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Sample records for plasmid vector subcloning

  1. Cloning of Dense Granular (GRA 7 Gene of Toxoplasma gondii into pTZ57RT Vectors for Sub-Cloning in Prokaryotic and Eukaryotic Plasmids

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    Zahra Arab-Mazar

    2014-11-01

    Full Text Available Background: Serological assay based on dense granular (GRA proteins of Toxoplasma gondii (T. gondii is actually the most popular laboratory diagnostic tool to detection of toxoplasmosis. We aimed to construct a recombinant GRA7-pTZ57RT plasmid vectors that it is suitable for sub-cloning and GRA7 protein production.Materials and Methods: Souris mice were used for maintaining of T. gondii tachyzoites by serial intraperitoneal passage. The tachyzoites’ DNA was extracted, and the GRA7 gene was amplified by PCR. The purified DNA was inserted into pTZ57RT cloning vectors, and then transformed into TOP10 competent cells. Finally, cloning and transformation were confirmed by restriction enzymatic digestion and gene sequencing.Results: Agarose gel electrophoresis analysis on PCR products of genomic DNA, revealed 726 bp bands that were equal to the GRA7 gene. Both white (recombinant and blue (non-recombinant colonies appeared on ampicillin-LB agar. Results of enzymatic digestion and gene sequencing confirmed successful cloning and transformation procedures.Conclusion: The GRA7 gene of T. gondii was cloned into pTZ57RT plasmid, which is suggested to be further used as DNA vaccine or sub-cloned for production of recombinant GRA7 protein.

  2. Subcloning plus insertion (SPI)--a novel recombineering method for the rapid construction of gene targeting vectors.

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    Reddy, Thimma R; Kelsall, Emma J; Fevat, Léna M S; Munson, Sarah E; Cowley, Shaun M

    2015-01-08

    Gene targeting refers to the precise modification of a genetic locus using homologous recombination. The generation of novel cell lines and transgenic mouse models using this method necessitates the construction of a 'targeting' vector, which contains homologous DNA sequences to the target gene, and has for many years been a limiting step in the process. Vector construction can be performed in vivo in Escherichia coli cells using homologous recombination mediated by phage recombinases using a technique termed recombineering. Recombineering is the preferred technique to subclone the long homology sequences (>4 kb) and various targeting elements including selection markers that are required to mediate efficient allelic exchange between a targeting vector and its cognate genomic locus. Typical recombineering protocols follow an iterative scheme of step-wise integration of the targeting elements and require intermediate purification and transformation steps. Here, we present a novel recombineering methodology of vector assembly using a multiplex approach. Plasmid gap repair is performed by the simultaneous capture of genomic sequence from mouse Bacterial Artificial Chromosome libraries and the insertion of dual bacterial and mammalian selection markers. This subcloning plus insertion method is highly efficient and yields a majority of correct recombinants. We present data for the construction of different types of conditional gene knockout, or knock-in, vectors and BAC reporter vectors that have been constructed using this method. SPI vector construction greatly extends the repertoire of the recombineering toolbox and provides a simple, rapid and cost-effective method of constructing these highly complex vectors.

  3. A cryptic promoter in potato virus X vector interrupted plasmid construction

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    Schultz Ronald D

    2007-03-01

    Full Text Available Abstract Background Potato virus X has been developed into an expression vector for plants. It is widely used to express foreign genes. In molecular manipulation, the foreign genes need to be sub-cloned into the vector. The constructed plasmid needs to be amplified. Usually, during amplification stage, the foreign genes are not expressed. However, if the foreign gene is expressed, the construction work could be interrupted. Two different viral genes were sub-cloned into the vector, but only one foreign gene was successfully sub-cloned. The other foreign gene, canine parvovirus type 2 (CPV-2 VP1 could not be sub-cloned into the vector and amplified without mutation (frame shift mutation. Results A cryptic promoter in the PVX vector was discovered with RT-PCR. The promoter activity was studied with Northern blots and Real-time RT-PCR. Conclusion It is important to recognize the homologous promoter sequences in the vector when a virus is developed as an expression vector. During the plasmid amplification stage, an unexpected expression of the CPV-2 VP1 gene (not in the target plants, but in E. coli can interrupt the downstream work.

  4. Molecular Epidemiology of KPC-Producing Escherichia coli: Occurrence of ST131-fimH30 Subclone Harboring pKpQIL-Like IncFIIk Plasmid

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    O'Hara, Jessica A.; Hu, Fupin; Ahn, Chulsoo; Nelson, Jeremy; Rivera, Jesabel I.; Pasculle, Anthony W.

    2014-01-01

    Of 20 Klebsiella pneumoniae carbapenemase (KPC)-producing Escherichia coli isolates identified at hospitals in western Pennsylvania, 60% belonged to the epidemic ST131-fimH30 subclone. IncFIIk was the most common replicon type for the blaKPC-carrying plasmids (n = 8). All IncFIIk plasmids possessed a scaffold similar to that of pKpQIL, and seven of them were borne by ST131-fimH30 isolates. IncN plasmids conferred resistance to trimethoprim-sulfamethoxazole, and IncA/C plasmids conferred resistance to gentamicin. Three blaKPC-carrying plasmids (IncA/C and IncN) possessed blaSHV-7/12 and qnrA1 or qnrS1. PMID:24820082

  5. An easy and versatile 2-step protocol for targeted modification and subcloning of DNA from bacterial artificial chromosomes using non-commercial plasmids

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    Hartwich Heiner

    2012-03-01

    Full Text Available Abstract Background Promoter-specific expression of foreign DNA in transgenic organisms often relies on bacterial artificial chromosomes (BACs. This approach requires modification and subcloning of BAC-DNA by recombineering technologies in Escherichia coli. Most current protocols rely on commercial kits or isolation of BACs, their transfer between different host strains, and their restriction. Findings In this report we present a 2-step protocol for efficient modification and subcloning of DNA from bacterial artificial chromosomes using the non-commercial plasmids pKM208 and pTP223, distributed from addgene.com. A targeting cassette was successfully integrated into a BAC and 42 kb of this construct were subcloned. Both a plasmid-derived substrate with longer homology arms and a PCR-generated substrate with short homology arms (50 bp were used for recombination. pKM208 and pTP223 contain all required genes for recombineering, but differ in their antibiotic resistance genes. This makes the system independent of the selection markers on the DNA molecules targeted for recombination. Conclusions The time and cost saving protocol presented here compares favorably to currently used systems. Using non-commercial plasmids, it allows targeted modification and cloning of large DNA (> 40 kb fragments in vivo without restriction and ligation. Furthermore, both steps are performed in the same host eliminating the need to isolate BAC DNA and to use different bacterial strains.

  6. The Inhibitory Effects of an Antisense u-PAR Vector on Invasion of Highly Invasive Human Prostate Carcinoma PC-3M Cell Subclones

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    廖国宁; 李清芬; 冯友梅; 邓耀祖; 李卓娅; 龚非力; 马丁

    2003-01-01

    Summary: To observe the inhibitory effects of an antisense u-PAR vector on invasion of highly inva-sive PC-3M cell subclones, the effects of the antisense u-PAR on activity of MMP-9 in those highlyinvasive cell subclones were detected by a quantitative RT-PCR and zymography. The monolayer in-vasion assay and colony formation assay in soft agar were used. And tumorigenesis rate and invasionsby the cell subclones with or without the antisense u-PAR were observed in nude mice. It was foundthat in vitro growth of highly invasive PC-3M cell subclones transfected with the antisense u-PARwas declined, and the ability of anchorage-independent growth of those cell subclones was found de-creased sharply, with the inhibiting rate becoming 79 % and 60 %, respectively. Although the anti-sense u-PAR didn't change MMP-9 gene transcription, they could inhibit the activation of MMP-9 ofhighly invasive PC-3M cell subclones. Moreover, the tumorigenesis rate of the cell subclones with theantisense u-PAR decreased and the growth of a neoplasm also slowed down. Thet tests showed thedifference between experimental and control groups was statistically significant (P<0. 01). The anti-sense u-PAR vector could not only inhibit the invasion ability of highly invasive PC-3M cell subclonesin vitro but also restrain the growth of those cell subclones in vivo.

