacterium attach to normal tobacco and carrot tissue culture cells. Kinetic studies showed that virulent strains of A. tumefaciens attach to the plant tissue culture cells in increasing numbers during the first 1 to 2 h of incubation of the bacteria with the plant cells. Five Ti-plasmid-containing virulent Agrobacterium strains showed greater attachment to tobacco cells than did five avirulent strains. Light and scanning electron microscopic observations confirmed that virulent strains showed little attachment. Bacterial attachment was blocked by prior incubation of the plant cells with lipopolysaccharide extracted from A. tumefaciens, but not from A. radiobacter, suggesting that bacterial lipopolysaccharide is one of the components involved in the attachment process. At least one other bacterial product may be required for attachment in tissue culture because the virulent A. tumefaciens NT1, which lacks the Ti plasmid, does not itself attach to tobacco cells, but its lipopolysaccharide does inhibit the attachment of virulent strains.

  19. Permissiveness of soil microbial communities towards broad host range plasmids

    DEFF Research Database (Denmark)

    Klümper, Uli

    . Plasmids are implicated in the rapid spread of antibiotic resistance and the emergence of multi-resistant pathogenic bacteria, making it crucial to be able to quantify, understand, and, ideally, control plasmid transfer in mixed microbial communities. The fate of plasmids in microbial communities...... of microbial communities may be directly interconnected through transfer of BHR plasmids at a so far unrecognized level. The developed method furthermore enabled me to explore how agronomic practices may affect gene transfer in soil microbial communities. I compared bacterial communities extracted from plots...

  20. Optimization‐based framework for resin selection strategies in biopharmaceutical purification process development

    Science.gov (United States)

    Liu, Songsong; Gerontas, Spyridon; Gruber, David; Turner, Richard; Titchener‐Hooker, Nigel J.

    2017-01-01

    This work addresses rapid resin selection for integrated chromatographic separations when conducted as part of a high‐throughput screening exercise during the early stages of purification process development. An optimization‐based decision support framework is proposed to process the data generated from microscale experiments to identify the best resins to maximize key performance metrics for a biopharmaceutical manufacturing process, such as yield and purity. A multiobjective mixed integer nonlinear programming model is developed and solved using the ε‐constraint method. Dinkelbach's algorithm is used to solve the resulting mixed integer linear fractional programming model. The proposed framework is successfully applied to an industrial case study of a process to purify recombinant Fc Fusion protein from low molecular weight and high molecular weight product related impurities, involving two chromatographic steps with eight and three candidate resins for each step, respectively. The computational results show the advantage of the proposed framework in terms of computational efficiency and flexibility. © 2017 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 33:1116–1126, 2017 PMID:28393478

  1. Drug resistance plasmids in Lactobacillus acidophilus and Lactobacillus reuteri.

    OpenAIRE

    Vescovo, M; Morelli, L; Bottazzi, V

    1982-01-01

    Sixteen strains of Lactobacillus reuteri and 20 strains of Lactobacillus acidophilus were tested for resistance to 22 antibiotics by using commercially available sensitivity disks. Evidence suggesting linkage of these resistances to plasmids was obtained by "curing" experiments with acridine dyes and high growth temperatures. Examination of plasmid patterns of agarose gel electrophoresis provided further evidence of loss in plasmid DNA under curing conditions in some of the strains examined.

  2. Damage of plasmid DNA by high energy ions

    International Nuclear Information System (INIS)

    Michaelidesova, A.; Pachnerova Brabcova, K.; Davidkova, M.

    2018-01-01

    The aim of the study was to determine the degree of direct DNA damage by high-energy ions, which are one of the components of cosmic rays, and therefore the knowledge of the biological effects of these ions is key to long-term space missions with human crew. The pBR322 plasmid containing 4361 base pairs was used in this study. The aqueous solution of plasmid pBR322 was transferred on ice to Japan to the Heavy Ion Medical Accelerator in Chiba, the Research Center for Charged Particle Therapy. Just before the experiment, the droplets of solution of known concentration were applied to the slides and the water was allowed to evaporate to produce dry DNA samples. Half of the slides were irradiated with 290 MeV/u of carbon ions and a dose rate of 20 Gy/min. The other half of the slides were irradiated with helium nuclei of 150 MeV/hr and a dose rate of 12.6 Gy/min. Both sets of slides were irradiated with doses of 0-1,400 Gy with a 200 Gy step. After irradiation, the samples were re-dissolved in distilled water, frozen and transported on ice to the Czech Republic for processing. Samples were analyzed by agarose gel electrophoresis. The plasmid was evaluated separately to determine the degree of radiation induced lesions and further to incubation with enzymes recognizing basal damage. (authors)

  3. Stress analysis of the O-element pipe during the process of flue gases purification

    Directory of Open Access Journals (Sweden)

    Nekvasil R.

    2008-11-01

    Full Text Available Equipment for flue gases purification from undesired substances is used throughout power and other types of industry. This paper deals with damaging of the O-element pipe designed to remove sulphur from the flue gases, i.e. damaging of the pipe during flue gases purification. This purification is conducted by spraying the water into the O-shaped pipe where the flue gases flow. Thus the sulphur binds itself onto the water and gets removed from the flue gas. Injection of cold water into hot flue gases, however, causes high stress on the inside of the pipe, which can gradually damage the O-element pipe. In this paper initial injection of water into hot pipe all the way to stabilization of temperature fields will be analyzed and the most dangerous places which shall be considered for fatigue will be determined.

  4. Plasmid construction using recombination activity in the fission yeast Schizosaccharomyces pombe.

    Directory of Open Access Journals (Sweden)

    Ayako Chino

    Full Text Available BACKGROUND: Construction of plasmids is crucial in modern genetic manipulation. As of now, the common method for constructing plasmids is to digest specific DNA sequences with restriction enzymes and to ligate the resulting DNA fragments with DNA ligase. Another potent method to construct plasmids, known as gap-repair cloning (GRC, is commonly used in the budding yeast Saccharomyces cerevisiae. GRC makes use of the homologous recombination activity that occurs within the yeast cells. Due to its flexible design and efficiency, GRC has been frequently used for constructing plasmids with complex structures as well as genome-wide plasmid collections. Although there have been reports indicating GRC feasibility in the fission yeast Schizosaccharomyces pombe, this species is not commonly used for GRC as systematic studies of reporting GRC efficiency in S. pombe have not been performed till date. METHODOLOGY/PRINCIPAL FINDINGS: We investigated GRC efficiency in S. pombe in this study. We first showed that GRC was feasible in S. pombe by constructing a plasmid that contained the LEU2 auxotrophic marker gene in vivo and showed sufficient efficiency with short homology sequences (>25 bp. No preference was shown for the sequence length from the cut site in the vector plasmid. We next showed that plasmids could be constructed in a proper way using 3 DNA fragments with 70% efficiency without any specific selections being made. The GRC efficiency with 3 DNA fragments was dramatically increased >95% in lig4Delta mutant cell, where non-homologous end joining is deficient. Following this approach, we successfully constructed plasmid vectors with leu1+, ade6+, his5+, and lys1+ markers with the low-copy stable plasmid pDblet as a backbone by applying GRC in S. pombe. CONCLUSIONS/SIGNIFICANCE: We concluded that GRC was sufficiently feasible in S. pombe for genome-wide gene functional analysis as well as for regular plasmid construction. Plasmids with different

  5. Brownian Ratchet Mechanism for Faithful Segregation of Low-Copy-Number Plasmids.

    Science.gov (United States)

    Hu, Longhua; Vecchiarelli, Anthony G; Mizuuchi, Kiyoshi; Neuman, Keir C; Liu, Jian

    2017-04-11

    Bacterial plasmids are extrachromosomal DNA that provides selective advantages for bacterial survival. Plasmid partitioning can be remarkably robust. For high-copy-number plasmids, diffusion ensures that both daughter cells inherit plasmids after cell division. In contrast, most low-copy-number plasmids need to be actively partitioned by a conserved tripartite ParA-type system. ParA is an ATPase that binds to chromosomal DNA; ParB is the stimulator of the ParA ATPase and specifically binds to the plasmid at a centromere-like site, parS. ParB stimulation of the ParA ATPase releases ParA from the bacterial chromosome, after which it takes a long time to reset its DNA-binding affinity. We previously demonstrated in vitro that the ParA system can exploit this biochemical asymmetry for directed cargo transport. Multiple ParA-ParB bonds can bridge a parS-coated cargo to a DNA carpet, and they can work collectively as a Brownian ratchet that directs persistent cargo movement with a ParA-depletion zone trailing behind. By extending this model, we suggest that a similar Brownian ratchet mechanism recapitulates the full range of actively segregated plasmid motilities observed in vivo. We demonstrate that plasmid motility is tuned as the replenishment rate of the ParA-depletion zone progressively increases relative to the cargo speed, evolving from diffusion to pole-to-pole oscillation, local excursions, and, finally, immobility. When the plasmid replicates, the daughters largely display motilities similar to that of their mother, except that when the single-focus progenitor is locally excursive, the daughter foci undergo directed segregation. We show that directed segregation maximizes the fidelity of plasmid partition. Given that local excursion and directed segregation are the most commonly observed modes of plasmid motility in vivo, we suggest that the operation of the ParA-type partition system has been shaped by evolution for high fidelity of plasmid segregation

  6. Plasmid P1 replication: negative control by repeated DNA sequences.

    OpenAIRE

    Chattoraj, D; Cordes, K; Abeles, A

    1984-01-01

    The incompatibility locus, incA, of the unit-copy plasmid P1 is contained within a fragment that is essentially a set of nine 19-base-pair repeats. One or more copies of the fragment destabilizes the plasmid when present in trans. Here we show that extra copies of incA interfere with plasmid DNA replication and that a deletion of most of incA increases plasmid copy number. Thus, incA is not essential for replication but is required for its control. When cloned in a high-copy-number vector, pi...

  7. The Purification of a Blood Group A Glycoprotein: An Affinity Chromatography Experiment.

    Science.gov (United States)

    Estelrich, J.; Pouplana, R.

    1988-01-01

    Describes a purification process through affinity chromatography necessary to obtain specific blood group glycoproteins from erythrocytic membranes. Discusses the preparation of erythrocytic membranes, extraction of glycoprotein from membranes, affinity chromatography purification, determination of glycoproteins, and results. (CW)

  8. Robotic high-throughput purification of affinity-tagged recombinant proteins.

    Science.gov (United States)

    Wiesler, Simone C; Weinzierl, Robert O J

    2015-01-01

    Affinity purification of recombinant proteins has become the method of choice to obtain good quantities and qualities of proteins for a variety of downstream biochemical applications. While manual or FPLC-assisted purification techniques are generally time-consuming and labor-intensive, the advent of high-throughput technologies and liquid handling robotics has simplified and accelerated this process significantly. Additionally, without the human factor as a potential source of error, automated purification protocols allow for the generation of large numbers of proteins simultaneously and under directly comparable conditions. The delivered material is ideal for activity comparisons of different variants of the same protein. Here, we present our strategy for the simultaneous purification of up to 24 affinity-tagged proteins for activity measurements in biochemical assays. The protocol described is suitable for the scale typically required in individual research laboratories.

  9. Characterization of a Large Antibiotic Resistance Plasmid Found in Enteropathogenic Escherichia coli Strain B171 and Its Relatedness to Plasmids of Diverse E. coli and Shigella Strains.

    Science.gov (United States)

    Hazen, Tracy H; Michalski, Jane; Nagaraj, Sushma; Okeke, Iruka N; Rasko, David A

    2017-09-01

    Enteropathogenic Escherichia coli (EPEC) is a leading cause of severe infantile diarrhea in developing countries. Previous research has focused on the diversity of the EPEC virulence plasmid, whereas less is known regarding the genetic content and distribution of antibiotic resistance plasmids carried by EPEC. A previous study demonstrated that in addition to the virulence plasmid, reference EPEC strain B171 harbors a second, larger plasmid that confers antibiotic resistance. To further understand the genetic diversity and dissemination of antibiotic resistance plasmids among EPEC strains, we describe the complete sequence of an antibiotic resistance plasmid from EPEC strain B171. The resistance plasmid, pB171_90, has a completed sequence length of 90,229 bp, a GC content of 54.55%, and carries protein-encoding genes involved in conjugative transfer, resistance to tetracycline ( tetA ), sulfonamides ( sulI ), and mercury, as well as several virulence-associated genes, including the transcriptional regulator hha and the putative calcium sequestration inhibitor ( csi ). In silico detection of the pB171_90 genes among 4,798 publicly available E. coli genome assemblies indicates that the unique genes of pB171_90 ( csi and traI ) are primarily restricted to genomes identified as EPEC or enterotoxigenic E. coli However, conserved regions of the pB171_90 plasmid containing genes involved in replication, stability, and antibiotic resistance were identified among diverse E. coli pathotypes. Interestingly, pB171_90 also exhibited significant similarity with a sequenced plasmid from Shigella dysenteriae type I. Our findings demonstrate the mosaic nature of EPEC antibiotic resistance plasmids and highlight the need for additional sequence-based characterization of antibiotic resistance plasmids harbored by pathogenic E. coli . Copyright © 2017 American Society for Microbiology.

  10. In Vivo Transmission of an IncA/C Plasmid in Escherichia coli Depends on Tetracycline Concentration, and Acquisition of the Plasmid Results in a Variable Cost of Fitness.

    Science.gov (United States)

    Johnson, Timothy J; Singer, Randall S; Isaacson, Richard E; Danzeisen, Jessica L; Lang, Kevin; Kobluk, Kristi; Rivet, Bernadette; Borewicz, Klaudyna; Frye, Jonathan G; Englen, Mark; Anderson, Janet; Davies, Peter R

    2015-05-15

    IncA/C plasmids are broad-host-range plasmids enabling multidrug resistance that have emerged worldwide among bacterial pathogens of humans and animals. Although antibiotic usage is suspected to be a driving force in the emergence of such strains, few studies have examined the impact of different types of antibiotic administration on the selection of plasmid-containing multidrug resistant isolates. In this study, chlortetracycline treatment at different concentrations in pig feed was examined for its impact on selection and dissemination of an IncA/C plasmid introduced orally via a commensal Escherichia coli host. Continuous low-dose administration of chlortetracycline at 50 g per ton had no observable impact on the proportions of IncA/C plasmid-containing E. coli from pig feces over the course of 35 days. In contrast, high-dose administration of chlortetracycline at 350 g per ton significantly increased IncA/C plasmid-containing E. coli in pig feces (P IncA/C plasmid to other indigenous E. coli hosts. There was no evidence of conjugal transfer of the IncA/C plasmid to bacterial species other than E. coli. In vitro competition assays demonstrated that bacterial host background substantially impacted the cost of IncA/C plasmid carriage in E. coli and Salmonella. In vitro transfer and selection experiments demonstrated that tetracycline at 32 μg/ml was necessary to enhance IncA/C plasmid conjugative transfer, while subinhibitory concentrations of tetracycline in vitro strongly selected for IncA/C plasmid-containing E. coli. Together, these experiments improve our knowledge on the impact of differing concentrations of tetracycline on the selection of IncA/C-type plasmids. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  11. Review of Membranes for Helium Separation and Purification

    Directory of Open Access Journals (Sweden)

    Colin A. Scholes

    2017-02-01

    Full Text Available Membrane gas separation has potential for the recovery and purification of helium, because the majority of membranes have selectivity for helium. This review reports on the current state of the research and patent literature for membranes undertaking helium separation. This includes direct recovery from natural gas, as an ancillary stage in natural gas processing, as well as niche applications where helium recycling has potential. A review of the available polymeric and inorganic membranes for helium separation is provided. Commercial gas separation membranes in comparable gas industries are discussed in terms of their potential in helium separation. Also presented are the various membrane process designs patented for the recovery and purification of helium from various sources, as these demonstrate that it is viable to separate helium through currently available polymeric membranes. This review places a particular focus on those processes where membranes are combined in series with another separation technology, commonly pressure swing adsorption. These combined processes have the most potential for membranes to produce a high purity helium product. The review demonstrates that membrane gas separation is technically feasible for helium recovery and purification, though membranes are currently only applied in niche applications focused on reusing helium rather than separation from natural sources.

  12. Plasmid-Mediated Quinolone Resistance in Shigella flexneri Isolated From Macaques

    Directory of Open Access Journals (Sweden)

    Anthony J. Mannion

    2018-03-01

    Full Text Available Non-human primates (NHPs for biomedical research are commonly infected with Shigella spp. that can cause acute dysentery or chronic episodic diarrhea. These animals are often prophylactically and clinically treated with quinolone antibiotics to eradicate these possible infections. However, chromosomally- and plasmid-mediated antibiotic resistance has become an emerging concern for species in the family Enterobacteriaceae. In this study, five individual isolates of multi-drug resistant Shigella flexneri were isolated from the feces of three macaques. Antibiotic susceptibility testing confirmed resistance or decreased susceptibility to ampicillin, amoxicillin-clavulanic acid, cephalosporins, gentamicin, tetracycline, ciprofloxacin, enrofloxacin, levofloxacin, and nalidixic acid. S. flexneri isolates were susceptible to trimethoprim-sulfamethoxazole, and this drug was used to eradicate infection in two of the macaques. Plasmid DNA from all isolates was positive for the plasmid-encoded quinolone resistance gene qnrS, but not qnrA and qnrB. Conjugation and transformation of plasmid DNA from several S. flexneri isolates into antibiotic-susceptible Escherichia coli strains conferred the recipients with resistance or decreased susceptibility to quinolones and beta-lactams. Genome sequencing of two representative S. flexneri isolates identified the qnrS gene on a plasmid-like contig. These contigs showed >99% homology to plasmid sequences previously characterized from quinolone-resistant Shigella flexneri 2a and Salmonella enterica strains. Other antibiotic resistance genes and virulence factor genes were also identified in chromosome and plasmid sequences in these genomes. The findings from this study indicate macaques harbor pathogenic S. flexneri strains with chromosomally- and plasmid-encoded antibiotic resistance genes. To our knowledge, this is the first report of plasmid-mediated quinolone resistance in S. flexneri isolated from NHPs and warrants

  13. Advanced purification of petroleum refinery wastewater by catalytic vacuum distillation.

    Science.gov (United States)

    Yan, Long; Ma, Hongzhu; Wang, Bo; Mao, Wei; Chen, Yashao

    2010-06-15

    In our work, a new process, catalytic vacuum distillation (CVD) was utilized for purification of petroleum refinery wastewater that was characteristic of high chemical oxygen demand (COD) and salinity. Moreover, various common promoters, like FeCl(3), kaolin, H(2)SO(4) and NaOH were investigated to improve the purification efficiency of CVD. Here, the purification efficiency was estimated by COD testing, electrolytic conductivity, UV-vis spectrum, gas chromatography-mass spectrometry (GC-MS) and pH value. The results showed that NaOH promoted CVD displayed higher efficiency in purification of refinery wastewater than other systems, where the pellucid effluents with low salinity and high COD removal efficiency (99%) were obtained after treatment, and the corresponding pH values of effluents varied from 7 to 9. Furthermore, environment estimation was also tested and the results showed that the effluent had no influence on plant growth. Thus, based on satisfied removal efficiency of COD and salinity achieved simultaneously, NaOH promoted CVD process is an effective approach to purify petroleum refinery wastewater. Copyright 2010 Elsevier B.V. All rights reserved.

  14. [Pilot-scale purification of lipopeptide from marine-derived Bacillus marinus].

    Science.gov (United States)

    Gu, Kangbo; Guan, Cheng; Xu, Jiahui; Li, Shulan; Luo, Yuanchan; Shen, Guomin; Zhang, Daojing; Li, Yuanguang

    2016-11-25

    This research was aimed at establishing the pilot-scale purification technology of lipopeptide from marine-derived Bacillus marinus. We studied lipopeptide surfactivity interferences on scale-up unit technologies including acid precipitation, methanol extraction, solvent precipitation, salting out, extraction, silica gel column chromatography and HZ806 macroporous absorption resin column chromatography. Then, the unit technologies were combined in a certain order, to remove the impurities gradually, and to gain purified lipopeptide finally, with high recovery rate throughout the whole process. The novel pilot-scale purification technology could effectively isolate and purify lipopeptide with 87.51% to 100% purity in hectograms from 1 ton of Bacillus marinus B-9987 fermentation broth with more than 81.73% recovery rate. The first practical hectogram production of highly purified lipopeptide derived from Bacillus marinus was achieved. With this new purification method, using complex media became possible in fermentation process to reduce the fermentation cost and scale-up the purification for lipopeptide production. For practicability and economy, foaming problem resulting from massive water evaporation was avoided in this technology.

  15. Complete sequencing of IncI1 sequence type 2 plasmid pJIE512b indicates mobilization of blaCMY-2 from an IncA/C plasmid.

    Science.gov (United States)

    Tagg, Kaitlin A; Iredell, Jonathan R; Partridge, Sally R

    2014-08-01

    Sequencing of pJIE512b, a 92.3-kb IncI1 sequence type 2 (ST2) plasmid carrying bla(CMY-2), revealed a bla(CMY-2) context that appeared to have been mobilized from an IncA/C plasmid by the insertion sequence IS1294. A comparison with published plasmids suggests that bla(CMY-2) has been mobilized from IncA/C to IncI1 plasmids more than once by IS1294-like elements. Alignment of pJIE512b with the only other available IncI1 ST2 plasmid revealed differences across the backbones, indicating variability within this sequence type. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  16. Polyether sulfone/hydroxyapatite mixed matrix membranes for protein purification

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Junfen, E-mail: junfensun@dhu.edu.cn [State Key Laboratory for Modification of Chemical Fibers and Polymer Materials, College of Material Science and Engineering, Donghua University, North People Road 2999, Shanghai 201620 (China); Wu, Lishun [Department of Chemistry and Chemical Engineering, Heze University, Daxue Road 2269, Heze, Shandong Province 274015 (China)

    2014-07-01

    This work proposes a novel approach for protein purification from solution using mixed matrix membranes (MMMs) comprising of hydroxyapatite (HAP) inside polyether sulfone (PES) matrix. The influence of HAP particle loading on membrane morphology is studied. The MMMs are further characterized concerning permeability and adsorption capacity. The MMMs show purification of protein via both diffusion as well as adsorption, and show the potential of using MMMs for improvements in protein purification techniques. The bovine serum albumin (BSA) was used as a model protein. The properties and structures of MMMs prepared by immersion phase separation process were characterized by pure water flux, BSA adsorption and scanning electron microscopy (SEM).

  17. Novel assay to measure the plasmid mobilizing potential of mixed microbial communities

    DEFF Research Database (Denmark)

    Klümper, Uli; Droumpali, Ariadni; Dechesne, Arnaud

    2014-01-01

    Mobilizable plasmids lack necessary genes for complete conjugation and are therefore non-self-transmissible. Instead, they rely on the conjugation system of conjugal plasmids to be horizontally transferred to new recipients. While community permissiveness, the fraction of a mixed microbial...... community that can receive self-transmissible conjugal plasmids, has been studied, the intrinsic ability of a community to mobilize plasmids that lack conjugation systems is unexplored. Here, we present a novel framework and experimental method to estimate the mobilization potential of mixed communities. We...... of the donors receiving the conjugal plasmid in the first step. Further work is needed to establish how plasmid mobilization potential varies within and across microbial communities....

  18. Development of a self-replicating plasmid system for Mycoplasma hyopneumoniae.

    Science.gov (United States)

    Maglennon, Gareth A; Cook, Beth S; Matthews, Dominic; Deeney, Alannah S; Bossé, Janine T; Langford, Paul R; Maskell, Duncan J; Tucker, Alexander W; Wren, Brendan W; Rycroft, Andrew N

    2013-07-29

    Mycoplasma hyopneumoniae is a prevalent swine respiratory pathogen that is a major cause of economic loss to pig producers. Control is achieved by a combination of antimicrobials, vaccination and management practices, but current vaccines offer only partial control and there is a need for improved preventative strategies. A major barrier to advances in understanding the pathogenesis of M. hyopneumoniae and in developing new vaccines is the lack of tools to genetically manipulate the organism. We describe the development and optimisation of the first successful plasmid-based system for the genetic manipulation of M. hyopneumoniae. Our artificial plasmids contain the origin of replication (oriC) of M. hyopneumoniae along with tetM, conferring resistance to tetracycline. With these plasmids, we have successfully transformed M. hyopneumoniae strain 232 by electroporation, generating tetracycline resistant organisms. The persistence of extrachromosomal plasmid and maintenance of plasmid DNA over serial passages shows that these artificial plasmids are capable of self-replication in M. hyopneumoniae. In addition to demonstrating the amenability of M. hyopneumoniae to genetic manipulation and in optimising the conditions necessary for successful transformation, we have used this system to determine the minimum functional oriC of M. hyopneumoniae. In doing so, we have developed a plasmid with a small oriC that is stably maintained over multiple passages that may be useful in generating targeted gene disruptions. In conclusion, we have generated a set of plasmids that will be valuable in studies of M. hyopneumoniae pathogenesis and provide a major step forward in the study of this important swine pathogen.

  19. Strand breaks and lethal damage in plasmid DNA subjected to 60CO-γirradiation

    International Nuclear Information System (INIS)

    Klimczak, U.

    1992-01-01

    Experiments with calf thymus DNA subjected to extracellular irradiation yield information on the role of direct and indirect effects in single-strand breakage, if this is evaluated with reference to the scavenger activity in respect of OH radicals. The role of the two processes in the occurrence of double-stand breaks and further damage leading to cell decay has so far remained largely obscure. It was the aim of the study described here to contribute to research in this field by performing in vitro experiments on biologically active DNA. For this purpose, DNA from pBR322 plasmids was irradiated in the presence of OH-radical scavengers. The number of single-strand and double-strand breaks was determined on the basis of the system's ability to eliminate OH radicals. In order to asses the influence of irradiation processes on the biological activity of DNA, investigations were carried out in E. coli for transformations caused by irradiated plasmid DNA. The results were interpreted in the light of theories about inhomogenous reaction kinetics put forward by Mark et al. (1989). It was finally discussed, which of the gamma-irradiation injuries occurring in DNA was to be held responsible for the inactivation of plasmid DNA and which enzymatic processes were additionally at work here. (orig./MG) [de

  20. Photoinduced silver nanoparticles/nanorings on plasmid DNA scaffolds.

    Science.gov (United States)

    Liu, Jianhua; Zhang, Xiaoliang; Yu, Mei; Li, Songmei; Zhang, Jindan

    2012-01-23

    Biological scaffolds are being actively explored for the synthesis of nanomaterials with novel structures and unexpected properties. Toroidal plasmid DNA separated from the Bacillus host is applied as a sacrificial mold for the synthesis of silver nanoparticles and nanorings. The photoirradiation method is applied to reduce Ag(I) on the plasmid. The nanoparticles are obtained by varying the concentration of the Ag(I) ion solution and the exposure time of the plasmid-Ag(I) complex under UV light at 254 nm and room temperature. It is found that the plasmid serves not only as a template but also as a reductant to drive the silver nucleation and deposition. The resulting nanoparticles have a face-centered cubic (fcc) crystal structure and 20-30 nm average diameter. The detailed mechanism is discussed, and other metals or alloys could also be synthesized with this method. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Plasmids and rickettsial evolution: insight from Rickettsia felis.

    Directory of Open Access Journals (Sweden)

    Joseph J Gillespie

    2007-03-01

    Full Text Available The genome sequence of Rickettsia felis revealed a number of rickettsial genetic anomalies that likely contribute not only to a large genome size relative to other rickettsiae, but also to phenotypic oddities that have confounded the categorization of R. felis as either typhus group (TG or spotted fever group (SFG rickettsiae. Most intriguing was the first report from rickettsiae of a conjugative plasmid (pRF that contains 68 putative open reading frames, several of which are predicted to encode proteins with high similarity to conjugative machinery in other plasmid-containing bacteria.Using phylogeny estimation, we determined the mode of inheritance of pRF genes relative to conserved rickettsial chromosomal genes. Phylogenies of chromosomal genes were in agreement with other published rickettsial trees. However, phylogenies including pRF genes yielded different topologies and suggest a close relationship between pRF and ancestral group (AG rickettsiae, including the recently completed genome of R. bellii str. RML369-C. This relatedness is further supported by the distribution of pRF genes across other rickettsiae, as 10 pRF genes (or inactive derivatives also occur in AG (but not SFG rickettsiae, with five of these genes characteristic of typical plasmids. Detailed characterization of pRF genes resulted in two novel findings: the identification of oriV and replication termination regions, and the likelihood that a second proposed plasmid, pRFdelta, is an artifact of the original genome assembly.Altogether, we propose a new rickettsial classification scheme with the addition of a fourth lineage, transitional group (TRG rickettsiae, that is unique from TG and SFG rickettsiae and harbors genes from possible exchanges with AG rickettsiae via conjugation. We offer insight into the evolution of a plastic plasmid system in rickettsiae, including the role plasmids may have played in the acquirement of virulence traits in pathogenic strains, and the

  2. Cadmium purification with a vibrating reactor

    International Nuclear Information System (INIS)

    Torres, N.; Esna-Ashari, M.; Biallas, H.; Kangas, K.

    1986-01-01

    While electrolytically producing zinc from sulfide concentrates, purification is the most significant step. Impurities such as Co, Sn, Ge, Ni and Sb can cause extensive redissolution of the electrodeposited zinc, thus diminishing current efficiency. Other metals, particularly cadmium, lead and copper, can negatively affect zinc properties by deposition on the cathode. It is standard practice to use atomized zinc dust as a reducing agent in the purification process, either alone or combined with additives. In conventional operations, special facilities are necessary to produce zinc dust in an amount close to 8wt% of cathode production. This paper examines a technique which makes use of zinc granules instead of dust

  3. Type 3 Fimbriae Encoded on Plasmids Are Expressed from a Unique Promoter without Affecting Host Motility, Facilitating an Exceptional Phenotype That Enhances Conjugal Plasmid Transfer

    DEFF Research Database (Denmark)

    Madsen, Jonas Stenlokke; Riber, Leise; Kot, Witold

    2016-01-01

    Horizontal gene transfer (HGT), the transmission of genetic material to a recipient that is not the progeny of the donor, is fundamental in bacterial evolution. HGT is often mediated by mobile genetic elements such as conjugative plasmids, which may be in conflict with the chromosomal elements...... of the genome because they are independent replicons that may petition their own evolutionary strategy. Here we study differences between type 3 fimbriae encoded on wild type plasmids and in chromosomes. Using known and newly characterized plasmids we show that the expression of type 3 fimbriae encoded...... on plasmids is systematically different, as MrkH, a c-di-GMP dependent transcriptional activator is not needed for strong expression of the fimbriae. MrkH is required for expression of type 3 fimbriae of the Klebsiella pneumoniae chromosome, wherefrom the fimbriae operon (mrkABCDF) of plasmids is believed...

  4. Resistant plasmid profile analysis of multidrug resistant Escherichia ...

    African Journals Online (AJOL)

    Multiple drug resistance isolates causing UTI has seri- ous implications for the empiric therapy against patho- genic isolates and for the possible co-selection of antimicrobial resistant mediated by multi drug resistant plasmids21,22. E. coli from clinical isolates are known to harbour plasmids of different molecular sizes23.

  5. Breathing Air Purification for Hyperbaric Purposes, Part II

    Directory of Open Access Journals (Sweden)

    Woźniak Arkadiusz

    2015-03-01

    Full Text Available Determining the efficiency of breathing air purification for hyperbaric purposes with the use of filtration systems is of a crucial importance. However, when the Polish Navy took samples of breathing air from their own filtration plant for quality purposes, these were found to not meet the required standard. The identification of this problem imposed the need to undertake actions aimed at the elimination of the identified disruptions in the process of breathing air production, with the objective of assuring its proper quality. This study presents the results of the initial tests on the air supply sources utilised by the Polish Navy, which were carried out for the purpose of setting a proper direction of future works and implementing corrective measures in order to optimise the breathing air production process. The obtained test results will be used in a subsequent publication devoted to the assessment of the level of efficiency of air purification with the use of a multifaceted approach consisting in the utilisation of various types of air supply sources and different configurations of purification systems.

  6. Addressing the medicinal chemistry bottleneck: a lean approach to centralized purification.

    Science.gov (United States)

    Weller, Harold N; Nirschl, David S; Paulson, James L; Hoffman, Steven L; Bullock, William H

    2012-09-10

    The use of standardized lean manufacturing principles to improve drug discovery productivity is often thought to be at odds with fostering innovation. This manuscript describes how selective implementation of a lean optimized process, in this case centralized purification for medicinal chemistry, can improve operational productivity and increase scientist time available for innovation. A description of the centralized purification process is provided along with both operational and impact (productivity) metrics, which indicate lower cost, higher output, and presumably more free time for innovation as a result of the process changes described.

  7. IncA/C plasmids: An emerging threat to human and animal health?

    Science.gov (United States)

    Johnson, Timothy J; Lang, Kevin S

    2012-01-01

    Incompatibility group IncA/C plasmids are large, low copy, theta-replicating plasmids that have been described in the literature for over 40 years. However, they have only recently been intensively studied on the genomic level because of their associations with the emergence of multidrug resistance in enteric pathogens of humans and animals. These plasmids are unique among other enterobacterial plasmids in many aspects, including their modular structure and gene content. While the IncA/C plasmid genome structure has now been well defined, many questions remain pertaining to their basic biological mechanisms of dissemination and regulation. Here, we discuss the history of IncA/C plasmids in light of our recent understanding of their population distribution, genomics, and effects on host bacteria.

  8. AAVS1-Targeted Plasmid Integration in AAV Producer Cell Lines.

    Science.gov (United States)

    Luo, Yuxia; Frederick, Amy; Martin, John M; Scaria, Abraham; Cheng, Seng H; Armentano, Donna; Wadsworth, Samuel C; Vincent, Karen A

    2017-06-01

    Adeno-associated virus (AAV) producer cell lines are created via transfection of HeLaS3 cells with a single plasmid containing three components (the vector sequence, the AAV rep and cap genes, and a selectable marker gene). As this plasmid contains both the cis (Rep binding sites) and trans (Rep protein encoded by the rep gene) elements required for site-specific integration, it was predicted that plasmid integration might occur within the AAVS1 locus on human chromosome 19 (chr19). The objective of this study was to investigate whether integration in AAVS1 might be correlated with vector yield. Plasmid integration sites within several independent cell lines were assessed via Southern, fluorescence in situ hybridization (FISH) and PCR analyses. In the Southern analyses, the presence of fragments detected by both rep- and AAVS1-specific probes suggested that for several mid- and high-producing lines, plasmid DNA had integrated into the AAVS1 locus. Analysis with puroR and AAVS1-specific probes suggested that integration in AAVS1 was a more widespread phenomenon. High-producing AAV2-secreted alkaline phosphatase (SEAP) lines (masterwell 82 [MW82] and MW278) were evaluated via FISH using probes specific for the plasmid, AAVS1, and a chr19 marker. FISH analysis detected two plasmid integration sites in MW278 (neither in AAVS1), while a total of three sites were identified in MW82 (two in AAVS1). An inverse PCR assay confirmed integration within AAVS1 for several mid- and high-producing lines. In summary, the FISH, Southern, and PCR data provide evidence of site-specific integration of the plasmid within AAVS1 in several AAV producer cell lines. The data also suggest that integration in AAVS1 is a general phenomenon that is not necessarily restricted to high producers. The results also suggest that plasmid integration within the AAVS1 locus is not an absolute requirement for a high vector yield.

  9. Functional analysis of three plasmids from Lactobacillus plantarum

    NARCIS (Netherlands)

    Kranenburg, R. van; Golic, N.; Bongers, R.; Leer, R.J.; Vos, W.M. de; Siezen, R.J.; Kleerebezem, M.

    2005-01-01

    Lactobacillus plantarum WCFS1 harbors three plasmids, pWCFS101, pWCFS102, and pWCFS103, with sizes of 1,917, 2,365, and 36,069 bp, respectively. The two smaller plasmids are of unknown function and contain replication genes that are likely to function via the rolling-circle replication mechanism.

  10. Purification of CD47-streptavidin fusion protein from bacterial lysate using biotin-agarose affinity chromatography.

    Science.gov (United States)

    Salehi, Nasrin; Peng, Ching-An

    2016-07-08

    CD47 is a widely expressed transmembrane glycoprotein that modulates the activity of a plethora of immune cells via its extracellular domain. Therefore, CD47 plays important roles in the regulation of immune responses and may serve as targets for the development of immunotherapeutic agents. To make sure CD47 functionality is intact under the process of protein conjugation, CD47-streptavidin fusion protein was expressed and purified because it can easily bind to biotin-tagged materials via the unique biotin-streptavidin affinity. In this study, gene sequences of CD47 extracellular domain (CD47ECD) and core streptavidin (coreSA) with a total 834 bp were inserted into pET20b plasmid to construct recombinant plasmid encoding CD47-SA fusion gene. After bacteria transformation, the CD47-SA fusion protein was expressed by isopropyl-β-d-thiogalactopyranoside (IPTG) induction. The collected bacteria lysate was loaded on biotinylated agarose to proceed the purification of CD47-SA fusion protein. Due to the unexpected high affinity between biotin and coreSA, standard washing and elution approaches (e.g., varying pH, using biotin, and applying guanidine hydrochloride) reported for biotin-streptavidin affinity chromatography were not able to separate the target fusion protein. Instead, using low concentration of the non-ionic detergent Triton X-100 followed with alkaline buffer could efficiently weaken the binding between biotin and coreSA, thereby eluting out CD47-SA fusion protein from the biotin agarose column. The purified CD47-SA fusion protein was further characterized by molecular biology methods and its antiphagocytic functionality was confirmed by the phagocytosis assay. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:949-958, 2016. © 2016 American Institute of Chemical Engineers.

  11. Role of plasmids in Lactobacillus brevis BSO 464 hop tolerance and beer spoilage.

    Science.gov (United States)

    Bergsveinson, Jordyn; Baecker, Nina; Pittet, Vanessa; Ziola, Barry

    2015-02-01

    Specific isolates of lactic acid bacteria (LAB) can grow in the harsh beer environment, thus posing a threat to brew quality and the economic success of breweries worldwide. Plasmid-localized genes, such as horA, horC, and hitA, have been suggested to confer hop tolerance, a trait required for LAB survival in beer. The presence and expression of these genes among LAB, however, do not universally correlate with the ability to grow in beer. Genome sequencing of the virulent beer spoilage organism Lactobacillus brevis BSO 464 revealed the presence of eight plasmids, with plasmids 1, 2, and 3 containing horA, horC, and hitA, respectively. To investigate the roles that these and the other five plasmids play in L. brevis BSO 464 growth in beer, plasmid curing with novobiocin was used to derive 10 plasmid variants. Multiplex PCRs were utilized to determine the presence or absence of each plasmid, and how plasmid loss affected hop tolerance and growth in degassed (noncarbonated) beer was assessed. Loss of three of the eight plasmids was found to affect hop tolerance and growth in beer. Loss of plasmid 2 (horC and 28 other genes) had the most dramatic effect, with loss of plasmid 4 (120 genes) and plasmid 8 (47 genes) having significant, but smaller, impacts. These results support the contention that genes on mobile genetic elements are essential for bacterial growth in beer and that beer spoilage ability is not dependent solely on the three previously described hop tolerance genes or on the chromosome of a beer spoilage LAB isolate.

  12. Purification and characterization of enterocin FH 99 produced by a faecal isolate Enterococcus faecium FH 99.

    Science.gov (United States)

    Gupta, H; Malik, R K; Bhardwaj, A; Kaur, G; De, S; Kaushik, J K

    2010-06-01

    Enterococcus faecium FH 99 was isolated from human faeces and selected because of its broad spectrum of inhibitory activity against several Gram-positive foodborne spoilage and pathogenic bacteria. Ent. faecium FH 99 accumulates enterocin in large number in early stationary phase of the growth. The enterocin FH 99 was stable over a wide pH range (2-10) and recovered activity even after treatment at high temperatures (10 min at 100°C). The enterocin was subjected to different purification techniques viz., gel filteration, cation exchange chromatography and reverse-phase high-performance liquid chromatography. The activity was eluted as one individual active fraction. SDSPAGE revealed a molecular weight of less than 6.5 kDa. Studies carried out to identify the genetic determinants for bacteriocin production showed that this trait may be plasmid encoded as loss in both of the plasmids (size>chromosomal DNA) led to loss in bacteriocin production by Ent. faecium FH 99. Ent. faecium strain FH 99 is a newly discovered high bacteriocin producer with Activity Units 1.8 × 10(5) AU ml(-1) and its characteristics indicate that it may have strong potential for application as a protective agent against pathogens and spoilage bacteria in foods.

  13. Future of antibody purification.

    Science.gov (United States)

    Low, Duncan; O'Leary, Rhona; Pujar, Narahari S

    2007-03-15

    Antibody purification seems to be safely ensconced in a platform, now well-established by way of multiple commercialized antibody processes. However, natural evolution compels us to peer into the future. This is driven not only by a large, projected increase in the number of antibody therapies, but also by dramatic improvements in upstream productivity, and process economics. Although disruptive technologies have yet escaped downstream processes, evolution of the so-called platform is already evident in antibody processes in late-stage development. Here we perform a wide survey of technologies that are competing to be part of that platform, and provide our [inherently dangerous] assessment of those that have the most promise.

  14. The method of purification of waste water of NPS from petroleum oil using UV-radiation

    International Nuclear Information System (INIS)

    Kulemin, V.V.; Kareta, V.I.

    1993-01-01

    The main methods of concentration and purification of radioactive waste water of russian NPS are distillation and ion exchange. When waste water containing petroleum oil and washing matter is distillated, part of petroleum and washing matters go to the condensate. The purification of this condensate leads to pollution of ion exchange resins by petroleum oil and reduction of the filter cycle number. The purification of condensate of Russian NPS from petroleum oil is carried out using active carbon and polymer filters, but this process is not effective and fails to give pure condensate. Therefore, the authors began to search for more effective methods of purification of waste water from petroleum oil. They found that UV-radiation makes it possible to purify water from petroleum matter to concentration of the organic phase less than 0.5 mg/dm3. In this process of purification the air, contained in the water phase, was used as an oxidant. When purification is carried out in the absence of sorbents, the quantity of radioactive solid waste, which have to be recovered, decreases. During the study of purification of waste water it was found that increasing of the temperature of the process increases the rate of UV-radiation-induced oxidation of organic phase. The increase in the initial concentration of petroleum products also increases the rate of petroleum oil decomposition. The content of ions in water phase decreases the purification rate. The investigations were carried out on the laboratory scale with water and condensate from Tver's NPS

  15. Liquid-liquid extraction for purification of wet process phosphoric acid

    International Nuclear Information System (INIS)

    Lotfollahi, Mohammad Nader; Bakshi, Mahdi

    2006-01-01

    The Wet Process Phosphoric Acid (WPA) is used to produce fertilizers and alimentary supplies for cattle. In each of these applications, the impurities contained in acid must be in standard range. In this paper purification of WPA by solvent extraction is performed and the effect of the mass ratio of solvent to feed on extraction efficiency is studied. The working solvents are Methyl Iso Butyl Ketone (MIBK), Iso Amyl Alcohol (IAA) and the mixture of them. The results show that the IAA is better than other solvents in extraction of WPA. This solvent can extract 82.2% of acid after two extraction stages but MIBK can extract only 73.5% of acid after three extraction stages. For all of these solvents, the Pb and Cd concentrations go down to trace. The experimental results show that the maximum separation of Mg with MIBK is 87.5% which occurs at the mass ratio of solvent to feed eual to 4. In the case of IAA solvent the percent is 91.7% and the ratio is 8

  16. [Efficiency of the preparation "Ekolan-M" for purification of oil polluted soil].

    Science.gov (United States)

    Nogina, T M; Dumanskaia, T U; Khomenko, L A; Podgorskiĭ, V S

    2012-01-01

    The efficiency of purification of oil contaminated loamy chernozem by the preparation "Ekolan-M" was investigated. During 12 months a complex soil bioremediation using the preparation and alfalfa, as the land-improving plant, at the final stage of purification resulted in the reduction of hydrocarbon content by 97.0%, and without the preparation - by 65.5 %. In the version of experiment with the preparation a 100% decrease of soil phytotoxicity was achieved and a significant stimulation of plant growth and development was observed. The process of soil purification was accompanied by intensive development of hydrocarbon-oxidizing microorganisms, the amount of which during the process of oil concentration gradually decreased, approaching the level in the control uncontaminated soil.