  7. Remarkable stability of an instability-prone lentiviral vector plasmid in Escherichia coli Stbl3

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    Al-Allaf, Faisal A.; Tolmachov, Oleg E.; Zambetti, Lia Paola; Tchetchelnitski, Viktoria; Mehmet, Huseyin

    2012-01-01

    Large-scale production of plasmid DNA to prepare therapeutic gene vectors or DNA-based vaccines requires a suitable bacterial host, which can stably maintain the plasmid DNA during industrial cultivation. Plasmid loss during bacterial cell divisions and structural changes in the plasmid DNA can dramatically reduce the yield of the desired recombinant plasmid DNA. While generating an HIV-based gene vector containing a bicistronic expression cassette 5′-Olig2cDNA-IRES-dsRed2-3′, we encountered ...

  8. A plasmid toolkit for cloning chimeric cDNAs encoding customized fusion proteins into any Gateway destination expression vector.

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    Buj, Raquel; Iglesias, Noa; Planas, Anna M; Santalucía, Tomàs

    2013-08-20

    Valuable clone collections encoding the complete ORFeomes for some model organisms have been constructed following the completion of their genome sequencing projects. These libraries are based on Gateway cloning technology, which facilitates the study of protein function by simplifying the subcloning of open reading frames (ORF) into any suitable destination vector. The expression of proteins of interest as fusions with functional modules is a frequent approach in their initial functional characterization. A limited number of Gateway destination expression vectors allow the construction of fusion proteins from ORFeome-derived sequences, but they are restricted to the possibilities offered by their inbuilt functional modules and their pre-defined model organism-specificity. Thus, the availability of cloning systems that overcome these limitations would be highly advantageous. We present a versatile cloning toolkit for constructing fully-customizable three-part fusion proteins based on the MultiSite Gateway cloning system. The fusion protein components are encoded in the three plasmids integral to the kit. These can recombine with any purposely-engineered destination vector that uses a heterologous promoter external to the Gateway cassette, leading to the in-frame cloning of an ORF of interest flanked by two functional modules. In contrast to previous systems, a third part becomes available for peptide-encoding as it no longer needs to contain a promoter, resulting in an increased number of possible fusion combinations. We have constructed the kit's component plasmids and demonstrate its functionality by providing proof-of-principle data on the expression of prototype fluorescent fusions in transiently-transfected cells. We have developed a toolkit for creating fusion proteins with customized N- and C-term modules from Gateway entry clones encoding ORFs of interest. Importantly, our method allows entry clones obtained from ORFeome collections to be used without prior

  9. Riesling Italico subclones

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    Ivanišević Dragoslav

    2012-01-01

    Full Text Available Clone selection in viticulture represents a way of eliminating the negative influence of mutation changes in the future vineyard, as well as preventing the virus-infected plants and plants infected with virus-related diseases from further propagation. Riesling Italico variety is an old variety. It has been grown in Vojvodina for centuries. The clone selection of Riesling Italico variety in Sremski Karlovci was started in 1975. In January 1991 three clones were recognized. Shortly after, the subclone selection of these three clones was initiated. This paper presents the resultants of subclone selection of Riesling Italico. Subclones SK 54-4 and SK 54-10 are characterized by good quality and the grapes of these subclones can be used for the production of high quality wines in most of the years.

  10. Remarkable stability of an instability-prone lentiviral vector plasmid in Escherichia coli Stbl3.

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    Al-Allaf, Faisal A; Tolmachov, Oleg E; Zambetti, Lia Paola; Tchetchelnitski, Viktoria; Mehmet, Huseyin

    2013-02-01

    Large-scale production of plasmid DNA to prepare therapeutic gene vectors or DNA-based vaccines requires a suitable bacterial host, which can stably maintain the plasmid DNA during industrial cultivation. Plasmid loss during bacterial cell divisions and structural changes in the plasmid DNA can dramatically reduce the yield of the desired recombinant plasmid DNA. While generating an HIV-based gene vector containing a bicistronic expression cassette 5'-Olig2cDNA-IRES-dsRed2-3', we encountered plasmid DNA instability, which occurred in homologous recombination deficient recA1 Escherichia coli strain Stbl2 specifically during large-scale bacterial cultivation. Unexpectedly, the new recombinant plasmid was structurally changed or completely lost in 0.5 L liquid cultures but not in the preceding 5 mL cultures. Neither the employment of an array of alternative recA1 E. coli plasmid hosts, nor the lowering of the culture incubation temperature prevented the instability. However, after the introduction of this instability-prone plasmid into the recA13E. coli strain Stbl3, the transformed bacteria grew without being overrun by plasmid-free cells, reduction in the plasmid DNA yield or structural changes in plasmid DNA. Thus, E. coli strain Stbl3 conferred structural and maintenance stability to the otherwise instability-prone lentivirus-based recombinant plasmid, suggesting that this strain can be used for the faithful maintenance of similar stability-compromised plasmids in large-scale bacterial cultivations. In contrast to Stbl2, which is derived wholly from the wild type isolate E. coli K12, E. coli Stbl3 is a hybrid strain of mixed E. coli K12 and E. coli B parentage. Therefore, we speculate that genetic determinants for the benevolent properties of E. coli Stbl3 for safe plasmid propagation originate from its E. coli B ancestor.

  11. Modular construction of plasmids by parallel assembly of linear vector components.

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    Gao, XinZheng; Yan, Pu; Shen, Wentao; Li, Xiaoying; Zhou, Peng; Li, Yuenan

    2013-06-15

    Construction of plasmids is the basic and pivotal technology in molecular biology. The common method for constructing plasmids is to cut DNA fragments by restriction enzymes and then join the resulting fragments using ligase. We present here a modified Golden Gate cloning method for modular construction of plasmids. Unlike the original Golden Gate cloning system for cloning from entry vector to expression vector, this method can be used to construct plasmids immediately from linear DNA fragments. After polymerase chain reaction (PCR) amplification for flanking with BsaI sites, multiple linear DNA components (modules) can be parallel assembled into a circle plasmid by a single restriction-ligation reaction using the method. This method is flexible to construct different types of plasmids because the modules can be freely selected and assembled in any combination. This method was applied successfully to construct a prokaryotic expression plasmid from four modules and a plant expression plasmid from five modules (fragments). The results suggest that this method provides a simple and flexible platform for modular construction of plasmids.

  12. Quantifying and resolving multiple vector transformants in S. cerevisiae plasmid libraries

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    Gray Elizabeth C

    2009-11-01

    Full Text Available Abstract Background In addition to providing the molecular machinery for transcription and translation, recombinant microbial expression hosts maintain the critical genotype-phenotype link that is essential for high throughput screening and recovery of proteins encoded by plasmid libraries. It is known that Escherichia coli cells can be simultaneously transformed with multiple unique plasmids and thusly complicate recombinant library screening experiments. As a result of their potential to yield misleading results, bacterial multiple vector transformants have been thoroughly characterized in previous model studies. In contrast to bacterial systems, there is little quantitative information available regarding multiple vector transformants in yeast. Saccharomyces cerevisiae is the most widely used eukaryotic platform for cell surface display, combinatorial protein engineering, and other recombinant library screens. In order to characterize the extent and nature of multiple vector transformants in this important host, plasmid-born gene libraries constructed by yeast homologous recombination were analyzed by DNA sequencing. Results It was found that up to 90% of clones in yeast homologous recombination libraries may be multiple vector transformants, that on average these clones bear four or more unique mutant genes, and that these multiple vector cells persist as a significant proportion of library populations for greater than 24 hours during liquid outgrowth. Both vector concentration and vector to insert ratio influenced the library proportion of multiple vector transformants, but their population frequency was independent of transformation efficiency. Interestingly, the average number of plasmids born by multiple vector transformants did not vary with their library population proportion. Conclusion These results highlight the potential for multiple vector transformants to dominate yeast libraries constructed by homologous recombination. The

  13. Expression of Hepatitis B Virus Surface Antigen Gene in Cultured Cells by Using Recombinant Plasmid Vectors

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    Siddiqui, Aleem

    1983-01-01

    By using a new host-vector system, expression of the gene coding for hepatitis B surface antigen has been studied. A subgenomic fragment of cloned hepatitis B viral DNA was inserted into the plasmid vector pSV010. Transfection of COS cells with the recombinant plasmid vector containing hepatitis sequences leads to the synthesis of hepatitis B surface antigen, which is released in the culture medium in the form of 22-nm particles similar to those found in the sera of hepatitis carriers.

  14. Effects of medium composition on the production of plasmid DNA vector potentially for human gene therapy

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    XU Zhi-nan; SHEN Wen-he; CHEN Hao; CEN Pei-lin

    2005-01-01

    Plasmid vector is increasingly applied to gene therapy or gene vaccine. The production of plasmid pCMV-AP3 for cancer gene therapy was conducted in a modified MBL medium using a recombinant E. coli BL21 system. The effects of different MMBL components on plasmid yield, cell mass and specific plasmid DNA productivity were evaluated on shake-flask scale. The results showed that glucose was the optimal carbon source. High plasmid yield (58.3 mg/L) was obtained when 5.0 g/L glucose was added to MMBL. Glycerol could be chosen as a complementary carbon source because of the highest specific plasmid productivity (37.9 mg DNA/g DCW). After tests of different levels of nitrogen source and inorganic phosphate, a modified MMBL medium was formulated for optimal plasmid production. Further study showed that the initial acetate addition (less than 4.0 g/L) in MMBL improved plasmid production significantly, although it inhibited cell growth. The results will be useful for large-scale plasmid production using recombinant E. coli system.

  15. Modular construction of plasmids through ligation-free assembly of vector components with oligonucleotide linkers.

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    Vroom, Jonathan A; Wang, Clifford L

    2008-06-01

    We have developed a modular method of plasmid construction that can join multiple DNA components in a single reaction. A nicking enzyme is used to create 5' and 3' overhangs on PCR-generated DNA components. Without the use of ligase or restriction enzymes, components are joined using oligonucleotide linkers that recognize the overhangs. By specifying the sequences of the linkers, desired components can be assembled in any combination and order to generate different plasmid vectors.

  16. New and Redesigned pRS Plasmid Shuttle Vectors for Genetic Manipulation of Saccharomycescerevisiae.

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    Chee, Mark K; Haase, Steven B

    2012-05-01

    We have constructed a set of 42 plasmid shuttle vectors based on the widely used pRS series for use in the budding yeast Saccharomyces cerevisiae and the bacterium Escherichia coli. This set of pRSII plasmids includes new shuttle vectors that can be used with histidine and adenine auxotrophic laboratory yeast strains carrying mutations in the genes HIS2 and ADE1, respectively. Our pRSII plasmids also include updated versions of commonly used pRS plasmids from which common restriction sites that occur within their yeast-selectable biosynthetic marker genes have been removed to increase the availability of unique restriction sites within their polylinker regions. Hence, our pRSII plasmids are a complete set of integrating, centromere and 2μ episomal plasmids with the biosynthetic marker genes ADE2, HIS3, TRP1, LEU2, URA3, HIS2, and ADE1 and a standardized selection of at least 16 unique restriction sites in their polylinkers. Additionally, we have expanded the range of drug selection options that can be used for PCR-mediated homologous replacement using pRS plasmid templates by replacing the G418-resistance kanMX4 cassette of pRS400 with MX4 cassettes encoding resistance to phleomycin, hygromycin B, nourseothricin, and bialaphos. Finally, in the process of generating the new plasmids, we have determined several errors in existing publicly available sequences for several commonly used yeast plasmids. Using our updated sequences, we constructed pRS plasmid backbones with a unique restriction site for inserting new markers to facilitate future expansion of the pRS series.

  17. Development of a novel plasmid as a shuttle vector for heterologous gene expression in Mycoplasma yeatsii.

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    Kent, Bethany N; Foecking, Mark F; Calcutt, Michael J

    2012-10-01

    A circular plasmid, pMyBK1, was detected in Mycoplasma yeatsii strain GIH(T). Analysis of the sequence of the 3432-bp replicon identified two predicted open reading frames (ORFs), one with sequence similarity to multiple plasmid mobilization proteins and one that matches only to hypothetical ORFs encoded by integrated chromosomal elements in the sequenced genomes of two Mycoplasma species. Shuttle vectors were constructed in Escherichia coli which could be introduced into M. yeatsii at high efficiency (10(4)-10(5) per μg DNA) by electroporation. Independent deletion analysis of the two ORFs disclosed that whereas mob was dispensable, orf2 was necessary for plasmid replication or maintenance. The absence of plasmid-encoded database matches for ORF2 indicates that pMyBK1 represents a novel plasmid family. One shuttle vector was used to demonstrate heterologous expression of the Mycoplasma fermentans malp gene and was stable during multiple passages. The host-plasmid system described has potential application for genetic manipulation in a genus for which few replicative vectors are available.

  18. Stable plasmid vectors for complementation of Xylella fastidiosa mutants in planta

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    Current understanding of the mechanisms of Pierce’s Disease development has been significantly advanced by molecular genetic studies of the causal agent, Xylella fastidiosa (Xf). Plasmid vectors are an essential tool for studies of bacterial genetics and pathogenesis. However, most commonly used pla...

  19. Ultrasound enhances the transfection of plasmid DNA by non-viral vectors.

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    Hosseinkhani, Hossein; Aoyama, Teruyoshi; Ogawa, Osamu; Tabata, Yasuhiko

    2003-04-01

    Increasing attention has been paid to technology used for the delivery of genetic materials into cells for gene therapy and the generation of genetically engineered cells. So far, viral vectors have been mainly used because of their inherently high transfection efficiency of gene. However, there are some problems to be resolved for the clinical applications, such as the pathogenicity and immunogenicity of viral vectors themselves. Therefore, many research trials with non-viral vectors have been performed to enhance their efficiency to a level comparable to the viral vector. Two directions of these trials exist: material improvement of non-viral vectors and their combination with various external physical stimuli. This paper reviews the latter research trials, with special attention paid to the enhancement of gene expression by ultrasound (US). The expression level of plasmid DNA by various cationized polymers and liposomes is promoted by US irradiation in vitro as well as in vivo. This US-enhanced expression of plasmid DNA will be discussed to emphasize the technical feasibility of US in gene therapy and biotechnology.