  17. Experimental Study on Purification of Low Grade Diatomite

    Science.gov (United States)

    Xiao, Liguang; Pang, Bo

    2017-04-01

    This paper presented an innovation for purification of low grade diatomite(DE) by grinding, ultrasonic pretreatment, acid leaching of closed stirring and calcination. The optimum process parameters of DE purification were obtained, the characterizations of original and purified DE were determined by SEM and BET. The results showed that the specific surface area of DE increased from 12.65m2/g to 23.23m2/g, which increased by 45.54%. SEM analysis revealed that the pore structure of purified DE was dredged highly.

  18. Comparative Sequence Analysis of Multidrug-Resistant IncA/C Plasmids from Salmonella enterica.

    Science.gov (United States)

    Hoffmann, Maria; Pettengill, James B; Gonzalez-Escalona, Narjol; Miller, John; Ayers, Sherry L; Zhao, Shaohua; Allard, Marc W; McDermott, Patrick F; Brown, Eric W; Monday, Steven R

    2017-01-01

    Determinants of multidrug resistance (MDR) are often encoded on mobile elements, such as plasmids, transposons, and integrons, which have the potential to transfer among foodborne pathogens, as well as to other virulent pathogens, increasing the threats these traits pose to human and veterinary health. Our understanding of MDR among Salmonella has been limited by the lack of closed plasmid genomes for comparisons across resistance phenotypes, due to difficulties in effectively separating the DNA of these high-molecular weight, low-copy-number plasmids from chromosomal DNA. To resolve this problem, we demonstrate an efficient protocol for isolating, sequencing and closing IncA/C plasmids from Salmonella sp. using single molecule real-time sequencing on a Pacific Biosciences (Pacbio) RS II Sequencer. We obtained six Salmonella enterica isolates from poultry, representing six different serovars, each exhibiting the MDR-Ampc resistance profile. Salmonella plasmids were obtained using a modified mini preparation and transformed with Escherichia coli DH10Br. A Qiagen Large-Construct kit™ was used to recover highly concentrated and purified plasmid DNA that was sequenced using PacBio technology. These six closed IncA/C plasmids ranged in size from 104 to 191 kb and shared a stable, conserved backbone containing 98 core genes, with only six differences among those core genes. The plasmids encoded a number of antimicrobial resistance genes, including those for quaternary ammonium compounds and mercury. We then compared our six IncA/C plasmid sequences: first with 14 IncA/C plasmids derived from S. enterica available at the National Center for Biotechnology Information (NCBI), and then with an additional 38 IncA/C plasmids derived from different taxa. These comparisons allowed us to build an evolutionary picture of how antimicrobial resistance may be mediated by this common plasmid backbone. Our project provides detailed genetic information about resistance genes in

  19. Comparative Sequence Analysis of Multidrug-Resistant IncA/C Plasmids from Salmonella enterica

    Directory of Open Access Journals (Sweden)

    Maria Hoffmann

    2017-08-01

    Full Text Available Determinants of multidrug resistance (MDR are often encoded on mobile elements, such as plasmids, transposons, and integrons, which have the potential to transfer among foodborne pathogens, as well as to other virulent pathogens, increasing the threats these traits pose to human and veterinary health. Our understanding of MDR among Salmonella has been limited by the lack of closed plasmid genomes for comparisons across resistance phenotypes, due to difficulties in effectively separating the DNA of these high-molecular weight, low-copy-number plasmids from chromosomal DNA. To resolve this problem, we demonstrate an efficient protocol for isolating, sequencing and closing IncA/C plasmids from Salmonella sp. using single molecule real-time sequencing on a Pacific Biosciences (Pacbio RS II Sequencer. We obtained six Salmonella enterica isolates from poultry, representing six different serovars, each exhibiting the MDR-Ampc resistance profile. Salmonella plasmids were obtained using a modified mini preparation and transformed with Escherichia coli DH10Br. A Qiagen Large-Construct kit™ was used to recover highly concentrated and purified plasmid DNA that was sequenced using PacBio technology. These six closed IncA/C plasmids ranged in size from 104 to 191 kb and shared a stable, conserved backbone containing 98 core genes, with only six differences among those core genes. The plasmids encoded a number of antimicrobial resistance genes, including those for quaternary ammonium compounds and mercury. We then compared our six IncA/C plasmid sequences: first with 14 IncA/C plasmids derived from S. enterica available at the National Center for Biotechnology Information (NCBI, and then with an additional 38 IncA/C plasmids derived from different taxa. These comparisons allowed us to build an evolutionary picture of how antimicrobial resistance may be mediated by this common plasmid backbone. Our project provides detailed genetic information about

  20. Extraction and purification of high added value compounds from by-products of the winemaking chain using alternative/nonconventional processes/technologies.

    Science.gov (United States)

    Yammine, Sami; Brianceau, Sylène; Manteau, Sébastien; Turk, Mohammad; Ghidossi, Rémy; Vorobiev, Eugène; Mietton-Peuchot, Martine

    2018-05-24

    Grape byproducts are today considered as a cheap source of valuable compounds since existent technologies allow the recovery of target compounds and their recycling. The goal of the current article is to explore the different recovery stages used by both conventional and alternative techniques and processes. Alternative pre-treatments techniques reviewed are: ultrasounds, pulsed electric fields and high voltage discharges. In addition, nonconventional solvent extraction under high pressure, specifically, supercritical fluid extraction and subcritical water extraction are discussed. Finally alternative purification technologies, for example membrane processing were also examined. The intent is to describe the mechanisms involved by these alternative technologies and to summarize the work done on the improvement of the extraction process of phenolic compounds from winery by-products. With a focus on the developmental stage of each technology, highlighting the research need and challenges to be overcome for an industrial implementation of these unitary operations in the overall extraction process. A critical comparison of conventional and alternative techniques will be reviewed for ethe pre-treatment of raw material, the diffusion of polyphenols and the purification of these high added value compounds. This review intends to give the reader some key answers (costs, advantages, drawbacks) to help in the choice of alternative technologies for extraction purposes.

  1. Bicistronic expression plasmid for the rapid production of recombinant fused proteins in Escherichia coli.

    Science.gov (United States)

    Yero, Daniel; Pajón, Rolando; Niebla, Olivia; Sardiñas, Gretel; Vivar, Isbel; Perera, Yasser; García, Darien; Delgado, Maité; Cobas, Karem

    2006-04-01

    In the post-genomic era, every aspect of the production of proteins must be accelerated. In this way, several vectors are currently exploited for rapid production of recombinant proteins in Escherichia coli. N-terminal fusions to the first 47 amino acids of the LpdA (dihydrolipoamide dehydrogenase A) protein of Neisseria meningitidis have been shown to increase the expression of recombinant proteins. Consequently, we have constructed a modified N-terminal LpdA fusion vector, introducing the blue/white colony selection by exploiting a bicistronic gene organization. In the new vector, the sequence encoding the first 47 amino acids of meningococcal LpdA and the alpha-peptide sequence of beta-galactosidase were connected via a ribosome-binding site, and two MCSs (multiple cloning sites) were located surrounding the latter, allowing efficient cloning by colour selection of recombinants. The vector was also improved with the addition of a C-terminal polyhistidine tag, and an EKS (enterokinase recognition sequence) immediately after the LpdA fusion sequence. The new plasmid was employed in the expression and purification of six different bacterial polypeptides. One of these recombinant proteins, P6 protein from Haemophilus influenzae, was used as a model and its N-terminal fusion sequence was totally removed from the recombinant version after incubation with the enterokinase protease, while the polyhistidine tail successfully allowed the purification of the unfused protein from the protease reaction. Two completely new neisserial vaccine candidates, NMB0088 and NMB1126 proteins, were cloned, expressed and purified using this system. To our knowledge, this constitutes the first report of the cloning and expression of these proteins in E. coli.

  2. Secretory immunoglobulin purification from whey by chromatographic techniques.

    Science.gov (United States)

    Matlschweiger, Alexander; Engelmaier, Hannah; Himmler, Gottfried; Hahn, Rainer

    2017-08-15

    Secretory immunoglobulins (SIg) are a major fraction of the mucosal immune system and represent potential drug candidates. So far, platform technologies for their purification do not exist. SIg from animal whey was used as a model to develop a simple, efficient and potentially generic chromatographic purification process. Several chromatographic stationary phases were tested. A combination of two anion-exchange steps resulted in the highest purity. The key step was the use of a small-porous anion exchanger operated in flow-through mode. Diffusion of SIg into the resin particles was significantly hindered, while the main impurities, IgG and serum albumin, were bound. In this step, initial purity was increased from 66% to 89% with a step yield of 88%. In a second anion-exchange step using giga-porous material, SIg was captured and purified by step or linear gradient elution to obtain fractions with purities >95%. For the step gradient elution step yield of highly pure SIg was 54%. Elution of SIgA and SIgM with a linear gradient resulted in a step yield of 56% and 35%, respectively. Overall yields for both anion exchange steps were 43% for the combination of flow-through and step elution mode. Combination of flow-through and linear gradient elution mode resulted in a yield of 44% for SIgA and 39% for SIgM. The proposed process allows the purification of biologically active SIg from animal whey in preparative scale. For future applications, the process can easily be adopted for purification of recombinant secretory immunoglobulin species. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. Long- term manure exposure increases soil bacterial community potential for plasmid uptake

    DEFF Research Database (Denmark)

    Musovic, Sanin; Klümper, Uli; Dechesne, Arnaud

    2014-01-01

    Microbial communities derived from soils subject to different agronomic treatments were challenged with three broad host range plasmids, RP4, pIPO2tet and pRO101, via solid surface filter matings to assess their permissiveness. Approximately 1 in 10 000 soil bacterial cells could receive and main......Microbial communities derived from soils subject to different agronomic treatments were challenged with three broad host range plasmids, RP4, pIPO2tet and pRO101, via solid surface filter matings to assess their permissiveness. Approximately 1 in 10 000 soil bacterial cells could receive...... and maintain the plasmids. The community permissiveness increased up to 100% in communities derived from manured soil. While the plasmid transfer frequency was significantly influenced by both the type of plasmid and the agronomic treatment, the diversity of the transconjugal pools was purely plasmid dependent...

  4. Bromelain: an overview of industrial application and purification strategies.

    Science.gov (United States)

    Arshad, Zatul Iffah Mohd; Amid, Azura; Yusof, Faridah; Jaswir, Irwandi; Ahmad, Kausar; Loke, Show Pau

    2014-09-01

    This review highlights the use of bromelain in various applications with up-to-date literature on the purification of bromelain from pineapple fruit and waste such as peel, core, crown, and leaves. Bromelain, a cysteine protease, has been exploited commercially in many applications in the food, beverage, tenderization, cosmetic, pharmaceutical, and textile industries. Researchers worldwide have been directing their interest to purification strategies by applying conventional and modern approaches, such as manipulating the pH, affinity, hydrophobicity, and temperature conditions in accord with the unique properties of bromelain. The amount of downstream processing will depend on its intended application in industries. The breakthrough of recombinant DNA technology has facilitated the large-scale production and purification of recombinant bromelain for novel applications in the future.

  5. Synthesis Gas Purification Purification des gaz de synthèse

    Directory of Open Access Journals (Sweden)

    Chiche D.

    2013-10-01

    Full Text Available Fischer-Tropsch (FT based B-XTL processes are attractive alternatives for future energy production. These processes aim at converting lignocellulosic biomass possibly in co-processing with petcoke, coal, or vacuum residues into synthetic biofuels. A gasification step converts the feed into a synthesis gas (CO and H2 mixture , which undergoes the Fischer-Tropsch reaction after H2/CO ratio adjustment and CO2 removal. However synthesis gas also contains various impurities that must be removed in order to prevent Fischer-Tropsch catalyst poisoning. Due to the large feedstocks variety that can be processed, significant variations of the composition of the synthesis gas are expected. Especially, this affects the nature of the impurities that are present (element, speciation, as well as their relative contents. Moreover, due to high FT catalyst sensitivity, severe syngas specifications regarding its purity are required. For these reasons, synthesis gas purification constitutes a major challenge for the development of B-XTL processes. In this article, we focus on these major hurdles that have to be overcome. The different kinds of syngas impurities are presented. The influence of the nature of feedstocks, gasification technology and operating conditions on the type and content of impurities is discussed. Highlight is given on the fate of sulfur compounds, nitrogen compounds, halides, transition and heavy metals. Main synthesis gas purification technologies (based on adsorption, absorption, catalytic reactions, etc. are finally described, as well as the related challenges. Les procédés de synthèse de biocarburants par voie Fischer-Tropsch (FT, voies B-XTL, représentent des alternatives prometteuses pour la production d’énergie. Ces procédés permettent la conversion en carburants de synthèse de biomasse lignocellulosique, éventuellement mise en oeuvre en mélange avec des charges fossiles telles que petcoke, charbons ou résidus sous vide. Pour

  6. Effect of charcoal on water purification

    OpenAIRE

    Suzuki, Hirotaka; Kawahigashi, Tatsuo

    2014-01-01

    [Abstract] A natural basin system purifies water through self-purification, but the water pollution load of a river might exceed its self-purification capacity. Charcoal, which is used for other uses aside from heating, such as air purification, was evaluated experimentally for water quality purification. The experiment described herein is based on simple water quality measurements. Some experimentally obtained results are discussed.

  7. Intensification of oily waste waters purification by means of liquid atomization

    Science.gov (United States)

    Eskin, A. A.; Tkach, N. S.; Kim, M. I.; Zakharov, G. A.

    2017-10-01

    In this research, a possibility of using liquid atomization for improving the efficiency of purification of wastewater by different methods has been studied. By the introduced method and an experimental setup for wastewater purification, saturation rate increases with its purification by means of dissolved air flotation. Liquid atomization under excess pressure allows to gain a large interfacial area between the saturated liquid and air, which may increase the rate of purified liquid saturation almost twice, compared to the existing methods of saturation. Current disadvantages of liquid atomization used for intensification of wastewater purification include high energy cost and secondary emulsion of polluting agents. It is also known that by means of liquid atomization a process of ozonizing can be intensified. Large contact surface between the purified liquid and ozone-air mixture increases the oxidizing efficiency, which allows to diminish ozone discharge. Liquid atomization may be used for purification of wastewaters by ultraviolet radiation. Small drops of liquid will be proportionally treated by ultraviolet, which makes it possible to do purification even of turbid wastewaters. High-speed liquid motion will prevent the pollution of quartz tubes of ultraviolet lamps.

  8. Prevalence of plasmid-bearing and plasmid-free Chlamydia trachomatis infection among women who visited obstetrics and gynecology clinics in Malaysia

    OpenAIRE

    Yeow, Tee Cian; Wong, Won Fen; Sabet, Negar Shafiei; Sulaiman, Sofiah; Shahhosseini, Fatemeh; Tan, Grace Min Yi; Movahed, Elaheh; Looi, Chung Yeng; Shankar, Esaki M.; Gupta, Rishien; Arulanandam, Bernard P.; Hassan, Jamiyah; Abu Bakar, Sazaly

    2016-01-01

    Background The 7.5?kb cryptic plasmid of Chlamydia trachomatis has been shown to be a virulence factor in animal models, but its significance in humans still remains unknown. The aim of this study was to investigate the prevalence and potential involvement of the C. trachomatis cryptic plasmid in causing various clinical manifestations; including infertility, reproductive tract disintegrity, menstrual disorder, and polycystic ovarian syndrome (PCOS) among genital C. trachomatis?infected patie...

  9. Genome Stability of Lyme Disease Spirochetes: Comparative Genomics of Borrelia burgdorferi Plasmids

    Energy Technology Data Exchange (ETDEWEB)

    Casjens S. R.; Dunn J.; Mongodin, E. F.; Qiu, W.-G.; Luft, B. J.; Schutzer, S. E.; Gilcrease, E. B.; Huang, W. M.; Vujadinovic, M.; Aron, J. K.; Vargas, L. C.; Freeman, S.; Radune, D.; Weidman, J. F.; Dimitrov, G. I.; Khouri, H. M.; Sosa, J. E.; Halpin, R. A.; Fraser, C. M.

    2012-03-14

    Lyme disease is the most common tick-borne human illness in North America. In order to understand the molecular pathogenesis, natural diversity, population structure and epizootic spread of the North American Lyme agent, Borrelia burgdorferi sensu stricto, a much better understanding of the natural diversity of its genome will be required. Towards this end we present a comparative analysis of the nucleotide sequences of the numerous plasmids of B. burgdorferi isolates B31, N40, JD1 and 297. These strains were chosen because they include the three most commonly studied laboratory strains, and because they represent different major genetic lineages and so are informative regarding the genetic diversity and evolution of this organism. A unique feature of Borrelia genomes is that they carry a large number of linear and circular plasmids, and this work shows that strains N40, JD1, 297 and B31 carry related but non-identical sets of 16, 20, 19 and 21 plasmids, respectively, that comprise 33-40% of their genomes. We deduce that there are at least 28 plasmid compatibility types among the four strains. The B. burgdorferi {approx}900 Kbp linear chromosomes are evolutionarily exceptionally stable, except for a short {le}20 Kbp plasmid-like section at the right end. A few of the plasmids, including the linear lp54 and circular cp26, are also very stable. We show here that the other plasmids, especially the linear ones, are considerably more variable. Nearly all of the linear plasmids have undergone one or more substantial inter-plasmid rearrangements since their last common ancestor. In spite of these rearrangements and differences in plasmid contents, the overall gene complement of the different isolates has remained relatively constant.

  10. Plasmid Conjugation in E. coli and Drug Resistance | Igwe ...

    African Journals Online (AJOL)

    This study aimed at determining the antibiotics susceptibility pattern of E. coli isolates claimed to be multidrug resistance using disc diffusion method. It also determined the presence of transferable resistance plasmids through conjugation and evaluated the medical significance of plasmid encoding E. coli and drug ...

  11. Assessing the performance of a Loop Mediated Isothermal Amplification (LAMP) assay for the detection and subtyping of high-risk suptypes of Human Papilloma Virus (HPV) for Oropharyngeal Squamous Cell Carcinoma (OPSCC) without DNA purification.

    Science.gov (United States)

    Rohatensky, Mitchell G; Livingstone, Devon M; Mintchev, Paul; Barnes, Heather K; Nakoneshny, Steven C; Demetrick, Douglas J; Dort, Joseph C; van Marle, Guido

    2018-02-08

    Oropharyngeal Squamous Cell Carcinoma (OPSCC) is increasing in incidence despite a decline in traditional risk factors. Human Papilloma Virus (HPV), specifically subtypes 16, 18, 31 and 35, has been implicated as the high-risk etiologic agent. HPV positive cancers have a significantly better prognosis than HPV negative cancers of comparable stage, and may benefit from different treatment regimens. Currently, HPV related carcinogenesis is established indirectly through Immunohistochemistry (IHC) staining for p16, a tumour suppressor gene, or polymerase chain reaction (PCR) that directly tests for HPV DNA in biopsied tissue. Loop mediated isothermal amplification (LAMP) is more accurate than IHC, more rapid than PCR and is significantly less costly. In previous work we showed that a subtype specific HPV LAMP assay performed similar to PCR on purified DNA. In this study we examined the performance of this LAMP assay without DNA purification. We used LAMP assays using established primers for HPV 16 and 18, and new primers for HPV 31 and 35. LAMP reaction conditions were tested on serial dilutions of plasmid HPV DNA to confirm minimum viral copy number detection thresholds. LAMP was then performed directly on different human cell line samples without DNA purification. Our LAMP assays could detect 10 5 , 10 3 , 10 4 , and 10 5 copies of plasmid DNA for HPV 16, 18, 31, and 35, respectively. All primer sets were subtype specific, with no cross-amplification. Our LAMP assays also reliably amplified subtype specific HPV DNA from samples without requiring DNA isolation and purification. The high risk OPSCC HPV subtype specific LAMP primer sets demonstrated, excellent clinically relevant, minimum copy number detection thresholds with an easy readout system. Amplification directly from samples without purification illustrated the robust nature of the assay, and the primers used. This lends further support HPV type specific LAMP assays, and these specific primer sets and assays

  12. Quantification of plasmid DNA reference materials for Shiga toxin-producing Escherichia coli based on UV, HR-ICP-MS and digital PCR.

    Science.gov (United States)

    Liang, Wen; Xu, Li; Sui, Zhiwei; Li, Yan; Li, Lanying; Wen, Yanli; Li, Chunhua; Ren, Shuzhen; Liu, Gang

    2016-01-01

    The accuracy and metrology traceability of DNA quantification is becoming a critical theme in many fields, including diagnosis, forensic analysis, microorganism detection etc. Thus the research of DNA reference materials (RMs) and consistency of DNA quantification methods has attracted considerable research interest. In this work, we developed 3 plasmid candidate RMs, containing 3 target genes of Escherichia coli O157:H7 (E. coli O157:H7) and other Shiga toxin-producing Escherichia coli (STEC): stx1, stx2, and fliC (h7) respectively. Comprehensive investigation of the plasmid RMs was performed for their sequence, purity, homogeneity and stability, and then the concentration was quantified by three different methods: ultraviolet spectrophotometer (UV), high resolution inductively coupled plasma mass spectrometry (HR-ICP-MS) and digital PCR. As a routinely applied method for DNA analysis, UV was utilized for the quantification (OD260) and purity analysis for the plasmids. HR-ICP-MS quantified the plasmid DNA through analysing the phosphorus in DNA molecules. Digital PCR distributed the DNA samples onto a microarray chip containing thousands of reaction chambers, and quantified the DNA copy numbers by analysing the number of positive signals without any calibration curves needed. Based on the high purification of the DNA reference materials and the optimization of dPCR analysis, we successfully achieved good consistency between UV, HR-ICP-MS and dPCR, with relative deviations lower than 10 %. We then performed the co-quantification of 3 DNA RMs with three different methods together, and the uncertainties of their concentration were evaluated. Finally, the certified values and expanded uncertainties for 3 DNA RMs (pFliC, pStx1 and pStx2) were (1.60 ± 0.10) × 10(10) copies/μL, (1.53 ± 0.10) × 10(10) copies/μL and (1.70 ± 0.11) × 10(10) copies/μL respectively.Graphical abstractWe developed 3 plasmid candidate RMs, containing 3 target genes of

  13. Moderate temperature gas purification system: Application to high calorific coal-derived fuel

    Energy Technology Data Exchange (ETDEWEB)

    Kobayashi, M.; Shirai, H.; Nunokawa, M. [Central Research Institute of Electric Power Industry, Kanagawa (Japan)

    2008-01-15

    Simultaneous removal of dust, alkaline and alkaline-earth metals, halides and sulfur compounds is required to enlarge application of coal-derived gas to the high-temperature fuel cells and the fuel synthesis through chemical processing. Because high calorific fuel gas, such as oxygen-blown coal gas, has high carbon monoxide content, high-temperature (above 450{sup o}C) gas purification system is always subjected to the carbon deposition. We suggest moderate temperature (around 300{sup o}C) operation of the gas purification system to avoid the harmful disproportionation reaction and efficient removal of the various contaminants. Because the reaction rate is predominant to the performance of contaminant removal in the moderate temperature gas purification system, we evaluated the chemical removal processes; performance of the removal processes for halides and sulfur compounds was experimentally evaluated. The halide removal process with sodium aluminate sorbent had potential performance at around 300{sup o}C. The sulfur removal process with zinc ferrite sorbent was also applicable to the temperature range, though the reaction kinetics of the sorbent is essential to be approved.

  14. INVESTIGATIONS ON BIOCHEMICAL PURIFICATION OF GROUND WATER FROM HYDROGEN SULFIDE

    Directory of Open Access Journals (Sweden)

    Yu. P. Sedlukho

    2015-01-01

    Full Text Available The paper considers problems and features of biochemical removal of hydrogen sulfide from ground water. The analysis of existing methods for purification of ground water from hydrogen sulfide has been given in the paper. The paper has established shortcomings of physical and chemical purification of ground water. While using aeration methods for removal of hydrogen sulfide formation of colloidal sulfur that gives muddiness and opalescence to water occurs due to partial chemical air oxidation. In addition to this violation of sulfide-carbonate equilibrium taking place in the process of aeration due to desorption of H2S and CO2, often leads to clogging of degasifier nozzles with formed CaCO3 that causes serious operational problems. Chemical methods require relatively large flow of complex reagent facilities, storage facilities and transportation costs.In terms of hydrogen sulfide ground water purification the greatest interest is given to the biochemical method. Factors deterring widespread application of the biochemical method is its insufficient previous investigation and necessity to execute special research in order to determine optimal process parameters while purifying groundwater of a particular water supply source. Biochemical methods for oxidation of sulfur compounds are based on natural biological processes that ensure natural sulfur cycle. S. Vinogradsky has established a two-stage mechanism for oxidation of hydrogen sulfide with sulfur bacteria (Beggiatoa. The first stage presupposes oxidation of hydrogen sulphide to elemental sulfur which is accumulating in the cytoplasm in the form of globules. During the second stage sulfur bacteria begin to oxidize intracellular sulfur to sulfuric acid due to shortage of hydrogen sulfide.The paper provides the results of technological tests of large-scale pilot plants for biochemical purification of groundwater from hydrogen sulfide in semi-industrial conditions. Dependences of water quality

  15. Plasmids of Staphylococcus cohnii isolated from the intensive-care unit.

    Science.gov (United States)

    Szewczyk, E M; Rózalska, M; Cieślikowski, T; Nowak, T

    2004-01-01

    Numerous isolates of both subspecies of Staphylococcus cohnii were found in the environment of the intensive-care unit of a pediatric hospital. These isolates carried in their cells many plasmids, up to fourteen, of a wide range of sizes ( 56 kb). Striking was the occurrence of large plasmids not very common in staphylococci. These were present in > 80% of S. cohnii isolates. Fifty-two different plasmid profiles were found in 79 investigated isolates belonging to S. cohnii ssp. cohnii and S. cohnii ssp. urealyticus. Isolates similar in plasmid profiles were grouped in antibiotic-resistance clusters established for 9 antibiotics (gentamicin, ciprofloxacin, clindamycin, erythromycin, tetracycline, chloramphenicol, mupirocin, trimethoprim-sulfamethoxazole, vancomycin) using the method of unweighted pair group mathematical averages (UPGMA). Many isolates were multiresistant to antibiotics and produced bacteriocins.

  16. Influence of the drying method in chitosans purification step

    International Nuclear Information System (INIS)

    Fonseca, Ana C.M.; Batista, Jorge G.S.; Bettega, Antonio; Lima, Nelson B. de

    2015-01-01

    Currently, the study of extracellular biopolymers properties has received prominence for being easy extraction and purification. Chitosan has been an attractive proposition for applications in various fields such as engineering, biotechnology, medicine and pharmacology. For such applications, it is necessary purification of chitosan to obtain a product more concentrated and free of undesirable impurities. However, at this stage of the process of obtaining the biopolymer may occur morphological and physicochemical changes. This study evaluated the influence of the drying process after purification of a commercial chitosan sample and the importance of this step and its cost/benefit in applications requiring a high degree of purity. The method of drying influenced in the organoleptic properties and in the main characteristics of material. Analysis of the crystal structure by X-ray diffraction showed that the degree of crystallinity, X (%), in the purified chitosan samples was lower when compared with the unpurified sample. The degree of acetylation, DA (%), was analyzed by spectroscopy infrared with no significant changes on the three drying methods assessed, unlike the viscosimetric molecular weight, M_v, determined by capillary viscometry. (author)

  17. Construction of Stable Fluorescent Reporter Plasmids for Use in Staphylococcus aureus

    Directory of Open Access Journals (Sweden)

    Michelle D. Rodriguez

    2017-12-01

    Full Text Available Here, the genes encoding three different fluorescent proteins were cloned into the stably maintained Staphylococcus aureus shuttle vector pKK30. The resulting plasmids were transformed into two S. aureus strains; SH1000 and RN4220. Stability assays illustrated that the three recombinant plasmids retained near 100% maintenance in vitro for 160 generations. S. aureus strain SH1000 expressing green fluorescent protein was then inoculated in an ovine model and in vivo stability for 6 days was demonstrated. In essence, these reporter plasmids represent a useful set of tools for dynamic imaging studies in S. aureus. These three reporter plasmids are available through BEI Resources.

  18. Construction of Stable Fluorescent Reporter Plasmids for Use in Staphylococcus aureus.

    Science.gov (United States)

    Rodriguez, Michelle D; Paul, Zubin; Wood, Charles E; Rice, Kelly C; Triplett, Eric W

    2017-01-01

    Here, the genes encoding three different fluorescent proteins were cloned into the stably maintained Staphylococcus aureus shuttle vector pKK30. The resulting plasmids were transformed into two S. aureus strains; SH1000 and RN4220. Stability assays illustrated that the three recombinant plasmids retained near 100% maintenance in vitro for 160 generations. S. aureus strain SH1000 expressing green fluorescent protein was then inoculated in an ovine model and in vivo stability for 6 days was demonstrated. In essence, these reporter plasmids represent a useful set of tools for dynamic imaging studies in S. aureus . These three reporter plasmids are available through BEI Resources.

  19. A purification process for an inert gas system

    International Nuclear Information System (INIS)

    Raj, S.S.; Samanta, S.K.; Jain, N.G.; Deshingkar, D.S.; Ramaswamy, M.

    1984-01-01

    Special inert atmosphere is desired inside hot cells used for handling radioactive materials. In this report, details of experiments conducted to generate data required for the design of a system for maintaining very low levels of organic and acid vapours, oxygen and moisture in a nitrogen gas inert atmosphere, are described. Several grades of activated charcoals impregnated with 1% KOH were studied for the adsorption of acidic and organic vapours. A Pd/Al 2 O 3 catalyst was developed to remove oxygen with greater than 90% efficiency. For the removal of moisture, a regenerable molecular sieve 4A dual-bed was provided. Based on the performance data thus generated, an integrated purification system for nitrogen gas is proposed. (author)

  20. Fast and efficient three-step target-specific curing of a virulence plasmid in Salmonella enterica.

    Science.gov (United States)

    de Moraes, Marcos H; Teplitski, Max

    2015-12-01

    Virulence plasmids borne by serovars of Salmonella enterica carry genes involved in its pathogenicity, as well as other functions. Characterization of phenotypes associated with virulence plasmids requires a system for efficiently curing strains of their virulence plasmids. Here, we developed a 3-step protocol for targeted curing of virulence plasmids. The protocol involves insertion of an I-SecI restriction site linked to an antibiotic resistance gene into the target plasmid using λ-Red mutagenesis, followed by the transformation with a temperature-sensitive auxiliary plasmid which carries I-SecI nuclease expressed from a tetracycline-inducible promoter. Finally, the auxiliary plasmid is removed by incubation at 42 °C and the plasmid-less strains are verified on antibiotic-containing media. This method is fast and very efficient: over 90 % of recovered colonies lacked their virulence plasmid.

  1. Broad host range plasmids can invade an unexpectedly diverse fraction of a soil bacterial community

    DEFF Research Database (Denmark)

    Klümper, Uli; Riber, Leise; Dechesne, Arnaud

    2014-01-01

    and Actinobacteria suggests that inter-Gram plasmid transfer of IncP-1 and IncPromA-type plasmids is a frequent phenomenon. While the plasmid receiving fractions of the community were both plasmid- and donor- dependent, we identified a core super-permissive fraction that could take up different plasmids from diverse...

  2. Purification of Sodium Phosphates as by Product of Rirang Ore Decomposition Process

    International Nuclear Information System (INIS)

    Sugeng-Walujo; Hafni-LN; Susilaningtyas; Mukhlis; Budi-Sarono; Widowati

    2004-01-01

    The aim of this experiment is to get purification condition of sodium phosphates from the filtration result of mixing mother liquor and filtrate of washing residue from Rirang monazite decomposition by alkaline. The method of purification which has been used is dissolved the precipitation of sodium phosphates into agitated water 5 minutes and solution settling for 12 hours until appear of sodium phosphate crystals. The variable of experiment included dissolution time and ratio of the amount precipitate sodium phosphate volume of water to solvent. Experimental data shown that the good temperature of dissolution is 70 o C with the ratio of precipitate sodium phosphate is 80 gram/ 40 ml to water. The recovery of sodium phosphate crystallisation is 87.4314 % with 54.0105 % pure of Na 3 PO 4 , U content is 0.0004%, NaOH content and other impurities is 45.9889%. (author)

  3. Plasmids in Vibrio parahemolyticus strains isolated in Japan and Bangladesh with special reference to different distributions.

    Science.gov (United States)

    Arai, T; Ando, T; Kusakabe, A; Ullah, M A

    1983-01-01

    We surveyed plasmids in naturally occurring Vibrio parahemolyticus strains isolated in Japan and Bangladesh. Among the strains isolated in Japan, about half of the strains isolated from stools of patients of domestic diarrhea outbreaks as well as of travelers returning from East Asia were found to have plasmids, but no strains from foods had plasmids. In contrast, among the strains isolated in Bangladesh, none of the four strains isolated from patients had plasmids, but two out of eight strains isolated from water had plasmids, suggesting that plasmids are common in strains from the water in Bangladesh. All plasmids so far reported in V. parahemolyticus were detected in strains isolated from stools of patients. Incidences of plasmids in this organism were not so high in either area. In Japan, all plasmids were detected in strains from human intestines at 37 C, but in Bangladesh, where the temperature is around 30-40 C, the plasmids were detected in strains from the natural environment. These results suggested the possibility that these plasmids can come from different bacteria under rather high temperatures and that incidences of plasmids are influenced by the incidences of plasmids in bacteria present in the vicinity of V. parahemolyticus strains. None of these plasmids were found to have any relation to the biological characters tested.

  4. Characterization of Plasmid pPO1 from the Hyperacidophile Picrophilus oshimae

    Directory of Open Access Journals (Sweden)

    Angel Angelov

    2011-01-01

    Full Text Available Picrophilus oshimae and Picrophilus torridus are free-living, moderately thermophilic and acidophilic organisms from the lineage of Euryarchaeota. With a pH optimum of growth at pH 0.7 and the ability to even withstand molar concentrations of sulphuric acid, these organisms represent the most extreme acidophiles known. So far, nothing is known about plasmid biology in these hyperacidophiles. Also, there are no genetic tools available for this genus. We have mobilized the 7.6 Kbp plasmid from P. oshimae in E. coli by introducing origin-containing transposons and described the plasmid in terms of its nucleotide sequence, copy number in the native host, mode of replication, and transcriptional start sites of the encoded ORFs. Plasmid pPO1 may encode a restriction/modification system in addition to its replication functions. The information gained from the pPO1 plasmid may prove useful in developing a cloning system for this group of extreme acidophiles.

  5. Sequential acquisition of R-plasmids in vivo by Salmonella typhimurium.

    Science.gov (United States)

    Platt, D J; Sommerville, J S; Gribben, J

    1984-01-01

    Salmonella typhimurium, resistant only to trimethoprim and sulphamethoxazole, was isolated from the faeces and blood of a chronic alcoholic patient in acute renal failure. The isolates harboured an 18 Md non-conjugative plasmid. He was dialysed peritoneally and treated with ampicillin; four days later there was no clinical improvement and his peritoneal dialysis fluid (PDF) had become infected. Salm. typhimurium was isolated from faeces and PDF. Both isolates were additionally resistant to ampicillin and contained two plasmids (55 Md and 18 Md). Therapy was changed to chloramphenicol and gentamicin was added to the PDF. Two weeks later Salm. typhimurium was again isolated from PDF and faeces. The PDF isolate was unchanged but 4% of the colonies isolated from this faecal specimen were resistant to chloramphenicol and had acquired an additional 62 Md plasmid. From all PDF and faecal specimens two different strains of Escherichia coli and one strain of Klebsiella pneumoniae were isolated which contained plasmids indistinguishable, on the basis of molecular weight and transferable resistance markers, from those acquired by Salm. typhimurium. The transferability of these plasmids in vitro to E. coli K12 and to the patient's initial Salm. typhimurium was studied and the results discussed.

  6. Process for recovery and purification of lithium from dilute sources

    International Nuclear Information System (INIS)

    Kunugita, Eiichi; Kim, Jong-Hwa; Komasawa, Isao

    1990-01-01

    A process was studied for recovery and purification of lithium from hot spring water and leachate of coal fly ash in which lithium is present at very low concentration in a mixture of high concentration of less desirable species such as alkali metals, alkaline earth metals and others. Lithium is, as a first step, adsorbed selectively by an ion-sieve type adsorbent, λ-MnO 2 , which can effect separation by adsorbing lithium alone in its intraparticle spaces while adsorbing most metals on its surface. Stagewise elutions are performed, employing two elutrients: NH 4 NO 3 and then HNO 3 solution. The concentration ratio of lithium and sodium is 1/70∼1/120 in the original feed stream, and this is upgraded to 300/1 in the pregnant elutrient. Some impurities in the pregnant elutrient is removed by a solvent extraction technique, employing a mixed extractant of a β-diketone (LIX51) and tri-n-octyl-phosphine oxide in kerosene. A lithium solution with an undetectable amount of impurities is obtained by single extraction followed by single scrubbing with dilute hydrochloric acid of carefully controlled concentration. (author)

  7. Characterization of plasmids in a human clinical strain of Lactococcus garvieae.

    Directory of Open Access Journals (Sweden)

    Mónica Aguado-Urda

    Full Text Available The present work describes the molecular characterization of five circular plasmids found in the human clinical strain Lactococcus garvieae 21881. The plasmids were designated pGL1-pGL5, with molecular sizes of 4,536 bp, 4,572 bp, 12,948 bp, 14,006 bp and 68,798 bp, respectively. Based on detailed sequence analysis, some of these plasmids appear to be mosaics composed of DNA obtained by modular exchange between different species of lactic acid bacteria. Based on sequence data and the derived presence of certain genes and proteins, the plasmid pGL2 appears to replicate via a rolling-circle mechanism, while the other four plasmids appear to belong to the group of lactococcal theta-type replicons. The plasmids pGL1, pGL2 and pGL5 encode putative proteins related with bacteriocin synthesis and bacteriocin secretion and immunity. The plasmid pGL5 harbors genes (txn, orf5 and orf25 encoding proteins that could be considered putative virulence factors. The gene txn encodes a protein with an enzymatic domain corresponding to the family actin-ADP-ribosyltransferases toxins, which are known to play a key role in pathogenesis of a variety of bacterial pathogens. The genes orf5 and orf25 encode two putative surface proteins containing the cell wall-sorting motif LPXTG, with mucin-binding and collagen-binding protein domains, respectively. These proteins could be involved in the adherence of L. garvieae to mucus from the intestine, facilitating further interaction with intestinal epithelial cells and to collagenous tissues such as the collagen-rich heart valves. To our knowledge, this is the first report on the characterization of plasmids in a human clinical strain of this pathogen.

  8. Non-additive costs and interactions alter the competitive dynamics of co-occurring ecologically distinct plasmids.

    Science.gov (United States)

    Morton, Elise R; Platt, Thomas G; Fuqua, Clay; Bever, James D

    2014-03-22

    Plasmids play an important role in shaping bacterial evolution and adaptation to heterogeneous environments. As modular genetic elements that are often conjugative, the selective pressures that act on plasmid-borne genes are distinct from those that act on the chromosome. Many bacteria are co-infected by multiple plasmids that impart niche-specific phenotypes. Thus, in addition to host-plasmid dynamics, interactions between co-infecting plasmids are likely to be important drivers of plasmid population dynamics, evolution and ecology. Agrobacterium tumefaciens is a facultative plant pathogen that commonly harbours two distinct megaplasmids. Virulence depends on the presence of the tumour-inducing (Ti) plasmid, with benefits that are primarily restricted to the disease environment. Here, we demonstrate that a second megaplasmid, the At plasmid, confers a competitive advantage in the rhizosphere. To assess the individual and interactive costs of these plasmids, we generated four isogenic derivatives: plasmidless, pAt only, pTi only and pAtpTi, and performed pairwise competitions under carbon-limiting conditions. These studies reveal a low cost to the virulence plasmid when outside of the disease environment, and a strikingly high cost to the At plasmid. In addition, the costs of pAt and pTi in the same host were significantly lower than predicted based on single plasmid costs, signifying the first demonstration of non-additivity between naturally occurring co-resident plasmids. Based on these empirically demonstrated costs and benefits, we developed a resource-consumer model to generate predictions about the frequencies of these genotypes in relevant environments, showing that non-additivity between co-residing plasmids allows for their stable coexistence across environments.

  9. Reconstructing the complex evolutionary history of mobile plasmids in red algal genomes

    Science.gov (United States)

    Lee, JunMo; Kim, Kyeong Mi; Yang, Eun Chan; Miller, Kathy Ann; Boo, Sung Min; Bhattacharya, Debashish; Yoon, Hwan Su

    2016-01-01

    The integration of foreign DNA into algal and plant plastid genomes is a rare event, with only a few known examples of horizontal gene transfer (HGT). Plasmids, which are well-studied drivers of HGT in prokaryotes, have been reported previously in red algae (Rhodophyta). However, the distribution of these mobile DNA elements and their sites of integration into the plastid (ptDNA), mitochondrial (mtDNA), and nuclear genomes of Rhodophyta remain unknown. Here we reconstructed the complex evolutionary history of plasmid-derived DNAs in red algae. Comparative analysis of 21 rhodophyte ptDNAs, including new genome data for 5 species, turned up 22 plasmid-derived open reading frames (ORFs) that showed syntenic and copy number variation among species, but were conserved within different individuals in three lineages. Several plasmid-derived homologs were found not only in ptDNA but also in mtDNA and in the nuclear genome of green plants, stramenopiles, and rhizarians. Phylogenetic and plasmid-derived ORF analyses showed that the majority of plasmid DNAs originated within red algae, whereas others were derived from cyanobacteria, other bacteria, and viruses. Our results elucidate the evolution of plasmid DNAs in red algae and suggest that they spread as parasitic genetic elements. This hypothesis is consistent with their sporadic distribution within Rhodophyta. PMID:27030297

  10. Strategies and approaches in plasmidome studies—uncovering plasmid diversity disregarding of linear elements?

    Science.gov (United States)

    Dib, Julián R.; Wagenknecht, Martin; Farías, María E.; Meinhardt, Friedhelm

    2015-01-01

    The term plasmid was originally coined for circular, extrachromosomal genetic elements. Today, plasmids are widely recognized not only as important factors facilitating genome restructuring but also as vehicles for the dissemination of beneficial characters within bacterial communities. Plasmid diversity has been uncovered by means of culture-dependent or -independent approaches, such as endogenous or exogenous plasmid isolation as well as PCR-based detection or transposon-aided capture, respectively. High-throughput-sequencing made possible to cover total plasmid populations in a given environment, i.e., the plasmidome, and allowed to address the quality and significance of self-replicating genetic elements. Since such efforts were and still are rather restricted to circular molecules, here we put equal emphasis on the linear plasmids which—despite their frequent occurrence in a large number of bacteria—are largely neglected in prevalent plasmidome conceptions. PMID:26074886

  11. Natural plasmid transformation in a high-frequency-of transformation marine Vibrio strain

    International Nuclear Information System (INIS)

    Frischer, M.E.; Thurmond, J.M.; Paul, J.H.