  20. Comprehensive set of integrative plasmid vectors for copper-inducible gene expression in Myxococcus xanthus.

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    Gómez-Santos, Nuria; Treuner-Lange, Anke; Moraleda-Muñoz, Aurelio; García-Bravo, Elena; García-Hernández, Raquel; Martínez-Cayuela, Marina; Pérez, Juana; Søgaard-Andersen, Lotte; Muñoz-Dorado, José

    2012-04-01

    Myxococcus xanthus is widely used as a model system for studying gliding motility, multicellular development, and cellular differentiation. Moreover, M. xanthus is a rich source of novel secondary metabolites. The analysis of these processes has been hampered by the limited set of tools for inducible gene expression. Here we report the construction of a set of plasmid vectors to allow copper-inducible gene expression in M. xanthus. Analysis of the effect of copper on strain DK1622 revealed that copper concentrations of up to 500 μM during growth and 60 μM during development do not affect physiological processes such as cell viability, motility, or aggregation into fruiting bodies. Of the copper-responsive promoters in M. xanthus reported so far, the multicopper oxidase cuoA promoter was used to construct expression vectors, because no basal expression is observed in the absence of copper and induction linearly depends on the copper concentration in the culture medium. Four different plasmid vectors have been constructed, with different marker selection genes and sites of integration in the M. xanthus chromosome. The vectors have been tested and gene expression quantified using the lacZ gene. Moreover, we demonstrate the functional complementation of the motility defect caused by lack of PilB by the copper-induced expression of the pilB gene. These versatile vectors are likely to deepen our understanding of the biology of M. xanthus and may also have biotechnological applications.

  1. Development of new plasmid DNA vaccine vectors with R1-based replicons

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    Bower Diana M

    2012-08-01

    Full Text Available Abstract Background There has been renewed interest in biopharmaceuticals based on plasmid DNA (pDNA in recent years due to the approval of several veterinary DNA vaccines, on-going clinical trials of human pDNA-based therapies, and significant advances in adjuvants and delivery vehicles that have helped overcome earlier efficacy deficits. With this interest comes the need for high-yield, cost-effective manufacturing processes. To this end, vector engineering is one promising strategy to improve plasmid production. Results In this work, we have constructed a new DNA vaccine vector, pDMB02-GFP, containing the runaway R1 origin of replication. The runaway replication phenotype should result in plasmid copy number amplification after a temperature shift from 30°C to 42°C. However, using Escherichia coli DH5α as a host, we observed that the highest yields of pDMB02-GFP were achieved during constant-temperature culture at 30°C, with a maximum yield of approximately 19 mg pDNA/g DCW being observed. By measuring mRNA and protein levels of the R1 replication initiator protein, RepA, we determined that RepA may be limiting pDMB02-GFP yield at 42°C. A mutant plasmid, pDMB-ATG, was constructed by changing the repA start codon from the sub-optimal GTG to ATG. In cultures of DH5α[pDMB-ATG], temperature-induced plasmid amplification was more dramatic than that observed with pDMB02-GFP, and RepA protein was detectable for several hours longer than in cultures of pDMB02-GFP at 42°C. Conclusions Overall, we have demonstrated that R1-based plasmids can produce high yields of high-quality pDNA without the need for a temperature shift, and have laid the groundwork for further investigation of this class of vectors in the context of plasmid DNA production.

  2. Advances in host and vector development for the production of plasmid DNA vaccines.

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    Mairhofer, Juergen; Lara, Alvaro R

    2014-01-01

    Recent developments in DNA vaccine research provide a new momentum for this rather young and potentially disruptive technology. Gene-based vaccines are capable of eliciting protective immunity in humans to persistent intracellular pathogens, such as HIV, malaria, and tuberculosis, for which the conventional vaccine technologies have failed so far. The recent identification and characterization of genes coding for tumor antigens has stimulated the development of DNA-based antigen-specific cancer vaccines. Although most academic researchers consider the production of reasonable amounts of plasmid DNA (pDNA) for immunological studies relatively easy to solve, problems often arise during this first phase of production. In this chapter we review the current state of the art of pDNA production at small (shake flasks) and mid-scales (lab-scale bioreactor fermentations) and address new trends in vector design and strain engineering. We will guide the reader through the different stages of process design starting from choosing the most appropriate plasmid backbone, choosing the right Escherichia coli (E. coli) strain for production, and cultivation media and scale-up issues. In addition, we will address some points concerning the safety and potency of the produced plasmids, with special focus on producing antibiotic resistance-free plasmids. The main goal of this chapter is to make immunologists aware of the fact that production of the pDNA vaccine has to be performed with as much as attention and care as the rest of their research.

  3. [Construction of genomic library of L. interrogans serovar lai using lambda gt11 as the vector and a study of recombiant plasmid pDL121].

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    Liu, H; Dai, B; Jing, B; Wu, W; Li, S; Fang, Z; Zhao, H; Ye, D; Yan, R; Liu, J; Song, S; Yang, Y; Zhang, Y; Liu, F; Tu, Y; Yang, H; Huang, Z; Liang, L; Hu, L; Zhao, M

    1997-03-01

    A genomic library of L. interrogans serovar lai strain 017 has been constructed using lambda gt11 as the vector. DNA was partially digested by two blunt-end restriction enzymes, then methylated with EcoR I methylase; after EcoR I linker was added to the DNA, the linker-ended DNA was ligated to the dephosphorylated EcoR I digested lambda gt11 arms. The recombined DNA was packaged in vitro, and used to transduct E. coli Y1090 for amplification. There were 2.1 x 10(6) recombinant bacteriophages as recognized by their ability to form white plaques plated on Lac host in the presence of both IPTG and X-Ga1. A positive clone, designated lambda DL12, was screened with a rabbit anti-serum against L. interrogans serovar lai from the genomic library. The DNA from lambda DL12 was subcloned into plasmid pUC18. A recombinant (designated as pDL121) was obtained. SDS-PAGE analysis indicated that a 23 kd was expressed in E. coli JM 103 harboring pDL121. Western blotting analysis showed that a specific protein band molecular weight of 23 kd could be recognized by the rabbit antiserum against L. interrogans serovar lai strain 017.

  4. Plasmid cloning vectors for the conjugal transfer of DNA from Escherichia coli to Streptomyces spp.

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    Bierman, M; Logan, R; O'Brien, K; Seno, E T; Rao, R N; Schoner, B E

    1992-07-01

    We have constructed cloning vectors for the conjugal transfer of DNA from Escherichia coli to Streptomyces spp. All vectors contain the 760-bp oriT fragment from the IncP plasmid, RK2. Transfer functions need to be supplied in trans by the E. coli donor strain. We have incorporated into these vectors selectable antibiotic-resistance markers (AmR, ThR, SpR) that function in Streptomyces spp. and other features that should allow for: (i) integration via homologous recombination between cloned DNA and the Streptomyces spp. chromosome, (ii) autonomous replication, or (iii) site-specific integration at the bacteriophage phi C31 attachment site. Shuttle cosmids for constructing genomic libraries and bacteriophage P1 cloning vector capable of accepting approx. 100-kb fragments are also described. A simple mating procedure has been developed for the conjugal transfer of these vectors from E. coli to Streptomyces spp. that involves plating of the donor strain and either germinated spores or mycelial fragments of the recipient strain. We have shown that several of these vectors can be introduced into Streptomyces fradiae, a strain that is notoriously difficult to transform by PEG-mediated protoplast transformation.

  5. A Transient Three-plasmid Expression System for the Production of Hepatocytes Targeting Retroviral Vectors

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    Peng QI; Jinxiang HAN; Yanqin LU; Chuanxi WANG; Bo ZHU

    2007-01-01

    Targeting of retroviral vectors to specific cells was attempted through modifying the surface protein of the murine leukemia viruses (MLVs), but in many cases the protein function was affected, and it is difficult to achieve the targeted delivery. In this study, we have tried to engineer ecotropic Moloney murine leukemia viruses (MoMLV)-based retroviral vectors to transduce hepatocytes. A chimeric envelope (Env)expression plasmid was constructed containing the hepatitis B virus PreS2 peptide fused to aa +1 at the Nterminus of Env. Following simultaneous transfection of pgag-pol, pLEGFP and chimeric env plasmids into 293T cells, helper-free retrovirus stocks with the titer of approximately 104 infectious units/ml were achieved at 48 h post-transfection. These pseudotype vectors showed the normal host range of retrovirus, infecting host NIH 3T3 cells, although the efficiency was reduced compared with that of virions carrying wild-type ecotropic MoMLV envelope. In addition, the resultant pseudotype viruses could transduce human hepatoma cells mediated by polymerized human serum albumin with relatively high titers in comparison with those transductions without polymerized human serum albumin. This approach can be used to target hepatocytes selectively.

  6. Mutations induced in a shuttle vector plasmid exposed to monofunctionally activated mitomycin C.

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    Maccubbin, A E; Mudipalli, A; Nadadur, S S; Ersing, N; Gurtoo, H L

    1997-01-01

    Reductive activation of mitomycin C leads to its covalent binding to DNA, forming monoadducts and cross-links. The cytotoxicity of mitomycin C has been attributed to cross-link formation, whereas monoadducts are assumed to cause mutagenicity. We have developed a 32P-postlabeling technique to measure mitomycin C DNA adducts. Using this technique, we have measured monoadduct formation in the shuttle vector plasmid pSP189 and have determined mutations induced by monoadduct formation. The shuttle vector plasmid was incubated with mitomycin C under conditions favoring monofunctional activation of mitomycin C. The plasmid was then replicated in human Ad293 cells, rescued in bacteria, and analyzed for mutations in the supF tRNA gene sequence of pSP189. One major mitomycin C/DNA adduct was observed by 32P-postlabeling and was characterized as a monoadduct of guanine. When pSP189 was exposed to monofunctionally activated mitomycin C, increases in adduct levels and mutation frequency were found to be related to mitomycin C concentration. The majority of the mutations involved single bases, with base substitutions making up 59.1% of the total mutations observed. Of the base substitutions, 67.2% were transversions and 32.8% were transitions, with nearly 80% of all base substitutions involving G:C base pairs. Deletions, either as single bases or large deletions, also involved G:C base pairs the majority of the time. The observed bias of mutations at G:C and the formation of a mitomycin C/DNA monoadduct involving guanine suggests that monoadduct formation may be responsible for the mutations.

  7. Cloning vectors based on cryptic plasmids isolated from lactic acid bacteria: their characteristics and potential applications in biotechnology.

    Science.gov (United States)

    Shareck, Julie; Choi, Young; Lee, Byong; Miguez, Carlos B

    2004-01-01

    Lactic acid bacteria (LAB) are Gram positive bacteria, widely distributed in nature, and industrially important as they are used in a variety of industrial food fermentations. The use of genetic engineering techniques is an effective means of enhancing the industrial applicability of LAB. However, when using genetic engineering technology, safety becomes an essential factor for the application of improved LAB to the food industry. Cloning and expression systems should be derived preferably from LAB cryptic plasmids that generally encode genes for which functions can be proposed, but no phenotypes can be observed. However, some plasmid-encoded functions have been discovered in cryptic plasmids originating from Lactobacillus, Streptococcus thermophilus, and Pediococcus spp. and can be used as selective marker systems in vector construction. This article presents information concerning LAB cryptic plasmids, and their structures, functions, and applications. A total of 134 cryptic plasmids collated are discussed.

  8. Identification of facultatively heterotrophic, N2-fixing cyanobacteria able to receive plasmid vectors from Escherichia coli by conjugation.

    OpenAIRE

    Flores, E; Wolk, C P

    1985-01-01

    Plasmid vectors transferable by conjugation from Escherichia coli to obligately photoautotrophic strains of Anabaena spp. are also transferred to and maintained in heterotrophic, filamentous cyanobacteria of the genus Nostoc. These organisms can be used for the genetic analysis of oxygenic photosynthesis, chromatic adaptation, nitrogen fixation, and heterocyst development.

  9. Transformation of Cowpea Vigna unguiculata Cells with an Antibiotic Resistance Gene Using a Ti-Plasmid-Derived Vector

    NARCIS (Netherlands)

    Hille, Jacques; Goldbach, Rob

    1986-01-01

    A chimaeric antibiotic resistance gene was transferred to cowpea (Vigna unguiculata), a member of the legume family. This transfer was established by inoculating cowpea leaf discs with an Agrobacterium tumefaciens strain harboring a Ti-plasmid-derived vector that contained two copies of a chimaeric

  10. Transformation of Cowpea Vigna unguiculata Cells with an Antibiotic Resistance Gene Using a Ti-Plasmid-Derived Vector

    NARCIS (Netherlands)

    Hille, Jacques; Goldbach, Rob

    1986-01-01

    A chimaeric antibiotic resistance gene was transferred to cowpea (Vigna unguiculata), a member of the legume family. This transfer was established by inoculating cowpea leaf discs with an Agrobacterium tumefaciens strain harboring a Ti-plasmid-derived vector that contained two copies of a chimaeric

  11. Analysis of comparative efficiencies of different transformation methods of E. coli using two common plasmid vectors.

    Science.gov (United States)

    Roychoudhury, Aryadeep; Basu, Supratim; Sengupta, Dibyendu N

    2009-10-01

    The efficiencies of different transformation methods of E. coli DH5Qalpha train, induced by several cations like Mg2+, Mn2+ Rb+ and especially Ca2+, with or without polyethylene glycol (PEG) and dimethyl sulfoxide (DMSO) were compared using the two commonly used plasmid vectors pCAMBIA1201 and pBI121. The widely used calcium chloride (CaCl2) method appeared to be the most efficient procedure, while rubidium chloride (RbCl) method was the least effective. The improvements in the classical CaCl2 method were found to further augment the transformation efficiency (TR)E for both the vectors like repeated alternate cycles of heat shock, followed by immediate cold, at least up to the third cycle; replacement of the heat shock step by a single microwave pulse and even more by double microwave treatment and administration of combined heat shock-microwave treatments. The pre-treatment of CaCl2-competent cells with 5% (v/v) ethanol, accompanied by single heat shock also triggered the (TR)E, which was further enhanced, when combined heat shock-microwave was applied. The minor alterations or improved approaches in CaCl2 method suggested in the present study may thus find use in more efficient E. coli transformation.

  12. Development of plasmids for quantitative detection of integrated lentiviral vectors and evaluation of culture time to perform vector titer by real-time quantitative polymerase chain reaction assay

    Directory of Open Access Journals (Sweden)

    Elena Baiamonte

    2014-09-01

    Full Text Available The accurate assessment of provirus copy number per cell (VCN/cell is a fundamental issue in transgenesis as well as in gene therapy studies based on stably integrated vectors. To this end, real-time quantitative polymerase chain reaction (qPCR is a powerful method but it is sensible to differences in quality or concentration of the two-plasmid preparations used for the construction of the standard curves. In order to minimize technical errors we included genome specific sequences (mouse or human and vector specific sequences in the same plasmid. We evaluated the specificity and sensitivity of these bivalent plasmids by qPCR analysis on mouse and human genomic DNA containing a known number of a reporter lentiviral vector and we found that the system is reliable to measure up to 0.1 VCN/cell. Here we have applied this assay to measure vector titer of virus stock preparations and to determine the optimal cell passages at which viral titration effectively reflects the number of integrated vectors.