    1990-01-01

    The estuarine bacterium Vibrio strain DI-9 has been shown to be naturally transformable with both broad host range plasmid multimers and homologous chromosomal DNA at average frequencies of 3.5 x 10 -9 and 3.4 x 10 -7 transformants per recipient, respectively. Growth of plasmid transformants in nonselective medium resulted in cured strains that transformed 6 to 42,857 times more frequently than the parental strain, depending on the type of transforming DNA. These high-frequency-of-transformation (HfT) strains were transformed at frequencies ranging from 1.1 x 10 -8 to 1.3 x 10 -4 transformants per recipient with plasmid DNA and at an average frequency of 8.3 x 10 -5 transformants per recipient with homologous chromosomal DNA. The highest transformation frequencies were observed by using multimers of an R1162 derivative carrying the transposon Tn5 (pQSR50). Probing of total DNA preparations from one of the cured strains demonstrated that no plasmid DNA remained in the cured strains which may have provided homology to the transforming DNA. All transformants and cured strains could be differentiated from the parental strains by colony morphology. DNA binding studies indicated that late-log-phase HfT strains bound [ 3 H]bacteriophage lambda DNA 2.1 times more rapidly than the parental strain. These results suggest that the original plasmid transformation event of strain DI-9 was the result of uptake and expression of plasmid DNA by a competent mutant (HfT strain). Additionally, it was found that a strain of Vibrio parahaemolyticus, USFS 3420, could be naturally transformed with plasmid DNA. Natural plasmid transformation by high-transforming mutants may be a means of plasmid acquisition by natural aquatic bacterial populations

  12. Heparin-binding peptide as a novel affinity tag for purification of recombinant proteins.

    Science.gov (United States)

    Morris, Jacqueline; Jayanthi, Srinivas; Langston, Rebekah; Daily, Anna; Kight, Alicia; McNabb, David S; Henry, Ralph; Kumar, Thallapuranam Krishnaswamy Suresh

    2016-10-01

    Purification of recombinant proteins constitutes a significant part of the downstream processing in biopharmaceutical industries. Major costs involved in the production of bio-therapeutics mainly depend on the number of purification steps used during the downstream process. Affinity chromatography is a widely used method for the purification of recombinant proteins expressed in different expression host platforms. Recombinant protein purification is achieved by fusing appropriate affinity tags to either N- or C- terminus of the target recombinant proteins. Currently available protein/peptide affinity tags have proved quite useful in the purification of recombinant proteins. However, these affinity tags suffer from specific limitations in their use under different conditions of purification. In this study, we have designed a novel 34-amino acid heparin-binding affinity tag (HB-tag) for the purification of recombinant proteins expressed in Escherichia coli (E. coli) cells. HB-tag fused recombinant proteins were overexpressed in E. coli in high yields. A one-step heparin-Sepharose-based affinity chromatography protocol was developed to purify HB-fused recombinant proteins to homogeneity using a simple sodium chloride step gradient elution. The HB-tag has also been shown to facilitate the purification of target recombinant proteins from their 8 M urea denatured state(s). The HB-tag has been demonstrated to be successfully released from the fusion protein by an appropriate protease treatment to obtain the recombinant target protein(s) in high yields. Results of the two-dimensional NMR spectroscopy experiments indicate that the purified recombinant target protein(s) exist in the native conformation. Polyclonal antibodies raised against the HB-peptide sequence, exhibited high binding specificity and sensitivity to the HB-fused recombinant proteins (∼10 ng) in different crude cell extracts obtained from diverse expression hosts. In our opinion, the HB-tag provides a

  13. Repair promoted by plasmid pKM101 is different from SOS repair

    International Nuclear Information System (INIS)

    Goze, A.; Devoret, R.

    1979-01-01

    In E. coli K12 bacteria carrying plasmid pKM101, prophage lambda was induced at UV doses higher than in plasmid-less parental bacteria. UV-induced reactivation per se was less effective. Bacteria with pKM101 showed no alteration in their division cycle. Plasmid PKM101 coded for a constitutive error-prone repair different from the inducible error-prone repair called SOS repair. Plasmid pKM101 protected E. coli bacteria from UV damage but slightly sensitized them to X-ray lesions. Protection against UV damage was effective in mutant bacteria deficient in DNA excision-repair provided that the recA, lexA and uvrE genes were functional. Survival of phages lambda and S13 after UV irradiation was enhanced in bacteria carrying plasmid pKM101; phage lambda mutagenesis was also increased. Plasmid pKM101 repaired potentially lethal DNA lesions, although Wild-type DNA sequences may not necessarily be restored; hence the mutations observed are the traces of the original DNA lesions. (Auth.)

  14. Purification of Water by Aquatic Plants

    OpenAIRE

    Morimitsu, Katsuhito; Kawahigashi, Tatsuo

    2013-01-01

    [Abstract] Water quality purification of many water systems including those occurring in rivers depends to a great degree on water quality purification activities of aquatic plants and microbes. This paper presents a discussion of results, based on laboratory experiments, of purification by aquatic plants.

  15. The complete sequence and comparative analysis of a multidrug- resistance and virulence multireplicon IncFII plasmid pEC302/04 from an extraintestinal pathogenic Escherichia coli EC302/04 indicate extensive diversity of IncFII plasmids

    Directory of Open Access Journals (Sweden)

    Wing Sze eHo

    2016-01-01

    Full Text Available Extraintestinal pathogenic Escherichia coli (ExPEC that causes extraintestinal infections often harbor plasmids encoding fitness traits such as resistance and virulence determinants that are of clinical importance. We determined the complete nucleotide sequence of plasmid pEC302/04 from a multidrug-resistant E. coli EC302/04 which was isolated from the tracheal aspirate of a patient in Malaysia. In addition, we also performed comparative sequence analyses of 18 related IncFIIA plasmids to determine the phylogenetic relationship and diversity of these plasmids. The 140,232 bp pEC302/04 is a multireplicon plasmid that bears three replication systems (FII, FIA and FIB with subtype of F2:A1:B1. The plasmid is self-transmissible with a complete transfer region. pEC302/04 also carries antibiotic resistance genes such as blaTEM-1 and a class I integron containing sul1, cml and aadA resistance genes, conferring multidrug resistance (MDR to its host, E. coli EC302/04. Besides, two iron acquisition systems (SitABCD and IutA-IucABCD which are the conserved virulence determinants of ExPEC-colicin V or B and M (ColV/ColBM-producing plasmids were identified in pEC302/04. Multiple toxin-antitoxin (TA-based addiction systems (i.e., PemI/PemK, VagC/VagD, CcdA/CcdB, and Hok/Sok and a plasmid partitioning system, ParAB and PsiAB, which are important for plasmid maintenance were also found.Comparative plasmid analysis revealed only one conserved gene, the repA1 as the core genome, showing that there is an extensive diversity among the IncFIIA plasmids. The phylogenetic relationship of 18 IncF plasmids based on the core regions revealed that ColV/ColBM-plasmids and non-ColV/ColBM plasmids were separated into two distinct groups. These plasmids, which carry highly diverse genetic contents, are also mosaic in nature. The atypical combination of genetic materials, i.e., the MDR- and ColV/ColBM-plasmid-virulence encoding regions in a single ExPEC plasmid is rare but of

  16. The Complete Sequence and Comparative Analysis of a Multidrug-Resistance and Virulence Multireplicon IncFII Plasmid pEC302/04 from an Extraintestinal Pathogenic Escherichia coli EC302/04 Indicate Extensive Diversity of IncFII Plasmids.

    Science.gov (United States)

    Ho, Wing Sze; Yap, Kien-Pong; Yeo, Chew Chieng; Rajasekaram, Ganeswrie; Thong, Kwai Lin

    2015-01-01

    Extraintestinal pathogenic Escherichia coli (ExPEC) that causes extraintestinal infections often harbor plasmids encoding fitness traits such as resistance and virulence determinants that are of clinical importance. We determined the complete nucleotide sequence of plasmid pEC302/04 from a multidrug-resistant E. coli EC302/04 which was isolated from the tracheal aspirate of a patient in Malaysia. In addition, we also performed comparative sequence analyses of 18 related IncFIIA plasmids to determine the phylogenetic relationship and diversity of these plasmids. The 140,232 bp pEC302/04 is a multireplicon plasmid that bears three replication systems (FII, FIA, and FIB) with subtype of F2:A1:B1. The plasmid is self-transmissible with a complete transfer region. pEC302/04 also carries antibiotic resistance genes such as bla TEM-1 and a class I integron containing sul1, cml and aadA resistance genes, conferring multidrug resistance (MDR) to its host, E. coli EC302/04. Besides, two iron acquisition systems (SitABCD and IutA-IucABCD) which are the conserved virulence determinants of ExPEC-colicin V or B and M (ColV/ColBM)-producing plasmids were identified in pEC302/04. Multiple toxin-antitoxin (TA)-based addiction systems (i.e., PemI/PemK, VagC/VagD, CcdA/CcdB, and Hok/Sok) and a plasmid partitioning system, ParAB, and PsiAB, which are important for plasmid maintenance were also found. Comparative plasmid analysis revealed only one conserved gene, the repA1 as the core genome, showing that there is an extensive diversity among the IncFIIA plasmids. The phylogenetic relationship of 18 IncF plasmids based on the core regions revealed that ColV/ColBM-plasmids and non-ColV/ColBM plasmids were separated into two distinct groups. These plasmids, which carry highly diverse genetic contents, are also mosaic in nature. The atypical combination of genetic materials, i.e., the MDR- and ColV/ColBM-plasmid-virulence encoding regions in a single ExPEC plasmid is rare but of clinical

  17. Novel archaeal plasmid pAH1 and its interactions with the lipothrixvirus AFV1

    DEFF Research Database (Denmark)

    Basta, Tamara; Smyth, John; Forterre, Patrick

    2009-01-01

    . Although nucleotide sequence comparisons revealed extensive intergenomic exchange during the evolution of archaeal conjugative plasmids, pAH1 was shown to be stably maintained suggesting that the host system is suitable for studying plasmid-virus interactions. AFV1 infection and propagation leads to a loss...... of the circular form of pAH1 and this effect correlates positively with the increase in the intracellular quantity of AFV1 DNA. We infer that the virus inhibits plasmid replication since no pAH1 degradation was observed. This mechanism of archaeal viral inhibition of plasmid propagation is not observed...... in bacteria where relevant bacteriophages either are dependent on a conjugative plasmid for successful infection or are excluded by a resident plasmid....

  18. Comparing Russian and Finnish standards of water purification

    OpenAIRE

    Maria, Pupkova

    2012-01-01

    The subject of this thesis is water purification. The first aim of this thesis is to consider different ways of water purification. The second aim is to compare Finnish and Russian standards of water purification. The third one is to show water purification methods on the pattern of Mikkeli water purification plan. Water purification methods of water intended for human consumption will be described.Combined tables will be done according to the quality requirement of drinking water of both,...

  19. Moderate temperature gas purification system: application to high calorific coal derived fuel

    Energy Technology Data Exchange (ETDEWEB)

    M. Kobayashi; H. Shirai; M. Nunokawa [Central Research Institute of Electric Power Industry (CRIEPI), Kanagawa (Japan)

    2005-07-01

    Simultaneous removal of dust, alkaline and alkaline-earth metals, halides and sulfur compounds is required to enlarge application of coal-derived gas to the high temperature fuel cells and the fuel synthesis through chemical processing. Because high calorific fuel gas, such as oxygen-blown coal gas, has high carbon monoxide content, high temperature gas purification system is always subjected to the carbon deposition and slippage of contaminant of high vapor pressure. It was suggested that moderate temperature operation of the gas purification system is applied to avoid the harmful disproportionation reaction and efficient removal of the various contaminants. To establish the moderate temperature gas purification system, the chemical-removal processes where the reaction rate is predominant to the performance of contaminant removal should be evaluated. Performance of the removal processes for halides and sulfur compounds were experimentally evaluated. The halide removal process with sodium based sorbent had potential good performance at around 300{sup o}C. The sulfur removal process was also applicable to the temperature range, although the improvement of the sulfidation reaction rate is considered to be essential. 11 refs., 8 figs., 1 tab.

  20. Strep-Tagged Protein Purification.

    Science.gov (United States)

    Maertens, Barbara; Spriestersbach, Anne; Kubicek, Jan; Schäfer, Frank

    2015-01-01

    The Strep-tag system can be used to purify recombinant proteins from any expression system. Here, protocols for lysis and affinity purification of Strep-tagged proteins from E. coli, baculovirus-infected insect cells, and transfected mammalian cells are given. Depending on the amount of Strep-tagged protein in the lysate, a protocol for batch binding and subsequent washing and eluting by gravity flow can be used. Agarose-based matrices with the coupled Strep-Tactin ligand are the resins of choice, with a binding capacity of up to 9 mg ml(-1). For purification of lower amounts of Strep-tagged proteins, the use of Strep-Tactin magnetic beads is suitable. In addition, Strep-tagged protein purification can also be automated using prepacked columns for FPLC or other liquid-handling chromatography instrumentation, but automated purification is not discussed in this protocol. The protocols described here can be regarded as an update of the Strep-Tag Protein Handbook (Qiagen, 2009). © 2015 Elsevier Inc. All rights reserved.

  1. Comparative metagenomic analysis of plasmid encoded functions in the human gut microbiome

    Directory of Open Access Journals (Sweden)

    Marchesi Julian R

    2010-01-01

    Full Text Available Abstract Background Little is known regarding the pool of mobile genetic elements associated with the human gut microbiome. In this study we employed the culture independent TRACA system to isolate novel plasmids from the human gut microbiota, and a comparative metagenomic analysis to investigate the distribution and relative abundance of functions encoded by these plasmids in the human gut microbiome. Results Novel plasmids were acquired from the human gut microbiome, and homologous nucleotide sequences with high identity (>90% to two plasmids (pTRACA10 and pTRACA22 were identified in the multiple human gut microbiomes analysed here. However, no homologous nucleotide sequences to these plasmids were identified in the murine gut or environmental metagenomes. Functions encoded by the plasmids pTRACA10 and pTRACA22 were found to be more prevalent in the human gut microbiome when compared to microbial communities from other environments. Among the most prevalent functions identified was a putative RelBE toxin-antitoxin (TA addiction module, and subsequent analysis revealed that this was most closely related to putative TA modules from gut associated bacteria belonging to the Firmicutes. A broad phylogenetic distribution of RelE toxin genes was observed in gut associated bacterial species (Firmicutes, Bacteroidetes, Actinobacteria and Proteobacteria, but no RelE homologues were identified in gut associated archaeal species. We also provide indirect evidence for the horizontal transfer of these genes between bacterial species belonging to disparate phylogenetic divisions, namely Gram negative Proteobacteria and Gram positive species from the Firmicutes division. Conclusions The application of a culture independent system to capture novel plasmids from the human gut mobile metagenome, coupled with subsequent comparative metagenomic analysis, highlighted the unexpected prevalence of plasmid encoded functions in the gut microbial ecosystem. In

  2. Partial purification and biochemical characterization of acid ...

    African Journals Online (AJOL)

    Mung bean (Vigna radiata) is one of the important crops of the North Eastern Region of India. In the present study, acid phosphatase enzyme was isolated and partially purified from germinated local mung bean seeds. The sequential partial purification process was performed using ammonium sulphate precipitation method.

  3. Plant-inducible virulence promoter of the Agrobacterium tumefaciens Ti plasmid

    NARCIS (Netherlands)

    Okker, Robert J.H.; Spaink, Herman; Hille, Jacques; Brussel, Ton A.N. van; Lugtenberg, Ben; Schilperoort, Rob A.

    1984-01-01

    Agrobacterium tumefaciens is the causative agent of crown gall, a plant tumour that can arise on most species of dicotyledonous plants. The tumour-inducing capacity of the bacterium requires the presence of a large plasmid, designated the Ti plasmid, which itself contains two regions essential for

  4. An analysis of main factors in electron beam flue gas purification

    International Nuclear Information System (INIS)

    Zhang Ming; Xu Guang

    2003-01-01

    Electron beam flue gas purification method is developing very quickly in recent years. Based on the experiment setting for electron beam flue gas purification in Institute of Nuclear Energy and Technology, Tsinghua University, how the technique factors affect the ratio of desulphurization and denitrogenation are described. Radiation dose (D), temperature (T), humidity (H), pour ammonia quantity (α) and initial concentration of SO 2 (C SO 2 ) and NO x (C NO x ) are main factors influencing flue gas purification. Using the methods of correlation analysis and regression analysis, the primary effect factors are found out and the regression equations are set to optimize the system process, predigest the system structure and to forecast the experimental results. (authors)

  5. High Prevalence of Plasmid-Mediated Quinolone Resistance and IncQ Plasmids Carrying qnrS2 Gene in Bacteria from Rivers near Hospitals and Aquaculture in China.

    Directory of Open Access Journals (Sweden)

    Yanping Wen

    Full Text Available Effluents from hospital and aquaculture are considered important sources of quinolone resistance. However, little information is available on the impact of this effluent on nearby rivers. In this study, 188 ciprofloxacin-resistant bacterial isolates obtained from rivers near hospitals and aquaculture were screened for plasmid-mediated quinolone resistance (PMQR genes. Species identification, antibiotic susceptibility testing, and PMQR gene transferability assessment were conducted for PMQR-positive bacteria. Representative qnrS2-encoding plasmids were subsequently sequenced using a primer-walking approach. In total, 44 isolates (23.4% were positive for qnr genes (16 qnrB2, 3 qnrS1, and 25 qnrS2 and 32 isolates (17.0% were positive for aac(6'-Ib-cr. Other PMQR genes were not detected. The qnrB2 and aac(6'-Ib-cr genes had a higher prevalence in aquaculture samples than in hospital samples, and were significantly associated with Enterobacteriaceae (p < 0.05. In contrast, the prevalence of qnrS2 was not site-related, but was significantly associated with Aeromonas spp. (p < 0.05. All PMQR isolates were resistant to three or more classes of antibiotics. Eleven qnrS2-harboring plasmids from Aeromonas spp., including a novel conjugative plasmid pHP18, were selected for sequencing. These plasmids were small in size (6,388-16,197 bp and belonged to the IncQ or IncU plasmid family, with qnrS2 being part of a mobile insertion cassette. Taken together, our findings suggest that aquaculture is a possible source for aac(6'-Ib-cr and qnrB2 dissemination, and demonstrate the ubiquity of qnrS2 in aquatic environments. Finally, Aeromonas spp. served as vectors for qnrS2 with the help of IncQ-type plasmids.

  6. Nanomaterials and Water Purification: Opportunities and Challenges

    Science.gov (United States)

    Savage, Nora; Diallo, Mamadou S.

    2005-10-01

    Advances in nanoscale science and engineering suggest that many of the current problems involving water quality could be resolved or greatly ameliorated using nanosorbents, nanocatalysts, bioactive nanoparticles, nanostructured catalytic membranes and nanoparticle enhanced filtration among other products and processes resulting from the development of nanotechnology. Innovations in the development of novel technologies to desalinate water are among the most exciting and promising. Additionally, nanotechnology-derived products that reduce the concentrations of toxic compounds to sub-ppb levels can assist in the attainment of water quality standards and health advisories. This article gives an overview of the use of nanomaterials in water purification. We highlight recent advances on the development of novel nanoscale materials and processes for treatment of surface water, groundwater and industrial wastewater contaminated by toxic metal ions, radionuclides, organic and inorganic solutes, bacteria and viruses. In addition, we discuss some challenges associated with the development of cost effective and environmentally acceptable functional nanomaterials for water purification.

  7. Nanomaterials and Water Purification: Opportunities and Challenges

    International Nuclear Information System (INIS)

    Savage, Nora; Diallo, Mamadou S.

    2005-01-01

    Advances in nanoscale science and engineering suggest that many of the current problems involving water quality could be resolved or greatly ameliorated using nanosorbents, nanocatalysts, bioactive nanoparticles, nanostructured catalytic membranes and nanoparticle enhanced filtration among other products and processes resulting from the development of nanotechnology. Innovations in the development of novel technologies to desalinate water are among the most exciting and promising. Additionally, nanotechnology-derived products that reduce the concentrations of toxic compounds to sub-ppb levels can assist in the attainment of water quality standards and health advisories. This article gives an overview of the use of nanomaterials in water purification. We highlight recent advances on the development of novel nanoscale materials and processes for treatment of surface water, groundwater and industrial wastewater contaminated by toxic metal ions, radionuclides, organic and inorganic solutes, bacteria and viruses. In addition, we discuss some challenges associated with the development of cost effective and environmentally acceptable functional nanomaterials for water purification

  8. Genetic characterization of blaNDM-harboring plasmids in carbapenem-resistant Escherichia coli from Myanmar.

    Directory of Open Access Journals (Sweden)

    Yo Sugawara

    Full Text Available The bacterial enzyme New Delhi metallo-β-lactamase hydrolyzes almost all β-lactam antibiotics, including carbapenems, which are drugs of last resort for severe bacterial infections. The spread of carbapenem-resistant Enterobacteriaceae that carry the New Delhi metallo-β-lactamase gene, blaNDM, poses a serious threat to public health. In this study, we genetically characterized eight carbapenem-resistant Escherichia coli isolates from a tertiary care hospital in Yangon, Myanmar. The eight isolates belonged to five multilocus-sequence types and harbored multiple antimicrobial-resistance genes, resulting in resistance against nearly all of the antimicrobial agents tested, except colistin and fosfomycin. Nine plasmids harboring blaNDM genes were identified from these isolates. Multiple blaNDM genes were found in the distinct Inc-replicon types of the following plasmids: an IncA/C2 plasmid harboring blaNDM-1 (n = 1, IncX3 plasmids harboring blaNDM-4 (n = 2 or blaNDM-7 (n = 1, IncFII plasmids harboring blaNDM-4 (n = 1 or blaNDM-5 (n = 3, and a multireplicon F plasmid harboring blaNDM-5 (n = 1. Comparative analysis highlighted the diversity of the blaNDM-harboring plasmids and their distinct characteristics, which depended on plasmid replicon types. The results indicate circulation of phylogenetically distinct strains of carbapenem-resistant E. coli with various plasmids harboring blaNDM genes in the hospital.

  9. Rapid purification of recombinant histones.

    Science.gov (United States)

    Klinker, Henrike; Haas, Caroline; Harrer, Nadine; Becker, Peter B; Mueller-Planitz, Felix

    2014-01-01

    The development of methods to assemble nucleosomes from recombinant histones decades ago has transformed chromatin research. Nevertheless, nucleosome reconstitution remains time consuming to this day, not least because the four individual histones must be purified first. Here, we present a streamlined purification protocol of recombinant histones from bacteria. We termed this method "rapid histone purification" (RHP) as it circumvents isolation of inclusion bodies and thereby cuts out the most time-consuming step of traditional purification protocols. Instead of inclusion body isolation, whole cell extracts are prepared under strongly denaturing conditions that directly solubilize inclusion bodies. By ion exchange chromatography, the histones are purified from the extracts. The protocol has been successfully applied to all four canonical Drosophila and human histones. RHP histones and histones that were purified from isolated inclusion bodies had similar purities. The different purification strategies also did not impact the quality of octamers reconstituted from these histones. We expect that the RHP protocol can be readily applied to the purification of canonical histones from other species as well as the numerous histone variants.

  10. Magnetic purification of curcumin from Curcuma longa rhizome by novel naked maghemite nanoparticles.

    Science.gov (United States)

    Magro, Massimiliano; Campos, Rene; Baratella, Davide; Ferreira, Maria Izabela; Bonaiuto, Emanuela; Corraducci, Vittorino; Uliana, Maíra Rodrigues; Lima, Giuseppina Pace Pereira; Santagata, Silvia; Sambo, Paolo; Vianello, Fabio

    2015-01-28

    Naked maghemite nanoparticles, namely, surface active maghemite nanoparticles (SAMNs), characterized by a diameter of about 10 nm, possessing peculiar colloidal stability, surface chemistry, and superparamagnetism, present fundamental requisites for the development of effective magnetic purification processes for biomolecules in complex matrices. Polyphenolic molecules presenting functionalities with different proclivities toward iron chelation were studied as probes for testing SAMN suitability for magnetic purification. Thus, the binding efficiency and reversibility on SAMNs of phenolic compounds of interest in the pharmaceutical and food industries, namely, catechin, tyrosine, hydroxytyrosine, ferulic acid, coumaric acid, rosmarinic acid, naringenin, curcumin, and cyanidin-3-glucoside, were evaluated. Curcumin emerged as an elective compound, suitable for magnetic purification by SAMNs from complex matrices. A combination of curcumin, demethoxycurcumin, and bis-demethoxycurcumin was recovered by a single magnetic purification step from extracts of Curcuma longa rhizomes, with a purity >98% and a purification yield of 45%, curcumin being >80% of the total purified curcuminoids.

  11. Effects of different replicons in conjugative plasmids on transformation efficiency, plasmid stability, gene expression and n-butanol biosynthesis in Clostridium tyrobutyricum

    Energy Technology Data Exchange (ETDEWEB)

    Yu, Mingrui; Du, Yinming; Jiang, Wenyan; Chang, Wei-Lun; Yang, Shang-Tian [Ohio State Univ., Columbus, OH (United States). William G. Lowrie Dept. of Chemical and Biomolecular Engineering; Tang, I-Ching [Bioprocessing Innovative Company, Dublin, OH (United States)

    2012-01-15

    Clostridium tyrobutyricum ATCC 25755 can produce butyric acid, acetic acid, and hydrogen as the main products from various carbon sources. In this study, C. tyrobutyricum was used as a host to produce n-butanol by expressing adhE2 gene under the control of a native thiolase promoter using four different conjugative plasmids (pMTL82151, 83151, 84151, and 85151) each with a different replicon (pBP1 from C. botulinum NCTC2916, pCB102 from C. butyricum, pCD6 from Clostridium difficile, and pIM13 from Bacillus subtilis). The effects of different replicons on transformation efficiency, plasmid stability, adhE2 expression and aldehyde/alcohol dehydrogenase activities, and butanol production by different mutants of C. tyrobutyricum were investigated. Among the four plasmids and replicons studied, pMTL82151 with pBP1 gave the highest transformation efficiency, plasmid stability, gene expression, and butanol biosynthesis. Butanol production from various substrates, including glucose, xylose, mannose, and mannitol were then investigated with the best mutant strain harboring adhE2 in pMTL82151. A high butanol titer of 20.5 g/L with 0.33 g/g yield and 0.32 g/L h productivity was obtained with mannitol as the substrate in batch fermentation with pH controlled at {proportional_to}6.0. (orig.)

  12. Insights into dynamics of mobile genetic elements in hyperthermophilic environments from five new Thermococcus plasmids.

    Directory of Open Access Journals (Sweden)

    Mart Krupovic

    Full Text Available Mobilome of hyperthermophilic archaea dwelling in deep-sea hydrothermal vents is poorly characterized. To gain insight into genetic diversity and dynamics of mobile genetic elements in these environments we have sequenced five new plasmids from different Thermococcus strains that have been isolated from geographically remote hydrothermal vents. The plasmids were ascribed to two subfamilies, pTN2-like and pEXT9a-like. Gene content and phylogenetic analyses illuminated a robust connection between pTN2-like plasmids and Pyrococcus abyssi virus 1 (PAV1, with roughly half of the viral genome being composed of genes that have homologues in plasmids. Unexpectedly, pEXT9a-like plasmids were found to be closely related to the previously sequenced plasmid pMETVU01 from Methanocaldococcus vulcanius M7. Our data suggests that the latter observation is most compatible with an unprecedented horizontal transfer of a pEXT9a-like plasmid from Thermococcales to Methanococcales. Gene content analysis revealed that thermococcal plasmids encode Hfq-like proteins and toxin-antitoxin (TA systems of two different families, VapBC and RelBE. Notably, although abundant in archaeal genomes, to our knowledge, TA and hfq-like genes have not been previously found in archaeal plasmids or viruses. Finally, the plasmids described here might prove to be useful in developing new genetic tools for hyperthermophiles.

  13. Effective removal of a range of Ti/Ri plasmids using a pBBR1-type vector having a repABC operon and a lux reporter system.

    Science.gov (United States)

    Yamamoto, Shinji; Sakai, Ayako; Agustina, Vita; Moriguchi, Kazuki; Suzuki, Katsunori

    2018-02-01

    Ti and Ri plasmids of pathogenic Agrobacterium strains are stably maintained by the function of a repABC operon and have been classified into four incompatibility groups, namely, incRh1, incRh2, incRh3, and incRh4. Removal of these plasmids from their bacterial cells is an important step in determining strain-specific virulence characteristics and to construct strains useful for transformation. Here, we developed two powerful tools to improve this process. We first established a reporter system to detect the presence and absence of Ti/Ri plasmids in cells by using an acetosyringone (AS)-inducible promoter of the Ti2 small RNA and luxAB from Vibrio harveyi. This system distinguished a Ti/Ri plasmid-free cell colony among plasmid-harboring cell colonies by causing the latter colonies to emit light in response to AS. We then constructed new "Ti/Ri eviction plasmids," each of which carries a repABC from one of four Ti/Ri plasmids that belonged to incRh1, incRh2, incRh3, and incRh4 groups in the suicidal plasmid pK18mobsacB and in a broad-host-range pBBR1 vector. Introduction of the new eviction plasmids into Agrobacterium cells harboring the corresponding Ti/Ri plasmids led to Ti/Ri plasmid-free cells in every incRh group. The Ti/Ri eviction was more effective by plasmids with the pBBR1 backbone than by those with the pK18mobsacB backbone. Furthermore, the highly stable cryptic plasmid pAtC58 in A. tumefaciens C58 was effectively evicted by the introduction of a pBBR1 vector containing the repABC of pAtC58. These results indicate that the set of pBBR1-repABC plasmids is a powerful tool for the removal of stable rhizobial plasmids.

  14. Two novel conjugative plasmids from a single strain of Sulfolobus

    NARCIS (Netherlands)

    Erauso, G.; Stedman, K.M.; Werken, van de H.J.G.; Zillig, W.; Oost, van der J.

    2006-01-01

    Two conjugative plasmids (CPs) were isolated and characterized from the same 'Sulfolobus islandicus' strain, SOG2/4, The plasmids were separated from each other and transferred into Sulfolobus soltataricus. One has a high copy number and is not stable (pSOG1) whereas the other has a low copy number

  15. Optimized Expression and Purification for High-Activity Preparations of Algal [FeFe]-Hydrogenase

    Energy Technology Data Exchange (ETDEWEB)

    Yacoby, I.; Tegler, L. T.; Pochekailov, S.; Zhang, S.; King, P. W.

    2012-04-01

    Recombinant expression and purification of metallo-enzymes, including hydrogenases, at high-yields is challenging due to complex, and enzyme specific, post-translational maturation processes. Low fidelities of maturation result in preparations containing a significant fraction of inactive, apo-protein that are not suitable for biophysical or crystallographic studies. We describe the construction, overexpression and high-yield purification of a fusion protein consisting of the algal [2Fe2S]-ferredoxin PetF (Fd) and [FeFe]-hydrogenase HydA1. The maturation of Fd-HydA1 was optimized through improvements in culture conditions and media components used for expression. We also demonstrated that fusion of Fd to the N-terminus of HydA1, in comparison to the C-terminus, led to increased expression levels that were 4-fold higher. Together, these improvements led to enhanced HydA1 activity and improved yield after purification. The strong binding-affinity of Fd for DEAE allowed for two-step purification by ion exchange and StrepTactin affinity chromatography. In addition, the incorporation of a TEV protease site in the Fd-HydA1 linker allowed for the proteolytic removal of Fd after DEAE step, and purification of HydA1 alone by StrepTactin. In combination, this process resulted in HydA1 purification yields of 5 mg L{sup -1} of culture from E. coli with specific activities of 1000 U (U = 1 {micro}mol hydrogen evolved mg{sup -1} min{sup -1}). The [FeFe]-hydrogenases are highly efficient enzymes and their catalytic sites provide model structures for synthetic efforts to develop robust hydrogen activation catalysts. In order to characterize their structure-function properties in greater detail, and to use hydrogenases for biotechnological applications, reliable methods for rapid, high-yield expression and purification are required.

  16. Mitochondrial pAL2-1 plasmid homologs are senescence factors in Podospora anserina independent of intrinsic senescence

    NARCIS (Netherlands)

    Diepeningen, van A.D.; Debets, A.J.M.; Slakhorst-Wandel, S.M.; Hoekstra, R.F.

    2008-01-01

    Since the first description of a linear mitochondrial plasmid in Podospora anserina, pAL2-1, and homologous plasmids have gone from being considered beneficial longevity plasmids, via neutral genetic elements, toward mutator plasmids causing senescence. The plasmid has an invertron structure, with

  17. Mitochondrial pAL2-1 plasmid homologs are senescence factors in Podospora anserina independent of intrinsic senescence

    NARCIS (Netherlands)

    van Diepeningen, Anne D; Debets, Alfons J M; Slakhorst, S Marijke; Hoekstra, Rolf F

    Since the first description of a linear mitochondrial plasmid in Podospora anserina, pAL2-1, and homologous plasmids have gone from being considered beneficial longevity plasmids, via neutral genetic elements, toward mutator plasmids causing senescence. The plasmid has an invertron structure, with

  18. Plasmid profiles and antibiotic susceptibility patterns of Staphylococcus aureus isolates from Nigeria.

    Science.gov (United States)

    Olukoya, D K; Asielue, J O; Olasupo, N A; Ikea, J K

    1995-06-01

    In an investigation into the problems of infections due to Staphylococcus aureus in Nigeria, 100 strains were isolated from various hospitals in Lagos. The strains were screened for the presence of plasmids and for susceptibility to antimicrobial agents. Plasmids were extracted by modification of the method of Takahashi and Nagono[1]. The plasmids were diverse in nature. The strains were found to be highly resistant to commonly prescribed antibiotics.

  19. Abundance and diversity of ammonia-oxidizing archaea and bacteria on granular activated carbon and their fates during drinking water purification process.

    Science.gov (United States)

    Niu, Jia; Kasuga, Ikuro; Kurisu, Futoshi; Furumai, Hiroaki; Shigeeda, Takaaki; Takahashi, Kazuhiko

    2016-01-01

    Ammonia is a precursor to trichloramine, which causes an undesirable chlorinous odor. Granular activated carbon (GAC) filtration is used to biologically oxidize ammonia during drinking water purification; however, little information is available regarding the abundance and diversity of ammonia-oxidizing archaea (AOA) and bacteria (AOB) associated with GAC. In addition, their sources and fates in water purification process remain unknown. In this study, six GAC samples were collected from five full-scale drinking water purification plants in Tokyo during summer and winter, and the abundance and community structure of AOA and AOB associated with GAC were studied in these two seasons. In summer, archaeal and bacterial amoA genes on GACs were present at 3.7 × 10(5)-3.9 × 10(8) gene copies/g-dry and 4.5 × 10(6)-4.2 × 10(8) gene copies/g-dry, respectively. In winter, archaeal amoA genes remained at the same level, while bacterial amoA genes decreased significantly for all GACs. No differences were observed in the community diversity of AOA and AOB from summer to winter. Phylogenetic analysis revealed high AOA diversity in group I.1a and group I.1b in raw water. Terminal-restriction fragment length polymorphism analysis of processed water samples revealed that AOA diversity decreased dramatically to only two OTUs in group I.1a after ozonation, which were identical to those detected on GAC. It suggests that ozonation plays an important role in determining AOA diversity on GAC. Further study on the cell-specific activity of AOA and AOB is necessary to understand their contributions to in situ nitrification performance.

  20. Computational design and characterization of a temperature-sensitive plasmid replicon for gram positive thermophiles

    Directory of Open Access Journals (Sweden)

    Olson Daniel G

    2012-05-01

    Full Text Available Abstract Background Temperature-sensitive (Ts plasmids are useful tools for genetic engineering, but there are currently none compatible with the gram positive, thermophilic, obligate anaerobe, Clostridium thermocellum. Traditional mutagenesis techniques yield Ts mutants at a low frequency, and therefore requires the development of high-throughput screening protocols, which are also not available for this organism. Recently there has been progress in the development of computer algorithms which can predict Ts mutations. Most plasmids currently used for genetic modification of C. thermocellum are based on the replicon of plasmid pNW33N, which replicates using the RepB replication protein. To address this problem, we set out to create a Ts plasmid by mutating the gene coding for the RepB replication protein using an algorithm designed by Varadarajan et al. (1996 for predicting Ts mutants based on the amino-acid sequence of the protein. Results A library of 34 mutant plasmids was designed, synthesized and screened, resulting in 6 mutants which exhibited a Ts phenotype. Of these 6, the one with the most temperature-sensitive phenotype (M166A was compared with the original plasmid. It exhibited lower stability at 48°C and was completely unable to replicate at 55°C. Conclusions The plasmid described in this work could be useful in future efforts to genetically engineer C. thermocellum, and the method used to generate this plasmid may be useful for others trying to make Ts plasmids.

  1. Using the CRISPR/Cas9 system to eliminate native plasmids of Zymomonas mobilis ZM4.

    Science.gov (United States)

    Cao, Qing-Hua; Shao, Huan-Huan; Qiu, Hui; Li, Tao; Zhang, Yi-Zheng; Tan, Xue-Mei

    2017-03-01

    The CRISPR/Cas system can be used to simply and efficiently edit the genomes of various species, including animals, plants, and microbes. Zymomonas mobilis ZM4 is a highly efficient, ethanol-producing bacterium that contains five native plasmids. Here, we constructed the pSUZM2a-Cas9 plasmid and a single-guide RNA expression plasmid. The pSUZM2a-Cas9 plasmid was used to express the Cas9 gene cloned from Streptococcus pyogenes CICC 10464. The single-guide RNA expression plasmid pUC-T7sgRNA, with a T7 promoter, can be used for the in vitro synthesis of single-guide RNAs. This system was successfully employed to knockout the upp gene of Escherichia coli and the replicase genes of native Z. mobilis plasmids. This is the first study to apply the CRISPR/Cas9 system of S. pyogenes to eliminate native plasmids in Z. mobilis. It provides a new method for plasmid curing and paves the way for the genomic engineering of Z. mobilis.

  2. Transfer of conjugative plasmids among bacteria under environmentally relevant conditions

    DEFF Research Database (Denmark)

    Musovic, Sanin

    Mobile genetiske elementer (f.eks. plasmider), der ofte bærer ekstra funktioner såsom antibiotikaresistens, eller kataboliske- og xenobiotiske nedbrydnings gener, antages at have en meget vigtigt evolutionær rolle for bakterier. I denne PhD afhandling undersøgte jeg størrelsen af plasmid overførs...

  3. Characterization of Endogenous Plasmids from Lactobacillus salivarius UCC118▿ †

    OpenAIRE

    Fang, Fang; Flynn, Sarah; Li, Yin; Claesson, Marcus J.; van Pijkeren, Jan-Peter; Collins, J. Kevin; van Sinderen, Douwe; O'Toole, Paul W.

    2008-01-01

    The genome of Lactobacillus salivarius UCC118 comprises a 1.83-Mb chromosome, a 242-kb megaplasmid (pMP118), and two smaller plasmids of 20 kb (pSF118-20) and 44 kb (pSF118-44). Annotation and bioinformatic analyses suggest that both of the smaller plasmids replicate by a theta replication mechanism. Furthermore, it appears that they are transmissible, although neither possesses a complete set of conjugation genes. Plasmid pSF118-20 encodes a toxin-antitoxin system composed of pemI and pemK h...

  4. Purification

    DEFF Research Database (Denmark)

    Andersen, Astrid Oberborbeck

    2017-01-01

    In Arequipa, Peru’s second largest city, engineers work hard to control water flows and provide different sectors with clean and sufficient water. In 2011, only 10 percent of the totality of water used daily by Arequipa’s then close to 1 million people—in households, tourism, industry, and mining......—was treated before it was returned to the river where it continues its flow downstream towards cultivated fields and, finally, into the Pacific Ocean. It takes specialized knowledge and manifold technologies to manage water and sustain life in Arequipa, and engineers are central actors for making water flow...... of categories can be understood as practices of purification. However, a purely technical grip on water is never possible. Unruly elements, like weather, contamination, urban dwellers, and competing interests, interfere and make processes of intervention unstable. Water is never completely cleaned, and, equally...

  5. Isolation and properties of plasmids from Deinococcus radiodurans Sark

    International Nuclear Information System (INIS)

    Sjarief, S.H.; Kikuchi, Masahiro; Kurita, Hiromi; Kitayama, Shigeru; Watanabe, Hiroshi.

    1990-05-01

    Radioresistant bacterium, Deinococcus radiodurans, can repair completely almost all of DNA damages including double strand breaks induced by gamma-rays up to about 5 kGy. In order to reveal the repair mechanism, it is necessary to develop a cloning vector available for the genetic analysis. We tried to isolate plasmids from D.radiodurans Sark strain. In the present paper the isolation and properties of plasmids were described. (author)

  6. Soft-Bake Purification of SWCNTs Produced by Pulsed Laser Vaporization

    Science.gov (United States)

    Yowell, Leonard; Nikolaev, Pavel; Gorelik, Olga; Allada, Rama Kumar; Sosa, Edward; Arepalli, Sivaram

    2013-01-01

    The "soft-bake" method is a simple and reliable initial purification step first proposed by researchers at Rice University for single-walled carbon nanotubes (SWCNT) produced by high-pressure carbon mon oxide disproportionation (HiPco). Soft-baking consists of annealing as-produced (raw) SWCNT, at low temperatures in humid air, in order to degrade the heavy graphitic shells that surround metal particle impurities. Once these shells are cracked open by the expansion and slow oxidation of the metal particles, the metal impurities can be digested through treatment with hydrochloric acid. The soft-baking of SWCNT produced by pulsed-laser vaporization (PLV) is not straightforward, because the larger average SWCNT diameters (.1.4 nm) and heavier graphitic shells surrounding metal particles call for increased temperatures during soft-bake. A part of the technology development focused on optimizing the temperature so that effective cracking of the graphitic shells is balanced with maintaining a reasonable yield, which was a critical aspect of this study. Once the ideal temperature was determined, a number of samples of raw SWCNT were purified using the soft-bake method. An important benefit to this process is the reduced time and effort required for soft-bake versus the standard purification route for SWCNT. The total time spent purifying samples by soft-bake is one week per batch, which equates to a factor of three reduction in the time required for purification as compared to the standard acid purification method. Reduction of the number of steps also appears to be an important factor in improving reproducibility of yield and purity of SWCNT, as small deviations are likely to get amplified over the course of a complicated multi-step purification process.

  7. An improved method for purification of recombinant truncated heme oxygenase-1 by expanded bed adsorption and gel filtration.

    Science.gov (United States)

    Hu, Hong-Bo; Wang, Wei; Han, Ling; Zhou, Wen-Pu; Zhang, Xue-Hong

    2007-03-01

    Recombinant truncated human heme oxygenase-1 (hHO-1) expressed in Escherichia coli was efficiently separated and purified from feedstock by DEAE-ion exchange expanded bed adsorption. Protocol optimization of hHO-1 on DEAE adsorbent resulted in adsorption in 0 M NaCl and elution in 150 mM NaCl at a pH of 8.5. The active enzyme fractions separated from the expanded bed column were further purified by a Superdex 75 gel filtration step. The specific hHO-1 activity increased from 0.82 +/- 0.05 to 24.8 +/- 1.8 U/mg during the whole purification steps. The recovery and purification factor of truncated hHO-1 of the whole purification were 72.7 +/- 4.7 and 30.2 +/- 2.3%, respectively. This purification process can decrease the demand on the preparation of feedstock and simplify the purification process.

  8. Item Purification Does Not Always Improve DIF Detection: A Counterexample with Angoff's Delta Plot

    Science.gov (United States)

    Magis, David; Facon, Bruno

    2013-01-01

    Item purification is an iterative process that is often advocated as improving the identification of items affected by differential item functioning (DIF). With test-score-based DIF detection methods, item purification iteratively removes the items currently flagged as DIF from the test scores to get purified sets of items, unaffected by DIF. The…

  9. Validation of 68Ge/68Ga generator processing by chemical purification for routine clinical application of 68Ga-DOTATOC

    International Nuclear Information System (INIS)

    Asti, Mattia; De Pietri, Giovanni; Fraternali, Alessandro; Grassi, Elisa; Sghedoni, Roberto; Fioroni, Federica; Roesch, Frank; Versari, Annibale; Salvo, Diana

    2008-01-01

    Introduction: Imaging of somatostatin receptor expressing tumours has been greatly enhanced by the use of 68 Ga-DOTATOC and PET/CT. Methods: In this work, a purification method for the 68 Ge/ 68 Ga generator eluate and a method to produce 68 Ga-DOTATOC suitable for clinical use were evaluated. The generator eluate was purified and concentrated on a cation-exchange cartridge in HCl/acetone media. The efficacy of this procedure in eliminating metal impurities from the 68 Ga solution was investigated by ICP-MS. The radiotracer quality was evaluated by radio-TLC, GC and γ-ray spectrometry. Results: 68 Ga-DOTATOC preparations (n=33) were carried out with a mean synthesis yield of 59.3±2.8% (not corrected for decay) and a batch activity ranging from 555 to 296 MBq. The radiochemical and radionuclidic purity were >98% and 99.9999%, respectively. With this purification process, >95% of the Fe(III), Zn(II) and Mn(II) were eliminated from the solution. Conclusions: 68 Ga-DOTATOC produced with this method can be efficiently used in nuclear medicine departments for PET evaluations

  10. Processing and removal of the Three Mile Island makeup and purification system resins

    International Nuclear Information System (INIS)

    Reilly, J.K.; McIntosh, T.W.; Northey, L.M.; GaTanto, J.J.; Osterhoudt, T.R.; Thompson, J.D.