  13. A Simple and Inexpensive Method for Sending Binary Vector Plasmid DNA by Mail

    Science.gov (United States)

    We describe a simple cost-effective technique for the transport of plasmid DNA by mail. Our results demonstrate that common multipurpose printing paper is a satisfactory substrate and superior to the more absorbent 3MM chromatography paper for the transport of plasmid DNA through the U.S. first clas...

  14. A novel peptide delivers plasmids across blood-brain barrier into neuronal cells as a single-component transfer vector.

    Directory of Open Access Journals (Sweden)

    Ailing Fu

    Full Text Available There is no data up to now to show that peptide can deliver plasmid into brain as a single-component transfer vector. Here we show that a novel peptide, RDP (consisted of 39 amino acids, can be exploited as an efficient plasmid vector for brain-targeting delivery. The plasmids containing Lac Z reporter gene (pVAX-Lac Z and BDNF gene (pVAX-BDNF are complexed with RDP and intravenously injected into mice. The results of gel retardation assay show that RDP enables to bind DNA in a dose-dependent manner, and the X-Gal staining identity that Lac Z is specifically expressed in the brain. Also, the results of Western blot and immunofluorescence staining of BDNF indicate that pVAX-BDNF complexed with RDP can be delivered into brain, and show neuroprotective properties in experimental Parkinson's disease (PD model. The results demonstrate that RDP enables to bind and deliver DNA into the brain, resulting in specific gene expression in the neuronal cells. This strategy provides a novel, simple and effective approach for non-viral gene therapy of brain diseases.

  15. A novel peptide delivers plasmids across blood-brain barrier into neuronal cells as a single-component transfer vector.

    Science.gov (United States)

    Fu, Ailing; Zhang, Miaomiao; Gao, Feiyan; Xu, Xingran; Chen, Zhangbao

    2013-01-01

    There is no data up to now to show that peptide can deliver plasmid into brain as a single-component transfer vector. Here we show that a novel peptide, RDP (consisted of 39 amino acids), can be exploited as an efficient plasmid vector for brain-targeting delivery. The plasmids containing Lac Z reporter gene (pVAX-Lac Z) and BDNF gene (pVAX-BDNF) are complexed with RDP and intravenously injected into mice. The results of gel retardation assay show that RDP enables to bind DNA in a dose-dependent manner, and the X-Gal staining identity that Lac Z is specifically expressed in the brain. Also, the results of Western blot and immunofluorescence staining of BDNF indicate that pVAX-BDNF complexed with RDP can be delivered into brain, and show neuroprotective properties in experimental Parkinson's disease (PD) model. The results demonstrate that RDP enables to bind and deliver DNA into the brain, resulting in specific gene expression in the neuronal cells. This strategy provides a novel, simple and effective approach for non-viral gene therapy of brain diseases.

  16. A new approach to produce amino-carbon nanotubes as plasmid transfection vector by [2 + 1] cycloaddition of nitrenes

    Science.gov (United States)

    Jiang, Yongjian; Jin, Chen; Yang, Feng; Yu, Xianjun; Wang, Guojian; Cheng, Si; Di, Yang; Li, Ji; Fu, Deliang; Ni, Quanxing

    2011-01-01

    Amino-carbon nanotubes (amino-CNTs) can conjugate with the DNA by electrostatic interactions and shuttle the DNA to the cell cytoplasm or even the nucleus. Here we report a new approach to produce amino-CNTs by cycloaddition of nitrenes. Fourier transform infrared spectroscopy was used to verify the success of the functionalization, and the functionalization degree was calculated by thermal gravity analysis. Transmission electron microscope (TEM) was used to observe the solubility of the CNTs and the interactions of the amino-CNTs with the plasmids. Cell ultrathin sections were made and observed under the TEM to confirm the amino-CNTs enter the cells. Transfection experiments ultimately verify the amino-CNTs produced through cycloadditions of nitrenes can serve as plasmid vector.

  17. [Construction and analysis of transgenic plants of Nicotiana tabacum L. expressing a bacterial gene for beta-1,3-glucanase. I. Construction of vector plasmids for transfer into plants and expression of a modified gene for beta-1,3-glucanase from Clostridium thermocellum in tobacco protoplasts].

    Science.gov (United States)

    Darbinian, N S; Popov, Iu G; Mochul'skiĭ, A V; Volkova, L V; Piruzian, E S; Vasilevko, V T

    1996-02-01

    We constructed two vectors, pC27-glc and pC29-glc, that allow expression of the beta-1,3-glucanase gene (glc) in plant cells. The glc gene was previously cloned from anaerobic thermophilous bacterium Clostridium thermocellum. To increase the efficiency of expression, the N-terminal fragment of the glc gene encoding bacterial transient peptide was deleted, and hybrid variants of lacZ-glc were obtained. Analysis of expression of the hybrid genes in Escherichia coli showed that deletion of the fragment corresponding to 31 amino acids (a.a.) of beta-glucanase affected neither activity nor thermostability of the enzyme. The modified gene was subcloned into two vectors, pC27 and pC29, in which its expression was controlled by the TR2' promoter of the 2' gene of T-DNA and the rbcS promoter from Arabidopsis, respectively. Each of the resulting plasmids, pC27-glc and pC29-glc, was transfected into protoplasts of Nicotiana plumbaginifolia. Both the plasmids were shown to allow a high level of activity of the thermostable beta-1,3-glucanase. We plan to use the vectors obtained for transformation of agrobacteria and construction of transgenic plants.

  18. A single-plasmid vector for transgene amplification using short hairpin RNA targeting the 3'-UTR of amplifiable dhfr.

    Science.gov (United States)

    Kang, Shin-Young; Kim, Yeon-Gu; Lee, Hong Weon; Lee, Eun Gyo

    2015-12-01

    Gene amplification using dihydrofolate reductase gene (dhfr) and methotrexate (MTX) is widely used for recombinant protein production in mammalian cells and is typically conducted in DHFR-deficient Chinese hamster ovary (CHO) cell lines. Generation of DHFR-deficient cells can be achieved by an expression vector incorporating short hairpin RNA (shRNA) that targets the 3'-untranslated region (UTR) of endogenous dhfr. Thus, shRNAs were designed to target the 3'-UTR of endogenous dhfr, and shRNA-2 efficiently down-regulated dhfr expression in CHO-K1 cells. A single gene copy of shRNA-2 also decreased the translational level of DHFR by 80% in Flp-In CHO cells. shRNA-2 was then incorporated into a plasmid vector expressing human erythropoietin (EPO) and an exogenous DHFR to develop EPO-producing cells in the Flp-In system. The specific EPO productivity (q EPO) was enhanced by stepwise increments of MTX concentration, and differences in the amplification rate were observed in Flp-In CHO cells that expressed shRNA-2. In addition, the q EPO increased by more than 2.5-fold in the presence of 500 nM MTX. The mRNA expression level and gene copy numbers of dhfr were correlated with increased productivity in the cells, which is influenced by inhibition of endogenous dhfr. This study reveals that an expression vector including shRNA that targets the 3'-UTR of endogenous dhfr can enhance the transgene amplification rate and productivity by generating DHFR-deficient cells. This approach may be applied for amplifying the foreign gene in wild-type cell lines as a versatile single-plasmid vector.

  19. IncP-1ε Plasmids are Important Vectors of Antibiotic Resistance Genes in Agricultural Systems: Diversification Driven by Class 1 Integron Gene Cassettes.