    1985-01-01

    The accident at Three Mile Island Unit 2 in March 1979 left the makeup and purification demineralizers inaccessible to workers. These demineralizers contained the highest concentrations of radioactive isotopes outside the Reactor Building. Additionally, unknown quantities of fuel were in the demineralizers. Other concerns centered on the unknown condition of the demineralizer resins, the possible loss of system integrity, and the presence of hydrogen due to the radiolytic breakdown of water contained in the demineralizers. GPU Nuclear, with the US Department of Energy and its contractors EG and G Idaho, Inc., Oak Ridge National Laboratory, and Westinghouse Hanford Company, remotely measured the fuel and fission product inventory of the demineralizers and demonstrated a practical chemical processing method using samples acquired from the demineralizers. Equipment was developed and installed to elute the radioactive cesium from the demineralizers. And elution, which began in September 1984 now nears completion

  11. Process development, design and operation of off-line purification system for oil-contaminated impure heavy water

    International Nuclear Information System (INIS)

    Bose, H.; Rakesh Kumar; Gandhi, H.C.; Unny, V.K.P.; Ghosh, S.K.; Mishra, Vivek; Shukla, D.K.; Duraisamy, S.; Agarwal, S.K.

    2004-01-01

    A large volume of degraded, tritiated heavy water contaminated with mineral oil and ionic impurities have accumulated at Dhruva in the past years of reactor operation as a result of routine operation and maintenance activities. The need was felt for a simple and efficient process that could be set up and operated locally at site using readily available materials, to purify the accumulated impure heavy waters at Dhruva so as to make them acceptable at the up gradation facilities. After a detailed laboratory study, a three stage clean-up process was developed which could purify a highly turbid oil-water emulsion to yield clear, oil-free and de-mineralized heavy water at reasonable rates of volume through-put. Based on the laboratory data, a suitably scaled up purification unit has been designed and commissioned which in the past few months has processed a sizeable volume of oil-contaminated heavy water waste from Dhruva, with most satisfactory results

  12. Analysis of plasmid profiling as a method for rapid differentiation of food-associated Clostridium perfringens strains.

    Science.gov (United States)

    Jones, M K; Iwanejko, L A; Longden, M S

    1989-09-01

    Plasmid analysis of over 120 strains of Clostridium perfringens, isolated during food-poisoning incidents and from animal carcasses and food constituents with no association with food poisoning, showed the potential of plasmid profiling as a means of differentiating epidemiologically related strains. On average 65% of freshly isolated strains contained one or more plasmids which could be used in the analysis. Comparison of profiles of strains from unrelated sources or unrelated strains from the same source showed a particularly wide variety of plasmid profiles. Thus the possibility that epidemiologically-unrelated strains might possess similar profiles appears to be very low in this organism. Analysis of serologically-related strains from the same source revealed similar plasmid profiles in all the plasmid-bearing strains examined. A high proportion (71%) of fresh and well-characterized food-poisoning strains possessed plasmids of 6.2 kb in size (compared with 19% of non-food-poisoning strains). The possible role of these plasmids is discussed, since the structural gene encoding the enterotoxin type A was not present on any of the plasmids in the food-poisoning strains tested.

  13. A comprehensive review on biodiesel purification and upgrading

    Directory of Open Access Journals (Sweden)

    Hamed Bateni

    2017-09-01

    Full Text Available Serious environmental concerns regarding the use of fossil-based fuels have raised awareness regarding the necessity of alternative clean fuels and energy carriers. Biodiesel is considered a clean, biodegradable, and non-toxic diesel substitute produced via the transesterification of triglycerides with an alcohol in the presence of a proper catalyst. After initial separation of the by-product (glycerol, the crude biodiesel needs to be purified to meet the standard specifications prior to marketing. The presence of impurities in the biodiesel not only significantly affects its engine performance but also complicates its handling and storage. Therefore, biodiesel purification is an essential step prior to marketing. Biodiesel purification methods can be classified based on the nature of the process into equilibrium-based, affinity-based, membrane-based, reaction-based, and solid-liquid separation processes. The main adverse properties of biodiesel – namely moisture absorption, corrosiveness, and high viscosity – primarily arise from the presence of oxygen. To address these issues, several upgrading techniques have been proposed, among which catalytic (hydrodeoxygenation using conventional hydrotreating catalysts, supported metallic materials, and most recently transition metals in various forms appear promising. Nevertheless, catalyst deactivation (via coking and/or inadequacy of product yields necessitate further research. This paper provides a comprehensive overview on the techniques and methods used for biodiesel purification and upgrading.

  14. Presence of Glycopeptide-Encoding Plasmids in Enterococcal Isolates from Food and Humans in Denmark

    DEFF Research Database (Denmark)

    Migura, Lourdes Garcia; Valenzuela, Antonio Jesus Sanchez; Jensen, Lars Bogø

    2011-01-01

    developed techniques for classification of plasmids. Replicons associated with sex pheromone-inducible plasmids were detected in all GR E. faecalis, whereas GR Enterococcus faecium contained plasmids known to be widely distributed among enterococci. vanA resistance is common in E. faecium isolates from meat...... and animals in Europe and is rarely found in E. faecalis. This article describes the first characterization of MGE from vanA mediated E. faecalis, thus linking this resistance genotype to pheromone responding plasmids....

  15. Infectious alphavirus production from a simple plasmid transfection+

    Directory of Open Access Journals (Sweden)

    Olson Ken E

    2011-07-01

    Full Text Available Abstract We have developed a new method for producing infectious double subgenomic alphaviruses from plasmids transfected into mammalian cells. A double subgenomic Sindbis virus (TE3'2J was transcribed from a cytomegalovirus PolII promoter, which results in the production of infectious virus. Transfection of as little as 125 ng of plasmid is able to produce 1 × 108 plaque forming units/ml (PFU/ml of infectious virus 48 hours post-transfection. This system represents a more efficient method for producing recombinant Sindbis viruses.

  16. Novel peptide ligand with high binding capacity for antibody purification

    DEFF Research Database (Denmark)

    Lund, L. N.; Gustavsson, P. E.; Michael, R.

    2012-01-01

    Small synthetic ligands for protein purification have become increasingly interesting with the growing need for cheap chromatographic materials for protein purification and especially for the purification of monoclonal antibodies (mAbs). Today, Protein A-based chromatographic resins are the most...... commonly used capture step in mAb down stream processing; however, the use of Protein A chromatography is less attractive due to toxic ligand leakage as well as high cost. Whether used as an alternative to the Protein A chromatographic media or as a subsequent polishing step, small synthetic peptide...... ligands have an advantage over biological ligands; they are cheaper to produce, ligand leakage by enzymatic degradation is either eliminated or significantly reduced, and they can in general better withstand cleaning in place (CIP) conditions such as 0.1 M NaOH. Here, we present a novel synthetic peptide...

  17. Contaminated nickel scrap processing

    International Nuclear Information System (INIS)

    Compere, A.L.; Griffith, W.L.; Hayden, H.W.; Johnson, J.S. Jr.; Wilson, D.F.

    1994-12-01

    The DOE will soon choose between treating contaminated nickel scrap as a legacy waste and developing high-volume nickel decontamination processes. In addition to reducing the volume of legacy wastes, a decontamination process could make 200,000 tons of this strategic metal available for domestic use. Contaminants in DOE nickel scrap include 234 Th, 234 Pa, 137 Cs, 239 Pu (trace), 60 Co, U, 99 Tc, and 237 Np (trace). This report reviews several industrial-scale processes -- electrorefining, electrowinning, vapormetallurgy, and leaching -- used for the purification of nickel. Conventional nickel electrolysis processes are particularly attractive because they use side-stream purification of process solutions to improve the purity of nickel metal. Additionally, nickel purification by electrolysis is effective in a variety of electrolyte systems, including sulfate, chloride, and nitrate. Conventional electrorefining processes typically use a mixed electrolyte which includes sulfate, chloride, and borate. The use of an electrorefining or electrowinning system for scrap nickel recovery could be combined effectively with a variety of processes, including cementation, solvent extraction, ion exchange, complex-formation, and surface sorption, developed for uranium and transuranic purification. Selected processes were reviewed and evaluated for use in nickel side-stream purification. 80 refs

  18. A novel multimodal chromatography based single step purification process for efficient manufacturing of an E. coli based biotherapeutic protein product.

    Science.gov (United States)

    Bhambure, Rahul; Gupta, Darpan; Rathore, Anurag S

    2013-11-01

    Methionine oxidized, reduced and fMet forms of a native recombinant protein product are often the critical product variants which are associated with proteins expressed as bacterial inclusion bodies in E. coli. Such product variants differ from native protein in their structural and functional aspects, and may lead to loss of biological activity and immunogenic response in patients. This investigation focuses on evaluation of multimodal chromatography for selective removal of these product variants using recombinant human granulocyte colony stimulating factor (GCSF) as the model protein. Unique selectivity in separation of closely related product variants was obtained using combined pH and salt based elution gradients in hydrophobic charge induction chromatography. Simultaneous removal of process related impurities was also achieved in flow-through leading to single step purification process for the GCSF. Results indicate that the product recovery of up to 90.0% can be obtained with purity levels of greater than 99.0%. Binding the target protein at pHproduct variants using the combined pH and salt based elution gradient and removal of the host cell impurities in flow-through are the key novel features of the developed multimodal chromatographic purification step. Copyright © 2013 Elsevier B.V. All rights reserved.

  19. Conjugal plasmid transfer (pAM beta 1) in Lactobacillus plantarum.

    OpenAIRE

    Shrago, A W; Chassy, B M; Dobrogosz, W J

    1986-01-01

    The streptococcal plasmid pAM beta 1 (erythromycin resistance) was transferred via conjugation from Streptococcus faecalis to Lactobacillus plantarum and was transferred among L. plantarum strains. Streptococcus sanguis Challis was transformed with pAM beta 1 isolated from these transconjugants, and transformants harboring intact pAM beta 1 could conjugate the plasmid back to L. plantarum.

  20. Plasmid profiling of bacterial isolates from confined environments

    Science.gov (United States)

    van Houdt, Rob; Provoost, Ann; Coninx, Ilse; Leys, Natalie; Mergeay, Max

    Plasmid profiling of bacterial isolates from confined environments R. Van Houdt, I. Coninx, A. Provoost, N. Leys, and M. Mergeay Expertise group for Molecular and Cellular Biology, Institute for Environment, Health and Safety, Belgian Nuclear Research Centre (SCK•CEN), Boeretang 200, B-2400 Mol, Belgium. Human exploration of extreme and isolated hostile environments such as space requires special confined small volume habitats to protect and house the crew. However, human confinement in such small volume habitats has restrictions on waste disposal and personal hygiene and inevitably generates a particular community of microorganisms within the habitat. These microorganisms are mainly originating from the crew (skin, mucous membranes, upper respiratory tract, mouth, and gastrointestinal tract) but also include the residing environmental microorganisms. Earth-based confined habitats such as the Antarctic Research Station Concordia are used as test beds for long-duration spaceflights to study the physiologic and psychological adaptation to isolated environments. The dynamics of the environmental microbial population in such a test bed could render additional insights in assessing the potential health risks in long-duration space missions. Not only total bacterial contamination levels are important, but it is essential to identify also the predominant microbial taxa and their mobile genetic elements (MGE). These MGEs could be exchanged between bacteria by horizontal gene transfer and may alter the pathogenic potential since they often carry antibiotic resistance or more in general adaptation-enhancing traits. In this study several bacterial strains isolated in the Concordia research station were examined for their plasmid content. An optimized protocol for extraction of large plasmids showed the present of at least one plasmid in 50% of the strains. For all strains the minimal inhibitory concentration of a range of antibiotics was determined indicating resistance to

  1. Survival and evolution of a large multidrug resistance plasmid in new clinical bacterial hosts

    DEFF Research Database (Denmark)

    Porse, Andreas; Schønning, Kristian; Munck, Christian

    2016-01-01

    sequencing to show that the long-term persistence and molecular integrity of the plasmid is highly influenced by multiple factors within a 25 kb plasmid region constituting a host-dependent burden. In the E. coli hosts investigated here, improved plasmid stability readily evolves via IS26 mediated deletions...... consistently followed by all evolved E. coli lineages exposes a trade-off between horizontal and vertical transmission that may ultimately limit the dissemination potential of clinical multidrug resistance plasmids in these hosts....

  2. Repair of UV-irradiated plasmid DNA in excision repair deficient mutants of Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    Ikai, K.; Tano, K.; Ohnishi, T.; Nozu, K.

    1985-01-01

    The repair of UV-irradiated DNA of plasmid YEp13 was studied in the incision defective strains by measurement of cell transformation frequency. In Saccharomyces cerevisiae, rad1,2,3 and 4 mutants could repair UV-damaged plasmid DNA. In Escherichia coli, uvrA mutant was unable to repair UV-damaged plasmid DNA; however, pretreatment of the plasmid with Micrococcus luteus endonuclease increased repair. It was concluded that all the mutations of yeast were probably limited only to the nuclear DNA. (author)

  3. Plasmid DNA Analysis of Pasteurella multocida Serotype B isolated from Haemorrhagic Septicaemia outbreaks in Malaysia

    Directory of Open Access Journals (Sweden)

    Jamal, H.

    2005-01-01

    Full Text Available A total of 150 purified isolates of Pasteurella multocida serotype B were used (Salmah, 2004 for plasmid DNA curing experiment to determine hyaluronidase activity, antibiotic resistance pattern (ARP and mice lethality test (LD50 for their role of pathogenicity. A plasmid curing experiment was carried out by using the intercalating agent; ethidium bromide and rifampicin, where it was found all the plasmids had been cured (plasmidless from Pasteurella multocida. All of these plasmidless isolates maintained their phenotypic characteristics. They showed the same antibiotic resistancepattern as before curing, produced hyaluronidase and possessed lethality activity in mice when injected intraperitoneally(i.p. Based on this observation, the antibiotic resistance, hyaluronidase activity and mice virulence could probably be chromosomal-mediated. Plasmids were detected 100% in all P. multocida isolates with identical profile of 2 plasmids size 3.0 and 5.5 kb. No large plasmids could be detected in all isolates. Since all the isolates appeared to have identicalplasmid profiles, they were subjected to restriction enzyme(RE analysis. From RE analysis results obtained, it can be concluded that the plasmid DNA in serotype B isolates are identical. Only 4 of 32 REs were found to cleave these plasmids with identical restriction fingerprints; BglII, HaeIII, RsaI and SspI. From RE analysis results, it can be concluded that the plasmid DNA isolates are identical. This plasmid might not played any role in pathogenicity of Pasteurella multocida serotype B, however this information is important for the construction of shuttle vectors in genetic studies of the pathogenicity of haemorrhagic septicaemia(HS.

  4. Exergy costs analysis of water desalination and purification techniques by transfer functions

    International Nuclear Information System (INIS)

    Carrasquer, Beatriz; Martínez-Gracia, Amaya; Uche, Javier

    2016-01-01

    Highlights: • A procedure to estimate the unit exergy cost of water treatment techniques is provided. • Unit exergy costs of water purification and desalination are given as a function of design and operating parameters. • Unit exergy costs range from 3.3 to 6.8 in purification and from 2 to 26 in desalination. • They could be used in their preliminary design as good indicators of their energy efficiency. - Abstract: The unit exergy costs of desalination and purification, which are two alternatives commonly used for water supply and treatment, have been characterized as a function of the energy efficiency of the process by combining the Exergy Cost Analysis with Transfer Function Analysis. An equation to assess the exergy costs of these alternatives is then proposed as a quick guide to know the energy efficiency of any water treatment process under different design and operating conditions. This combination, was satisfactory applied to groundwaters and water transfers. After identifying the boundaries of the system, input and output flows are calculated in exergy values. Next, different examples are analyzed in order to propose a generic equation to assess the exergy cost of the water restoration technologies, attending to their main features. Recovery ratio, energy requirements and salts concentrations (for desalination), and plant capacity and organic matter recovery (for water purification) are introduced in the calculations as their main endogenous parameters. Values obtained for typical operation ranges of commercial plants showed that unit exergy costs of water purification ranged from 3.3 to 6.8; maximum values, as expected, were found at low plant capacities and high organic matter removal ratios. For water desalination, values varied from 2 to 7 in membrane technologies and from 10 to 26 in thermal processes. The recovery ratio and salts concentration in raw water increased the unit exergy costs in membrane techniques. In distillation processes

  5. Plasmid ColE1 as a Molecular Vehicle for Cloning and Amplification of DNA

    Science.gov (United States)

    Hershfield, Vickers; Boyer, Herbert W.; Yanofsky, Charles; Lovett, Michael A.; Helinski, Donald R.

    1974-01-01

    DNA fragments obtained from EcoRI endonuclease digestion of bacteriophage ϕ80pt190 (trp+) and the plasmid ColE1 were covalently joined with polynucleotide ligase. Transformation of Escherichia coli trp- strains to tryptophan independence with the recombined DNA selected for reconstituted ColE1 plasmids containing the tryptophan operon and the ϕ80 immunity region. Similarly, an EcoRI endonuclease generated fragment of plasmid pSC105 DNA containing the genetic determinant of kanamycin resistance was inserted into the ColE1 plasmid and recovered in E. coli. The plasmids containing the trp operon (ColE1-trp) and the kanamycin resistance gene were maintained under logarithmic growth conditions at a level of 25-30 copies per cell and accumulate to the extent of several hundred copies per cell in the presence of chloramphenicol. Cells carrying the ColE1-trp plasmid determined the production of highly elevated levels of trp operon-specific mRNA and tryptophan biosynthetic enzymes. Images PMID:4610576

  6. Centromere pairing by a plasmid-encoded type I ParB protein

    DEFF Research Database (Denmark)

    Ringgaard, Simon; Löwe, Jan; Gerdes, Kenn

    2007-01-01

    The par2 locus of Escherichia coli plasmid pB171 encodes two trans-acting proteins, ParA and ParB, and two cis-acting sites, parC1 and parC2, to which ParB binds cooperatively. ParA is related to MinD and oscillates in helical structures and thereby positions ParB/parC-carrying plasmids regularly......, hence identifying the N terminus of ParB as a requirement for ParB-mediated centromere pairing. These observations suggest that centromere pairing is an important intermediate step in plasmid partitioning mediated by the common type I loci....

  7. An Enterobacter plasmid as a new genetic background for the transposon Tn1331

    Directory of Open Access Journals (Sweden)

    Alavi MR

    2011-11-01

    Full Text Available Mohammad R Alavi1,2, Vlado Antonic2, Adrien Ravizee1, Peter J Weina3, Mina Izadjoo1,2, Alexander Stojadinovic21Division of Wound Biology and Translational Research, Armed Forces Institute of Pathology and American Registry of Pathology, Washington DC, 2Combat Wound Initiative Program, Walter Reed Army Medical Center, Washington DC, 3The Walter Reed Army Institute of Research, Silver Spring, MD, USABackground: Genus Enterobacter includes important opportunistic nosocomial pathogens that could infect complex wounds. The presence of antibiotic resistance genes in these microorganisms represents a challenging clinical problem in the treatment of these wounds. In the authors’ screening of antibiotic-resistant bacteria from complex wounds, an Enterobacter species was isolated that harbors antibiotic-resistant plasmids conferring resistance to Escherichia coli. The aim of this study was to identify the resistance genes carried by one of these plasmids.Methods: The plasmids from the Enterobacter isolate were propagated in E. coli and one of the plasmids, designated as pR23, was sequenced by the Sanger method using fluorescent dye-terminator chemistry on a genetic analyzer. The assembled sequence was annotated by search of the GenBank database.Results: Plasmid pR23 is composed of the transposon Tn1331 and a backbone plasmid that is identical to the plasmid pPIGDM1 from Enterobacter agglomerans. The multidrug-resistance transposon Tn1331, which confers resistance to aminoglycoside and beta lactam antibiotics, has been previously isolated only from Klebsiella. The Enterobacter plasmid pPIGDM1, which carries a ColE1-like origin of replication and has no apparent selective marker, appears to provide a backbone for propagation of Tn1331 in Enterobacter. The recognition sequence of Tn1331 transposase for insertion into pPIGDM1 is the pentanucleotide TATTA, which occurs only once throughout the length of this plasmid.Conclusion: Transposition of Tn1331 into

  8. Quorum-Dependent Mannopine-Inducible Conjugative Transfer of an Agrobacterium Opine-Catabolic Plasmid

    Science.gov (United States)

    Wetzel, Margaret E.; Kim, Kun-Soo; Miller, Marilyn; Olsen, Gary J.

    2014-01-01

    The Ti plasmid in Agrobacterium tumefaciens strain 15955 carries two alleles of traR that regulate conjugative transfer. The first is a functional allele, called traR, that is transcriptionally induced by the opine octopine. The second, trlR, is a nonfunctional, dominant-negative mutant located in an operon that is inducible by the opine mannopine (MOP). Based on these findings, we predicted that there exist wild-type agrobacterial strains harboring plasmids in which MOP induces a functional traR and, hence, conjugation. We analyzed 11 MOP-utilizing field isolates and found five where MOP induced transfer of the MOP-catabolic element and increased production of the acyl-homoserine lactone (acyl-HSL) quormone. The transmissible elements in these five strains represent a set of highly related plasmids. Sequence analysis of one such plasmid, pAoF64/95, revealed that the 176-kb element is not a Ti plasmid but carries genes for catabolism of MOP, mannopinic acid (MOA), agropinic acid (AGA), and the agrocinopines. The plasmid additionally carries all of the genes required for conjugative transfer, including the regulatory genes traR, traI, and traM. The traR gene, however, is not located in the MOP catabolism region. The gene, instead, is monocistronic and located within the tra-trb-rep gene cluster. A traR mutant failed to transfer the plasmid and produced little to no quormone even when grown with MOP, indicating that TraRpAoF64/95 is the activator of the tra regulon. A traM mutant was constitutive for transfer and acyl-HSL production, indicating that the anti-activator function of TraM is conserved. PMID:24363349

  9. Plasmids which make their host bacteria mutable as well as resistant to ultraviolet irradiation

    International Nuclear Information System (INIS)

    Arai, Toshihiko; Ando, Takao

    1980-01-01

    Some of the naturally occurring Iα, I zeta, M, N, O and T group plasmids increase both the mutability and UV resistance of their host bacteria, while group H and S plasmids only increase mutability. This suggests that these two plasmid-mediated repair functions are separable. The two functions have no direct relation to their restriction-modification systems and nitrofuran resistant functions. In addition, the close linking between the restriction-modification genes and these repair function genes was suggested in group N plasmids. (author)

  10. Partial Purification and Characterization of Extracellular Protease ...

    African Journals Online (AJOL)

    Nigerian Journal of Basic and Applied Sciences ... Purification of the enzyme by gel filtration chromatography on Sephadex G75 following ammonium sulphate precipitation gave 2.26 fold increase in purification with specific activity of 46.13 units/mg protein while purification on Sephadex CM50 resulted in reduced ...

  11. Antibiotic resistance and plasmid carriage among Escherichia coli isolates from chicken meat in Malaysia

    International Nuclear Information System (INIS)

    Tin Tin Myaing; Saleha, A.A.; Arifah, A.K.; Raha, A.R.

    2005-01-01

    Escherichia coli isolates from 131 raw chicken meat samples were tested for susceptibility to 12 antibiotics. Plasmids were isolated from many samples and their DNA molecular weight calculated. An 81.7% plasmid occurrence rate was observed among the isolates, ranging from 0 to 8 in number and with sizes from 1.2 to 118.6 MDa. Plasmids were detected in 93.8% of E. coIi isolates resistant to all 12 antibiotics, and in 90.5% of E. coli isolates resistant to 11. Three (2.8%) isolates harboured 8 plasmids and were resistant to all 12 antibiotics. Antibiotic resistant genes in bacteria are usually carried in extrachromosomal DNA and it is postulated that E. coli with a high number of plasmids possesses wider resistance to antibiotics. (author)

  12. Expansion of a plasmid classification system for Gram-positive bacteria and determination of the diversity of plasmids in Staphylococcus aureus strains of human, animal, and food origins

    DEFF Research Database (Denmark)

    Lozano, C.; Garcia-Migura, L.; Aspiroz, C.

    2012-01-01

    An expansion of a previously described plasmid classification was performed and used to reveal the plasmid content of a collection of 92 Staphylococcus aureus strains of different origins. rep genes of other genera were detected in Staphylococcus. S1 pulsed-field gel electrophoresis (PFGE) hybrid...

  13. Specific structural probing of plasmid-coded ribosomal RNAs from Escherichia coli

    DEFF Research Database (Denmark)

    Aagaard, C; Rosendahl, G; Dam, M

    1991-01-01

    The preferred method for construction and in vivo expression of mutagenised Escherichia coli ribosomal RNAs (rRNAs) is via high copy number plasmids. Transcription of wild-type rRNA from the seven chromosomal rrn operons in strains harbouring plasmid-coded mutant rRNAs leads to a heterogeneous...

  14. Utilization of internal purification rejects; Sisaeisen puhdistuksen rejektikonsentraattien kelvollistaminen - KLT 02

    Energy Technology Data Exchange (ETDEWEB)

    Manner, H.; Nissen, M.

    1998-12-31

    This was a preliminary study which is part of a larger programme. The aim of the programme is to determine the properties and process ability of the concentrates which come from the internal purification of waters from the papermaking process. It is very important to know the properties and process ability of these purification concentrates in order to find the best methods of separating, reprocessing and utilizing them. The objective of this preliminary study was to ascertain the basic properties of these internal purification concentrates. It was also of interest to analyse the properties of papermaking waters and the state of internal purification today in paper mills. The state of papermaking waters and their internal purification were clarified by a literature review and by analyses of different types of waters. It was found that in mechanical pulping organic dissolved and colloidal substances were present in the water. Also there was a lot of dissolved and colloidal substances in waters from machines producing wood-containing paper grades. The salt content and chemical oxygen demand are critical values concerning the reuse of circulation waters. In mechanical pulping the convection of dissolved and colloidal substances to the paper machine can be reduced by the washing stage. Thus, the amount of dissolved and colloidal substances in the paper machine circulation waters can be reduced. In a paper machine, a disk filter removes fibers and fines from the circulation waters, but dissolved and colloidal substances are not removed. Also the properties of different kind of membrane filtration concentrates were analyzed. The total residue of membrane concentrates is low. For example, they can not be burned purely. The chemical oxygen demand of membrane concentrates is high. The most important subjects for further investigation are the improvement of fractionation and condensability. Furthermore procedures must be found to lower the chemical oxygen demand. One

  15. Utilization of internal purification rejects; Sisaeisen puhdistuksen rejektikonsentraattien kelvollistaminen - KLT 02

    Energy Technology Data Exchange (ETDEWEB)

    Manner, H; Nissen, M

    1999-12-31

    This was a preliminary study which is part of a larger programme. The aim of the programme is to determine the properties and process ability of the concentrates which come from the internal purification of waters from the papermaking process. It is very important to know the properties and process ability of these purification concentrates in order to find the best methods of separating, reprocessing and utilizing them. The objective of this preliminary study was to ascertain the basic properties of these internal purification concentrates. It was also of interest to analyse the properties of papermaking waters and the state of internal purification today in paper mills. The state of papermaking waters and their internal purification were clarified by a literature review and by analyses of different types of waters. It was found that in mechanical pulping organic dissolved and colloidal substances were present in the water. Also there was a lot of dissolved and colloidal substances in waters from machines producing wood-containing paper grades. The salt content and chemical oxygen demand are critical values concerning the reuse of circulation waters. In mechanical pulping the convection of dissolved and colloidal substances to the paper machine can be reduced by the washing stage. Thus, the amount of dissolved and colloidal substances in the paper machine circulation waters can be reduced. In a paper machine, a disk filter removes fibers and fines from the circulation waters, but dissolved and colloidal substances are not removed. Also the properties of different kind of membrane filtration concentrates were analyzed. The total residue of membrane concentrates is low. For example, they can not be burned purely. The chemical oxygen demand of membrane concentrates is high. The most important subjects for further investigation are the improvement of fractionation and condensability. Furthermore procedures must be found to lower the chemical oxygen demand. One

  16. Gram-scale production of plasmid pUDK-HGF with current good manufacturing practices for gene therapy of critical limb ischemia.

    Science.gov (United States)

    Hu, ChunSheng; Cheng, XiaoChen; Lu, YuXin; Wu, ZuZe; Zhang, QingLin

    2016-11-16

    The demand of a plasmid encoding human hepatocyte growth factor gene (pUDK-HGF) in large quantities at high purity and concentration has increased for gene therapy of critical limb ischemia (CLI) in clinical trials. In this article, we produced pUDK-HGF in compliance with current good manufacturing practices at gram scale. The process included a 50-L batch fermentation, continuous alkaline lysis, and integrated three-step chromatography on Sepharose 6 Fast Flow, PlasmidSelect Xtra, and Source 15Q. The production process has been scaled up to yield 4.24 ± 0.41 g of pharmaceutical pUDK-HGF from 1.0 kg bacterial cell paste and the overall yield reached range from 58.37 to 66.70%. The final pUDK-HGF product exhibited high purity with supercoiled percentage of > 95.8% and undetectable residual RNA, contaminated protein, and bacterial endotoxin. The phase I clinical study indicates that intramuscular injection of pUDK-HGF is safe, well tolerated, and may provide symptomatic relief to CLI patients. These results show that our manufacturing process of pUDK-HGF is efficient in producing pharmaceutical-grade plasmid DNA and is safe for clinical applications.

  17. Chromatographic and electrophoretic methods for nanodisc purification and analysis

    DEFF Research Database (Denmark)

    Justesen, Bo Højen; Günther-Pomorski, Thomas

    2014-01-01

    Soluble nanoscale lipid bilayers, termed nanodiscs, are widely used in science for studying the membrane-anchored and integral membrane protein complexes under defined experimental conditions. Although their formation occurs by a self-assembly process, nanodisc purification and the verification...

  18. Design of functional guanidinium ionic liquid aqueous two-phase systems for the efficient purification of protein

    Energy Technology Data Exchange (ETDEWEB)

    Ding, Xueqin; Wang, Yuzhi, E-mail: wyzss@hnu.edu.cn; Zeng, Qun; Chen, Jing; Huang, Yanhua; Xu, Kaijia

    2014-03-01

    Graphical abstract: - Highlights: • A series of novel cationic functional hexaalkylguanidinium ionic liquids and anionic functional tetraalkylguanidinium ionic liquids have been synthesized. • Functional guanidinium ionic liquid aqueous two-phase systems have been first designed for the purification of protein. • Mechanisms and performances of the process were researched. • Simple, green, safety and presents better purified ability than ordinary process. • A potential efficient platform for protein purification and related studies. - Abstract: A series of novel cationic functional hexaalkylguanidinium ionic liquids and anionic functional tetraalkylguanidinium ionic liquids have been devised and synthesized based on 1,1,3,3-tetramethylguanidine. The structures of the ionic liquids (ILs) were confirmed by {sup 1}H nuclear magnetic resonance ({sup 1}H NMR) and 13C nuclear magnetic resonance (13C NMR) and the production yields were all above 90%. Functional guanidinium ionic liquid aqueous two-phase systems (FGIL-ATPSs) have been first designed with these functional guanidinium ILs and phosphate solution for the purification of protein. After phase separation, proteins had transferred into the IL-rich phase and the concentrations of proteins were determined by measuring the absorbance at 278 nm using an ultra violet visible (UV–vis) spectrophotometer. The advantages of FGIL-ATPSs were compared with ordinary ionic liquid aqueous two-phase systems (IL-ATPSs). The proposed FGIL-ATPS has been applied to purify lysozyme, trypsin, ovalbumin and bovine serum albumin. Single factor experiments were used to research the effects of the process, such as the amount of ionic liquid (IL), the concentration of salt solution, temperature and the amount of protein. The purification efficiency reaches to 97.05%. The secondary structure of protein during the experimental process was observed upon investigation using UV–vis spectrophotometer, Fourier-transform infrared

  19. Novel Plasmid Transformation Method Mediated by Chrysotile, Sliding Friction, and Elastic Body Exposure

    Directory of Open Access Journals (Sweden)

    Naoto Yoshida

    2007-01-01

    Full Text Available Escherichia coli as a plasmid recipient cell was dispersed in a chrysotile colloidal solution, containing chrysotile adsorbed to plasmid DNA (chrysotile-plasmid cell mixture. Following this, the chrysotile-plasmid cell mixture was dropped onto the surface of an elastic body, such as agarose, and treated physically by sliding a polystyrene streak bar over the elastic body to create friction. Plasmid DNA was easily incorporated into E. coli, and antibiotic resistance was conferred by transformation. The transformation efficiency of E. coli cultured in solid medium was greater than that of E. coli cultured in broth. To obtain greater transformation efficiency, we attempted to determine optimal transformation conditions. The following conditions resulted in the greatest transformation efficiency: the recipient cell concentration within the chrysotileplasmid cell mixture had an optical density greater than or equal to 2 at 550 nm, the vertical reaction force applied to the streak bar was greater than or equal to 40 g, and the rotation speed of the elastic body was greater than or equal to 34 rpm. Under these conditions, we observed a transformation efficiency of 107 per μg plasmid DNA. The advantage of achieving bacterial transformation using the elastic body exposure method is that competent cell preparation of the recipient cell is not required. In addition to E. coli, other Gram negative bacteria are able to acquire plasmid DNA using the elastic body exposure method.

  20. Plasmid Transfer in the Ocean – A Case Study from the Roseobacter Group

    Directory of Open Access Journals (Sweden)

    Jörn Petersen

    2017-07-01

    Full Text Available Plasmid mediated horizontal gene transfer (HGT has been speculated to be one of the prime mechanisms for the adaptation of roseobacters (Rhodobacteraceae to their ecological niches in the marine habitat. Their plasmids contain ecologically crucial functional modules of up to ∼40-kb in size, e.g., for aerobic anoxygenic photosynthesis, flagellar formation and the biosynthesis of the antibiotic tropodithietic acid. Furthermore, the widely present type four secretion system (T4SS of roseobacters has been shown to mediate conjugation across genus barriers, albeit in the laboratory. Here we discovered that Confluentimicrobium naphthalenivorans NS6T, a tidal flat bacterium isolated in Korea, carries a 185-kb plasmid, which exhibits a long-range synteny with the conjugative 126-kb plasmid of Dinoroseobacter shibae DFL12T. Both replicons are stably maintained by RepABC operons of the same compatibility group (-2 and they harbor a homologous T4SS. Principal component analysis of the codon usage shows a large similarity between the two plasmids, while the chromosomes are very distinct, showing that neither of the two bacterial species represents the original host of those RepABC-2 type plasmids. The two species do not share a common habitat today and they are phylogenetically only distantly related. Our finding demonstrates the first clear-cut evidence for conjugational plasmid transfer across biogeographical and phylogenetic barriers in Rhodobacteraceae and documents the importance of conjugative HGT in the ocean.

  1. Plasmid mediated quinolone resistance in Enterobacteriaceae

    NARCIS (Netherlands)

    Veldman, K.T.; LS Klinisch Onderzoek Wagenaar

    2014-01-01

    This thesis describes the occurrence of Plasmid Mediated Quinolone Resistance (PMQR) in Salmonella and E. coli from The Netherlands and other European countries. Furthermore, the genetic background of these genes was characterized. Fluoroquinolones are widely used antibiotics in both human and

  2. Plasmid and chromosome segregation in prokaryotes

    DEFF Research Database (Denmark)

    Møller-Jensen, Jakob; Bugge Jensen, Rasmus; Gerdes, Kenn

    2000-01-01

    Recent major advances in the understanding of prokaryotic DNA segregation have been achieved by using fluorescence microscopy to visualize the localization of cellular components. Plasmids and bacterial chromosomes are partitioned in a highly dynamic fashion, suggesting the presence of a mitotic...

  3. Integration of replication-defective R68.45-like plasmids into the Pseudomonas aeruginosa chromosome.

    Science.gov (United States)

    Reimmann, C; Rella, M; Haas, D

    1988-06-01

    R68.45 and other similar broad-host-range (IncP) plasmids carrying a tandem repeat of the 2.1 kb insertion element IS21 mobilize the chromosome of many different Gram-negative bacteria. To analyse the structure of R68.45-chromosome cointegrates, whose involvement in the mobilization process had been postulated previously, we selected for the stable integration of R68.45-like plasmids into the Pseudomonas aeruginosa chromosome. Two plasmids were chosen: pME28, a transfer-deficient, mobilizable RP1 derivative with an inactive replication control (trfA) gene, and pME487, an R68.45 derivative with a trfA(ts) mutation causing temperature-sensitive replication. Chromosomally integrated pME28 and pME487 were found to be flanked by single IS21 elements. This structure is in agreement with a 'cut-and-paste' mode of R68.45 transposition. pME28 and pME487 showed a low specificity of insertion but rarely (less than 0.1%) induced auxotrophic mutations. Hfr (high-frequency-of-recombination) donors of P. aeruginosa could be obtained by chromosomal integration of pME487 or pME28; in the latter case, the transfer functions lacking from pME28 had to be provided in trans on an autonomous plasmid. Hfr donors gave higher conjugational linkage and transferred longer stretches of the P. aeruginosa chromosome than did R68.45 donors. This suggests that the integration of R68.45 into the donor chromosome is short-lived in P. aeruginosa.

  4. Degenerate primer MOB typing of multiresistant clinical isolates of E. coli uncovers new plasmid backbones.

    Science.gov (United States)

    Garcillán-Barcia, M Pilar; Ruiz del Castillo, Belén; Alvarado, Andrés; de la Cruz, Fernando; Martínez-Martínez, Luis

    2015-01-01

    Degenerate Primer MOB Typing is a PCR-based protocol for the classification of γ-proteobacterial transmissible plasmids in five phylogenetic relaxase MOB families. It was applied to a multiresistant E. coli collection, previously characterized by PCR-based replicon-typing, in order to compare both methods. Plasmids from 32 clinical isolates of multiresistant E. coli (19 extended spectrum beta-lactamase producers and 13 non producers) and their transconjugants were analyzed. A total of 95 relaxases were detected, at least one per isolate, underscoring the high potential of these strains for antibiotic-resistance transmission. MOBP12 and MOBF12 plasmids were the most abundant. Most MOB subfamilies detected were present in both subsets of the collection, indicating a shared mobilome among multiresistant E. coli. The plasmid profile obtained by both methods was compared, which provided useful data upon which decisions related to the implementation of detection methods in the clinic could be based. The phylogenetic depth at which replicon and MOB-typing classify plasmids is different. While replicon-typing aims at plasmid replication regions with non-degenerate primers, MOB-typing classifies plasmids into relaxase subfamilies using degenerate primers. As a result, MOB-typing provides a deeper phylogenetic depth than replicon-typing and new plasmid groups are uncovered. Significantly, MOB typing identified 17 plasmids and an integrative and conjugative element, which were not detected by replicon-typing. Four of these backbones were different from previously reported elements. Copyright © 2014 Elsevier Inc. All rights reserved.

  5. Cefotaxime resistant Escherichia coli collected from a healthy volunteer; characterisation and the effect of plasmid loss.

    Directory of Open Access Journals (Sweden)

    Miranda Kirchner

    Full Text Available In this study 6 CTX-M positive E. coli isolates collected during a clinical study examining the effect of antibiotic use in a human trial were analysed. The aim of the study was to analyse these isolates and assess the effect of full or partial loss of plasmid genes on bacterial fitness and pathogenicity. A DNA array was utilised to assess resistance and virulence gene carriage. Plasmids were characterised by PCR-based replicon typing and addiction system multiplex PCR. A phenotypic array and insect virulence model were utilised to assess the effect of plasmid-loss in E. coli of a large multi-resistance plasmid. All six E. coli carrying bla CTX-M-14 were detected from a single participant and were identical by pulse field gel electrophoresis and MLST. Plasmid profiling and arrays indicated absence of a large multi-drug resistance (MDR F-replicon plasmid carrying blaTEM, aadA4, strA, strB, dfrA17/19, sul1, and tetB from one isolate. Although this isolate partially retained the plasmid it showed altered fitness characteristics e.g. inability to respire in presence of antiseptics, similar to a plasmid-cured strain. However, unlike the plasmid-cured or plasmid harbouring strains, the survival rate for Galleria mellonella infected by the former strain was approximately 5-times lower, indicating other possible changes accompanying partial plasmid loss. In conclusion, our results demonstrated that an apparently healthy individual can harbour bla CTX-M-14 E. coli strains. In one such strain, isolated from the same individual, partial absence of a large MDR plasmid resulted in altered fitness and virulence characteristics, which may have implications in the ability of this strain to infect and any subsequent treatment.

  6. Complete sequences of IncHI1 plasmids carrying blaCTX-M-1 and qnrS1 in equine Escherichia coli provide new insights into plasmid evolution

    DEFF Research Database (Denmark)

    Dolejska, Monika; Villa, Laura; Minoia, Marco

    2014-01-01

    OBJECTIVES: To determine the structure of two multidrug-resistant IncHI1 plasmids carrying blaCTX-M-1 in Escherichia coli isolates disseminated in an equine clinic in the Czech Republic. METHODS: A complete nucleotide sequencing of 239 kb IncHI1 (pEQ1) and 287 kb IncHI1/X1 (pEQ2) plasmids was per...... highlight the structure and evolution of IncHI1 from equine E. coli. A plasmid-mediated sugar metabolic element could play a key role in strain fitness, contributing to the successful dissemination and maintenance of these plasmids in the intestinal microflora of horses....

  7. High quality protein microarray using in situ protein purification

    Directory of Open Access Journals (Sweden)

    Fleischmann Robert D

    2009-08-01

    Full Text Available Abstract Background In the postgenomic era, high throughput protein expression and protein microarray technologies have progressed markedly permitting screening of therapeutic reagents and discovery of novel protein functions. Hexa-histidine is one of the most commonly used fusion tags for protein expression due to its small size and convenient purification via immobilized metal ion affinity chromatography (IMAC. This purification process has been adapted to the protein microarray format, but the quality of in situ His-tagged protein purification on slides has not been systematically evaluated. We established methods to determine the level of purification of such proteins on metal chelate-modified slide surfaces. Optimized in situ purification of His-tagged recombinant proteins has the potential to become the new gold standard for cost-effective generation of high-quality and high-density protein microarrays. Results Two slide surfaces were examined, chelated Cu2+ slides suspended on a polyethylene glycol (PEG coating and chelated Ni2+ slides immobilized on a support without PEG coating. Using PEG-coated chelated Cu2+ slides, consistently higher purities of recombinant proteins were measured. An optimized wash buffer (PBST composed of 10 mM phosphate buffer, 2.7 mM KCl, 140 mM NaCl and 0.05% Tween 20, pH 7.4, further improved protein purity levels. Using Escherichia coli cell lysates expressing 90 recombinant Streptococcus pneumoniae proteins, 73 proteins were successfully immobilized, and 66 proteins were in situ purified with greater than 90% purity. We identified several antigens among the in situ-purified proteins via assays with anti-S. pneumoniae rabbit antibodies and a human patient antiserum, as a demonstration project of large scale microarray-based immunoproteomics profiling. The methodology is compatible with higher throughput formats of in vivo protein expression, eliminates the need for resin-based purification and circumvents

  8. A conjugative 38 kB plasmid is present in multiple subspecies of Xylella fastidiosa.

    Science.gov (United States)

    Rogers, Elizabeth E; Stenger, Drake C

    2012-01-01

    A ≈ 38kB plasmid (pXF-RIV5) was present in the Riv5 strain of Xylella fastidiosa subsp. multiplex isolated from ornamental plum in southern California. The complete nucleotide sequence of pXF-RIV5 is almost identical to that of pXFAS01 from X. fastidiosa subsp. fastidiosa strain M23; the two plasmids vary at only 6 nucleotide positions. BLAST searches and phylogenetic analyses indicate pXF-RIV5 and pXFAS01 share some similarity to chromosomal and plasmid (pXF51) sequences of X. fastidiosa subsp. pauca strain 9a5c and more distant similarity to plasmids from a wide variety of bacteria. Both pXF-RIV5 and pXFAS01 encode homologues of a complete Type IV secretion system involved in conjugation and DNA transfer among bacteria. Mating pair formation proteins (Trb) from Yersinia pseudotuberculosis IP31758 are the mostly closely related non-X. fastidiosa proteins to most of the Trb proteins encoded by pXF-RIV5 and pXFAS01. Unlike many bacterial conjugative plasmids, pXF-RIV5 and pXFAS01 do not carry homologues of known accessory modules that confer selective advantage on host bacteria. However, both plasmids encode seven hypothetical proteins of unknown function and possess a small transposon-associated region encoding a putative transposase and associated factor. Vegetative replication of pXF-RIV5 and pXFAS01 appears to be under control of RepA protein and both plasmids have an origin of DNA replication (oriV) similar to that of pRP4 and pR751 from Escherichia coli. In contrast, conjugative plasmids commonly encode TrfA and have an oriV similar to those found in IncP-1 incompatibility group plasmids. The presence of nearly identical plasmids in single strains from two distinct subspecies of X. fastidiosa is indicative of recent horizontal transfer, probably subsequent to the introduction of subspecies fastidiosa to the United States in the late 19(th) century.

  9. A conjugative 38 kB plasmid is present in multiple subspecies of Xylella fastidiosa.

    Directory of Open Access Journals (Sweden)

    Elizabeth E Rogers

    Full Text Available A ≈ 38kB plasmid (pXF-RIV5 was present in the Riv5 strain of Xylella fastidiosa subsp. multiplex isolated from ornamental plum in southern California. The complete nucleotide sequence of pXF-RIV5 is almost identical to that of pXFAS01 from X. fastidiosa subsp. fastidiosa strain M23; the two plasmids vary at only 6 nucleotide positions. BLAST searches and phylogenetic analyses indicate pXF-RIV5 and pXFAS01 share some similarity to chromosomal and plasmid (pXF51 sequences of X. fastidiosa subsp. pauca strain 9a5c and more distant similarity to plasmids from a wide variety of bacteria. Both pXF-RIV5 and pXFAS01 encode homologues of a complete Type IV secretion system involved in conjugation and DNA transfer among bacteria. Mating pair formation proteins (Trb from Yersinia pseudotuberculosis IP31758 are the mostly closely related non-X. fastidiosa proteins to most of the Trb proteins encoded by pXF-RIV5 and pXFAS01. Unlike many bacterial conjugative plasmids, pXF-RIV5 and pXFAS01 do not carry homologues of known accessory modules that confer selective advantage on host bacteria. However, both plasmids encode seven hypothetical proteins of unknown function and possess a small transposon-associated region encoding a putative transposase and associated factor. Vegetative replication of pXF-RIV5 and pXFAS01 appears to be under control of RepA protein and both plasmids have an origin of DNA replication (oriV similar to that of pRP4 and pR751 from Escherichia coli. In contrast, conjugative plasmids commonly encode TrfA and have an oriV similar to those found in IncP-1 incompatibility group plasmids. The presence of nearly identical plasmids in single strains from two distinct subspecies of X. fastidiosa is indicative of recent horizontal transfer, probably subsequent to the introduction of subspecies fastidiosa to the United States in the late 19(th century.

  10. Fitness Advantage of mcr-1–Bearing IncI2 and IncX4 Plasmids in Vitro

    Directory of Open Access Journals (Sweden)

    Renjie Wu

    2018-02-01

    Full Text Available The objective of this study was to assess the impact of diverse plasmids bearing colistin resistance gene mcr-1 on host fitness. Forty-seven commensal E. coli isolates recovered from the pig farm where mcr-1 was first identified were screened for mcr-1. mcr-1-bearing plasmids were characterized by sequencing. The fitness impact of mcr-1-bearing plasmids was evaluated by in vitro competition assays. Twenty-seven (57.5% E. coli isolates were positive for mcr-1. The mcr-1 genes were mainly located on plasmids belonging to IncI2 (n = 5, IncX4 (n = 11, IncHI2/ST3 (n = 8, IncFII (n = 2, and IncY (n = 2. InHI2 plasmids also carried other resistance genes (floR, blaCTX−M, and fosA3 and were only detected in isolates from nursery pigs. Sequences of the representative mcr-1–bearing plasmids were almost identical to those of the corresponding plasmid types reported previously. An increase in the fitness of IncI2- and IncX4-carrying strains was observed, while the presence of IncHI2, IncFII and IncY plasmids showed a fitness cost although an insignificant fitness increase was initially observed in IncFII or IncY plasmids-containing strains. Acquisition of IncI2-type plasmid was more beneficial for host E. coli DH5α than either IncHI2 or IncX4 plasmid, while transformants with IncHI2-type plasmid presented a competitive disadvantage against IncI2 or IncX4 plasmid containing strains. In conclusion, IncI2, IncX4, and IncHI2 were the major plasmid types driving the dissemination of mcr-1 in this farm. Increased fitness or co-selection by other antimicrobials might contribute to the further dissemination of the three epidemic mcr-1–positive plasmids (IncI2, IncX4, and IncHI2 in this farm and worldwide.

  11. The complex between SOS3 and SOS2 regulatory domain from Arabidopsis thaliana: cloning, expression, purification, crystallization and preliminary X-ray analysis

    Energy Technology Data Exchange (ETDEWEB)

    Sánchez-Barrena, María José; Moreno-Pérez, Sandra; Angulo, Iván; Martínez-Ripoll, Martín; Albert, Armando, E-mail: xalbert@iqfr.csic.es [Grupo de Cristalografía Macromolecular y Biología Estructural, Instituto de Química Física ‘Rocasolano’, Consejo Superior de Investigaciones Científicas, Serrano 119, E-28006 Madrid (Spain)

    2007-07-01

    Recombinant SOS3 and SOS2 regulatory domain from A. thaliana have been coexpressed in E. coli, purified and crystallized by the hanging-drop vapour-diffusion method. An X-ray data set has been collected at 2.0 Å resolution. The salt-tolerance genes SOS3 (salt overly sensitive 3) and SOS2 (salt overly sensitive 2) regulatory domain of Arabidopsis thaliana were cloned into a polycistronic plasmid and the protein complex was expressed in Escherichia coli, allowing purification to homogeneity in three chromatographic steps. Crystals were grown using vapour-diffusion techniques. The crystals belonged to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 44.14, b = 57.39, c = 141.90 Å.

  12. Imipenem-resistance in Serratia marcescens is mediated by plasmid expression of KPC-2.

    Science.gov (United States)

    Su, W-Q; Zhu, Y-Q; Deng, N-M; Li, L

    2017-04-01

    Imipenem is a broad-spectrum carbapenem antibiotic with applications against severe bacterial infections. Here, we describe the identification of imipenem-resistant Serratia marcescens in our hospital and the role of plasmid-mediated KPC-2 expression in imipenem resistance. We used the modified Hodge test to detect carbapenemase produced in imipenem-resistant strains. His resistance can be transferred to E. coli in co-culture tests, which implicates the plasmid in imipenem resistance. PCR amplification from the plasmid identified two products consistent with KPC-2 of 583 and 1050 bp that were also present in E. coli after co-culture. The restriction pattern for both plasmids was identical, supporting the transfer from the S. marcescens isolate to E. coli. Finally, gene sequencing confirmed KPC-2 in the plasmid. Due to the presence of KPC-2 in the imipenem-resistant S. marcescens, we propose that KPC-2 mediates antibiotic resistance in the S. marcescens isolate.

  13. Novel heat-pump-assisted extractive distillation for bioethanol purification

    NARCIS (Netherlands)

    Luo, Hao; Bildea, Costin Sorin; Kiss, Anton A.

    2015-01-01

    The purification of bioethanol fuel involves an energy-intensive separation process to concentrate the diluted streams obtained in the fermentation stage and to overcome the azeotropic behavior of the ethanol-water mixture. The conventional separation sequence employs three distillation columns that

  14. Effect of the atmospheric pressure nonequilibrium plasmas on the conformational changes of plasmid DNA

    International Nuclear Information System (INIS)

    Yan Xu; He Guangyuan; Shi Mengjun; Gao Xuan; Li Yin; Ma Fengyun; Yu Men; Wang Changdong; Wang Yuesheng; Yang Guangxiao; Zou Fei; Lu Xinpei; Xiong Qing; Xiong Zilan

    2009-01-01

    The cold atmospheric pressure plasma, which has been widely used for biomedical applications, may potentially affect the conformation of DNA. In this letter, an atmospheric pressure plasma plume is used to investigate its effects on the conformational changes of DNA of plasmid pAHC25. It is found that the plasma plume could cause plasmid DNA topology alteration, resulting in the percentage of the supercoiled plasmid DNA form decreased while that of the open circular and linearized form of plasmid DNA increased as detected by agrose gel electrophoresis. On the other hand, further investigation by using polymerase chain reaction method shows that the atmospheric pressure plasma jet treatments under proper conditions does not affect the genes of the plasmid DNA, which may have potential application in increasing the transformation frequency by genetic engineering.

  15. Contaminated nickel scrap processing

    Energy Technology Data Exchange (ETDEWEB)

    Compere, A.L.; Griffith, W.L.; Hayden, H.W.; Johnson, J.S. Jr.; Wilson, D.F.

    1994-12-01

    The DOE will soon choose between treating contaminated nickel scrap as a legacy waste and developing high-volume nickel decontamination processes. In addition to reducing the volume of legacy wastes, a decontamination process could make 200,000 tons of this strategic metal available for domestic use. Contaminants in DOE nickel scrap include {sup 234}Th, {sup 234}Pa, {sup 137}Cs, {sup 239}Pu (trace), {sup 60}Co, U, {sup 99}Tc, and {sup 237}Np (trace). This report reviews several industrial-scale processes -- electrorefining, electrowinning, vapormetallurgy, and leaching -- used for the purification of nickel. Conventional nickel electrolysis processes are particularly attractive because they use side-stream purification of process solutions to improve the purity of nickel metal. Additionally, nickel purification by electrolysis is effective in a variety of electrolyte systems, including sulfate, chloride, and nitrate. Conventional electrorefining processes typically use a mixed electrolyte which includes sulfate, chloride, and borate. The use of an electrorefining or electrowinning system for scrap nickel recovery could be combined effectively with a variety of processes, including cementation, solvent extraction, ion exchange, complex-formation, and surface sorption, developed for uranium and transuranic purification. Selected processes were reviewed and evaluated for use in nickel side-stream purification. 80 refs.

  16. 3G vector-primer plasmid for constructing full-length-enriched cDNA libraries.

    Science.gov (United States)

    Zheng, Dong; Zhou, Yanna; Zhang, Zidong; Li, Zaiyu; Liu, Xuedong

    2008-09-01

    We designed a 3G vector-primer plasmid for the generation of full-length-enriched complementary DNA (cDNA) libraries. By employing the terminal transferase activity of reverse transcriptase and the modified strand replacement method, this plasmid (assembled with a polydT end and a deoxyguanosine [dG] end) combines priming full-length cDNA strand synthesis and directional cDNA cloning. As a result, the number of steps involved in cDNA library preparation is decreased while simplifying downstream gene manipulation, sequencing, and subcloning. The 3G vector-primer plasmid method yields fully represented plasmid primed libraries that are equivalent to those made by the SMART (switching mechanism at 5' end of RNA transcript) approach.

  17. Rap phosphatase of virulence plasmid pXO1 inhibits Bacillus anthracis sporulation.

    Science.gov (United States)

    Bongiorni, Cristina; Stoessel, Ricarda; Shoemaker, Dorinda; Perego, Marta

    2006-01-01

    This study shows that the Bacillus anthracis pXO1 virulence plasmid carries a Rap-Phr system, BXA0205, which regulates sporulation initiation in this organism. The BXA0205Rap protein was shown to dephosphorylate the Spo0F response regulator intermediate of the phosphorelay signal transduction system that regulates the initiation of the developmental pathway in response to environmental, metabolic, and cell cycle signals. The activity of the Rap protein was shown to be inhibited by the carboxy-terminal pentapeptide generated through an export-import processing pathway from the associated BXA0205Phr protein. Deregulation of the Rap activity by either overexpression or lack of the Phr pentapeptide resulted in severe inhibition of sporulation. Five additional Rap-Phr encoding systems were identified on the chromosome of B. anthracis, one of which, BA3790-3791, also affected sporulation initiation. The results suggest that the plasmid-borne Rap-Phr system may provide a selective advantage to the virulence of B. anthracis.

  18. Rap Phosphatase of Virulence Plasmid pXO1 Inhibits Bacillus anthracis Sporulation†

    Science.gov (United States)

    Bongiorni, Cristina; Stoessel, Ricarda; Shoemaker, Dorinda; Perego, Marta

    2006-01-01

    This study shows that the Bacillus anthracis pXO1 virulence plasmid carries a Rap-Phr system, BXA0205, which regulates sporulation initiation in this organism. The BXA0205Rap protein was shown to dephosphorylate the Spo0F response regulator intermediate of the phosphorelay signal transduction system that regulates the initiation of the developmental pathway in response to environmental, metabolic, and cell cycle signals. The activity of the Rap protein was shown to be inhibited by the carboxy-terminal pentapeptide generated through an export-import processing pathway from the associated BXA0205Phr protein. Deregulation of the Rap activity by either overexpression or lack of the Phr pentapeptide resulted in severe inhibition of sporulation. Five additional Rap-Phr encoding systems were identified on the chromosome of B. anthracis, one of which, BA3790-3791, also affected sporulation initiation. The results suggest that the plasmid-borne Rap-Phr system may provide a selective advantage to the virulence of B. anthracis. PMID:16385039

  19. Cloning, over-expression and purification of Pseudomonas aeruginosa murC encoding uridine diphosphate N-acetylmuramate: L-alanine ligase.

    Science.gov (United States)

    El Zoeiby, A; Sanschagrin, F; Lamoureux, J; Darveau, A; Levesque, R C

    2000-02-15

    We cloned and sequenced the murC gene from Pseudomonas aeruginosa encoding a protein of 53 kDa. Multiple alignments with 20 MurC peptide sequences from different bacteria confirmed the presence of highly conserved regions having sequence identities ranging from 22-97% including conserved motifs for ATP-binding and the active site of the enzyme. Genetic complementation was done in Escherichia coli (murCts) suppressing the lethal phenotype. The murC gene was subcloned into the expression vector pET30a and overexpressed in E. coli BL21(lambdaDE3). Three PCR cloning strategies were used to obtain the three recombinant plasmids for expression of the native MurC, MurC His-tagged at N-terminal and at C-terminal, respectively. MurC His-tagged at C-terminal was chosen for large scale production and protein purification in the soluble form. The purification was done in a single chromatographic step on an affinity nickel column and obtained in mg quantities at 95% homogeneity. MurC protein was used to produce monoclonal antibodies for epitope mapping and for assay development in high throughput screenings. Detailed studies of MurC and other genes of the bacterial cell cycle will provide the reagents and strain constructs for high throughput screening and for design of novel antibacterials.

  20. Uranium hexafluoride purification; Purificacao de hexafluoreto de uranio

    Energy Technology Data Exchange (ETDEWEB)

    Araujo, Eneas F. de

    1986-07-01

    Uranium hexafluoride might contain a large amount of impurities after manufacturing or handling. Three usual methods of purification of uranium hexafluoride were presented: selective sorption, sublimation, and distillation. Since uranium hexafluoride usually is contaminated with hydrogen fluoride, a theoretical study of the phase equilibrium properties was performed for the binary system UF{sub 6}-HF. A large deviation from the ideal solution behaviour was observed. A purification unity based on a constant reflux batch distillation process was developed. A procedure was established in order to design the re boiler, condenser and packed columns for the UF{sub 6}-HF mixture separation. A bench scale facility for fractional distillation of uranium hexafluoride was described. Basic operations for that facility and results extracted from several batches were discussed. (author)

  1. Comparative Genomics of Rhodococcus equi Virulence Plasmids Indicates Host-Driven Evolution of the vap Pathogenicity Island.

    Science.gov (United States)

    MacArthur, Iain; Anastasi, Elisa; Alvarez, Sonsiray; Scortti, Mariela; Vázquez-Boland, José A

    2017-05-01

    The conjugative virulence plasmid is a key component of the Rhodococcus equi accessory genome essential for pathogenesis. Three host-associated virulence plasmid types have been identified the equine pVAPA and porcine pVAPB circular variants, and the linear pVAPN found in bovine (ruminant) isolates. We recently characterized the R. equi pangenome (Anastasi E, et al. 2016. Pangenome and phylogenomic analysis of the pathogenic actinobacterium Rhodococcus equi. Genome Biol Evol. 8:3140-3148.) and we report here the comparative analysis of the virulence plasmid genomes. Plasmids within each host-associated type were highly similar despite their diverse origins. Variation was accounted for by scattered single nucleotide polymorphisms and short nucleotide indels, while larger indels-mostly in the plasticity region near the vap pathogencity island (PAI)-defined plasmid genomic subtypes. Only one of the plasmids analyzed, of pVAPN type, was exceptionally divergent due to accumulation of indels in the housekeeping backbone. Each host-associated plasmid type carried a unique PAI differing in vap gene complement, suggesting animal host-specific evolution of the vap multigene family. Complete conservation of the vap PAI was observed within each host-associated plasmid type. Both diversity of host-associated plasmid types and clonality of specific chromosomal-plasmid genomic type combinations were observed within the same R. equi phylogenomic subclade. Our data indicate that the overall strong conservation of the R. equi host-associated virulence plasmids is the combined result of host-driven selection, lateral transfer between strains, and geographical spread due to international livestock exchanges. © The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  2. Characterization of Endogenous Plasmids from Lactobacillus salivarius UCC118▿ †

    Science.gov (United States)

    Fang, Fang; Flynn, Sarah; Li, Yin; Claesson, Marcus J.; van Pijkeren, Jan-Peter; Collins, J. Kevin; van Sinderen, Douwe; O'Toole, Paul W.

    2008-01-01

    The genome of Lactobacillus salivarius UCC118 comprises a 1.83-Mb chromosome, a 242-kb megaplasmid (pMP118), and two smaller plasmids of 20 kb (pSF118-20) and 44 kb (pSF118-44). Annotation and bioinformatic analyses suggest that both of the smaller plasmids replicate by a theta replication mechanism. Furthermore, it appears that they are transmissible, although neither possesses a complete set of conjugation genes. Plasmid pSF118-20 encodes a toxin-antitoxin system composed of pemI and pemK homologs, and this plasmid could be cured when PemI was produced in trans. The minimal replicon of pSF118-20 was determined by deletion analysis. Shuttle vector derivatives of pSF118-20 were generated that included the replication region (pLS203) and the replication region plus mobilization genes (pLS208). The plasmid pLS203 was stably maintained without selection in Lactobacillus plantarum, Lactobacillus fermentum, and the pSF118-20-cured derivative strain of L. salivarius UCC118 (strain LS201). Cloning in pLS203 of genes encoding luciferase and green fluorescent protein, and expression from a constitutive L. salivarius promoter, demonstrated the utility of this vector for the expression of heterologous genes in Lactobacillus. This study thus expands the knowledge base and vector repertoire of probiotic lactobacilli. PMID:18390685

  3. Enriched Water-H2 18O Purification to be Used in Routine 18FDG Production

    International Nuclear Information System (INIS)

    Al Rayyes, A.H.

    2009-01-01

    Oxygen-18 enriched water has been recovered from IBA (Ion Beam Applications) recovery system followed by purification and then used in the production of 18 F-. The purification process has been carried out by irradiation with UV followed by a distillation under vacuum. After purification, 95% of water is recovered and organic compounds, radioisotopes, trace metals and gases are eliminated efficiently. Results show that there are no significant differences in (2-deoxy-2-[ 18 F]fluoro-D-glucose ([ 18 F]FDG) production yield using purified water by the proposed method and new enriched water. Tritium was detected in the irradiated enriched water. Contamination precautions during purification should be considered. Tritium was not present in 18 FDG or Na- 18 F final products. (author)

  4. Plasmid fingerprinting and virulence gene detection among indigenous strains of salmonella enterica serovar enteritidis

    International Nuclear Information System (INIS)

    Sajid, S.U.; Schwarz, S.

    2009-01-01

    Salmonella enterica serovar Enteritidis is an important frequently reported zoonotic pathogen and a common cause of human gastroenteritis worldwide. The highly conserved Serospecific plasmids (SSPs) and Salmonella plasmid virulence (Spv) genes have been shown to mediate extra-intestinal colonization and systemic infection. The objective of current study was to document the presence of SSPs and SpvB/SpvC genes prevailing in the indigenous population of serovar Enteritidis. A total of 48 epidemiologically unrelated strains of Salmonella enteritidis were included in the study. Preparation of plasmids DNA suitable for endonuclease digestion and separation of respective fragments by agarose gel electrophoresis followed previously described protocols. The plasmids of Escherichia coli V517, 1-kbp ladder, and lambda DNA HindIII fragments served as DNA size standards. Transfer of DNA fragments from agarose gels to nitrocellulose membranes was achieved by capillary blot procedure. An ECL labeled 3.6 kbp HindIII fragment of plasmid PRQ 51 was used as probe for SpvB/SpvC gene detection. Plasmid DNA fingerprinting revealed the presence of two different profiles of approximately 55 kbp and 90 kbp and were identified as virulence plasmids by DNA hybridization. The SpvB/SpvC genes were located on HindIII fragments of 3.6 kbp in each of the two types of virulence plasmids. The study confirms the presence of SSPs and SpvB/SpvC genes in indigenous strains of S. enteritidis isolated from Northern Punjab area of Pakistan and substantiate the previous data on such findings from other parts of the world. (author)

  5. Salmonella Typhimurium ST213 is associated with two types of IncA/C plasmids carrying multiple resistance determinants.

    Science.gov (United States)

    Wiesner, Magdalena; Calva, Edmundo; Fernández-Mora, Marcos; Cevallos, Miguel A; Campos, Freddy; Zaidi, Mussaret B; Silva, Claudia

    2011-01-11

    Salmonella Typhimurium ST213 was first detected in the Mexican Typhimurium population in 2001. It is associated with a multi-drug resistance phenotype and a plasmid-borne blaCMY-2 gene conferring resistance to extended-spectrum cephalosporins. The objective of the current study was to examine the association between the ST213 genotype and blaCMY-2 plasmids. The blaCMY-2 gene was carried by an IncA/C plasmid. ST213 strains lacking the blaCMY-2 gene carried a different IncA/C plasmid. PCR analysis of seven DNA regions distributed throughout the plasmids showed that these IncA/C plasmids were related, but the presence and absence of DNA stretches produced two divergent types I and II. A class 1 integron (dfrA12, orfF and aadA2) was detected in most of the type I plasmids. Type I contained all the plasmids carrying the blaCMY-2 gene and a subset of plasmids lacking blaCMY-2. Type II included all of the remaining blaCMY-2-negative plasmids. A sequence comparison of the seven DNA regions showed that both types were closely related to IncA/C plasmids found in Escherichia, Salmonella, Yersinia, Photobacterium, Vibrio and Aeromonas. Analysis of our Typhimurium strains showed that the region containing the blaCMY-2 gene is inserted between traA and traC as a single copy, like in the E. coli plasmid pAR060302. The floR allele was identical to that of Newport pSN254, suggesting a mosaic pattern of ancestry with plasmids from other Salmonella serovars and E. coli. Only one of the tested strains was able to conjugate the IncA/C plasmid at very low frequencies (10-7 to 10-9). The lack of conjugation ability of our IncA/C plasmids agrees with the clonal dissemination trend suggested by the chromosomal backgrounds and plasmid pattern associations. The ecological success of the newly emerging Typhimurium ST213 genotype in Mexico may be related to the carriage of IncA/C plasmids. We conclude that types I and II of IncA/C plasmids originated from a common ancestor and that the

  6. Optimized conditions for high-level solubilization and purification of ...

    African Journals Online (AJOL)

    NH Computers

    2011-10-10

    Oct 10, 2011 ... filtration chromatography using Sephadex G-50 column. The purified protein was refolded by .... blocking with BSA, the nitrocellulose membrane was processed using rabbit monoclonal antibody raised ..... recombinant human growth hormone using radial flow chromatography. Protein Exp. Purif., 68: 54-59.

  7. Differences in the stability of the plasmids of Yersinia pestis cultures in vitro: impact on virulence

    Directory of Open Access Journals (Sweden)

    TC Leal-Balbino

    2004-11-01

    Full Text Available Plasmid and chromosomal genes encode determinants of virulence for Yersinia pestis, the causative agent of plague. However, in vitro, Y. pestis genome is very plastic and several changes have been described. To evaluate the alterations in the plasmid content of the cultures in vitro and the impact of the alterations to their pathogenicity, three Y. pestis isolates were submitted to serial subculture, analysis of the plasmid content, and testing for the presence of characteristic genes in each plasmid of colonies selected after subculture. Different results were obtained with each strain. The plasmid content of one of them was shown to be stable; no apparent alteration was produced through 32 subcultures. In the other two strains, several alterations were observed. LD50 in mice of the parental strains and the derived cultures with different plasmid content were compared. No changes in the virulence plasmid content could be specifically correlated with changes in the LD50.

  8. Studies on the expression of plasmid-borne genes in the endosymbiotic state of Rhizobium leguminosarum

    NARCIS (Netherlands)

    Krol, A.J.M.

    1982-01-01

    The subject matter of the research reported in this thesis is the role of plasmid-borne genes of Rhizobium in symbiosis and nitrogen fixation. Plasmid DNA was isolated from Rhizobium leguminosarum strain PRE and the expression of plasmid DNA in nitrogen

  9. Identification of a Novel Conjugative Plasmid in Mycobacteria That Requires Both Type IV and Type VII Secretion

    KAUST Repository

    Ummels, R.

    2014-09-23

    Conjugative plasmids have been identified in a wide variety of different bacteria, ranging from proteobacteria to firmicutes, and conjugation is one of the most efficient routes for horizontal gene transfer. The most widespread mechanism of plasmid conjugation relies on different variants of the type IV secretion pathway. Here, we describe the identification of a novel type of conjugative plasmid that seems to be unique for mycobacteria. Interestingly, while this plasmid is efficiently exchanged between different species of slow-growing mycobacteria, including Mycobacterium tuberculosis, it could not be transferred to any of the fast-growing mycobacteria tested. Genetic analysis of the conjugative plasmid showed the presence of a locus containing homologues of three type IV secretion system components and a relaxase. In addition, a new type VII secretion locus was present. Using transposon insertion mutagenesis, we show that in fact both these secretion systems are essential for conjugation, indicating that this plasmid represents a new class of conjugative plasmids requiring two secretion machineries. This plasmid could form a useful new tool to exchange or introduce DNA in slow-growing mycobacteria. IMPORTANCE: Conjugative plasmids play an important role in horizontal gene transfer between different bacteria and, as such, in their adaptation and evolution. This effect is most obvious in the spread of antibiotic resistance genes. Thus far, conjugation of natural plasmids has been described only rarely for mycobacterial species. In fact, it is generally accepted that M. tuberculosis does not show any recent sign of horizontal gene transfer. In this study, we describe the identification of a new widespread conjugative plasmid that can also be efficiently transferred to M. tuberculosis. This plasmid therefore poses both a threat and an opportunity. The threat is that, through the acquisition of antibiotic resistance markers, this plasmid could start a rapid spread of

  10. Purification processes of cadmium based quantum dots in aqueous medium: a comparative study

    Science.gov (United States)

    Petris, Dimitri; Freitas, Denilson V.; dos Santos, Rayany K. V.; Dias, Jéssica M. M.; Navarro, Marcelo

    2017-07-01

    Three different purification methods for CdTe-MPA quantum dot (QD) were performed in aqueous medium: acid titration (HCl, HClO4, H2SO4 and CH3COOH), non-selective precipitation by addition of acetone and co-precipitation of the QD in the presence of inorganic salts. The QD stock solutions were prepared by an electrochemical method of synthesis, in four different heating times (1 h, 4 h, 8 h and 12 h). After purifications, the QD solids were redispersed in distilled water and analyzed by absorption and emission spectra. The λ abs and λ em of the purified QDs showed similar data observed for QD stock solutions, and the recovery rate varied from 71% to 99%. Co-precipitation method showed some advantages: quantum yield maintenance of the QD redispersed solution, longer period of storage (over 6 months) in solution and in solid state (QD embedded into the KCl crystal lattice). CdSe-MPA and CdS-MPA solutions were also purified by co-precipitation method with KCl, showing good results as observed for CdTe-MPA.

  11. TOL plasmid transfer during bacterial conjugation in vitro and rhizoremediation of oil compounds in vivo

    International Nuclear Information System (INIS)

    Jussila, Minna M.; Zhao, Ji; Suominen, Leena; Lindstroem, Kristina

    2007-01-01

    Molecular profiling methods for horizontal transfer of aromatics-degrading plasmids were developed and applied during rhizoremediation in vivo and conjugations in vitro. pWW0 was conjugated from Pseudomonas to Rhizobium. The xylE gene was detected both in Rhizobium galegae bv. officinalis and bv. orientalis, but it was neither stably maintained in orientalis nor functional in officinalis. TOL plasmids were a major group of catabolic plasmids among the bacterial strains isolated from the oil-contaminated rhizosphere of Galega orientalis. A new finding was that some Pseudomonas migulae and Pseudomonas oryzihabitans strains harbored a TOL plasmid with both pWW0- and pDK1-type xylE gene. P. oryzihabitans 29 had received the archetypal TOL plasmid pWW0 from Pseudomonas putida PaW85. As an application for environmental biotechnology, the biodegradation potential of oil-polluted soil and the success of bioremediation could be estimated by monitoring changes not only in the type and amount but also in transfer of degradation plasmids. - Horizontal transfer of degradation plasmids in the oil-contaminated rhizosphere reveals the dynamic nature of the intrinsic biodegradation potential

  12. Recombinogenic engineering of conjugative plasmids with fluorescent marker cassettes

    DEFF Research Database (Denmark)

    Reisner, A.; Molin, Søren; Zechner, E.L.

    2002-01-01

    An efficient approach for the insertion of fluorescent marker genes with sequence specificity into conjugative plasmids in Escherichia coli is described. For this purpose, homologous recombination of linear double-stranded targeting DNA was mediated by the bacteriophage lambda recombination...... resistance genes and fluorescent markers. The choice of 5' non-homologous extensions in primer pairs used for amplifying the marker cassettes determines the site specificity of the targeting DNA. This methodology is applicable to the modification of all plasmids that replicate in E coli and is not restricted...

  13. Novel immobilization process of a thermophilic catalase: efficient purification by heat treatment and subsequent immobilization at high temperature.

    Science.gov (United States)

    Xu, Juan; Luo, Hui; López, Claudia; Xiao, Jing; Chang, Yanhong

    2015-10-01

    The main goal of the present work is to investigate a novel process of purification and immobilization of a thermophilic catalase at high temperatures. The catalase, originated from Bacillus sp., was overexpressed in a recombinant Escherichia coli BL21(DE3)/pET28-CATHis and efficiently purified by heat treatment, achieving a threefold purification. The purified catalase was then immobilized onto an epoxy support at different temperatures (25, 40, and 55 °C). The immobilizate obtained at higher temperatures reached its maximum activity in a shorter time than that obtained at lower temperatures. Furthermore, immobilization at higher temperatures required a lower ionic strength than immobilization at lower temperatures. The characteristics of immobilized enzymes prepared at different temperatures were investigated. The high-temperature immobilizate (55 °C) showed the highest thermal stability, followed by the 40 °C immobilizate. And the high-temperature immobilizate (55 °C) had slightly higher operational stability than the 25 °C immobilizate. All of the immobilized catalase preparations showed higher stability than the free enzyme at alkaline pH 10.0, while the alkali resistance of the 25 °C immobilizate was slightly better than that of the 40 and 55 °C immobilizates.

  14. Entanglement purification of multi-mode quantum states

    International Nuclear Information System (INIS)

    Clausen, J; Knoell, L; Welsch, D-G

    2003-01-01

    An iterative random procedure is considered allowing entanglement purification of a class of multi-mode quantum states. In certain cases, complete purification may be achieved using only a single signal state preparation. A physical implementation based on beam splitter arrays and non-linear elements is suggested. The influence of loss is analysed in the example of purification of entangled N-mode coherent states

  15. Investigation of Various LiCl Waste Salt Purification Technologies

    International Nuclear Information System (INIS)

    Yung-Zun Cho; Hee-Chul Yang; Han-Soo Lee; In-Tae Kim

    2008-01-01

    Various purification research of LiCl waste molten salt generated from electroreduction process were tested. The purification of the LiCl waste salt very important in a various aspects, where the purification means separation of cesium and strontium form LiCl salt melts. In this study, for the separation of cesium and strontium from LiCl salt melts, precipitant agent addition techniques such as sulfate and carbonate addition method and, as a new attempt, zone freezing technique for concentration of cesium and strontium elements was investigated. As a results of this research, only strontium was carbonated by reaction with Li 2 CO 3 (cesium did not react with Li 2 CO 3 ). In case of sulfate addition method, both cesium and strontium were converted into their sulfate that is Cs 2 S 2 O 6 and SrSO 4 and maximum sulfate efficiency of cesium and strontium were about 72% and 95%, respectively. Cesium and strontium involved in LiCl molten salt could be concentrated in the molten salt by using zone freezing method. (authors)

  16. Automated genomic DNA purification options in agricultural applications using MagneSil paramagnetic particles

    Science.gov (United States)

    Bitner, Rex M.; Koller, Susan C.

    2002-06-01

    The automated high throughput purification of genomic DNA form plant materials can be performed using MagneSil paramagnetic particles on the Beckman-Coulter FX, BioMek 2000, and the Tecan Genesis robot. Similar automated methods are available for DNA purifications from animal blood. These methods eliminate organic extractions, lengthy incubations and cumbersome filter plates. The DNA is suitable for applications such as PCR and RAPD analysis. Methods are described for processing traditionally difficult samples such as those containing large amounts of polyphenolics or oils, while still maintaining a high level of DNA purity. The robotic protocols have ben optimized for agricultural applications such as marker assisted breeding, seed-quality testing, and SNP discovery and scoring. In addition to high yield purification of DNA from plant samples or animal blood, the use of Promega's DNA-IQ purification system is also described. This method allows for the purification of a narrow range of DNA regardless of the amount of additional DNA that is present in the initial sample. This simultaneous Isolation and Quantification of DNA allows the DNA to be used directly in applications such as PCR, SNP analysis, and RAPD, without the need for separate quantitation of the DNA.

  17. TIO2 based photocatalytic gas purification: the effects of co-catalysts and process conditions

    NARCIS (Netherlands)

    Fraters, B.D.

    2015-01-01

    Photocatalysis is highly promising as a technology to mitigate environmental pollution. In this thesis the focus will be on air purification by photocatalytic oxidation of volatile organic compounds (VOC’s). A major challenge in photocatalysis is the low photonic efficiency, due to high electron

  18. An automated synthesis-purification-sample-management platform for the accelerated generation of pharmaceutical candidates.

    Science.gov (United States)

    Sutherland, J David; Tu, Noah P; Nemcek, Thomas A; Searle, Philip A; Hochlowski, Jill E; Djuric, Stevan W; Pan, Jeffrey Y

    2014-04-01

    A flexible and integrated flow-chemistry-synthesis-purification compound-generation and sample-management platform has been developed to accelerate the production of small-molecule organic-compound drug candidates in pharmaceutical research. Central to the integrated system is a Mitsubishi robot, which hands off samples throughout the process to the next station, including synthesis and purification, sample dispensing for purity and quantification analysis, dry-down, and aliquot generation.

  19. Enterococcus faecalis Sex Pheromone cCF10 Enhances Conjugative Plasmid Transfer In Vivo.

    Science.gov (United States)

    Hirt, Helmut; Greenwood-Quaintance, Kerryl E; Karau, Melissa J; Till, Lisa M; Kashyap, Purna C; Patel, Robin; Dunny, Gary M

    2018-02-13

    Cell-cell communication mediated by peptide pheromones (cCF10 [CF]) is essential for high-frequency plasmid transfer in vitro in Enterococcus faecalis To examine the role of pheromone signaling in vivo , we established either a CF-producing (CF+) recipient or a recipient producing a biologically inactive variant of CF (CF- recipient) in a germfree mouse model 3 days before donor inoculation and determined transfer frequencies of the pheromone-inducible plasmid pCF10. Plasmid transfer was detected in the upper and middle sections of the intestinal tract 5 h after donor inoculation and was highly efficient in the absence of antibiotic selection. The transconjugant/donor ratio reached a maximum level approaching 1 on day 4 in the upper intestinal tract. Plasmid transfer was significantly lower with the CF- recipient. While rescue of the CF- mating defect by coculture with CF+ recipients is easily accomplished in vitro , no extracellular complementation occurred in vivo This suggests that most pheromone signaling in the gut occurs between recipient and donor cells in very close proximity. Plasmid-bearing cells (donors plus transconjugants) steadily increased in the population from 0.1% after donor inoculation to about 10% at the conclusion of the experiments. This suggests a selective advantage of pCF10 carriage distinct from antibiotic resistance or bacteriocin production. Our results demonstrate that pheromone signaling is required for efficient pCF10 transfer in vivo In the absence of CF+ recipients, a low level of transfer to CF- recipients occurred in the gut. This may result from low-level host-mediated induction of the donors in the gastrointestinal (GI) tract, similar to that previously observed in serum. IMPORTANCE Horizontal gene transfer is a major factor in the biology of Enterococcus faecalis , an important nosocomial pathogen. Previous studies showing efficient conjugative plasmid transfer in the gastrointestinal (GI) tracts of experimental animals did

  20. Standardized Cloning and Curing of Plasmids

    DEFF Research Database (Denmark)

    Lauritsen, Ida; Kim, Se Hyeuk; Porse, Andreas

    2018-01-01

    and exchange of genetic parts in the Standard European Vectors Architecture (SEVA) vector system. Additionally, to facilitate rapid testing and iterative bioengineering using different vector designs, we provide a one-step protocol for a universal CRISPR-Cas9-based plasmid curing system (pFREE) and demonstrate...

  1. Strong ion exchange in centrifugal partition extraction (SIX-CPE): effect of partition cell design and dimensions on purification process efficiency.

    Science.gov (United States)

    Hamzaoui, Mahmoud; Hubert, Jane; Reynaud, Romain; Marchal, Luc; Foucault, Alain; Renault, Jean-Hugues

    2012-07-20

    The aim of this article was to evaluate the influence of the column design of a hydrostatic support-free liquid-liquid chromatography device on the process efficiency when the strong ion-exchange (SIX) development mode is used. The purification of p-hydroxybenzylglucosinolate (sinalbin) from a crude aqueous extract of white mustard seeds (Sinapis alba L.) was achieved on two types of devices: a centrifugal partition chromatograph (CPC) and a centrifugal partition extractor (CPE). They differ in the number, volume and geometry of their partition cells. The SIX-CPE process was evaluated in terms of productivity and sinalbin purification capability as compared to previously optimized SIX-CPC protocols that were carried out on columns of 200 mL and 5700 mL inner volume, respectively. The objective was to determine whether the decrease in partition cell number, the increase in their volume and the use of a "twin cell" design would induce a significant increase in productivity by applying higher mobile phase flow rate while maintaining a constant separation quality. 4.6g of sinalbin (92% recovery) were isolated from 25 g of a crude white mustard seed extract, in only 32 min and with a purity of 94.7%, thus corresponding to a productivity of 28 g per hour and per liter of column volume (g/h/LV(c)). Therefore, the SIX-CPE process demonstrates promising industrial technology transfer perspectives for the large-scale isolation of ionized natural products. Copyright © 2012 Elsevier B.V. All rights reserved.

  2. Microfluidic cartridges for DNA purification and genotyping processed in standard laboratory instruments

    Science.gov (United States)

    Focke, Maximilian; Mark, Daniel; Stumpf, Fabian; Müller, Martina; Roth, Günter; Zengerle, Roland; von Stetten, Felix

    2011-06-01

    Two microfluidic cartridges intended for upgrading standard laboratory instruments with automated liquid handling capability by use of centrifugal forces are presented. The first microfluidic cartridge enables purification of DNA from human whole blood and is operated in a standard laboratory centrifuge. The second microfluidic catridge enables genotyping of pathogens by geometrically multiplexed real-time PCR. It is operated in a slightly modified off-the-shelf thermal cycler. Both solutions aim at smart and cost-efficient ways to automate work flows in laboratories. The DNA purification cartridge automates all liquid handling steps starting from a lysed blood sample to PCR ready DNA. The cartridge contains two manually crushable glass ampoules with liquid reagents. The DNA yield extracted from a 32 μl blood sample is 192 +/- 30 ng which corresponds to 53 +/- 8% of a reference extraction. The genotyping cartridge is applied to analyse isolates of the multi-resistant Staphyloccus aureus (MRSA) by real-time PCR. The wells contain pre-stored dry reagents such as primers and probes. Evaluation of the system with 44 genotyping assays showed a 100% specificity and agreement with the reference assays in standard tubes. The lower limit of detection was well below 10 copies of DNA per reaction.

  3. Characterization of a plasmid carrying cat, ermB and tetS genes in a foodborne Listeria monocytogenes strain and uptake of the plasmid by cariogenic Streptococcus mutans

    DEFF Research Database (Denmark)

    Li, Lili; Olsen, Rikke Heidemann; Shi, Lei

    2016-01-01

    A multi-drug resistant (MDR) Listeria monocytogenes isolate (serotype 1/2c) was recovered from a quick-frozen rice flour product collected from Langfang city in northern China. PCR screening identified the presence of cat, ermB and tetS genes. The plasmid profile of the strain showed the presence...... of an approximately 22.4-kb plasmid. Curing of this plasmid resulted in the loss of cat, ermB and tetS genes and increased susceptibility to several antibiotics, suggesting the involvement of the plasmid in multiple antibiotic resistances. Moreover, the plasmid was able to be uptaken by human oral pathogen...

  4. Autonomous replication of plasmids bearing monkey DNA origin-enriched sequences

    International Nuclear Information System (INIS)

    Frappier, L.; Zannis-Hadjopoulos, M.

    1987-01-01

    Twelve clones of origin-enriched sequences (ORS) isolated from early replicating monkey (CV-1) DNA were examined for transient episomal replication in transfected CV-1, COS-7, and HeLa cells. Plasmid DNA was isolated at time intervals after transfection and screened by the Dpn I resistance assay or by the bromodeoxyuridine substitution assay to differentiate between input and replicated DNA. The authors have identified four monkey ORS (ORS3, -8, -9, and -12) that can support plasmid replication in mammalian cells. This replication is carried out in a controlled and semiconservative manner characteristic of mammalian replicons. ORS replication was most efficient in HeLa cells. Electron microscopy showed ORS8 and ORS12 plasmids of the correct size with replication bubbles. Using a unique restriction site in ORS12, we have mapped the replication bubble within the monkey DNA sequence

  5. Plasmids replicatable in Bacillus subtilis, E. coli and lactic acid streptococcus bacteria

    NARCIS (Netherlands)

    Kok, Jan; Maat, Jan; van der Vossen, Josephus Mauritius; Venema, Gerard

    1997-01-01

    The claimed invention is drawn to a recombinant plasmid which can replicate in Bacillus subtilis, Escherichia coli, and lactic acid Streptococcus bacteria comprising the replication of origin from Streptococcus cremoris plasmid pWV01 as its origin of replication, in addition to coding marker genes

  6. Plasma exhaust purification by thermal swing adsorption: Experimental results and modeling

    International Nuclear Information System (INIS)

    Ricapito, I.; Malara, R.C.