    Science.gov (United States)

    Heuer, Holger; Binh, Chu T T; Jechalke, Sven; Kopmann, Christoph; Zimmerling, Ute; Krögerrecklenfort, Ellen; Ledger, Thomas; González, Bernardo; Top, Eva; Smalla, Kornelia

    2012-01-01

    The role of broad-host range IncP-1ε plasmids in the dissemination of antibiotic resistance in agricultural systems has not yet been investigated. These plasmids were detected in total DNA from all of 16 manure samples and in arable soil based on a novel 5'-nuclease assay for real-time PCR. A correlation between IncP-1ε plasmid abundance and antibiotic usage was revealed. In a soil microcosm experiment the abundance of IncP-1ε plasmids was significantly increased even 127 days after application of manure containing the antibiotic compound sulfadiazine, compared to soil receiving only manure, only sulfadiazine, or water. Fifty IncP-1ε plasmids that were captured in E. coli CV601gfp from bacterial communities of manure and arable soil were characterized by PCR and hybridization. All plasmids carried class 1 integrons with highly varying sizes of the gene cassette region and the sul1 gene. Three IncP-1ε plasmids captured from soil bacteria and one from manure were completely sequenced. The backbones were nearly identical to that of the previously described IncP-1ε plasmid pKJK5. The plasmids differed mainly in the composition of a Tn402-like transposon carrying a class 1 integron with varying gene cassettes, IS1326, and in three of the plasmids the tetracycline resistance transposon Tn1721 with various truncations. Diverse Beta- and Gammaproteobacteria were revealed as hosts of one of the IncP-1ε plasmids in soil microcosms. Our data suggest that IncP-1ε plasmids are important vectors for horizontal transfer of antibiotic resistance in agricultural systems.

  20. IncP-1ε plasmids are important vectors of antibiotic resistance genes in agricultural systems: diversification driven by class 1 integron gene cassettes

    Directory of Open Access Journals (Sweden)

    Holger eHeuer

    2012-01-01

    Full Text Available The role of broad host range IncP-1ε plasmids in the dissemination of antibiotic resistance in agricultural systems has not yet been investigated. These plasmids were detected in total DNA from all of 16 manure samples and in arable soil based on a novel 5’-nuclease assay for real time PCR. A correlation between IncP-1ε plasmid abundance and antibiotic usage was revealed. In a soil microcosm experiment the abundance of IncP-1ε plasmids was significantly increased even 127 days after application of manure containing the antibiotic compound sulfadiazine, compared to soil receiving only manure, only sulfadiazine, or water. Fifty IncP-1ε plasmids that were captured in E. coli CV601gfp from bacterial communities of manure and arable soil were characterized by PCR and hybridisation. All plasmids carried class 1 integrons with highly varying sizes of the gene cassette region and the sul1 gene. Three IncP-1ε plasmids captured from soil bacteria and one from manure were completely sequenced. The backbones were nearly identical to that of the previously described IncP-1ε plasmid pKJK5. The plasmids differed mainly in the composition of a Tn402-like transposon carrying a class 1 integron with varying gene cassettes, IS1326, and in three of the plasmids the tetracycline resistance transposon Tn1721 with various truncations. Diverse Beta- and Gammaproteobacteria were revealed as hosts of one of the IncP-1ε plasmids in soil microcosms. Our data suggest that IncP-1ε plasmids are important vectors for horizontal transfer of antibiotic resistance in agricultural systems.

  1. Small multicopy, non-integrative shuttle vectors based on the plasmid pRN1 for Sulfolobus acidocaldarius and Sulfolobus solfataricus, model organisms of the (cren-)archaea.

    Science.gov (United States)

    Berkner, Silvia; Grogan, Dennis; Albers, Sonja-Verena; Lipps, Georg

    2007-01-01

    The extreme thermoacidophiles of the genus Sulfolobus are among the best-studied archaea but have lacked small, reliable plasmid vectors, which have proven extremely useful for manipulating and analyzing genes in other microorganisms. Here we report the successful construction of a series of Sulfolobus-Escherichia coli shuttle vectors based on the small multicopy plasmid pRN1 from Sulfolobus islandicus. Selection in suitable uracil auxotrophs is provided through inclusion of pyrEF genes in the plasmid. The shuttle vectors do not integrate into the genome and do not rearrange. The plasmids allow functional overexpression of genes, as could be demonstrated for the beta-glycosidase (lacS) gene of S. solfataricus. In addition, we demonstrate that this beta-glycosidase gene could function as selectable marker in S. solfataricus. The shuttle plasmids differ in their interruption sites within pRN1 and allowed us to delineate functionally important regions of pRN1. The orf56/orf904 operon appears to be essential for pRN1 replication, in contrast interruption of the highly conserved orf80/plrA gene is tolerated. The new vector system promises to facilitate genetic studies of Sulfolobus and to have biotechnological uses, such as the overexpression or optimization of thermophilic enzymes that are not readily performed in mesophilic hosts.

  2. Optimizing a Method for the Quantification by Quantitative Real-Time Polymerase Chain Reaction of Host Cell DNA in Plasmid Vector Batches Used in Human Gene Therapy.

    Science.gov (United States)

    Ferro, Serge; Fabre, Isabelle; Chenivesse, Xavier

    2016-08-01

    Gene therapy products are very complex advanced therapy medicinal products produced using different processes that require many chemical and biological reagents and production intermediates, such as producing cells. The quantification of residual impurities in gene therapy vectors is a major quality control step when these vectors are used for therapeutic purposes, whether or not they are derived from viruses. Indeed, in nonviral gene therapy products, particularly plasmid vectors used to transfer genetic material, the presence of host-cell DNA (HCDNA) from the bacterial cells used for the vector production is an important concern because of the risk of immunogenicity and insertional mutagenesis. Several methods have been developed to quantify residual HCDNA, but real-time quantitative polymerase chain reaction (qPCR) seems to be most suitable because it allows detecting traces of "contaminating" DNA. The French National Agency for Medicines and Health Products Safety (ANSM) ensures the quality and safety of gene transfer medicinal products and must be able to quantify, in its own laboratories, the amount of HCDNA present in plasmid vector batches. Therefore, we developed and validated a qPCR method to quantify at the femtogram level the presence of Escherichia coli residual DNA in plasmid vectors. This approach uses the capillary-based LightCycler 1.5 System (Roche) with SYBR Green I, a primer pair against the E. coli 23S ribosomal RNA gene and different concentrations of a linearized plasmid that contains the 23S target sequence, as standard. This qPCR method is linear on an 8-decade logarithmic scale, accurate, reproducible, and sensitive (quantification of up to 10 copies of 23S target sequence per reaction, or 1.4 E. coli genome, or 7 fg of bacterial DNA). This technique allows ensuring that batches of plasmid vectors to be used in clinical trials comply with the specifications on HCDNA content.

  3. Molecular cloning with bifunctional plasmid vectors in Bacillus subtilis: isolation of a spontaneous mutant of Bacillus subtilis with enhanced transformability for Escherichia coli-propagated chimeric plasmid DNA.

    OpenAIRE

    Ostroff, G. R.; Pène, J. J.

    1983-01-01

    Hybrid plasmid DNA cloned in Escherichia coli undergoes deletions when returned to competent Bacillus subtilis, even in defined restriction and modification mutants of strain 168. We have isolated a mutant of B. subtilis MI112 which is stably transformed at high frequency by chimeric plasmid DNA propagated in E. coli.