    1996-01-01

    For several years at the Joint Research Centre-Ispra laboratories, cyclic adsorption processes have been developed for the purification of the plasma exhaust stream of a deuterium-tritium fusion reactor. A purification process consisting of two coupled thermal swing adsorption systems seemed to be the most convenient process. In this context, a screening study was carried out to select the most suitable adsorbent materials and appropriate working temperatures. This was mainly done by experimental measurements of adsorption isotherms of the single components of the plasma exhaust stream and by a careful evaluation of the multicomponent adsorption equilibria. Experiments on adsorption dynamics were carried out in a pilot plant to demonstrate the feasibility and to evaluate the performance of the process. The experimental apparatus was designed to treat gas mixture flow rates up to 20 to 30 standard temperature and pressure l/h. A mathematical model was developed and tested against the experimental results to describe the adsorption process and, in particular, to evaluate and to optimize the process cycle time. 27 refs., 4 figs., 9 tabs

  7. Construction of pTM series plasmids for gene expression in Brucella species.

    Science.gov (United States)

    Tian, Mingxing; Qu, Jing; Bao, Yanqing; Gao, Jianpeng; Liu, Jiameng; Wang, Shaohui; Sun, Yingjie; Ding, Chan; Yu, Shengqing

    2016-04-01

    Brucellosis, the most common widespread zoonotic disease, is caused by Brucella spp., which are facultative, intracellular, Gram-negative bacteria. With the development of molecular biology techniques, more and more virulence-associated factors have been identified in Brucella spp. A suitable plasmid system is an important tool to study virulence genes in Brucella. In this study, we constructed three constitutive replication plasmids (pTM1-Cm, pTM2-Amp, and pTM3-Km) using the replication origin (rep) region derived from the pBBR1-MCS vector. Also, a DNA fragment containing multiple cloning sites (MCSs) and a terminator sequence derived from the pCold vector were produced for complementation of the deleted genes. Besides pGH-6×His, a plasmid containing the groE promoter of Brucella spp. was constructed to express exogenous proteins in Brucella with high efficiency. Furthermore, we constructed the inducible expression plasmid pZT-6×His, containing the tetracycline-inducible promoter pzt1, which can induce expression by the addition of tetracycline in the Brucella culture medium. The constructed pTM series plasmids will play an important role in the functional investigation of Brucella spp. Copyright © 2016 Elsevier B.V. All rights reserved.

  8. Phytofilter - environmental friendly solution for purification of surface plate from urbanized territories

    Science.gov (United States)

    Ruchkinova, O.; Shchuckin, I.

    2017-06-01

    Its proved, that phytofilters are environmental friendly solution of problem of purification of surface plate from urbanized territories. Phytofilters answer the nowadays purposes to systems of purification of land drainage. The main problem of it is restrictions, connecter with its use in the conditions of cold temperature. Manufactured a technology and mechanism, which provide a whole-year purification of surface plate and its storage. Experimentally stated optimal makeup of filtering load: peat, zeolite and sand in per cent of volume, which provides defined hydraulic characteristics. Stated sorbate and ion-selective volume of complex filtering load of ordered composition in dynamic conditions. Estimated dependences of exit concentrations of oil products and heavy metals on temperature by filtering through complex filtering load of ordered composition. Defined effectiveness of purification at phytofiltering installation. Fixed an influence of embryophytes on process of phytogeneration and capacity of filtering load. Recommended swamp iris, mace reed and reed grass. Manufactured phytofilter calculation methodology. Calculated economic effect from use of phytofiltration technology in comparison with traditional block-modular installations.

  9. Structural and functional analysis of the kid toxin protein from E. coli Plasmid R1

    NARCIS (Netherlands)

    Hargreaves, D.; Santos-Sierra, S.; Giraldo, R.; Sabariegos-Jareño, R.; de la Cueva-Méndez, G.; Boelens, R.|info:eu-repo/dai/nl/070151407; Díaz-Orejas, R.; Rafferty, J.B.

    2002-01-01

    We have determined the structure of Kid toxin protein from E. coli plasmid R1 involved in stable plasmid inheritance by postsegregational killing of plasmid-less daughter cells. Kid forms a two-component system with its antagonist, Kis antitoxin. Our 1.4 Å crystal structure of Kid reveals a 2-fold

  10. APPLICATION OF PAN/PANI COMPOSITE MEMBRANES IN PURIFICATION OF INDUSTRIAL WASTEWATER GENERATED DURING PROCESSING OF METALS

    Directory of Open Access Journals (Sweden)

    Beata Fryczkowska

    2017-04-01

    Full Text Available The paper presents results of research on the use of composite membranes of polyacrylonitrile (PAN doped polyaniline (PANI to remove contaminations of industrial wastewater generated during the processing of metals. Wastewater obtained from industry was pre-treated with the flocculant Magnafloc®336, and then the supernatant solution was introduced into the ultrafiltration cell, AMICON (Millipore equipped in the previously prepared polymer membrane. Using spectrophotometer UV-Vis (HACH and atomic absorption spectrometry (AAS pollution indicators was marked before and after the integrated purification proces, to determine the degree of removal of selected ions from wastewater. As a result of flocculation from wastewater there have been removed phosphates (79%, chlorides (11-14%, sulfates (2-10% and iron (36-92%, cobalt (~ 80%, cadmium (~ 31% and nickel (~ 25%. However, the pressure membrane process almost completely removed zinc, copper and cadmium (~ 100%, iron (by a further 43-69% and phosphate anions, which was a little.

  11. Electron-stimulated purification of platinum nanostructures grown via focused electron beam induced deposition

    Directory of Open Access Journals (Sweden)

    Brett B. Lewis

    2015-04-01

    Full Text Available Platinum–carbon nanostructures deposited via electron beam induced deposition from MeCpPt(IVMe3 are purified during a post-deposition electron exposure treatment in a localized oxygen ambient at room temperature. Time-dependent studies demonstrate that the process occurs from the top–down. Electron beam energy and current studies demonstrate that the process is controlled by a confluence of the electron energy loss and oxygen concentration. Furthermore, the experimental results are modeled as a 2nd order reaction which is dependent on both the electron energy loss density and the oxygen concentration. In addition to purification, the post-deposition electron stimulated oxygen purification process enhances the resolution of the EBID process due to the isotropic carbon removal from the as-deposited materials which produces high-fidelity shape retention.

  12. Evaluation of the effect of non-B DNA structures on plasmid integrity via accelerated stability studies.

    Science.gov (United States)

    Ribeiro, S C; Monteiro, G A; Prazeres, D M F

    2009-04-01

    Plasmid biopharmaceuticals are a new class of medicines with an enormous potential. Attempts to increase the physical stability of highly purified supercoiled (SC) plasmid DNA in pharmaceutical aqueous solutions have relied on: (i) changing the DNA sequence, (ii) improving manufacturing to reduce deleterious impurities and initial DNA damage, and (iii) controlling the storage medium characteristics. In this work we analyzed the role of secondary structures on the degradation of plasmid molecules. Accelerated stability experiments were performed with SC, open circular (OC) and linear (L) isoforms of three plasmids which differed only in the "single-strandlike" content of their polyadenylation (poly A) signals. We have proved that the presence of more altered or interrupted (non-B) DNA secondary structures did not directly translate into an easier strand scission of the SC isoforms. Rather, those unusual structures imposed a lower degree of SC in the plasmids, leading to an increase in their resistance to thermal degradation. However, this behavior was reversed when the relaxed or L isoforms were tested, in which case the absence of SC rendered the plasmids essentially double-stranded. Overall, this work suggests that plasmid DNA sequence and secondary structures should be taken into account in future investigations of plasmid stability during prolonged storage.

  13. trans-Acting Virulence Functions of the Octopine Ti Plasmid from Agrobacterium tumefaciens

    NARCIS (Netherlands)

    Hille, Jacques; Kan, Jan van; Schilperoort, Rob

    1984-01-01

    All Ti plasmid-encoded virulence functions that were studied act in trans. An octopine Ti plasmid-specific vir operon, called vir-O, located on an EcoRI restriction fragment has been characterized. Sequences with promoter activity in Escherichia coli were identified for a second vir operon, called

  14. Photocatalytic materials and technologies for air purification.

    Science.gov (United States)

    Ren, Hangjuan; Koshy, Pramod; Chen, Wen-Fan; Qi, Shaohua; Sorrell, Charles Christopher

    2017-03-05

    Since there is increasing concern for the impact of air quality on human health, the present work surveys the materials and technologies for air purification using photocatalytic materials. The coverage includes (1) current photocatalytic materials for the decomposition of chemical contaminants and disinfection of pathogens present in air and (2) photocatalytic air purification systems that are used currently and under development. The present work focuses on five main themes. First, the mechanisms of photodegradation and photodisinfection are explained. Second, system designs for photocatalytic air purification are surveyed. Third, the photocatalytic materials used for air purification and their characteristics are considered, including both conventional and more recently developed photocatalysts. Fourth, the methods used to fabricate these materials are discussed. Fifth, the most significant coverage is devoted to materials design strategies aimed at improving the performance of photocatalysts for air purification. The review concludes with a brief consideration of promising future directions for materials research in photocatalysis. Copyright © 2016 Elsevier B.V. All rights reserved.

  15. Phase equilibria at the Zirconium metal purification

    International Nuclear Information System (INIS)

    Dwiretnani-Sudjoko; Busron-Masduki; Sunardjo; Budi-Sulistyo

    1996-01-01

    It was investigated the research in the purification of zirconium metal, which was results from the reduction process, by adding heat in the vacuum environment. The process was done in batch in the stainless steel reactor, equiped with vacuum pump and electric heater. The investigated variable were process temperature and pressure. From this research it was obtained that equilibrium constant for MgCl 2 and Mg were expressed in the equation K M g C l 2 = 0.9011 P 1 .3779 1.06552 T and K M g = 6.0115P + 1.35256T - 6.93912

  16. Purification of HGI2 for nuclear detector fabrication

    International Nuclear Information System (INIS)

    Schieber, M.M.

    1978-01-01

    A process for purification of mercuric iodide (HgI 2 ) to be used as a source material for the growth of detector quality crystals. The high purity HgI 2 raw material is produced by a combination of three stages: synthesis of HgI 2 from Hg and I 2 , repeated sublimation, and zone refining

  17. Tn5-induced pBS286 plasmid mutations blocking early stages of napthalene oxidation

    International Nuclear Information System (INIS)

    Kosheleva, I.A.; Tsoi, T.V.; Ivashina, T.V.; Selifonov, S.A.; Starovoitov, I.I.; Boronin, A.M.

    1988-01-01

    The authors present data on the further analysis of the structural and functional organization of the nah region of plasmid pBS286 controlling the constitutive oxidation of naphthalene by Pseudomonas putida cells. They have studied Tn5-induced mutations blocking early stages of naphthalene oxidation. They present and discuss data providing evidence that, in contrast to plasmid NAH7, the mechanism of regulation of the nahl operon of plasmid NPL-1, the parent plasmid of plasmid pBS286, with inducible synthesis of naphthalene dioxygenase can include elements of a negative control with participation of the regulatory locus R, located proximal to the structural nah genes and closely linked to or overlapped by the inverted control DNA segment (4.2 kb). They also present data on the possibility of regulation of the activity of the catechol-splitting meta-pathway genes with the participation of products of early stages of naphthalene oxidation

  18. Minimal and contributing sequence determinants of the cis-acting locus of transfer (clt) of streptomycete plasmid pIJ101 occur within an intrinsically curved plasmid region.

    Science.gov (United States)

    Ducote, M J; Prakash, S; Pettis, G S

    2000-12-01

    Efficient interbacterial transfer of streptomycete plasmid pIJ101 requires the pIJ101 tra gene, as well as a cis-acting plasmid function known as clt. Here we show that the minimal pIJ101 clt locus consists of a sequence no greater than 54 bp in size that includes essential inverted-repeat and direct-repeat sequences and is located in close proximity to the 3' end of the korB regulatory gene. Evidence that sequences extending beyond the minimal locus and into the korB open reading frame influence clt transfer function and demonstration that clt-korB sequences are intrinsically curved raise the possibility that higher-order structuring of DNA and protein within this plasmid region may be an inherent feature of efficient pIJ101 transfer.

  19. Cloning in Streptococcus lactis of plasmid-mediated UV resistance and effect on prophage stability

    International Nuclear Information System (INIS)

    Chopin, M.C.; Chopin, A.; Rouault, A.; Simon, D.

    1986-01-01

    Plasmid pIL7 (33 kilobases) from Streptococcus lactis enhances UV resistance and prophage stability. A 5.4-kilobase pIL7 fragment carrying genes coding for both characters was cloned into S. lactis, using plasmid pHV1301 as the cloning vector. The recombinant plasmid was subsequently transferred to three other S. lactis strains by transformation or protoplast fusion. Cloned genes were expressed in all tested strains

  20. Human Intestinal Cells Modulate Conjugational Transfer of Multidrug Resistance Plasmids between Clinical Escherichia coli Isolates

    DEFF Research Database (Denmark)

    Machado, Ana Manuel; Sommer, Morten

    2014-01-01

    Bacterial conjugation in the human gut microbiota is believed to play a major role in the dissemination of antibiotic resistance genes and virulence plasmids. However, the modulation of bacterial conjugation by the human host remains poorly understood and there is a need for controlled systems...... to study this process. We established an in vitro co-culture system to study the interaction between human intestinal cells and bacteria. We show that the conjugation efficiency of a plasmid encoding an extended spectrum beta-lactamase is reduced when clinical isolates of Escherichia coli are co...... of the intestinal cells exposed to bacteria leading to a two-fold reduction in conjugation efficiency. These results show that human gut epithelial cells can modulate bacterial conjugation and may have relevance to gene exchange in the gut....

  1. Effects of Two Purification Pretreatments on Electroless Copper Coating over Single-Walled Carbon Nano tubes

    International Nuclear Information System (INIS)

    Zheng, Z.; Li, L.; Dong, Sh.; Li, Sh.; Xiao, A.; Sun, Sh.

    2014-01-01

    To achieve the reinforcement of copper matrix composite by single-walled carbon nano tubes, a three-step-refluxing purification of carbon nano tubes sample with HNO 3 -NaOH-HCl was proposed and demonstrated. A previously reported purification process using an electromagnetic stirring with H 2 O 2 /HCl mixture was also repeated. Then, the purified carbon nano tubes were coated with copper by the same electroless plating process. At the end, the effects of the method on carbon nano tubes themselves and on copper coating were determined by transmission electron microscope spectroscopy, scanning electron microscope spectroscopy, X-ray diffractometry, thermogravimetric analysis, Fourier transformed infrared spectroscopy, and energy dispersive spectrometry. It was clearly confirmed that both of the two processes could remove most of iron catalyst particles and carbonaceous impurities without significant damage to carbon nano tubes. The thermal stability of the sample purified by H 2 O 2 /HCl treatment was slightly higher than that purified by HNO 3 -NaOH-HCl treatment. Nevertheless, the purification by HNO 3 -NaOH-HCl treatment was more effective for carboxyl functionalization on nano tubes than that by H 2 O 2 /HCl treatment. The Cu-coating on carbon nano tubes purified by both purification processes was complete, homogenous, and continuous. However, the Cu-coating on carbon nano tubes purified by H 2 O 2 /HCl was oxidized more seriously than those on carbon nano tubes purified by HNO 3 -NaOH-HCl treatment.

  2. Plasmid DNA damage by heavy ions at spread-out Bragg peak energies

    NARCIS (Netherlands)

    Dang, H. M.; van Goethem, M. J.; van der Graaf, E. R.; Brandenburg, S.; Hoekstra, R.; Schlatholter, T.

    2010-01-01

    Interaction of ionizing radiation with plasmid DNA can lead to formation of single strand breaks, double strand breaks and clustered lesions. We have investigated the response of the synthetic plasmid pBR322 in aqueous solution upon irradiation with (12)C ions under spread-out Bragg peak conditions

  3. Presence and analysis of plasmids in human and animal associated Arcobacter species

    DEFF Research Database (Denmark)

    Douidah, Laid; De Zutter, Lieven; Van Nieuwerburgh, Filip

    2014-01-01

    coding information, which contrasts to the 20 to 30% for the small plasmids. Some of the open reading frames showed a high homology to putative conserved domains found in other related organisms, such as replication, mobilization and genes involved in type IV secretion system. The large plasmid carried...

  4. High-throughput screening of cellulase F mutants from multiplexed plasmid sets using an automated plate assay on a functional proteomic robotic workcell

    Directory of Open Access Journals (Sweden)

    Qureshi Nasib

    2006-05-01

    Full Text Available Abstract Background The field of plasmid-based functional proteomics requires the rapid assay of proteins expressed from plasmid libraries. Automation is essential since large sets of mutant open reading frames are being cloned for evaluation. To date no integrated automated platform is available to carry out the entire process including production of plasmid libraries, expression of cloned genes, and functional testing of expressed proteins. Results We used a functional proteomic assay in a multiplexed setting on an integrated plasmid-based robotic workcell for high-throughput screening of mutants of cellulase F, an endoglucanase from the anaerobic fungus Orpinomyces PC-2. This allowed us to identify plasmids containing optimized clones expressing mutants with improved activity at lower pH. A plasmid library of mutagenized clones of the celF gene with targeted variations in the last four codons was constructed by site-directed PCR mutagenesis and transformed into Escherichia coli. A robotic picker integrated into the workcell was used to inoculate medium in a 96-well deep well plate, combining the transformants into a multiplexed set in each well, and the plate was incubated on the workcell. Plasmids were prepared from the multiplexed culture on the liquid handler component of the workcell and used for in vitro transcription/translation. The multiplexed expressed recombinant proteins were screened for improved activity and stability in an azo-carboxymethylcellulose plate assay. The multiplexed wells containing mutants with improved activity were identified and linked back to the corresponding multiplexed cultures stored in glycerol. Spread plates were prepared from the glycerol stocks and the workcell was used to pick single colonies from the spread plates, prepare plasmid, produce recombinant protein, and assay for activity. The screening assay and subsequent deconvolution of the multiplexed wells resulted in identification of improved Cel

  5. Virus-sized self-assembling lamellar complexes between plasmid DNA and cationic micelles promote gene transfer

    Science.gov (United States)

    Pitard, Bruno; Aguerre, Olivier; Airiau, Marc; Lachagès, Anne-Marie; Boukhnikachvili, Tsiala; Byk, Gérardo; Dubertret, Catherine; Herviou, Christian; Scherman, Daniel; Mayaux, Jean-François; Crouzet, Joël

    1997-01-01

    Gene therapy is based on the vectorization of genes to target cells and their subsequent expression. Cationic amphiphile-mediated delivery of plasmid DNA is the nonviral gene transfer method most often used. We examined the supramolecular structure of lipopolyamine/plasmid DNA complexes under various condensing conditions. Plasmid DNA complexation with lipopolyamine micelles whose mean diameter was 5 nm revealed three domains, depending on the lipopolyamine/plasmid DNA ratio. These domains respectively corresponded to negatively, neutrally, and positively charged complexes. Transmission electron microscopy and x-ray scattering experiments on complexes originating from these three domains showed that although their morphology depends on the lipopolyamine/plasmid DNA ratio, their particle structure consists of ordered domains characterized by even spacing of 80 Å, irrespective of the lipid/DNA ratio. The most active lipopolyamine/DNA complexes for gene transfer were positively charged. They were characterized by fully condensed DNA inside spherical particles (diameter: 50 nm) sandwiched between lipid bilayers. These results show that supercoiled plasmid DNA is able to transform lipopolyamine micelles into a supramolecular organization characterized by ordered lamellar domains. PMID:9405626

  6. Sequence analysis and characterization of rolling-circle replicating plasmid pVCM01 from Salmonella enterica

    Directory of Open Access Journals (Sweden)

    Penido, A. F. B.

    2013-12-01

    Full Text Available Aims: Characterization of cryptic plasmid pVCM01 (accession number JX133088 isolated from Salmonella enterica Enteritidis. Methodology and results: The complete sequence of pVCM01 was obtained. This plasmid possesses 1981 bp, with G+C content of 57% in agreement of the range of Salmonella genomic DNA. pVCM01 has a high degree of similarity to pB and pJ plasmids. It possesses six main open reading frames, only one have a very high degree of amino acid identity with protein involved in the rolling-circle-like replication (RCR. Based on the sequence similarities, pVCM01 plasmid belonged to the pC194/pUB110 rolling-circle replicating plasmid family. The Rep pVCM01 possesses the motifs: FLTLTVRN, HPHFHTL, SGDGYVKHERW, which were present in all Rep proteins. Conclusion, significance and impact of study: The small size of pVCM01 plasmid and its stability in E. coli cells, make it an attractive candidate to develop new vectors, such as cloning and/or expression vector.

  7. Energy Efficient Bioethanol Purification by Heat Pump Assisted Extractive Distillation

    NARCIS (Netherlands)

    Kiss, Anton A.; Luo, Hao; Bildea, Costin Sorin

    2015-01-01

    The purification of bioethanol fuel requires an energy demanding separation process to concentrate the diluted streams obtained in the fermentation stage and to overcome the azeotropic behaviour of ethanol-water mixture. The classic separation sequence consists of three distillation columns that

  8. The large universal Pantoea plasmid LPP-1 plays a major role in biological and ecological diversification

    Directory of Open Access Journals (Sweden)

    De Maayer Pieter

    2012-11-01

    Full Text Available Abstract Background Pantoea spp. are frequently isolated from a wide range of ecological niches and have various biological roles, as plant epi- or endophytes, biocontrol agents, plant-growth promoters or as pathogens of both plant and animal hosts. This suggests that members of this genus have undergone extensive genotypic diversification. One means by which this occurs among bacteria is through the acquisition and maintenance of plasmids. Here, we have analyzed and compared the sequences of a large plasmid common to all sequenced Pantoea spp. Results and discussion The Large PantoeaPlasmids (LPP-1 of twenty strains encompassing seven different Pantoea species, including pathogens and endo-/epiphytes of a wide range of plant hosts as well as insect-associated strains, were compared. The LPP-1 plasmid sequences range in size from ~281 to 794 kb and carry between 238 and 750 protein coding sequences (CDS. A core set of 46 proteins, encompassing 2.2% of the total pan-plasmid (2,095 CDS, conserved among all LPP-1 plasmid sequences, includes those required for thiamine and pigment biosynthesis. Phylogenetic analysis reveals that these plasmids have arisen from an ancestral plasmid, which has undergone extensive diversification. Analysis of the proteins encoded on LPP-1 also showed that these plasmids contribute to a wide range of Pantoea phenotypes, including the transport and catabolism of various substrates, inorganic ion assimilation, resistance to antibiotics and heavy metals, colonization and persistence in the host and environment, pathogenesis and antibiosis. Conclusions LPP-1 is universal to all Pantoea spp. whose genomes have been sequenced to date and is derived from an ancestral plasmid. LPP-1 encodes a large array of proteins that have played a major role in the adaptation of the different Pantoea spp. to their various ecological niches and their specialization as pathogens, biocontrol agents or benign saprophytes found in many diverse

  9. [Isolation and characterization of petroleum catabolic broad-host-range plasmids from Shen-Fu wastewater irrigation zone].

    Science.gov (United States)

    Wang, Ya-Fei; Wang, Ya-Fei; Li, Hui; Li, Xiao-Bin

    2013-11-01

    Based on triparental mating, we isolated a total of eight broad host range (BHR) petroleum hydrocarbon catabolic plasmids from the soils, sediments, and wastewater samples in the Shen-Fu irrigation zone. The antibiotic resistance of the plasmids was tested, and then, the plasmids were transferred to Escherichia coli EC100. The plasmids carrying no antibiotic resistance were tagged by miniTn5 transposon consisting of antibiotic resistant genes. The PCR-based incompatibility test revealed that the pS3-2C and pS4-6G belonged to Inc P group, the pS3-2G, pW22-3G, and pA15-7G belonged to Inc N group, the pS7-2G was identified as Inc W plasmid, and the pA23-1G and pA10-1C were placed into Inc Q group. By adopting the reported PCR amplification methods of petroleum hydrocarbon-degrading catabolic genes, the petroleum-degrading capability of these BHR plasmids were preliminarily analyzed. The plasmids pS3-2G, pS7-2G, pA23-1G, pW22-3G, and pA10-1C carried aromatic ring- hydroxylating dioxygenase gene phdA and toluene monooxygenase gene touA; the plasmid pA15-7G carried touA and toluene dioxygenase gene tod; the plasmid pS3-2C carried ben, phdA, and tod; whereas the pS4-6G only carried ben. The host range test showed that all the isolated plasmids except pS3-2C could be transferred and maintained stably in the representative strains Agrobacterium tumefaciens C58, Cupriavidus necator JMP228, and E. coli EC100 of the alpha-, beta-, and gamma-Proteobacteria, respectively.

  10. Very low-energy and low-fluence ion beam bombardment of naked plasmid DNA

    International Nuclear Information System (INIS)

    Norarat, R.; Semsang, N.; Anuntalabhochai, S.; Yu, L.D.

    2009-01-01

    Ion beam bombardment of biological organisms has been recently applied to mutation breeding of both agricultural and horticultural plants. In order to explore relevant mechanisms, this study employed low-energy ion beams to bombard naked plasmid DNA. The study aimed at simulation of the final stage of the process of the ion beam bombardment of real cells to check whether and how very low-energy and low-fluence of ions can induce mutation. Argon and nitrogen ions at 5 keV and 2.5 keV respectively bombarded naked plasmid DNA pGFP to very low-fluences, an order of 10 13 ions/cm 2 . Subsequently, DNA states were analyzed using electrophoresis. Results provided evidences that the very low-energy and low-fluence ion bombardment indeed altered the DNA structure from supercoil to short linear fragments through multiple double strand breaks and thus induced mutation, which was confirmed by transfer of the bombarded DNA into bacteria Escherichia coli and subsequent expression of the marker gene.

  11. Transformation of UV-hypersensitive Chinese hamster ovary cell mutants with UV-irradiated plasmids

    International Nuclear Information System (INIS)

    Nairn, R.S.; Humphrey, R.M.; Adair, G.M.

    1988-01-01

    Transfection of UV-hypersensitive, DNA repair-deficient Chinese hamster ovary (CHO) cell lines and parental, repair-proficient CHO cells with UV-irradiated pHaprt-1 or pSV2gpt plasmids resulted in different responses by recipient cell lines to UV damage in transfected DNA. Unlike results reported for human cells, UV irradiation of transfecting DNA did not stimulate genetic transformation of CHO recipient cells. In repair-deficient CHO cells, proportionally fewer transformants were produced with increasing UV damage than in repair-proficient cells in transfections with UV-irradiated hamster adenine phosphoribosyltransferase (APRT) gene contained in plasmid pHaprt-1. Transfection of CHO cells with UV-irradiated pSV2gpt resulted in neither decline in transformation frequencies in repair-deficient cell lines relative to repair-proficient cells nor stimulation of genetic transformation by UV damage in the plasmid. Blot hybridization analysis of DNA samples isolated from transformed cells showed no dramatic changes in copy number or arrangement of transfected plasmid DNA with increasing UV dose. The authors conclude responses of recipient cells to UV-damaged transfecting plasmids depend on type of recipient cell and characteristics of the genetic sequence used for transfection. (author)

  12. Structures of actin-like ParM filaments show architecture of plasmid-segregating spindles.

    Science.gov (United States)

    Bharat, Tanmay A M; Murshudov, Garib N; Sachse, Carsten; Löwe, Jan

    2015-07-02

    Active segregation of Escherichia coli low-copy-number plasmid R1 involves formation of a bipolar spindle made of left-handed double-helical actin-like ParM filaments. ParR links the filaments with centromeric parC plasmid DNA, while facilitating the addition of subunits to ParM filaments. Growing ParMRC spindles push sister plasmids to the cell poles. Here, using modern electron cryomicroscopy methods, we investigate the structures and arrangements of ParM filaments in vitro and in cells, revealing at near-atomic resolution how subunits and filaments come together to produce the simplest known mitotic machinery. To understand the mechanism of dynamic instability, we determine structures of ParM filaments in different nucleotide states. The structure of filaments bound to the ATP analogue AMPPNP is determined at 4.3 Å resolution and refined. The ParM filament structure shows strong longitudinal interfaces and weaker lateral interactions. Also using electron cryomicroscopy, we reconstruct ParM doublets forming antiparallel spindles. Finally, with whole-cell electron cryotomography, we show that doublets are abundant in bacterial cells containing low-copy-number plasmids with the ParMRC locus, leading to an asynchronous model of R1 plasmid segregation.

  13. Two highly divergent lineages of exfoliative toxin B-encoding plasmids revealed in impetigo strains of Staphylococcus aureus.

    Science.gov (United States)

    Botka, Tibor; Růžičková, Vladislava; Svobodová, Karla; Pantůček, Roman; Petráš, Petr; Čejková, Darina; Doškař, Jiří

    2017-09-01

    Exfoliative toxin B (ETB) encoded by some large plasmids plays a crucial role in epidermolytic diseases caused by Staphylococcus aureus. We have found as yet unknown types of etb gene-positive plasmids isolated from a set of impetigo strains implicated in outbreaks of pemphigus neonatorum in Czech maternity hospitals. Plasmids from the strains of clonal complex CC121 were related to archetypal plasmid pETB TY4 . Sharing a 33-kb core sequence including virulence genes for ETB, EDIN C, and lantibiotics, they were assigned to a stand-alone lineage, named pETB TY4 -based plasmids. Differing from each other in the content of variable DNA regions, they formed four sequence types. In addition to them, a novel unique plasmid pETB608 isolated from a strain of ST130 was described. Carrying conjugative cluster genes, as well as new variants of etb and edinA genes, pETB608 could be regarded as a source of a new lineage of ETB plasmids. We have designed a helpful detection assay, which facilitates the precise identification of the all described types of ETB plasmids. Copyright © 2017 Elsevier GmbH. All rights reserved.

  14. Growth dependence of conjugation explains limited plasmid invasion in biofilms: an individual‐based modelling study

    DEFF Research Database (Denmark)

    Merkey, Brian; Lardon, Laurent; Seoane, Jose Miguel

    2011-01-01

    Plasmid invasion in biofilms is often surprisingly limited in spite of the close contact of cells in a biofilm. We hypothesized that this poor plasmid spread into deeper biofilm layers is caused by a dependence of conjugation on the growth rate (relative to the maximum growth rate) of the donor......, we find that invasion of a resident biofilm is indeed limited when plasmid transfer depends on growth, but not so in the absence of growth dependence. Using sensitivity analysis we also find that parameters related to timing (i.e. a lag before the transconjugant can transfer, transfer proficiency...... and scan speed) and spatial reach (EPS yield, conjugal pilus length) are more important for successful plasmid invasion than the recipients' growth rate or the probability of segregational loss. While this study identifies one factor that can limit plasmid invasion in biofilms, the new individual...

  15. Development and application of a general plasmid reference material for GMO screening.

    Science.gov (United States)

    Wu, Yuhua; Li, Jun; Wang, Yulei; Li, Xiaofei; Li, Yunjing; Zhu, Li; Li, Jun; Wu, Gang

    The use of analytical controls is essential when performing GMO detection through screening tests. Additionally, the presence of taxon-specific sequences is analyzed mostly for quality control during GMO detection. In this study, 11 commonly used genetic elements involving three promoters (P-35S, P-FMV35S and P-NOS), four marker genes (Bar, NPTII, HPT and Pmi), and four terminators (T-NOS, T-35S, T-g7 and T-e9), together with the reference gene fragments from six major crops of maize, soybean, rapeseed, rice, cotton and wheat, were co-integrated into the same single plasmid to construct a general reference plasmid pBI121-Screening. The suitability test of pBI121-Screening plasmid as reference material indicated that the non-target sequence on the pBI121-Screening plasmid did not affect the PCR amplification efficiencies of screening methods and taxon-specific methods. The sensitivity of screening and taxon-specific assays ranged from 5 to 10 copies of pBI121-Screening plasmid, meeting the sensitivity requirement of GMO detection. The construction of pBI121-Screening solves the lack of a general positive control for screening tests, thereby reducing the workload and cost of preparing a plurality of the positive control. Copyright © 2016 Elsevier B.V. All rights reserved.

  16. Effect of Different Purification Techniques on the Characteristics of Heteropolysaccharide-Protein Biopolymer from Durian (Durio zibethinus Seed

    Directory of Open Access Journals (Sweden)

    Hamed Mirhosseini

    2012-09-01

    Full Text Available Natural biopolymers from plant sources contain many impurities (e.g., fat, protein, fiber, natural pigment and endogenous enzymes, therefore, an efficient purification process is recommended to minimize these impurities and consequently improve the functional properties of the biopolymer. The main objective of the present study was to investigate the effect of different purification techniques on the yield, protein content, solubility, water- and oil-holding capacity of a heteropolysaccharide-protein biopolymer obtained from durian seed. Four different purification methods using different chemicals and solvents (i.e., A (isopropanol and ethanol, B (isopropanol and acetone, C (saturated barium hydroxide, and D (Fehling solution] to liberate the purified biopolymer from its crude form were compared. In most cases, the purification process significantly (p < 0.05 improved the physicochemical properties of heteropolysaccharide-protein biopolymer from durian fruit seed. The present work showed that the precipitation using isopropanol and acetone (Method B resulted in the highest purification yield among all the tested purification techniques. The precipitation using saturated barium hydroxide (Method C led to induce the highest solubility and relatively high capacity of water absorption. The current study reveals that the precipitation using Fehling solution (Method D most efficiently eliminates the protein fraction, thus providing more pure biopolymer suitable for biological applications.

  17. [Construction and identification of eukaryotic plasmid pGC-silencer-U6/Neo/GFP/ABCG2].

    Science.gov (United States)

    Yu, Yanping; Zhang, Song; Kong, Weijia

    2010-09-01

    To construct three short hairpin RNA (shRNA) interference expression plasmid vectors of human ABCG2 gene, to assay the expression of ABCG2 in a human nasopharyngeal carcinoma (NPC) cell line, CEN-2 cell line, and to detect the RNAi effect of shRNA. Targeting ABCG2 gene sequence, three plasmid expression vectors coding for shRNA and a control vector containing random DNA fragment were constructed. The recombinant plasmids were amplified in Ecoli. DH5 and then identified by restriction digestion, PCR and sequencing. The recombinant plasmids were transfected into CEN-2 cells. ABCG2 expression was assayed by real-time quantitative PCR and Western blot. The construction of pGC-silencer-U6/Neo/GFP/ABCG2 was succeed. The shRNA plasmids significantly down-regulated the ABCG2 expression in CEN-2 cells, at both mRNA level and protein level. Recombinant plasmid 1 had the strongest effect compared with plasmids 2 and 3 (P < 0.05), with an inhibition ratio of 75% at the mRNA level and 68% at the protein level. pGC-silencer-U6/Neo/GFP/ABCG2 has been successfully constructed and it can down-regulate ABCG2 expression after transfected into CEN-2 cells, which could help further studies of ABCG2 functions CEN-2 cell line and contribute to the NPC gene therapy.

  18. Bacterial mitosis: Partitioning protein ParA oscillates in spiral-shaped structures and positions plasmids at mid-cell

    DEFF Research Database (Denmark)

    Ebersbach, G.; Gerdes, Kenn

    2004-01-01

    The par2 locus of Escherichia coli plasmid pB171 encodes oscillating ATPase ParA, DNA binding protein ParB and two cis-acting DNA regions to which ParB binds (parC1 and parC2). Three independent techniques were used to investigate the subcellular localization of plasmids carrying par2. In cells......A-GFP oscillated in spiral-shaped structures. Amino acid substitutions in ParA simultaneously abolished ParA spiral formation, oscillation and either plasmid localization or plasmid separation at mid-cell. Therefore, our results suggest that ParA spirals position plasmids at the middle of the bacterial nucleoid...

  19. Case studies on the physical-chemical parameters' variation during three different purification approaches destined to treat wastewaters from food industry.

    Science.gov (United States)

    Ghimpusan, Marieta; Nechifor, Gheorghe; Nechifor, Aurelia-Cristina; Dima, Stefan-Ovidiu; Passeri, Piero

    2017-12-01

    The paper presents a set of three interconnected case studies on the depuration of food processing wastewaters by using aeration & ozonation and two types of hollow-fiber membrane bioreactor (MBR) approaches. A secondary and more extensive objective derived from the first one is to draw a clearer, broader frame on the variation of physical-chemical parameters during the purification of wastewaters from food industry through different operating modes with the aim of improving the management of water purification process. Chemical oxygen demand (COD), pH, mixed liquor suspended solids (MLSS), total nitrogen, specific nitrogen (NH 4 + , NO 2 - , NO 3 - ) total phosphorous, and total surfactants were the measured parameters, and their influence was discussed in order to establish the best operating mode to achieve the purification performances. The integrated air-ozone aeration process applied in the second operating mode lead to a COD decrease by up to 90%, compared to only 75% obtained in a conventional biological activated sludge process. The combined purification process of MBR and ozonation produced an additional COD decrease of 10-15%, and made the Total Surfactants values to comply to the specific legislation. Copyright © 2016 Elsevier Ltd. All rights reserved.

  20. Radiation-adsorption purification of effluents containing pesticides

    International Nuclear Information System (INIS)

    Brusentseva, S.A.; Shubin, V.N.; Nikonorova, G.K.; Zorin, D.M.; Sosnovskaya, A.A.; Petryaev, E.P.; Vlasova, V.I.; Edimicheva, I.P.; Subbotina, N.N.; Belorusskij Gosudarstvennyj Univ., Minsk)

    1986-01-01

    The radiation-adsorption purification is one of the new direction in the radiation purification of natural wastes and effluents containing pesticides. This method combines the conventional adsorption purification with radiation treatment of the sorbent, and the result the protection time of the sorbent increases due to the radiation regeneration of carbon. In present work the method was used for purification of effluents from pesticides, such as 4,4'Dichlorodiphenyltrichloroethane /DDT/, 1,2,3,4,5,6-hexachlorocyclohexane /HCCH/, dimethyl 2,2-dichlorovinylphosphate /DDVF/ and petroleum products (a mixture of kerosene and xylene in ratio 7:1). Such effluents are formed at factories producing an insecticide aerosol 'Prime-71'. Three investigations were carried out on model with a solution similar composition to industrial effluents. (author)

  1. Low temperature techniques for natural gas purification and LNG production: An energy and exergy analysis

    International Nuclear Information System (INIS)

    Baccanelli, Margaret; Langé, Stefano; Rocco, Matteo V.; Pellegrini, Laura A.; Colombo, Emanuela

    2016-01-01

    Highlights: • Low-temperature processes for of high CO_2 content natural gas have been modelled. • Energy and exergy analyses have been performed. • The Dual Pressure distillation scheme has the best thermodynamic performances. • There is a synergy between cryogenic natural gas purification and LNG production. - Abstract: Due to the rapid increase of the World’s primary energy demand of the last decades, low-temperature processes for the purification of natural gas streams with high carbon dioxide content has gained interest, since they allow to make profitable exploitation of low-quality gas reserves. Low temperature purification processes allow the direct production of a methane stream at high purity and at low-temperature, suitable conditions for the direct synergistic integration with natural gas cryogenic liquefaction processes, while CO_2 is obtained in liquid phase and under pressure. In this way, it can be pumped for transportation, avoiding significant compression costs as for classical CO_2 capture units (where carbon dioxide is discharged in gas phase and at atmospheric pressure), and further uses such as Enhanced Oil Recovery (EOR) or underground storage. In this paper, the three most common natural gas low-temperature purification techniques have been modelled and their performances have been evaluated through energy and exergy analyses. Specifically, the dual pressure low-temperature distillation process, the anti-sublimation process and a hybrid configuration have been considered. It is found that the dual pressure low-temperature distillation scheme reach the highest thermodynamic performances, resulting in the best values of exergy efficiency and equivalent methane requirements with respect to the other configurations. This is mainly due to the distributed temperature profile along a distillation column, resulting in a less irreversible heat exchanging process.

  2. Plasmid Complement of Lactococcus lactis NCDO712 Reveals a Novel Pilus Gene Cluster.

    Science.gov (United States)

    Tarazanova, Mariya; Beerthuyzen, Marke; Siezen, Roland; Fernandez-Gutierrez, Marcela M; de Jong, Anne; van der Meulen, Sjoerd; Kok, Jan; Bachmann, Herwig

    2016-01-01

    Lactococcus lactis MG1363 is an important gram-positive model organism. It is a plasmid-free and phage-cured derivative of strain NCDO712. Plasmid-cured strains facilitate studies on molecular biological aspects, but many properties which make L. lactis an important organism in the dairy industry are plasmid encoded. We sequenced the total DNA of strain NCDO712 and, contrary to earlier reports, revealed that the strain carries 6 rather than 5 plasmids. A new 50-kb plasmid, designated pNZ712, encodes functional nisin immunity (nisCIP) and copper resistance (lcoRSABC). The copper resistance could be used as a marker for the conjugation of pNZ712 to L. lactis MG1614. A genome comparison with the plasmid cured daughter strain MG1363 showed that the number of single nucleotide polymorphisms that accumulated in the laboratory since the strains diverted more than 30 years ago is limited to 11 of which only 5 lead to amino acid changes. The 16-kb plasmid pSH74 was found to contain a novel 8-kb pilus gene cluster spaCB-spaA-srtC1-srtC2, which is predicted to encode a pilin tip protein SpaC, a pilus basal subunit SpaB, and a pilus backbone protein SpaA. The sortases SrtC1/SrtC2 are most likely involved in pilus polymerization while the chromosomally encoded SrtA could act to anchor the pilus to peptidoglycan in the cell wall. Overexpression of the pilus gene cluster from a multi-copy plasmid in L. lactis MG1363 resulted in cell chaining, aggregation, rapid sedimentation and increased conjugation efficiency of the cells. Electron microscopy showed that the over-expression of the pilus gene cluster leads to appendices on the cell surfaces. A deletion of the gene encoding the putative basal protein spaB, by truncating spaCB, led to more pilus-like structures on the cell surface, but cell aggregation and cell chaining were no longer observed. This is consistent with the prediction that spaB is involved in the anchoring of the pili to the cell.

  3. Characterization and plasmid elimination of NDM-1-producing Acinetobacter calcoaceticus from China.

    Directory of Open Access Journals (Sweden)

    Yang Sun

    Full Text Available The presence of multidrug-resistant bacterial pathogens in the environment poses a serious threat to public health. The opportunistic Acinetobacter spp. are among the most prevalent causes of nosocomial infections. Here, we performed complete genome sequencing of the Acinetobacter calcoaceticus strain XM1570, which was originally cultivated from the sputum of a patient diagnosed with pneumonia in Xiamen in 2010. We identified carbapenem resistance associated gene bla(NDM-1 located on a 47.3-kb plasmid. Three methods--natural reproduction, sodium dodecyl sulfate treatment and nalidixic acid treatment--were used to eliminate the bla(NDM-1-encoding plasmid, which achieved elimination rates of 3.32% (10/301, 83.78% (278/332, and 84.17% (298/354, respectively. Plasmid elimination dramatically increased antibiotic sensitivity, reducing the minimum bacteriostatic concentration of meropenem from 256 µg/ml in the clinical strain to 0.125 µg/ml in the plasmid-eliminated strain. Conjugation transfer assays showed that the bla(NDM-1-containing plasmid could be transferred into Escherichia coli DH5α:pBR322 in vitro as well as in vivo in mice. The bla(NDM-1 genetic environment was in accordance with that of other bla(NDM-1 genes identified from India, Japan, and Hong-Kong. The multilocus sequence type of the isolate was identified as ST-70. Two novel genes encoding intrinsic OXA and ADC were identified and named as OXA-417 and ADC-72. The finding of bla(NDM-1 in species like A. calcoaceticus demonstrates the wide spread of this gene in gram-negative bacteria which is possible by conjugative plasmid transfer. The results of this study may help in the development of a treatment strategy for controlling NDM-1 bacterial infection and transmission.

  4. Identification of dfrA14 in two distinct plasmids conferring trimethoprim resistance in Actinobacillus pleuropneumoniae.

    Science.gov (United States)

    Bossé, Janine T; Li, Yanwen; Walker, Stephanie; Atherton, Tom; Fernandez Crespo, Roberto; Williamson, Susanna M; Rogers, Jon; Chaudhuri, Roy R; Weinert, Lucy A; Oshota, Olusegun; Holden, Matt T G; Maskell, Duncan J; Tucker, Alexander W; Wren, Brendan W; Rycroft, Andrew N; Langford, Paul R

    2015-08-01

    The objective of this study was to determine the distribution and genetic basis of trimethoprim resistance in Actinobacillus pleuropneumoniae isolates from pigs in England. Clinical isolates collected between 1998 and 2011 were tested for resistance to trimethoprim and sulphonamide. The genetic basis of trimethoprim resistance was determined by shotgun WGS analysis and the subsequent isolation and sequencing of plasmids. A total of 16 (out of 106) A. pleuropneumoniae isolates were resistant to both trimethoprim (MIC >32 mg/L) and sulfisoxazole (MIC ≥256 mg/L), and a further 32 were resistant only to sulfisoxazole (MIC ≥256 mg/L). Genome sequence data for the trimethoprim-resistant isolates revealed the presence of the dfrA14 dihydrofolate reductase gene. The distribution of plasmid sequences in multiple contigs suggested the presence of two distinct dfrA14-containing plasmids in different isolates, which was confirmed by plasmid isolation and sequencing. Both plasmids encoded mobilization genes, the sulphonamide resistance gene sul2, as well as dfrA14 inserted into strA, a streptomycin-resistance-associated gene, although the gene order differed between the two plasmids. One of the plasmids further encoded the strB streptomycin-resistance-associated gene. This is the first description of mobilizable plasmids conferring trimethoprim resistance in A. pleuropneumoniae and, to our knowledge, the first report of dfrA14 in any member of the Pasteurellaceae. The identification of dfrA14 conferring trimethoprim resistance in A. pleuropneumoniae isolates will facilitate PCR screens for resistance to this important antimicrobial. © The Author 2015. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy.

  5. Protein-Nanocrystal Conjugates Support a Single Filament Polymerization Model in R1 Plasmid Segregation

    Energy Technology Data Exchange (ETDEWEB)

    Choi, Charina L.; Claridge, Shelley A.; Garner, Ethan C.; Alivisatos, A. Paul; Mullins, R. Dyche

    2008-07-15

    To ensure inheritance by daughter cells, many low-copy number bacterial plasmids, including the R1 drug-resistance plasmid, encode their own DNA segregation systems. The par operon of plasmid R1 directs construction of a simple spindle structure that converts free energy of polymerization of an actin-like protein, ParM, into work required to move sister plasmids to opposite poles of rod-shaped cells. The structures of individual components have been solved, but little is known about the ultrastructure of the R1 spindle. To determine the number of ParM filaments in a minimal R1 spindle, we used DNA-gold nanocrystal conjugates as mimics of the R1 plasmid. Wefound that each end of a single polar ParM filament binds to a single ParR/parC-gold complex, consistent with the idea that ParM filaments bind in the hollow core of the ParR/parC ring complex. Our results further suggest that multifilament spindles observed in vivo are associated with clusters of plasmidssegregating as a unit.

  6. High-Yield Purification, Preservation, and Serial Transplantation of Human Satellite Cells

    Directory of Open Access Journals (Sweden)

    Steven M. Garcia

    2018-03-01

    Full Text Available Summary: Investigation of human muscle regeneration requires robust methods to purify and transplant muscle stem and progenitor cells that collectively constitute the human satellite cell (HuSC pool. Existing approaches have yet to make HuSCs widely accessible for researchers, and as a result human muscle stem cell research has advanced slowly. Here, we describe a robust and predictable HuSC purification process that is effective for each human skeletal muscle tested and the development of storage protocols and transplantation models in dystrophin-deficient and wild-type recipients. Enzymatic digestion, magnetic column depletion, and 6-marker flow-cytometric purification enable separation of 104 highly enriched HuSCs per gram of muscle. Cryostorage of HuSCs preserves viability, phenotype, and transplantation potential. Development of enhanced and species-specific transplantation protocols enabled serial HuSC xenotransplantation and recovery. These protocols and models provide an accessible system for basic and translational investigation and clinical development of HuSCs. : Garcia and colleagues report methods for efficient purification of satellite cells from human skeletal muscle. They use their approaches to demonstrate stem cell functions of endogenous satellite cells and to make human satellite cells accessible for sharing among researchers. Keywords: human satellite cell purification, serial transplantation, satellite cell cryopreservation

  7. Purification Simulation With Vapor Permeation and Distillation-Adsorption In Bioethanol Plant

    Directory of Open Access Journals (Sweden)

    Misri Gozan

    2017-04-01

    Full Text Available High purity of Bioethanol is required in biofuel mixing with gasoline (EXX. In bioethanol production line, the azeotropic property of ethanol-water becomes the barrier for purification process. This study examined two bioethanol separation processes by support of simulation tools, Superpro Designer 9.0 software. Ethanol purity and a low costeconomical process were the major considerations. Purification method of vapor permeation membrane technology was compared with distillation-adsorption method. Data from previous lab experiments and some literatures were used. The results showed that distillation-adsorption method is more economical compared to vapor permeation technology. Payback period of the simulation is 3.9 years and 4.3 years to distillation adsorption and vapor permeation respectively with each IRR value is 20.23% and 17.89%. Initial investment value of vapor permeation is 9.6% higher than distillation method. Significant difference observed in operating costs, since more units involved in vapor permeation require more labors to operate.

  8. [Purification of arsenic-binding proteins in hamster plasma after oral administration of arsenite].

    Science.gov (United States)

    Wang, Wenwen; Zhang, Min; Li, Chunhui; Qin, Yingjie; Hua, Naranmandura

    2013-01-01

    To purify the arsenic-binding proteins (As-BP) in hamster plasma after a single oral administration of arsenite (iAs(III)). Arsenite was given to hamsters in a single dose. Three types of HPLC columns, size exclusion, gel filtration and anion exchange columns, combined with an inductively coupled argon plasma mass spectrometer (ICP MS) were used to purify the As-BP in hamster plasma. SDS-PAGE was used to confirm the arsenic-binding proteins at each purification step. The three-step purification process successfully separated As-BP from other proteins (ie, arsenic unbound proteins) in hamster plasma. The molecular mass of purified As-BP in plasma was approximately 40-50 kD on SDS-PAGE. The three-step purification method is a simple and fast approach to purify the As-BP in plasma samples.

  9. Spontaneous mutability and light-induced mutagenesis in Salmonella typhimurium: effects of an R-plasmid

    International Nuclear Information System (INIS)

    Valdivia, L.

    1979-01-01

    The UV-protecting plasmid R46 was transferred by conjugation to a genetically marked mouse-virulent Salmonella typhimurium strain, not derived from LT2; in this host the plasmid conferred UV protection and enhanced UV mutagenesis just as it does in LT2 lines. Tra - derivatives of R46 encountered during transduction retained UV-protecting and mutagenesis-enhancing ability. Stored strains carrying the R46-derived plasmids with strong mutator effect but not UV-protecting had lost most of their original streptomycin resistance but were slightly resistant to spectinomycin; attempts to transfer such plasmids failed. R46 enhanced the weak mutagenic effect of visible light on several his and trp mutants of strain LT2, including some whose frequency of spontaneous reversion was not increased by the plasmid. A mutagenic effect was produced by visible-light irradiation of hisG46(R46), either growing cells or nonmultiplying (histidine-deprived cells at 10 0 C). Presence of catalase or cyanide during irradiation did not prevent mutagenesis, which excludes some hypothetical mechanisms. Visible-light irradiation of hisG46 or hisG46(R46) under strict anaerobiosis had little or no mutagenic effect (controls showed that revertants if produced would have been detected). This is as expected if visible-light irradiation in air causes photodynamic damage to DNA and mutations are produced during error-prone, plasmid-enhanced repair

  10. Preparative Purification of Recombinant Proteins: Current Status and Future Trends

    Directory of Open Access Journals (Sweden)

    Mayank Saraswat

    2013-01-01

    Full Text Available Advances in fermentation technologies have resulted in the production of increased yields of proteins of economic, biopharmaceutical, and medicinal importance. Consequently, there is an absolute requirement for the development of rapid, cost-effective methodologies which facilitate the purification of such products in the absence of contaminants, such as superfluous proteins and endotoxins. Here, we provide a comprehensive overview of a selection of key purification methodologies currently being applied in both academic and industrial settings and discuss how innovative and effective protocols such as aqueous two-phase partitioning, membrane chromatography, and high-performance tangential flow filtration may be applied independently of or in conjunction with more traditional protocols for downstream processing applications.

  11. Purification of simple substances by distillation with impurity hydrothermal oxidation

    International Nuclear Information System (INIS)

    Kalashnik, O.N.; Nisel'son, L.A.

    1987-01-01

    A possibility of applying distillation method in water vapours for purification of simple substances from impurities is studied. Based on thermodynamic analysis of interaction processes in E-H 2 O system, conducted using a computer, it is as certained that SS, Se, Te, As, Cd, Hg can be purified from the majority of the impurities analysed by distillation in a water vapour flow. Behaviour of Zn, C, Ge, Al, Sb characteristic impurities under cadmium, arsenic and tellurium distillation is studied. Experiments on cadmium, arsenic and tellurium purification have confirmed, that distillation with hydrothermal oxidation of Zn, C, Ge impurities sometimes appears to be a more effective method as compared to distillation in a hydrogen flow

  12. Isolation of a minireplicon of the plasmid pG6303 of Lactobacillus ...

    Indian Academy of Sciences (India)

    is a new mode of plasmid replication. [Fan J., Xi X., ... coli using the BioTeKe plasmid extraction kit (BioTeKe, Beijing, China) according .... media and incubated at 37◦C for three days. The methods of ..... Each experiment was repeated five times. Journal of ..... Cold Spring Harbor Laboratory Press, New York, USA. Soler N.

  13. Nanocellulose-Based Materials for Water Purification.

    Science.gov (United States)

    Voisin, Hugo; Bergström, Lennart; Liu, Peng; Mathew, Aji P

    2017-03-05

    Nanocellulose is a renewable material that combines a high surface area with high strength, chemical inertness, and versatile surface chemistry. In this review, we will briefly describe how nanocellulose is produced, and present-in particular, how nanocellulose and its surface modified versions affects the adsorption behavior of important water pollutants, e.g., heavy metal species, dyes, microbes, and organic molecules. The processing of nanocellulose-based membranes and filters for water purification will be described in detail, and the uptake capacity, selectivity, and removal efficiency will also be discussed. The processing and performance of nanocellulose-based membranes, which combine a high removal efficiency with anti-fouling properties, will be highlighted.

  14. Characterization of Plasmid DNA Location within Chitosan/PLGA/pDNA Nanoparticle Complexes Designed for Gene Delivery

    Directory of Open Access Journals (Sweden)

    Hali Bordelon

    2011-01-01

    Full Text Available Poly(D,L-lactide-co-glycolide- (PLGA-chitosan nanoparticles are becoming an increasingly common choice for the delivery of nucleic acids to cells for various genetic manipulation techniques. These particles are biocompatible, with tunable size and surface properties, possessing an overall positive charge that promotes complex formation with negatively charged nucleic acids. This study examines properties of the PLGA-chitosan nanoparticle/plasmid DNA complex after formation. Specifically, the study aims to determine the optimal ratio of plasmid DNA:nanoparticles for nucleic acid delivery purposes and to elucidate the location of the pDNA within these complexes. Such characterization will be necessary for the adoption of these formulations in a clinical setting. The ability of PLGA-chitosan nanoparticles to form complexes with pDNA was evaluated by using the fluorescent intercalating due OliGreen to label free plasmid DNA. By monitoring the fluorescence at different plasmid: nanoparticle ratios, the ideal plasmid:nanoparticle ration for complete complexation of plasmid was determined to be 1:50. Surface-Enhanced Raman Spectroscopy and gel digest studies suggested that even at these optimal complexation ratios, a portion of the plasmid DNA was located on the outer complex surface. This knowledge will facilitate future investigations into the functionality of the system in vitro and in vivo.

  15. STUDY REGARDING EFFICIENCY OF INDUCED GENETIC TRANSFORMATION IN BACILLUS LICHENIFORMIS WITH PLASMID DNA

    Directory of Open Access Journals (Sweden)

    T. VINTILĂ

    2007-05-01

    Full Text Available A strain of Bacillus licheniformis was subject to genetic transformation with plasmid vectors (pLC1 and pNC61, using electroporation technique, protoplast transformation and bivalent cations (CaCl2 mediated transformation. In the case of transformation by electroporation of Bacillus licheniformis B40, the highest number of transformed colonies (3 were obtained only after a 1,79 KV electric shock, for 2,2 milliseconds. Using this transformation technique we have obtained six kanamycin resistant transformants. The frequency of Bacillus licheniformis B40 protoplasts transformation using pLC1 and pNC61 plasmid vectors is approximately 10% (TF = 10%. As a result of pLC1 plasmid integration in Bacillus licheniformis protoplasts, six kanamycin resistant transformants were obtained. The pNC61 plasmid, which confers trimethoprim resistance, does not integrate in receiver cells by protoplast transformation. The direct genetic transformation in the presence of bivalent cations (CaCl2, mediated by pLC1 and pNC61 plasmid vectors, produce a low transformation frequency. Using this technique, we have obtained three trimethoprim resistant colonies and four kanamycin resistant colonies. The chemical way of transformation is the only technique, which realizes the integration of pNC61 in B. licheniformis B40 cells.

  16. CO 2-scrubbing and methanation as purification system for PEFC

    Science.gov (United States)

    Ledjeff-Hey, K.; Roes, J.; Wolters, R.

    Hydrogen is usually produced by steam reforming of natural gas in large-scale processes. The reformate consists of hydrogen, carbon dioxide, carbon monoxide, and residues of hydrocarbons. Since the anode catalyst of a polymer electrolyte membrane fuel cell (PEFC) is usually based on platinum, which is easily poisoned by carbon monoxide, the conditioned feed gas should contain less than 100 ppmv CO, and preferably, less than 10 ppmv. Depending on the design and operating conditions of the hydrogen production process, the CO content of a typical reformate gas, even after the CO shift reactor may be in the range of 0.2-1.0 vol.%; this is far higher than a PEFC can tolerate. A CO management system is required to lower the CO concentration to acceptable levels. In many cases, the CO purification system consists of a combination of physical or chemical processes to achieve the necessary reduction in CO content. A promising alternative for hydrogen purification is a combined process consisting of a carbon dioxide scrubber with subsequent methanation to reduce the carbon monoxide content to an acceptable level of less than 10 ppmv.

  17. Dynamics in copy numbers of five plasmids of a dairy Lactococcus lactis in dairy-related conditions including near-zero growth rates.

    Science.gov (United States)

    van Mastrigt, Oscar; Lommers, Marcel M A N; de Vries, Yorick C; Abee, Tjakko; Smid, Eddy J

    2018-03-23

    Lactic acid bacteria can carry multiple plasmids affecting their performance in dairy fermentations. The expression of plasmid-encoded genes and the activity of the corresponding proteins is severely affected by changes in the number of plasmid copies. We studied the impact of growth rate on dynamics of plasmid copy numbers at high growth rates in chemostat cultures and down to near-zero growth rates in retentostat cultures. Five plasmids of the dairy strain Lactococcus lactis FM03-V1 were selected which varied in size (3 to 39 kb), in replication mechanism (theta or rolling-circle) and in putative (dairy-associated) functions. Copy numbers ranged from 1.5 to 40.5 and the copy number of theta-type replicating plasmids were negatively correlated to the plasmid size. Despite the extremely wide range of growth rates (0.0003 h -1 to 0.6 h -1 ), copy numbers of the five plasmids were stable and only slightly increased at near-zero growth rates showing that the plasmid replication rate was strictly controlled. One low-copy number plasmid, carrying a large exopolysaccharide gene cluster, was segregationally unstable during retentostat cultivations reflected in complete loss of the plasmid in one of the retentostat cultures. The copy number of the five plasmids was also hardly affected by varying the pH value, nutrient limitation or presence of citrate (maximum 2.2-fold) signifying the stability in copy number of the plasmids. Importance Lactococcus lactis is extensively used in starter cultures for dairy fermentations. Important traits for growth and survival of L. lactis in dairy fermentations are encoded by genes located on plasmids, such as genes involved in lactose and citrate metabolism, protein degradation and oligopeptide uptake and bacteriophage resistance. Because the number of plasmid copies could affect the expression of plasmid-encoded genes, it is important to know the factors that influence the plasmid copy numbers. We monitored plasmid copy numbers of L

  18. 21 CFR 876.5665 - Water purification system for hemodialysis.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Water purification system for hemodialysis. 876... SERVICES (CONTINUED) MEDICAL DEVICES GASTROENTEROLOGY-UROLOGY DEVICES Therapeutic Devices § 876.5665 Water purification system for hemodialysis. (a) Identification. A water purification system for hemodialysis is a...

  19. The development and testing of the new flowsheets for the plutonium purification of the Purex process

    Energy Technology Data Exchange (ETDEWEB)

    Bugrov, K.V.; Korotaev, V.G.; Korchenkin, K.K.; Logunov, M.V.; Ludin, S.A.; Mashkin, A.N.; Melentev, A.B.; Samarina, N.S. [FSUE ' PAMayak' , Lenin st., 35, Ozersk 456780 (Russian Federation)

    2016-07-01

    In order to improve the extraction flowsheet of RT-1 Plant two versions of plutonium purification unit flowsheet were developed: a flowsheet with stabilization of Pu(IV)-Np(IV) valence pair and Pu, Np co-recovery, and a flowsheet with stabilization of Pu(IV)-Np(V) valence pair and Pu recovery. The task related to stabilization of the valence pair of the target components in the required state was solved with the use of reactants already applied at RT-1 Plant, namely, hydrogen peroxide, hydrazine nitrate and catalyst (Fe). Both flowsheets were adapted for the plant purification facility with minimum modifications of the equipment, and passed the full scale industrial testing. As a result of this work, reduction in volume and salt content of the raffinate was achieved. (authors)

  20. Sodium purification in Rapsodie; La purification du sodium a Rapsodie

    Energy Technology Data Exchange (ETDEWEB)

    Giraud, B [Commissariat a l' Energie Atomique, Dir. des Piles Atomiques, Cadarache (France). Centre d' Etudes Nucleaires

    1968-07-01

    This report is one of a series of publications presenting the main results of tests carried out during the start-up of the first french fast neutron reactor: Rapsodie. The article presents the sodium purification techniques used in the reactor cooling circuits both from the constructional point of view and with respect to results obtained during the first years working. (author) [French] Ce rapport fait partie d'une serie de publications presentant l'essentiel des resultats des essais effectues a l'occasion du demarrage du premier reacteur francais a neutrons rapides: RAPSODIE. Cet article expose les techniques de la purification du sodium utilise dans les circuits de refroidissement du reacteur tant au point de vue de leur realisation technologique, que des resultats obtenus pendant la premiere annee de fonctionnement. (auteur)

  1. Nonaqueous processing methods

    International Nuclear Information System (INIS)

    Coops, M.S.; Bowersox, D.F.

    1984-09-01

    A high-temperature process utilizing molten salt extraction from molten metal alloys has been developed for purification of spent power reactor fuels. Experiments with laboratory-scale processing operations show that purification and throughput parameters comparable to the Barnwell Purex process can be achieved by pyrochemical processing in equipment one-tenth the size, with all wastes being discharged as stable metal alloys at greatly reduced volume and disposal cost. This basic technology can be developed for large-scale processing of spent reactor fuels. 13 references, 4 figures

  2. PURIFICATION OF TRANSFORMER OIL in PT. PJB UP PAITON

    OpenAIRE

    Gusti Wahdaniyah*, Purnomo Tri Prasetyo, Arif Setiabudi, Totok R. Biyanto

    2016-01-01

    The purpose of this paper is to describe the filtration or purification of transformer oil. One of the main equipment in coal-fired power generation unit is transformer. When the transformer fail to operate properly, the continuity of distribution system become interrupted. As a part of transformer, transformer oil contribute the failure of transformer due to the aging. To solve this problem, several methods is applied starting from dehydration process, degasification process, oxidation remov...

  3. Plasmid transfer by conjugation in Xylella fastidiosa.

    Science.gov (United States)

    Recombination and horizontal gene transfer have been implicated in the adaption of Xylella fastidiosa (Xf) to infect a wide variety of different plant species. There is evidence that certain strains of Xf carry native plasmids equipped with transfer and mobilization genes, suggesting conjugation as ...

  4. Resveratrol production in bioreactor: Assessment of cell physiological states and plasmid segregational stability

    Directory of Open Access Journals (Sweden)

    Margarida S. Afonso

    2015-03-01

    Full Text Available Resveratrol is a plant secondary metabolite commonly found in peanuts and grapevines with significant health benefits. Recombinant organisms can produce large amounts of resveratrol and, in this work, Escherichia coli BW27784 was used to produce resveratrol in bioreactors while monitoring cell physiology and plasmid stability through flow cytometry and real-time qPCR, respectively. Initially, the influence of culture conditions and precursor addition was evaluated in screening assays and the data gathered was used to perform the bioreactor assays, allowing the production of 160 μg/mL of resveratrol. Cellular physiology and plasmid instability affected the final resveratrol production, with lower viability and plasmid copy numbers associated with lower yields. In sum, this study describes new tools to monitor the bioprocess, evaluating the effect of culture conditions, and its correlation with cell physiology and plasmid segregational stability, in order to define a viable and scalable bioprocess to fulfill the need for larger quantities of resveratrol.

  5. Identification of a Novel Conjugative Plasmid in Mycobacteria That Requires Both Type IV and Type VII Secretion

    KAUST Repository

    Ummels, R.; Abdallah, A. M.; Kuiper, V.; Aajoud, A.; Sparrius, M.; Naeem, R.; Spaink, H. P.; van Soolingen, D.; Pain, Arnab; Bitter, W.

    2014-01-01

    Conjugative plasmids play an important role in horizontal gene transfer between different bacteria and, as such, in their adaptation and evolution. This effect is most obvious in the spread of antibiotic resistance genes. Thus far, conjugation of natural plasmids has been described only rarely for mycobacterial species. In fact, it is generally accepted that M. tuberculosis does not show any recent sign of horizontal gene transfer. In this study, we describe the identification of a new widespread conjugative plasmid that can also be efficiently transferred to M. tuberculosis. This plasmid therefore poses both a threat and an opportunity. The threat is that, through the acquisition of antibiotic resistance markers, this plasmid could start a rapid spread of antibiotic resistance genes between pathogenic mycobacteria. The opportunity is that we could use this plasmid to generate new tools for the efficient introduction of foreign DNA in slow-growing mycobacteria.

  6. Co-evolution of genomes and plasmids within Chlamydia trachomatis and the emergence in Sweden of a new variant strain

    Directory of Open Access Journals (Sweden)

    Skilton Rachel J

    2009-05-01

    Full Text Available Abstract Background Chlamydia trachomatis is the most common cause of sexually transmitted infections globally and the leading cause of preventable blindness in the developing world. There are two biovariants of C. trachomatis: 'trachoma', causing ocular and genital tract infections, and the invasive 'lymphogranuloma venereum' strains. Recently, a new variant of the genital tract C. trachomatis emerged in Sweden. This variant escaped routine diagnostic tests because it carries a plasmid with a deletion. Failure to detect this strain has meant it has spread rapidly across the country provoking a worldwide alert. In addition to being a key diagnostic target, the plasmid has been linked to chlamydial virulence. Analysis of chlamydial plasmids and their cognate chromosomes was undertaken to provide insights into the evolutionary relationship between chromosome and plasmid. This is essential knowledge if the plasmid is to be continued to be relied on as a key diagnostic marker, and for an understanding of the evolution of Chlamydia trachomatis. Results The genomes of two new C. trachomatis strains were sequenced, together with plasmids from six C. trachomatis isolates, including the new variant strain from Sweden. The plasmid from the new Swedish variant has a 377 bp deletion in the first predicted coding sequence, abolishing the site used for PCR detection, resulting in negative diagnosis. In addition, the variant plasmid has a 44 bp duplication downstream of the deletion. The region containing the second predicted coding sequence is the most highly conserved region of the plasmids investigated. Phylogenetic analysis of the plasmids and chromosomes are fully congruent. Moreover this analysis also shows that ocular and genital strains diverged from a common C. trachomatis progenitor. Conclusion The evolutionary pathways of the chlamydial genome and plasmid imply that inheritance of the plasmid is tightly linked with its cognate chromosome. These data

  7. Development and Host Compatibility of Plasmids for Two Important Ruminant Pathogens, Mycoplasma bovis and Mycoplasma agalactiae

    Science.gov (United States)

    Sharma, Shukriti; Citti, Chistine; Sagné, Eveline; Marenda, Marc S.

    2015-01-01

    Mycoplasma bovis is a cause of pneumonia, mastitis, arthritis and otitis media in cattle throughout the world. However, despite its clinical significance, there is a paucity of tools to genetically manipulate it, impeding our capacity to further explore the molecular basis of its virulence. To address this limitation, we developed a series of homologous and heterologous replicable plasmids from M. bovis and M. agalactiae. The shortest replicable oriC plasmid based on the region downstream of dnaA in M. bovis was 247 bp and contained two DnaA boxes, while oriC plasmids based on the region downstream of dnaA in M. agalactiae strains 5632 and PG2 were 219 bp and 217 bp in length, respectively, and contained only a single DnaA box. The efficiency of transformation in M. bovis and M. agalactiae was inversely correlated with the size of the oriC region in the construct, and, in general, homologous oriC plasmids had a higher transformation efficiency than heterologous oriC plasmids. The larger pWholeoriC45 and pMM21-7 plasmids integrated into the genomic oriC region of M. bovis, while the smaller oriC plasmids remained extrachromosomal for up to 20 serial passages in selective media. Although specific gene disruptions were not be achieved in M. bovis in this study, the oriC plasmids developed here could still be useful as tools in complementation studies and for expression of exogenous genes in both M. bovis and M. agalactiae. PMID:25746296

  8. Development and host compatibility of plasmids for two important ruminant pathogens, Mycoplasma bovis and Mycoplasma agalactiae.

    Directory of Open Access Journals (Sweden)

    Shukriti Sharma

    Full Text Available Mycoplasma bovis is a cause of pneumonia, mastitis, arthritis and otitis media in cattle throughout the world. However, despite its clinical significance, there is a paucity of tools to genetically manipulate it, impeding our capacity to further explore the molecular basis of its virulence. To address this limitation, we developed a series of homologous and heterologous replicable plasmids from M. bovis and M. agalactiae. The shortest replicable oriC plasmid based on the region downstream of dnaA in M. bovis was 247 bp and contained two DnaA boxes, while oriC plasmids based on the region downstream of dnaA in M. agalactiae strains 5632 and PG2 were 219 bp and 217 bp in length, respectively, and contained only a single DnaA box. The efficiency of transformation in M. bovis and M. agalactiae was inversely correlated with the size of the oriC region in the construct, and, in general, homologous oriC plasmids had a higher transformation efficiency than heterologous oriC plasmids. The larger pWholeoriC45 and pMM21-7 plasmids integrated into the genomic oriC region of M. bovis, while the smaller oriC plasmids remained extrachromosomal for up to 20 serial passages in selective media. Although specific gene disruptions were not be achieved in M. bovis in this study, the oriC plasmids developed here could still be useful as tools in complementation studies and for expression of exogenous genes in both M. bovis and M. agalactiae.

  9. Plasmid Vectors for Xylella fastidiosa Utilizing a Toxin-Antitoxin System for Stability in the Absence of Antibiotic Selection.

    Science.gov (United States)

    Burbank, Lindsey P; Stenger, Drake C

    2016-08-01

    The phytopathogen Xylella fastidiosa causes disease in a variety of important crop and landscape plants. Functional genetic studies have led to a broader understanding of virulence mechanisms used by this pathogen in the grapevine host. Plasmid shuttle vectors are important tools in studies of bacterial genetics but there are only a limited number of plasmid vectors available that replicate in X. fastidiosa, and even fewer that are retained without antibiotic selection. Two plasmids are described here that show stable replication in X. fastidiosa and are effective for gene complementation both in vitro and in planta. Plasmid maintenance is facilitated by incorporation of the PemI/PemK plasmid addiction system, consisting of PemK, an endoribonuclease toxin, and its cognate antitoxin, PemI. Vector pXf20pemIK utilizes a native X. fastidiosa replication origin as well as a high-copy-number pUC origin for propagation in Escherichia coli cloning strains. Broad-host-range vector pBBR5pemIK is a medium- to low-copy-number plasmid based on the pBBR1 backbone. Both plasmids are maintained for extended periods of time in the absence of antibiotic selection, as well as up to 14 weeks in grapevine, without affecting bacterial fitness. These plasmids present an alternative to traditional complementation and expression vectors which rely on antibiotic selection for plasmid retention.

  10. Purification of functionalized DNA origami nanostructures.

    Science.gov (United States)

    Shaw, Alan; Benson, Erik; Högberg, Björn

    2015-05-26

    The high programmability of DNA origami has provided tools for precise manipulation of matter at the nanoscale. This manipulation of matter opens up the possibility to arrange functional elements for a diverse range of applications that utilize the nanometer precision provided by these structures. However, the realization of functionalized DNA origami still suffers from imperfect production methods, in particular in the purification step, where excess material is separated from the desired functionalized DNA origami. In this article we demonstrate and optimize two purification methods that have not previously been applied to DNA origami. In addition, we provide a systematic study comparing the purification efficacy of these and five other commonly used purification methods. Three types of functionalized DNA origami were used as model systems in this study. DNA origami was patterned with either small molecules, antibodies, or larger proteins. With the results of our work we aim to provide a guideline in quality fabrication of various types of functionalized DNA origami and to provide a route for scalable production of these promising tools.

  11. Biodiesel separation and purification: A review

    International Nuclear Information System (INIS)

    Atadashi, I.M.; Aroua, M.K.; Aziz, A. Abdul

    2011-01-01

    Biodiesel as a biodegradable, sustainable and clean energy has worldwide attracted renewed and growing interest in topical years, chiefly due to development in biodiesel fuel and ecological pressures which include climatic changes. In the production of biodiesel from biomass, separation and purification of biodiesel is a critical technology. Conventional technologies used for biodiesel separation such as gravitational settling, decantation, filtration and biodiesel purification such as water washing, acid washing, and washing with ether and absorbents have proven to be inefficient, time and energy consumptive, and less cost effective. The involvement of membrane reactor and separative membrane shows great promise for the separation and purification of biodiesel. Membrane technology needs to be explored and exploited to overcome the difficulties usually encountered in the separation and purification of biodiesel. In this paper both conventional and most recent membrane technologies used in refining biodiesel have been critically reviewed. The effects of catalysts, free fatty acids, water content and oil to methanol ratios on the purity and quality of biodiesel are also examined. (author)

  12. Synergistic effect of Brønsted acid and platinum on purification of automobile exhaust gases.

    Science.gov (United States)

    Fu, Wei; Li, Xin-Hao; Bao, Hong-Liang; Wang, Kai-Xue; Wei, Xiao; Cai, Yi-Yu; Chen, Jie-Sheng

    2013-01-01

    The catalytic purification of automobile exhaust gases (CO, NOx and hydrocarbons) is one of the most practiced conversion processes used to lower the emissions and to reduce the air pollution. Nevertheless, the good performance of exhaust gas purification catalysts often requires the high consumption of noble metals such as platinum. Here we report that the Brønsted acid sites on the external surface of a microporous silicoaluminophosphate (SAPO) act as a promoter for exhaust gas purification, effectively cutting the loading amount of platinum in the catalyst without sacrifice of performance. It is revealed that in the Pt-loaded SAPO-CHA catalyst, there exists a remarkable synergistic effect between the Brønsted acid sites and the Pt nanoparticles, the former helping to adsorb and activate the hydrocarbon molecules for NO reduction during the catalytic process. The thermal stability of SAPO-CHA also makes the composite catalyst stable and reusable without activity decay.

  13. Synergistic effect of Brønsted acid and platinum on purification of automobile exhaust gases

    Science.gov (United States)

    Fu, Wei; Li, Xin-Hao; Bao, Hong-Liang; Wang, Kai-Xue; Wei, Xiao; Cai, Yi-Yu; Chen, Jie-Sheng

    2013-01-01

    The catalytic purification of automobile exhaust gases (CO, NOx and hydrocarbons) is one of the most practiced conversion processes used to lower the emissions and to reduce the air pollution. Nevertheless, the good performance of exhaust gas purification catalysts often requires the high consumption of noble metals such as platinum. Here we report that the Brønsted acid sites on the external surface of a microporous silicoaluminophosphate (SAPO) act as a promoter for exhaust gas purification, effectively cutting the loading amount of platinum in the catalyst without sacrifice of performance. It is revealed that in the Pt-loaded SAPO-CHA catalyst, there exists a remarkable synergistic effect between the Brønsted acid sites and the Pt nanoparticles, the former helping to adsorb and activate the hydrocarbon molecules for NO reduction during the catalytic process. The thermal stability of SAPO-CHA also makes the composite catalyst stable and reusable without activity decay. PMID:23907148

  14. A genetic study of a Staphylococus aureus plasmid involving cure and transference

    Directory of Open Access Journals (Sweden)

    Ana Lúcia Costa Darini

    Full Text Available High frequency transfer and elimination of drug resistance may indicate an extrachromosomal inheritance of genetic determinants. This study shows the cure and transfer of a small plasmid and tetracycline resistance in Staphylococcus aureus 1030 (55TetR strains. Several methods are available for plasmid elimination. We used ethidium bromide, an agent that binds to DNA, and thus inhibits DNA polymerase. This caused a high frequency of loss of the small plasmid and resistance to tetracycline. Transfer of tetracycline resistance was done in a mixed culture at a frequency of 10-6. This type of study is very important to physicians and epidemiology investigators and provides better knowledge on antibiotic-resistance mechanisms that may occur in vivo in a hospital environment.

  15. Regular cellular distribution of plasmids by oscillating and filament-forming ParA ATPase of plasmid pB171

    DEFF Research Database (Denmark)

    Ebersbach, Gitte; Ringgaard, Simon; Møller-Jensen, Jakob

    2006-01-01

    with each other in a bacterial two-hybrid assay but do not interact with FtsZ, eight other essential cell division proteins or MreB actin. Based on these observations, we propose a simple model for how oscillating ParA filaments can mediate regular cellular distribution of plasmids. The model functions...

  16. Sequencing of IncX-plasmids suggests ubiquity of mobile forms of a biofilm-promoting gene cassette recruited from Klebsiella pneumoniae.

    Directory of Open Access Journals (Sweden)

    Mette Burmølle

    Full Text Available Plasmids are a highly effective means with which genetic traits that influence human health, such as virulence and antibiotic resistance, are disseminated through bacterial populations. The IncX-family is a hitherto sparsely populated group of plasmids that are able to thrive within Enterobacteriaceae. In this study, a replicon-centric screening method was used to locate strains from wastewater sludge containing plasmids belonging to the IncX-family. A transposon aided plasmid capture method was then employed to transport IncX-plasmids from their original hosts (and co-hosted plasmids into a laboratory strain (Escherichia coli Genehogs® for further study. The nucleotide sequences of the three newly isolated IncX-plasmids (pLN126_33, pMO17_54, pMO440_54 and the hitherto un-sequenced type-plasmid R485 revealed a remarkable occurrence of whole or partial gene cassettes that promote biofilm-formation in Klebsiella pneumonia or E. coli, in all four instances. Two of the plasmids (R485 and pLN126_33 were shown to directly induce biofilm formation in a crystal violet retention assay in E. coli. Sequence comparison revealed that all plasmid-borne forms of the type 3 fimbriae encoding gene cassette mrkABCDF were variations of a composite transposon Tn6011 first described in the E. coli IncX plasmid pOLA52. In conclusion, IncX-plasmids isolated from Enterobacteriaceae over almost 40 years and on three different continents have all been shown to carry a type 3 fimbriae gene cassette mrkABCDF stemming from pathogenic K. pneumoniae. Apart from contributing general knowledge about IncX-plasmids, this study also suggests an apparent ubiquity of a mobile form of an important virulence factor and is an illuminating example of the recruitment, evolution and dissemination of genetic traits through plasmid-mediated horizontal gene transfer.

  17. Sequencing of IncX-plasmids suggests ubiquity of mobile forms of a biofilm-promoting gene cassette recruited from Klebsiella pneumoniae.

    Science.gov (United States)

    Burmølle, Mette; Norman, Anders; Sørensen, Søren J; Hansen, Lars Hestbjerg

    2012-01-01

    Plasmids are a highly effective means with which genetic traits that influence human health, such as virulence and antibiotic resistance, are disseminated through bacterial populations. The IncX-family is a hitherto sparsely populated group of plasmids that are able to thrive within Enterobacteriaceae. In this study, a replicon-centric screening method was used to locate strains from wastewater sludge containing plasmids belonging to the IncX-family. A transposon aided plasmid capture method was then employed to transport IncX-plasmids from their original hosts (and co-hosted plasmids) into a laboratory strain (Escherichia coli Genehogs®) for further study. The nucleotide sequences of the three newly isolated IncX-plasmids (pLN126_33, pMO17_54, pMO440_54) and the hitherto un-sequenced type-plasmid R485 revealed a remarkable occurrence of whole or partial gene cassettes that promote biofilm-formation in Klebsiella pneumonia or E. coli, in all four instances. Two of the plasmids (R485 and pLN126_33) were shown to directly induce biofilm formation in a crystal violet retention assay in E. coli. Sequence comparison revealed that all plasmid-borne forms of the type 3 fimbriae encoding gene cassette mrkABCDF were variations of a composite transposon Tn6011 first described in the E. coli IncX plasmid pOLA52. In conclusion, IncX-plasmids isolated from Enterobacteriaceae over almost 40 years and on three different continents have all been shown to carry a type 3 fimbriae gene cassette mrkABCDF stemming from pathogenic K. pneumoniae. Apart from contributing general knowledge about IncX-plasmids, this study also suggests an apparent ubiquity of a mobile form of an important virulence factor and is an illuminating example of the recruitment, evolution and dissemination of genetic traits through plasmid-mediated horizontal gene transfer.

  18. Ordering the mob: Insights into replicon and MOB typing schemes from analysis of a curated dataset of publicly available plasmids.

    Science.gov (United States)

    Orlek, Alex; Phan, Hang; Sheppard, Anna E; Doumith, Michel; Ellington, Matthew; Peto, Tim; Crook, Derrick; Walker, A Sarah; Woodford, Neil; Anjum, Muna F; Stoesser, Nicole

    2017-05-01

    Plasmid typing can provide insights into the epidemiology and transmission of plasmid-mediated antibiotic resistance. The principal plasmid typing schemes are replicon typing and MOB typing, which utilize variation in replication loci and relaxase proteins respectively. Previous studies investigating the proportion of plasmids assigned a type by these schemes ('typeability') have yielded conflicting results; moreover, thousands of plasmid sequences have been added to NCBI in recent years, without consistent annotation to indicate which sequences represent complete plasmids. Here, a curated dataset of complete Enterobacteriaceae plasmids from NCBI was compiled, and used to assess the typeability and concordance of in silico replicon and MOB typing schemes. Concordance was assessed at hierarchical replicon type resolutions, from replicon family-level to plasmid multilocus sequence type (pMLST)-level, where available. We found that 85% and 65% of the curated plasmids could be replicon and MOB typed, respectively. Overall, plasmid size and the number of resistance genes were significant independent predictors of replicon and MOB typing success. We found some degree of non-concordance between replicon families and MOB types, which was only partly resolved when partitioning plasmids into finer-resolution groups (replicon and pMLST types). In some cases, non-concordance was attributed to ambiguous boundaries between MOBP and MOBQ types; in other cases, backbone mosaicism was considered a more plausible explanation. β-lactamase resistance genes tended not to show fidelity to a particular plasmid type, though some previously reported associations were supported. Overall, replicon and MOB typing schemes are likely to continue playing an important role in plasmid analysis, but their performance is constrained by the diverse and dynamic nature of plasmid genomes. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  19. Comparative assessment of plasmid DNA delivery by encapsulation ...

    African Journals Online (AJOL)

    Tropical Journal of Pharmaceutical Research January 2018; 17 (1): 1-10 ... Purpose: To compare the gene delivery effectiveness of plasmid DNA (pDNA) ..... Intramuscular delivery of DNA ... copolymeric system for gene delivery in complete.

  20. Protection from ischemic heart injury by a vigilant heme oxygenase-1 plasmid system.

    Science.gov (United States)

    Tang, Yao Liang; Tang, Yi; Zhang, Y Clare; Qian, Keping; Shen, Leping; Phillips, M Ian

    2004-04-01

    Although human heme oxygenase-1 (hHO-1) could provide a useful approach for cellular protection in the ischemic heart, constitutive overexpression of hHO-1 may lead to unwanted side effects. To avoid this, we designed a hypoxia-regulated hHO-1 gene therapy system that can be switched on and off. This vigilant plasmid system is composed of myosin light chain-2v promoter and a gene switch that is based on an oxygen-dependent degradation domain from the hypoxia inducible factor-1-alpha. The vector can sense ischemia and switch on the hHO-1 gene system, specifically in the heart. In an in vivo experiment, the vigilant hHO-1 plasmid or saline was injected intramyocardially into myocardial infarction mice or sham operation mice. After gene transfer, expression of hHO-1 was only detected in the ischemic heart treated with vigilant hHO-1 plasmids. Masson trichrome staining showed significantly fewer fibrotic areas in vigilant hHO-1 plasmids-treated mice compared with saline control (43.0%+/-4.8% versus 62.5%+/-3.3%, PhHO-1 expression in peri-infarct border areas, concomitant with higher Bcl-2 levels and lower Bax, Bak, and caspase 3 levels in the ischemic myocardium compared with saline control. By use of a cardiac catheter, heart from vigilant hHO-1 plasmids-treated mice showed improved recovery of contractile and diastolic performance after myocardial infarction compared with saline control. This study documents the beneficial regulation and therapeutic potential of vigilant plasmid-mediated hHO-1 gene transfer. This novel gene transfer strategy can provide cardiac-specific protection from future repeated bouts of ischemic injury.

  1. Plasmid vectors for Xylella fastidiosa utilizing a toxin-antitoxin system for plasmid stability in the absence of antibiotic selection

    Science.gov (United States)

    The phytopathogen Xylella fastidiosa causes disease in a variety of important crop and landscape plants. Functional genetic studies have led to a broader understanding of virulence mechanisms used by this pathogen in the grapevine host. Plasmid shuttle vectors are important tools in studies of bacte...

  2. Prevalence and characterization of plasmids carrying sulfonamide resistance genes among Escherichia coli from pigs, pig carcasses and human.

    Science.gov (United States)

    Wu, Shuyu; Dalsgaard, Anders; Hammerum, Anette M; Porsbo, Lone J; Jensen, Lars B

    2010-07-30

    Sulfonamide resistance is very common in Escherichia coli. The aim of this study was to characterize plasmids carrying sulfonamide resistance genes (sul1, sul2 and sul3) in E. coli isolated from pigs and humans with a specific objective to assess the genetic diversity of plasmids involved in the mobility of sul genes. A total of 501 E. coli isolates from pig feces, pig carcasses and human stools were tested for their susceptibility to selected antimicrobial. Multiplex PCR was conducted to detect the presence of three sul genes among the sulfonamide-resistant E. coli isolates. Fifty-seven sulfonamide-resistant E. coli were selected based on presence of sul resistance genes and subjected to conjugation and/or transformation experiments. S1 nuclease digestion followed by pulsed-field gel electrophoresis was used to visualize and determine the size of plasmids. Plasmids carrying sul genes were characterized by PCR-based replicon typing to allow a comparison of the types of sul genes, the reservoir and plasmid present. A total of 109/501 isolates exhibited sulfonamide resistance. The relative prevalences of sul genes from the three reservoirs (pigs, pig carcasses and humans) were 65%, 45% and 12% for sul2, sul1, and sul3, respectively. Transfer of resistance through conjugation was observed in 42/57 isolates. Resistances to streptomycin, ampicillin and trimethoprim were co-transferred in most strains. Class 1 integrons were present in 80% of sul1-carrying plasmids and 100% of sul3-carrying plasmids, but only in 5% of sul2-carrying plasmids. The sul plasmids ranged from 33 to 160-kb in size and belonged to nine different incompatibility (Inc) groups: FII, FIB, I1, FIA, B/O, FIC, N, HI1 and X1. IncFII was the dominant type in sul2-carrying plasmids (52%), while IncI1 was the most common type in sul1 and sul3-carrying plasmids (33% and 45%, respectively). Multireplicons were found associated with all three sul genes. Sul genes were distributed widely in E. coli isolated

  3. Prevalence and characterization of plasmids carrying sulfonamide resistance genes among Escherichia coli from pigs, pig carcasses and human

    Directory of Open Access Journals (Sweden)

    Hammerum Anette M

    2010-07-01

    Full Text Available Abstract Background Sulfonamide resistance is very common in Escherichia coli. The aim of this study was to characterize plasmids carrying sulfonamide resistance genes (sul1, sul2 and sul3 in E. coli isolated from pigs and humans with a specific objective to assess the genetic diversity of plasmids involved in the mobility of sul genes. Methods A total of 501 E. coli isolates from pig feces, pig carcasses and human stools were tested for their susceptibility to selected antimicrobial. Multiplex PCR was conducted to detect the presence of three sul genes among the sulfonamide-resistant E. coli isolates. Fifty-seven sulfonamide-resistant E. coli were selected based on presence of sul resistance genes and subjected to conjugation and/or transformation experiments. S1 nuclease digestion followed by pulsed-field gel electrophoresis was used to visualize and determine the size of plasmids. Plasmids carrying sul genes were characterized by PCR-based replicon typing to allow a comparison of the types of sul genes, the reservoir and plasmid present. Results A total of 109/501 isolates exhibited sulfonamide resistance. The relative prevalences of sul genes from the three reservoirs (pigs, pig carcasses and humans were 65%, 45% and 12% for sul2, sul1, and sul3, respectively. Transfer of resistance through conjugation was observed in 42/57 isolates. Resistances to streptomycin, ampicillin and trimethoprim were co-transferred in most strains. Class 1 integrons were present in 80% of sul1-carrying plasmids and 100% of sul3-carrying plasmids, but only in 5% of sul2-carrying plasmids. The sul plasmids ranged from 33 to 160-kb in size and belonged to nine different incompatibility (Inc groups: FII, FIB, I1, FIA, B/O, FIC, N, HI1 and X1. IncFII was the dominant type in sul2-carrying plasmids (52%, while IncI1 was the most common type in sul1 and sul3-carrying plasmids (33% and 45%, respectively. Multireplicons were found associated with all three sul genes

  4. Optimization of plasmid electrotransformation into Escherichia coli ...

    African Journals Online (AJOL)

    In order to improve electroporation, optical density of bacteria, recovery time and electrical parameter (field strength and capacitance) were optimized using the Taguchi statistical method. ANOVA of obtained data indicated that the optimal conditions of electrotransformation of pET-28a (+) plasmid into Escherichia coli ...

  5. Antimicrobial resistance patterns and plasmid profiles of ...

    African Journals Online (AJOL)

    Objectives: To determine the frequency of resistance of Staphylococcus aureus to various antimicrobial agents, and the relationship between antimicrobial resistance of the isolates and carriage of plasmids. Design: A random sampling of milk and meat samples was carried out. Setting: Milk was collected from various dairy ...

  6. A degenerate primer MOB typing (DPMT method to classify gamma-proteobacterial plasmids in clinical and environmental settings.

    Directory of Open Access Journals (Sweden)

    Andrés Alvarado

    Full Text Available Transmissible plasmids are responsible for the spread of genetic determinants, such as antibiotic resistance or virulence traits, causing a large ecological and epidemiological impact. Transmissible plasmids, either conjugative or mobilizable, have in common the presence of a relaxase gene. Relaxases were previously classified in six protein families according to their phylogeny. Degenerate primers hybridizing to coding sequences of conserved amino acid motifs were designed to amplify related relaxase genes from γ-Proteobacterial plasmids. Specificity and sensitivity of a selected set of 19 primer pairs were first tested using a collection of 33 reference relaxases, representing the diversity of γ-Proteobacterial plasmids. The validated set was then applied to the analysis of two plasmid collections obtained from clinical isolates. The relaxase screening method, which we call "Degenerate Primer MOB Typing" or DPMT, detected not only most known Inc/Rep groups, but also a plethora of plasmids not previously assigned to any Inc group or Rep-type.

  7. Antimicrobial Peptide Production and Purification.

    Science.gov (United States)

    Suda, Srinivas; Field, Des; Barron, Niall

    2017-01-01

    Antimicrobial peptides (AMPs) are natural defense compounds which are synthesized as ribosomal gene-encoded pre-peptides and produced by all living organisms. AMPs are small peptides, usually cationic and typically have hydrophobic residues which interact with cell membranes and have either a narrow or broad spectrum of biological activity. AMPs are isolated from the natural host or heterologously expressed in other hosts such as Escherichia coli. The proto-typical lantibiotic Nisin is a widely used AMP that is produced by the food-grade organism Lactococcus lactis. Although AMP production and purification procedures require optimization for individual AMPs, the Nisin production and purification protocol outlined in this chapter can be easily applied with minor modifications for the production and purification of other lantibiotics or AMPs. While Nisin is produced and secreted into the supernatant, steps to recover Nisin from both cell-free supernatant and cell pellet are outlined in detail.

  8. Purification of bone morphogenetic protein-2 from refolding mixtures using mixed-mode membrane chromatography.

    Science.gov (United States)

    Gieseler, Gesa; Pepelanova, Iliyana; Stuckenberg, Lena; Villain, Louis; Nölle, Volker; Odenthal, Uwe; Beutel, Sascha; Rinas, Ursula; Scheper, Thomas

    2017-01-01

    In this study, we present the development of a process for the purification of recombinant human bone morphogenetic protein-2 (rhBMP-2) using mixed-mode membrane chromatography. RhBMP-2 was produced as inclusion bodies in Escherichia coli. In vitro refolding using rapid dilution was carried out according to a previously established protocol. Different membrane chromatography phases were analyzed for their ability to purify BMP-2. A membrane phase with salt-tolerant properties resulting from mixed-mode ligand chemistry was able to selectively purify BMP-2 dimer from refolding mixtures. No further purification or polishing steps were necessary and high product purity was obtained. The produced BMP-2 exhibited a biological activity of 7.4 × 10 5  U/mg, comparable to commercial preparations. Mixed-mode membrane chromatography can be a valuable tool for the direct purification of proteins from solutions with high-conductivity, for example refolding buffers. In addition, in this particular case, it allowed us to circumvent the use of heparin-affinity chromatography, thus allowing the design of an animal-component-free process.

  9. Protocol for Evaluating the Permissiveness of Bacterial Communities Toward Conjugal Plasmids by Quantification and Isolation of Transconjugants

    DEFF Research Database (Denmark)

    Klümper, Uli; Dechesne, Arnaud; Smets, Barth F.

    2014-01-01

    may encode catabolic pathways, virulence factors, and antibiotic or metal resistances, it is of environmental, evolutionary, and medical relevance to track and monitor the fate of plasmids in mixed microbial community. When assessing the short-term and long-term implications of conjugal plasmid...... a gfp-tagged plasmid in a mCherry red fluorescently tagged donor strain repressing gfp expression. We take advantage of fluorescent marker genes to microscopically detect plasmid transfer events and use subsequent high-throughput fluorescence-activated cell sorting (FACS) to isolate...

  10. Survival and evolution of a large multidrug resistance plasmid in new clinical bacterial hosts

    DEFF Research Database (Denmark)

    Porse, Andreas; Schønning, Kristian; Munck, Christian

    2016-01-01

    Large conjugative plasmids are important drivers of bacterial evolution and contribute significantly to the dissemination of antibiotic resistance. Although plasmid borne multidrug resistance is recognized as one of the main challenges in modern medicine, the adaptive forces shaping the evolution...

  11. A novel pAA virulence plasmid encoding toxins and two distinct variants of the fimbriae of enteroaggregative Escherichia coli

    DEFF Research Database (Denmark)

    Jønsson, Rie; Struve, Carsten; Boll, Erik J.

    2017-01-01

    phylogenetically distinct, strains harboring the major pilin subunits from both AAF/III and AAF/V. Whole-genome and plasmid sequencing revealed that in these six strains the agg3A and agg5A genes were located on a novel pAA plasmid variant. Moreover, the plasmid also encoded several other virulence genes including...... some not previously found on pAA plasmids. Thus, this plasmid endows the host strains with a remarkably high number of EAEC associated virulence genes hereby likely promoting strain pathogenicity....

  12. The sudden dominance of blaCTX-M harbouring plasmids in Shigella spp. Circulating in Southern Vietnam.

    Directory of Open Access Journals (Sweden)

    Nhu Thi Khanh Nguyen

    2010-06-01

    Full Text Available Plasmid mediated antimicrobial resistance in the Enterobacteriaceae is a global problem. The rise of CTX-M class extended spectrum beta lactamases (ESBLs has been well documented in industrialized countries. Vietnam is representative of a typical transitional middle income country where the spectrum of infectious diseases combined with the spread of drug resistance is shifting and bringing new healthcare challenges.We collected hospital admission data from the pediatric population attending the hospital for tropical diseases in Ho Chi Minh City with Shigella infections. Organisms were cultured from all enrolled patients and subjected to antimicrobial susceptibility testing. Those that were ESBL positive were subjected to further investigation. These investigations included PCR amplification for common ESBL genes, plasmid investigation, conjugation, microarray hybridization and DNA sequencing of a bla(CTX-M encoding plasmid.We show that two different bla(CTX-M genes are circulating in this bacterial population in this location. Sequence of one of the ESBL plasmids shows that rather than the gene being integrated into a preexisting MDR plasmid, the bla(CTX-M gene is located on relatively simple conjugative plasmid. The sequenced plasmid (pEG356 carried the bla(CTX-M-24 gene on an ISEcp1 element and demonstrated considerable sequence homology with other IncFI plasmids.The rapid dissemination, spread of antimicrobial resistance and changing population of Shigella spp. concurrent with economic growth are pertinent to many other countries undergoing similar development. Third generation cephalosporins are commonly used empiric antibiotics in Ho Chi Minh City. We recommend that these agents should not be considered for therapy of dysentery in this setting.

  13. CAPRRESI: Chimera Assembly by Plasmid Recovery and Restriction Enzyme Site Insertion.

    Science.gov (United States)

    Santillán, Orlando; Ramírez-Romero, Miguel A; Dávila, Guillermo

    2017-06-25

    Here, we present chimera assembly by plasmid recovery and restriction enzyme site insertion (CAPRRESI). CAPRRESI benefits from many strengths of the original plasmid recovery method and introduces restriction enzyme digestion to ease DNA ligation reactions (required for chimera assembly). For this protocol, users clone wildtype genes into the same plasmid (pUC18 or pUC19). After the in silico selection of amino acid sequence regions where chimeras should be assembled, users obtain all the synonym DNA sequences that encode them. Ad hoc Perl scripts enable users to determine all synonym DNA sequences. After this step, another Perl script searches for restriction enzyme sites on all synonym DNA sequences. This in silico analysis is also performed using the ampicillin resistance gene (ampR) found on pUC18/19 plasmids. Users design oligonucleotides inside synonym regions to disrupt wildtype and ampR genes by PCR. After obtaining and purifying complementary DNA fragments, restriction enzyme digestion is accomplished. Chimera assembly is achieved by ligating appropriate complementary DNA fragments. pUC18/19 vectors are selected for CAPRRESI because they offer technical advantages, such as small size (2,686 base pairs), high copy number, advantageous sequencing reaction features, and commercial availability. The usage of restriction enzymes for chimera assembly eliminates the need for DNA polymerases yielding blunt-ended products. CAPRRESI is a fast and low-cost method for fusing protein-coding genes.

  14. Combination of membrane technologies for purification of L (+) - lactic acid from juice of banana (Musa AAA, variety Cavendish cultivar Gram naine) obtained from an agroindustrial waste

    International Nuclear Information System (INIS)

    Murillo Viera, Esteban

    2013-01-01

    The process that has allowed recovery and purification of the L (+)-acid present in the juice fermented waste produced from banana was developed, treated enzymatically, using tangential nanofiltration. The effect of the enzymatic treatment was evaluated on physical chemical parameters of fermented banana juice. The process parameters of centrifugal clarification and microfiltration were characterized on banana juice as activities prior operations to recovery and purification of lactic acid. The temperature and the transmembrane pressure on the permeate flow and the performance of recovery and purification of lactic acid were evaluated by the ultrafiltration and nanofiltration processes. The properties physico-chemical the banana juice fermented and of the liquid filtrate obtained at the stage recovery and purification of lactic acid were compared by ultrafiltration [es

  15. Plasmid DNA loaded chitosan nanoparticles for nasal mucosal immunization against hepatitis B.

    Science.gov (United States)

    Khatri, Kapil; Goyal, Amit K; Gupta, Prem N; Mishra, Neeraj; Vyas, Suresh P

    2008-04-16

    This work investigates the preparation and in vivo efficacy of plasmid DNA loaded chitosan nanoparticles for nasal mucosal immunization against hepatitis B. Chitosan pDNA nanoparticles were prepared using a complex coacervation process. Prepared nanoparticles were characterized for size, shape, surface charge, plasmid loading and ability of nanoparticles to protect DNA against nuclease digestion and for their transfection efficacy. Nasal administration of nanoparticles resulted in serum anti-HBsAg titre that was less compared to that elicited by naked DNA and alum adsorbed HBsAg, but the mice were seroprotective within 2 weeks and the immunoglobulin level was above the clinically protective level. However, intramuscular administration of naked DNA and alum adsorbed HBsAg did not elicit sIgA titre in mucosal secretions that was induced by nasal immunization with chitosan nanoparticles. Similarly, cellular responses (cytokine levels) were poor in case of alum adsorbed HBsAg. Chitosan nanoparticles thus produced humoral (both systemic and mucosal) and cellular immune responses upon nasal administration. The study signifies the potential of chitosan nanoparticles as DNA vaccine carrier and adjuvant for effective immunization through non-invasive nasal route.

  16. Nanocellulose-Based Materials for Water Purification

    Directory of Open Access Journals (Sweden)

    Hugo Voisin

    2017-03-01

    Full Text Available Nanocellulose is a renewable material that combines a high surface area with high strength, chemical inertness, and versatile surface chemistry. In this review, we will briefly describe how nanocellulose is produced, and present—in particular, how nanocellulose and its surface modified versions affects the adsorption behavior of important water pollutants, e.g., heavy metal species, dyes, microbes, and organic molecules. The processing of nanocellulose-based membranes and filters for water purification will be described in detail, and the uptake capacity, selectivity, and removal efficiency will also be discussed. The processing and performance of nanocellulose-based membranes, which combine a high removal efficiency with anti-fouling properties, will be highlighted.

  17. Purification and concentration of uranium-bearing solutions at the plants of the Societe industrielle des minerais de l'Ouest

    International Nuclear Information System (INIS)

    Vollerin, G.

    1980-01-01

    The author describes the various processes for purification of uranium-bearing solutions used at the plants of the Societe industrielle des minerais de l'Ouest (SIMO) from their commissioning up to the present time, together with the purification circuit adopted at the two plants at present operating in Niger. (author)

  18. Diversity of Clostridium perfringens isolates from various sources and prevalence of conjugative plasmids.

    Science.gov (United States)

    Park, Miseon; Deck, Joanna; Foley, Steven L; Nayak, Rajesh; Songer, J Glenn; Seibel, Janice R; Khan, Saeed A; Rooney, Alejandro P; Hecht, David W; Rafii, Fatemeh

    2016-04-01

    Clostridium perfringens is an important pathogen, causing food poisoning and other mild to severe infections in humans and animals. Some strains of C. perfringens contain conjugative plasmids, which may carry antimicrobial resistance and toxin genes. We studied genomic and plasmid diversity of 145 C. perfringens type A strains isolated from soils, foods, chickens, clinical samples, and domestic animals (porcine, bovine and canine), from different geographic areas in the United States between 1994 and 2006, using multiple-locus variable-number tandem repeat analysis (MLVA) and/or pulsed-field gel electrophoresis (PFGE). MLVA detected the genetic diversity in a majority of the isolates. PFGE, using SmaI and KspI, confirmed the MLVA results but also detected differences among the strains that could not be differentiated by MLVA. All of the PFGE profiles of the strains were different, except for a few of the epidemiologically related strains, which were identical. The PFGE profiles of strains isolated from the same domestic animal species were clustered more closely with each other than with other strains. However, a variety of C. perfringens strains with distinct genetic backgrounds were found among the clinical isolates. Variation was also observed in the size and number of plasmids in the strains. Primers for the internal fragment of a conjugative tcpH gene of C. perfringens plasmid pCPF4969 amplified identical size fragments from a majority of strains tested; and this gene hybridized to the various-sized plasmids of these strains. The sequences of the PCR-amplified tcpH genes from 12 strains showed diversity among the tcpH genes. Regardless of the sources of the isolates, the genetic diversity of C. perfringens extended to the plasmids carrying conjugative genes. Published by Elsevier Ltd.

  19. Characterization of replication and conjugation of plasmid pWTY27 from a widely distributed Streptomyces species

    Directory of Open Access Journals (Sweden)

    Wang Tao

    2012-11-01

    Full Text Available Abstract Background Streptomyces species are widely distributed in natural habitats, such as soils, lakes, plants and some extreme environments. Replication loci of several Streptomyces theta-type plasmids have been reported, but are not characterized in details. Conjugation loci of some Streptomyces rolling-circle-type plasmids are identified and mechanism of conjugal transferring are described. Results We report the detection of a widely distributed Streptomyces strain Y27 and its indigenous plasmid pWTY27 from fourteen plants and four soil samples cross China by both culturing and nonculturing methods. The complete nucleotide sequence of pWTY27 consisted of 14,288 bp. A basic locus for plasmid replication comprised repAB genes and an adjacent iteron sequence, to a long inverted-repeat (ca. 105 bp of which the RepA protein bound specifically in vitro, suggesting that RepA may recognize a second structure (e.g. a long stem-loop of the iteron DNA. A plasmid containing the locus propagated in linear mode when the telomeres of a linear plasmid were attached, indicating a bi-directional replication mode for pWTY27. As for rolling-circle plasmids, a single traA gene and a clt sequence (covering 16 bp within traA and its adjacent 159 bp on pWTY27 were required for plasmid transfer. TraA recognized and bound specifically to the two regions of the clt sequence, one containing all the four DC1 of 7 bp (TGACACC and one DC2 (CCCGCCC and most of IC1, and another covering two DC2 and part of IC1, suggesting formation of a high-ordered DNA-protein complex. Conclusions This work (i isolates a widespread Streptomyces strain Y27 and sequences its indigenous theta-type plasmid pWTY27; (ii identifies the replication and conjugation loci of pWTY27 and; (iii characterizes the binding sequences of the RepA and TraA proteins.

  20. Characterization of replication and conjugation of plasmid pWTY27 from a widely distributed Streptomyces species

    Science.gov (United States)

    2012-01-01

    Background Streptomyces species are widely distributed in natural habitats, such as soils, lakes, plants and some extreme environments. Replication loci of several Streptomyces theta-type plasmids have been reported, but are not characterized in details. Conjugation loci of some Streptomyces rolling-circle-type plasmids are identified and mechanism of conjugal transferring are described. Results We report the detection of a widely distributed Streptomyces strain Y27 and its indigenous plasmid pWTY27 from fourteen plants and four soil samples cross China by both culturing and nonculturing methods. The complete nucleotide sequence of pWTY27 consisted of 14,288 bp. A basic locus for plasmid replication comprised repAB genes and an adjacent iteron sequence, to a long inverted-repeat (ca. 105 bp) of which the RepA protein bound specifically in vitro, suggesting that RepA may recognize a second structure (e.g. a long stem-loop) of the iteron DNA. A plasmid containing the locus propagated in linear mode when the telomeres of a linear plasmid were attached, indicating a bi-directional replication mode for pWTY27. As for rolling-circle plasmids, a single traA gene and a clt sequence (covering 16 bp within traA and its adjacent 159 bp) on pWTY27 were required for plasmid transfer. TraA recognized and bound specifically to the two regions of the clt sequence, one containing all the four DC1 of 7 bp (TGACACC) and one DC2 (CCCGCCC) and most of IC1, and another covering two DC2 and part of IC1, suggesting formation of a high-ordered DNA-protein complex. Conclusions This work (i) isolates a widespread Streptomyces strain Y27 and sequences its indigenous theta-type plasmid pWTY27; (ii) identifies the replication and conjugation loci of pWTY27 and; (iii) characterizes the binding sequences of the RepA and TraA proteins. PMID:23134842

  1. Purification-Free, Target-Selective Immobilization of a Protein from Cell Lysates.

    Science.gov (United States)

    Cha, Jaehyun; Kwon, Inchan

    2018-02-27

    Protein immobilization has been widely used for laboratory experiments and industrial processes. Preparation of a recombinant protein for immobilization usually requires laborious and expensive purification steps. Here, a novel purification-free, target-selective immobilization technique of a protein from cell lysates is reported. Purification steps are skipped by immobilizing a target protein containing a clickable non-natural amino acid (p-azidophenylalanine) in cell lysates onto alkyne-functionalized solid supports via bioorthogonal azide-alkyne cycloaddition. In order to achieve a target protein-selective immobilization, p-azidophenylalanine was introduced into an exogenous target protein, but not into endogenous non-target proteins using host cells with amber codon-free genomic DNAs. Immobilization of superfolder fluorescent protein (sfGFP) from cell lysates is as efficient as that of the purified sfGFP. Using two fluorescent proteins (sfGFP and mCherry), the authors also demonstrated that the target proteins are immobilized with a minimal immobilization of non-target proteins (target-selective immobilization). © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Optimization of a micro-scale, high throughput process development tool and the demonstration of comparable process performance and product quality with biopharmaceutical manufacturing processes.

    Science.gov (United States)

    Evans, Steven T; Stewart, Kevin D; Afdahl, Chris; Patel, Rohan; Newell, Kelcy J

    2017-07-14

    In this paper, we discuss the optimization and implementation of a high throughput process development (HTPD) tool that utilizes commercially available micro-liter sized column technology for the purification of multiple clinically significant monoclonal antibodies. Chromatographic profiles generated using this optimized tool are shown to overlay with comparable profiles from the conventional bench-scale and clinical manufacturing scale. Further, all product quality attributes measured are comparable across scales for the mAb purifications. In addition to supporting chromatography process development efforts (e.g., optimization screening), comparable product quality results at all scales makes this tool is an appropriate scale model to enable purification and product quality comparisons of HTPD bioreactors conditions. The ability to perform up to 8 chromatography purifications in parallel with reduced material requirements per run creates opportunities for gathering more process knowledge in less time. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  3. Drinking water purification in the Czech Republic and worldwide

    International Nuclear Information System (INIS)

    Krmela, Jan; Beckova, Vera; Vlcek, Jaroslav; Marhol, Milan

    2012-06-01

    The report is structured as follows: (i) Legislative (hygienic) requirements for technologies applied to drinking water purification with focus on uranium elimination; (ii) Technological drinking water treatment processes (settling, filtration, precipitation, acidification, iron and manganese removal) ; (iii) State Office for Nuclear Safety requirements for the operation of facilities to separate uranium from drinking water and for the handling of saturated ionexes from such facilities; (iv) Material requirements for the operation of ionex filters serving to separate uranium from drinking water; (v) Effect of enhanced uranium concentrations in drinking waters on human body; (vi) Uranium speciation in ground waters; (vii) Brief description of technologies which are used worldwide for uranium removal; (viii) Technologies which are usable and are used in the Czech Republic for drinking water purification from uranium; (ix) Inorganic and organic ion exchangers and sorbents. (P.A.)

  4. Persistence of plasmids, cholera toxin genes, and prophage DNA in classical Vibrio cholerae O1.

    OpenAIRE

    Cook, W L; Wachsmuth, K; Johnson, S R; Birkness, K A; Samadi, A R

    1984-01-01

    Plasmid profiles, the location of cholera toxin subunit A genes, and the presence of the defective VcA1 prophage genome in classical Vibrio cholerae isolated from patients in Bangladesh in 1982 were compared with those in older classical strains isolated during the sixth pandemic and with those in selected eltor and nontoxigenic O1 isolates. Classical strains typically had two plasmids (21 and 3 megadaltons), eltor strains typically had no plasmids, and nontoxigenic O1 strains had zero to thr...

  5. Destabilization of IncA and IncC plasmids by SGI1 and SGI2 type Salmonella genomic islands.

    Science.gov (United States)

    Harmer, Christopher J; Hamidian, Mohammad; Ambrose, Stephanie J; Hall, Ruth M

    Both the Salmonella genomic islands (SGI) and the conjugative IncC plasmids are known to contribute substantially to the acquisition of resistance to multiple antibiotics, and plasmids in the A/C group are known to mobilize the Salmonella genomic island SGI1, which also carries multiple antibiotic resistance genes. Plasmid pRMH760 (IncC; A/C 2 ) was shown to mobilize SGI1 variants SGI1-I, SGI1-F, SGI1-K and SGI2 from Salmonella enterica to Escherichia coli where it was integrated at the preferred location, at the end of the trmE (thdF) gene. The plasmid was transferred at a similar frequency. However, we observed that co-transfer of the SGI and the plasmid was rarer. In E. coli to E. coli transfer, the frequency of transfer of the IncC plasmid pRMH760 was at least 1000-fold lower when the donor carried SGI1-I or SGI1-K, indicating that the SGI suppresses transfer of the plasmid. In addition, pRMH760 was rapidly lost from both E. coli and S. enterica strains that also carried SGI1-I, SGI1-F or SGI2. However, plasmid loss was not seen when the SGI1 variant was SGI1-K, which lacks two segments of the SGI1 backbone. The complete sequence of the SGI1-I and SGI1-F were determined and SGI1-K also carries two single base substitutions relative to SGI1-I. The IncA (A/C 1 ) plasmid RA1 was also shown to mobilize SGI2-A and though there are significant differences between the backbones of IncA and IncC plasmids, RA1 was also rapidly lost when SGI2-A was present in the same cell. We conclude that there are multiple interactions, both cooperative and antagonistic, between an IncA or IncC plasmid and the SGI1 and SGI2 family genomic islands. Copyright © 2016 Elsevier B.V. All rights reserved.

  6. RELIGION AND PURIFICATION OF SOUL

    Directory of Open Access Journals (Sweden)

    Azam Khodashenas Pelko

    2010-11-01

    Full Text Available The Jainism emphasizes three major teachings about the purification of the soul (jiva, Ahimsa, Aparigrapha and anekantwad. Jainism, The focus of this religion has been purification of the soul by means of right conduct, right faith and right knowledge. The ultimate goal of Hinduism is Moksha or liberation (total freedom. In Hinduism, purification of the soul is a goal that one must work to attain. The Buddhism is the science of pursuing the aim of making the human mind perfect, and of purifying the human soul. The knowledge of purifying of the soul and softening of the hearts is as essential for human. They having the correct motivations means purifying our souls from hypocrisy, caprice, and heedlessness. The primary goal of Taoism may be described as the mystical intuition of the Tao, which is the way, the undivided unity, and the ultimate Reality. According to the Christianity access to truth cannot be conceived without purity of the soul

  7. Comparative genomics of multidrug resistance-encoding IncA/C plasmids from commensal and pathogenic Escherichia coli from multiple animal sources.

    Science.gov (United States)

    Fernández-Alarcón, Claudia; Singer, Randall S; Johnson, Timothy J

    2011-01-01

    Incompatibility group A/C (IncA/C) plasmids have received recent attention for their broad host range and ability to confer resistance to multiple antimicrobial agents. Due to the potential spread of multidrug resistance (MDR) phenotypes from foodborne pathogens to human pathogens, the dissemination of these plasmids represents a public health risk. In this study, four animal-source IncA/C plasmids isolated from Escherichia coli were sequenced and analyzed, including isolates from commercial dairy cows, pigs and turkeys in the U.S. and Chile. These plasmids were initially selected because they either contained the floR and tetA genes encoding for florfenicol and tetracycline resistance, respectively, and/or the bla(CMY-2) gene encoding for extended spectrum β-lactamase resistance. Overall, sequence analysis revealed that each of the four plasmids retained a remarkably stable and conserved backbone sequence, with differences observed primarily within their accessory regions, which presumably have evolved via horizontal gene transfer events involving multiple modules. Comparison of these plasmids with other available IncA/C plasmid sequences further defined the core and accessory elements of these plasmids in E. coli and Salmonella. Our results suggest that the bla(CMY-2) plasmid lineage appears to have derived from an ancestral IncA/C plasmid type harboring floR-tetAR-strAB and Tn21-like accessory modules. Evidence is mounting that IncA/C plasmids are widespread among enteric bacteria of production animals and these emergent plasmids have flexibility in their acquisition of MDR-encoding modules, necessitating further study to understand the evolutionary mechanisms involved in their dissemination and stability in bacterial populations.

  8. Comparative genomics of multidrug resistance-encoding IncA/C plasmids from commensal and pathogenic Escherichia coli from multiple animal sources.

    Directory of Open Access Journals (Sweden)

    Claudia Fernández-Alarcón

    Full Text Available Incompatibility group A/C (IncA/C plasmids have received recent attention for their broad host range and ability to confer resistance to multiple antimicrobial agents. Due to the potential spread of multidrug resistance (MDR phenotypes from foodborne pathogens to human pathogens, the dissemination of these plasmids represents a public health risk. In this study, four animal-source IncA/C plasmids isolated from Escherichia coli were sequenced and analyzed, including isolates from commercial dairy cows, pigs and turkeys in the U.S. and Chile. These plasmids were initially selected because they either contained the floR and tetA genes encoding for florfenicol and tetracycline resistance, respectively, and/or the bla(CMY-2 gene encoding for extended spectrum β-lactamase resistance. Overall, sequence analysis revealed that each of the four plasmids retained a remarkably stable and conserved backbone sequence, with differences observed primarily within their accessory regions, which presumably have evolved via horizontal gene transfer events involving multiple modules. Comparison of these plasmids with other available IncA/C plasmid sequences further defined the core and accessory elements of these plasmids in E. coli and Salmonella. Our results suggest that the bla(CMY-2 plasmid lineage appears to have derived from an ancestral IncA/C plasmid type harboring floR-tetAR-strAB and Tn21-like accessory modules. Evidence is mounting that IncA/C plasmids are widespread among enteric bacteria of production animals and these emergent plasmids have flexibility in their acquisition of MDR-encoding modules, necessitating further study to understand the evolutionary mechanisms involved in their dissemination and stability in bacterial populations.

  9. Mechanisms Involved in Acquisition of blaNDM Genes by IncA/C2 and IncFIIY Plasmids.

    Science.gov (United States)

    Wailan, Alexander M; Sidjabat, Hanna E; Yam, Wan Keat; Alikhan, Nabil-Fareed; Petty, Nicola K; Sartor, Anna L; Williamson, Deborah A; Forde, Brian M; Schembri, Mark A; Beatson, Scott A; Paterson, David L; Walsh, Timothy R; Partridge, Sally R

    2016-07-01

    blaNDM genes confer carbapenem resistance and have been identified on transferable plasmids belonging to different incompatibility (Inc) groups. Here we present the complete sequences of four plasmids carrying a blaNDM gene, pKP1-NDM-1, pEC2-NDM-3, pECL3-NDM-1, and pEC4-NDM-6, from four clinical samples originating from four different patients. Different plasmids carry segments that align to different parts of the blaNDM region found on Acinetobacter plasmids. pKP1-NDM-1 and pEC2-NDM-3, from Klebsiella pneumoniae and Escherichia coli, respectively, were identified as type 1 IncA/C2 plasmids with almost identical backbones. Different regions carrying blaNDM are inserted in different locations in the antibiotic resistance island known as ARI-A, and ISCR1 may have been involved in the acquisition of blaNDM-3 by pEC2-NDM-3. pECL3-NDM-1 and pEC4-NDM-6, from Enterobacter cloacae and E. coli, respectively, have similar IncFIIY backbones, but different regions carrying blaNDM are found in different locations. Tn3-derived inverted-repeat transposable elements (TIME) appear to have been involved in the acquisition of blaNDM-6 by pEC4-NDM-6 and the rmtC 16S rRNA methylase gene by IncFIIY plasmids. Characterization of these plasmids further demonstrates that even very closely related plasmids may have acquired blaNDM genes by different mechanisms. These findings also illustrate the complex relationships between antimicrobial resistance genes, transposable elements, and plasmids and provide insights into the possible routes for transmission of blaNDM genes among species of the Enterobacteriaceae family. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  10. Single-step affinity purification for fungal proteomics.

    Science.gov (United States)

    Liu, Hui-Lin; Osmani, Aysha H; Ukil, Leena; Son, Sunghun; Markossian, Sarine; Shen, Kuo-Fang; Govindaraghavan, Meera; Varadaraj, Archana; Hashmi, Shahr B; De Souza, Colin P; Osmani, Stephen A

    2010-05-01

    A single-step protein affinity purification protocol using Aspergillus nidulans is described. Detailed protocols for cell breakage, affinity purification, and depending on the application, methods for protein release from affinity beads are provided. Examples defining the utility of the approaches, which should be widely applicable, are included.

  11. Design of functional guanidinium ionic liquid aqueous two-phase systems for the efficient purification of protein.

    Science.gov (United States)

    Ding, Xueqin; Wang, Yuzhi; Zeng, Qun; Chen, Jing; Huang, Yanhua; Xu, Kaijia

    2014-03-07

    A series of novel cationic functional hexaalkylguanidinium ionic liquids and anionic functional tetraalkylguanidinium ionic liquids have been devised and synthesized based on 1,1,3,3-tetramethylguanidine. The structures of the ionic liquids (ILs) were confirmed by (1)H nuclear magnetic resonance ((1)H NMR) and 13C nuclear magnetic resonance (13C NMR) and the production yields were all above 90%. Functional guanidinium ionic liquid aqueous two-phase systems (FGIL-ATPSs) have been first designed with these functional guanidinium ILs and phosphate solution for the purification of protein. After phase separation, proteins had transferred into the IL-rich phase and the concentrations of proteins were determined by measuring the absorbance at 278 nm using an ultra violet visible (UV-vis) spectrophotometer. The advantages of FGIL-ATPSs were compared with ordinary ionic liquid aqueous two-phase systems (IL-ATPSs). The proposed FGIL-ATPS has been applied to purify lysozyme, trypsin, ovalbumin and bovine serum albumin. Single factor experiments were used to research the effects of the process, such as the amount of ionic liquid (IL), the concentration of salt solution, temperature and the amount of protein. The purification efficiency reaches to 97.05%. The secondary structure of protein during the experimental process was observed upon investigation using UV-vis spectrophotometer, Fourier-transform infrared spectroscopy (FT-IR) and circular dichroism spectrum (CD spectrum). The precision, stability and repeatability of the process were investigated. The mechanisms of purification were researched by dynamic light scattering (DLS), determination of the conductivity and transmission electron microscopy (TEM). It was suggested that aggregation and embrace phenomenon play a significant role in the purification of proteins. All the results show that FGIL-ATPSs have huge potential to offer new possibility in the purification of proteins. Copyright © 2014 Elsevier B.V. All rights

  12. CHARACTERIZATION OF SINGLE-STRAND ORIGINS OF CRYPTIC ROLLING-CIRCLE PLASMIDS FROM BACILLUS-SUBTILIS

    NARCIS (Netherlands)

    MEIJER, WJJ; VENEMA, G; BRON, S

    1995-01-01

    In this paper we describe the isolation and characterization of single strand origins (SSOs) of several cryptic Bacillus subtilis plasmids which use the rolling-circle mechanism of replication, The plasmids used in this study involved pTA1015, pTA1020, pTA1030, pTA1040, pTA1050 and pTA1060, The SSO

  13. Modeling Xenon Purification Systems in a Laser Inertial Fusion Engine

    Science.gov (United States)

    Hopkins, Ann; Gentile, Charles

    2011-10-01

    A Laser Inertial Fusion Engine (LIFE) is a proposed method to employ fusion energy to produce electricity for consumers. However, before it can be built and used as such, each aspect of a LIFE power plant must first be meticulously planned. We are in the process of developing and perfecting models for an exhaust processing and fuel recovery system. Such a system is especially essential because it must be able to recapture and purify expensive materials involved in the reaction so they may be reused. One such material is xenon, which is to be used as an intervention gas in the target chamber. Using Aspen HYSYS, we have modeled several subsystems for exhaust processing, including a subsystem for xenon recovery and purification. After removing hydrogen isotopes using lithium bubblers, we propose to use cryogenic distillation to purify the xenon from remaining contaminants. Aspen HYSYS allows us to analyze predicted flow rates, temperatures, pressures, and compositions within almost all areas of the xenon purification system. Through use of Aspen models, we hope to establish that we can use xenon in LIFE efficiently and in a practical manner.

  14. Development of the protocol for purification of artemisinin based on combination of commercial and computationally designed adsorbents.

    Science.gov (United States)

    Piletska, Elena V; Karim, Kal; Cutler, Malcolm; Piletsky, Sergey A

    2013-01-01

    A polymeric adsorbent for extraction of the antimalarial drug artemisinin from Artemisia annua L. was computationally designed. This polymer demonstrated a high capacity for artemisinin (120 mg g(-1) ), quantitative recovery (87%) and was found to be an effective material for purification of artemisinin from complex plant matrix. The artemisinin quantification was conducted using an optimised HPLC-MS protocol, which was characterised by high precision and linearity in the concentration range between 0.05 and 2 μg mL(-1) . Optimisation of the purification protocol also involved screening of commercial adsorbents for the removal of waxes and other interfering natural compounds, which inhibit the crystallisation of artemisinin. As a result of a two step-purification protocol crystals of artemisinin were obtained, and artemisinin purity was evaluated as 75%. By performing the second stage of purification twice, the purity of artemisinin can be further improved to 99%. The developed protocol produced high-purity artemisinin using only a few purification steps that makes it suitable for large scale industrial manufacturing process. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Air Stripping Designs and Reactive Water Purification Processes for the Lunar Surface

    Science.gov (United States)

    Boul, Peter J.; Lange, Kevin; Conger, Bruce; Anderson, Molly

    2010-01-01

    Air stripping designs are considered to reduce the presence of volatile organic compounds in the purified water. Components of the wastewater streams are ranked by Henry's Law Constant and the suitability of air stripping in the purification of wastewater in terms of component removal is evaluated. Distillation processes are modeled in tandem with air stripping to demonstrate the potential effectiveness and utility of these methods in recycling wastewater on the Moon. Scaling factors for distillation and air stripping columns are presented to account for the difference in the lunar gravitation environment. Commercially available distillation and air stripping units which are considered suitable for Exploration Life Support are presented. The advantages to the various designs are summarized with respect to water purity levels, power consumption, and processing rates. An evaluation of reactive distillation and air stripping is presented with regards to the reduction of volatile organic compounds in the contaminated water and air. Among the methods presented, an architecture is presented for the evaluation of the simultaneous oxidation of organics in air and water. These and other designs are presented in light of potential improvements in power consumptions and air and water purities for architectures which include catalytic activity integrated into the water processor. In particular, catalytic oxidation of organics may be useful as a tool to remove contaminants that more traditional distillation and/or air stripping columns may not remove. A review of the current leading edge at the commercial level and at the research frontier in catalytically active materials is presented. Themes and directions from the engineering developments in catalyst design are presented conceptually in light of developments in the nanoscale chemistry of a variety of catalyst materials.

  16. Cloning, expression, and purification of recombinant protein MPT-64 from a virulent strain of Mycobacterium bovis in a prokaryotic system.

    Science.gov (United States)

    Tashakkori, Maryam Mohammadi; Tebianian, Majid; Tabatabaei, Mohammad; Mosavari, Nader

    2016-12-01

    Tuberculosis (TB) is a zoonotic infectious disease common to humans and animals that is caused by the rod-shaped acid-fast bacterium Mycobacterium bovis. Rapid and sensitive detection of TB is promoted by specific antigens. Virulent strains of the TB complex from M. bovis contain 16 regions of difference (RD) in their genome that encode important proteins, including major protein of Mycobacterium Tuberculosis 64 (MBT-64, which is a primary immune-stimulating antigen encoded by RD-2. In this study, we cloned, expressed, and purified MPT-64 as a potent M. bovis antigen in a prokaryotic system for use in future diagnostic studies. The antigenic region of the Mpt64 gene was investigated by bioinformatics methods, cloned into the PQE-30 plasmid, and expressed in Escherichia coli M15 cells, followed by isopropyl β-d-1-thiogalactopyranoside induction. The expressed protein was analyzed sodium dodecyl sulfate polyacrylamide gel electrophoresis and purified using a nickel-affinity column. Biological activity was confirmed by western blot using specific antibodies. Our data verified the successful cloning of the Mpt64 gene (687-bp segment) via the expression vector and purification of recombinant MPT-64 as a 24-kDa protein. These results indicated successful expression and purification of recombinant MPT-64 protein in a prokaryotic system. This protein can be used for serological diagnosis, improved detection of pathogenicity and non-pathogenicity between infected cattle, and for verification of suspected cases of bovine TB. Copyright © 2016.

  17. Characteristics of plasmids in multi-drug-resistant Enterobacteriaceae isolated during prospective surveillance of a newly opened hospital in Iraq.

    Directory of Open Access Journals (Sweden)

    Xiao-Zhe Huang

    Full Text Available BACKGROUND: Gram-negative multidrug-resistant (MDR bacteria are major causes of nosocomial infections, and antibiotic resistance in these organisms is often plasmid mediated. Data are scarce pertaining to molecular mechanisms of antibiotic resistance in resource constrained areas such as Iraq. METHODOLOGY/PRINCIPAL FINDINGS: In this study, all MDR Enterobacteriaceae (n = 38 and randomly selected non-MDR counterparts (n = 41 isolated from patients, healthcare workers and environmental surfaces in a newly opened hospital in Iraq were investigated to characterize plasmids found in these isolates and determine their contribution to antibiotic resistance. Our results demonstrated that MDR E. coli and K. pneumoniae isolates harbored significantly more (≥ 3 plasmids compared to their non-MDR counterparts, which carried ≤ 2 plasmids (p<0.01. Various large plasmids (~52 to 100 kb from representative isolates were confirmed to contain multiple resistance genes by DNA microarray analysis. Aminoglycoside (acc, aadA, aph, strA/B, and ksgA, β-lactam (bla(TEM1, bla(AMPC, bla(CTX-M-15, bla(OXA-1, bla(VIM-2 and bla(SHV, sulfamethoxazole/trimethoprim (sul/dfr, tetracycline (tet and chloramphenicol (cat resistance genes were detected on these plasmids. Additionally, multiple plasmids carrying multiple antibiotic resistance genes were found in the same host strain. Genetic transfer-associated genes were identified on the plasmids from both MDR and non-MDR isolates. Seven plasmid replicon types (FII, FIA, FIB, B/O, K, I1 and N were detected in the isolates, while globally disseminated IncA/C and IncHI1 plasmids were not detected in these isolates. CONCLUSIONS/SIGNIFICANCE: This is the first report of the characteristics of the plasmids found in Enterobacteriaceae isolated following the opening of a new hospital in Iraq. The information provided here furthers our understanding of the mechanisms of drug resistance in this specific region and their evolutionary

  18. Gene electrotransfer of plasmid antiangiogenic metargidin peptide (AMEP) in disseminated melanoma

    DEFF Research Database (Denmark)

    Spanggaard, Iben; Snoj, Marko; Cavalcanti, Andrea

    2013-01-01

    ). In each patient, two cutaneous lesions were identified (one treated and one control). At day 1 and day 8, plasmid AMEP was injected intratumorally followed by electrotransfer. Patients were monitored weekly until day 29, and at day 64. Local efficacy was assessed at day 29 by direct measurement...... lesions increased more than 20%. No response occurred in distant lesions. This first-in-man study on electrotransfer of plasmid AMEP into cutaneous melanoma shows that the procedure and drug are safe and that local transfection was obtained....

  19. Plasmids from Food Lactic Acid Bacteria: Diversity, Similarity, and New Developments

    Science.gov (United States)

    Cui, Yanhua; Hu, Tong; Qu, Xiaojun; Zhang, Lanwei; Ding, Zhongqing; Dong, Aijun

    2015-01-01

    Plasmids are widely distributed in different sources of lactic acid bacteria (LAB) as self-replicating extrachromosomal genetic materials, and have received considerable attention due to their close relationship with many important functions as well as some industrially relevant characteristics of the LAB species. They are interesting with regard to the development of food-grade cloning vectors. This review summarizes new developments in the area of lactic acid bacteria plasmids and aims to provide up to date information that can be used in related future research. PMID:26068451

  20. Antimicrobial resistance and plasmid profiles of Aeromonas ...

    African Journals Online (AJOL)

    The purpose of this study was to investigate the presence of Aeromonas hydrophila at commonly used water collection points on the River Njoro and to determine the in-vitro antimicrobial susceptibility and plasmid profiles of isolates. In total, 126 samples were collected and 36.5% of them were positive for A. hydrophila.