  4. Plasmid Biopharmaceuticals.

    Science.gov (United States)

    Prazeres, Duarte Miguel F; Monteiro, Gabriel A

    2014-12-01

    Plasmids are currently an indispensable molecular tool in life science research and a central asset for the modern biotechnology industry, supporting its mission to produce pharmaceutical proteins, antibodies, vaccines, industrial enzymes, and molecular diagnostics, to name a few key products. Furthermore, plasmids have gradually stepped up in the past 20 years as useful biopharmaceuticals in the context of gene therapy and DNA vaccination interventions. This review provides a concise coverage of the scientific progress that has been made since the emergence of what are called today plasmid biopharmaceuticals. The most relevant topics are discussed to provide researchers with an updated overview of the field. A brief outline of the initial breakthroughs and innovations is followed by a discussion of the motivation behind the medical uses of plasmids in the context of therapeutic and prophylactic interventions. The molecular characteristics and rationale underlying the design of plasmid vectors as gene transfer agents are described and a description of the most important methods used to deliver plasmid biopharmaceuticals in vivo (gene gun, electroporation, cationic lipids and polymers, and micro- and nanoparticles) is provided. The major safety issues (integration and autoimmunity) surrounding the use of plasmid biopharmaceuticals is discussed next. Aspects related to the large-scale manufacturing are also covered, and reference is made to the plasmid products that have received marketing authorization as of today.

  5. The Cloning of the Human Tumor Supressor Gene INGI: DNA Cloning into Plasmid Vector and DNA Analysis by Restriction Enzymes

    Directory of Open Access Journals (Sweden)

    Elza Ibrahim Auerkari

    2015-11-01

    Full Text Available DNA cloning is one of the most important techniques In the field of molecular biology, with a critical role in analyzing the structure and function of genes and their adjacent regulatory regions. DNA cloning is helpful in learning fundamental molecular biological techniques, since DNA cloning involves a series of them, such as polymerase chain reaction (PCR, DNA ligation, bacterial transformation, bacterial culture, plasmid DNA extraction, DNA digestion with restriction enzymes and agarose gel electrophoresis. In this paper the cloning of the human tumor suppressor gene INGI has been used to illustrate the methodology. The gene was amplified by PCR, cloned into a TA-cloning vectore, and restriction enzyme mapping was used to distinguish the sense INGI construct from the antisense INGI construct.

  6. Development of electrochemical reporter assay using HeLa cells transfected with vector plasmids encoding various responsive elements

    Energy Technology Data Exchange (ETDEWEB)

    Shiku, Hitoshi, E-mail: shiku@bioinfo.che.tohoku.ac.jp [Graduate School of Environmental Studies, Tohoku University, 6-6-11-604 Aramaki-Aoba, Sendai 980-8579 (Japan); Takeda, Michiaki; Murata, Tatsuya [Graduate School of Environmental Studies, Tohoku University, 6-6-11-604 Aramaki-Aoba, Sendai 980-8579 (Japan); Akiba, Uichi; Hamada, Fumio [Graduate School of Engineering and Resource Science, Akita University, 1-1 Tegata gakuen-machi, Akita 010-8502 (Japan); Matsue, Tomokazu, E-mail: matsue@bioinfo.che.tohoku.ac.jp [Graduate School of Environmental Studies, Tohoku University, 6-6-11-604 Aramaki-Aoba, Sendai 980-8579 (Japan)

    2009-04-27

    Electrochemical assay using HeLa cell lines transfected with various plasmid vectors encoding SEAP (secreted alkaline phosphatase) as the reporter has been performed by using SECM (scanning electrochemical microscopy). The plasmid vector contains different responsive elements that include GRE (glucocorticoid response elements), CRE (cAMP responsive elements), or {kappa}B (binding site for NF{kappa}B (nuclear factor kappa B)) upstream of the SEAP sequence. The transfected HeLa cells were patterned on a culture dish in a 4 x 4 array of circles of diameter 300 {mu}m by using the PDMS (poly(dimethylsiloxane)) stencil technique. The cellular array was first exposed to 100 ng mL{sup -1} dexamethasone, 10 ng mL{sup -1} forskolin, or 100 ng mL{sup -1} TNF-{alpha} (tumor necrosis factor {alpha}) after which it was further cultured in an RPMI culture medium for 6 h. After incubation, the cellular array was soaked in a measuring solution containing 4.7 mM PAPP (p-aminophenylphosphate) at pH 9.5, following which electrochemical measurements were performed immediately within 40 min. The SECM method allows parallel evaluation of different cell lines transfected with pGRE-SEAP, pCRE-SEAP, and pNF{kappa}B-SEAP patterned on the same solid support for detection of the oxidation current of PAP (p-aminophenol) flux produced from only 300 HeLa cells in each stencil pattern. The results of the SECM method were highly sensitive as compared to those obtained from the conventional CL (chemiluminescence) protocol with at least 5 x 10{sup 4} cells per well.

  7. CONSTRUCTION AND EXPRESSION OF ADENOASSOCIATED VIRUS- BASED PLASMID EXPRESSING VECTORS CONTAINING hIL- 2 GENE OR mIFN-γ GENE

    Institute of Scientific and Technical Information of China (English)

    张景迎; 梁宏立; 陈诗书

    2000-01-01

    Objective To improve the plasmid vectors in gene therapy, adeno - associated virus (AA V) based plasmid expressing vectors containing hIL-2 gene or mIFN-γ gene were constructed and its expression in transfected cells was studied. Methods By means of step to step cloning, promoter CMVp was placed at the downstream of 5' inverted terminal repeat from AA V (AA V- ITR) of pAP, hIL- 2 gene or mIFN- γ gene inserted into pAC between CMVp and poly A. Then intron A was inserted into pAC- hIL - 2 or pAC- mIFN- γ between CMVp and IL - 2 gene or IFNγ gene to construct pAI- hIL - 2 or pAI- mIFN - γ. Liposome -plasmid complexes were formed by mixing Dosper with these AAV-based plasmids containing hIL-2 gene or mIFN-γgene. Results High biological activities of IL - 2 or IFN- γ could be detected in the supernatants of NIH3T3 and MM45T. Li cells after transfection. Insertion of intron A into pAC-hIL-2 or pAC-mIFN-γ improved the expression of IL- 2 or IFN- γ. Conclusion These data demonstrated that the constructed AA V- based plasmid expressing vectors could efficiently express therapeutic genes in cultured cells and could be used as a nonviral gene transfer system in human gene therapy.

  8. Small multicopy, non-integrative shuttle vectors based on the plasmid pRN1 for Sulfolobus acidocaldarius and Sulfolobus solfataricus, model organisms of the (cren-)archaea

    NARCIS (Netherlands)

    Berkner, Silvia; Grogan, Dennis; Albers, Sonja-Verena; Lipps, Georg

    2007-01-01

    The extreme thermoacidophiles of the genus Sulfolobus are among the best-studied archaea but have lacked small, reliable plasmid vectors, which have proven extremely useful for manipulating and analyzing genes in other microorganisms. Here we report the successful construction of a series of Sulfolo

  9. Delivery of plasmid DNA expression vector for keratinocyte growth factor-1 using electroporation to improve cutaneous wound healing in a septic rat model.

    Science.gov (United States)

    Lin, Michael P; Marti, Guy P; Dieb, Rami; Wang, Jiaai; Ferguson, Mark; Qaiser, Rabia; Bonde, Pramod; Duncan, Mark D; Harmon, John W

    2006-01-01

    We have previously shown that wound healing was improved in a diabetic mouse model of impaired wound healing following transfection with keratinocyte growth factor-1 (KGF-1) cDNA. We now extend these findings to the characterization of the effects of DNA plasmid vectors delivered to rats using electroporation (EP) in vivo in a sepsis-based model of impaired wound healing. To assess plasmid transfection and wound healing, gWIZ luciferase and PCDNA3.1/KGF-1 expression vectors were used, respectively. Cutaneous wounds were produced using an 8 mm-punch biopsy in Sprague-Dawley rats in which healing was impaired by cecal ligation-induced sepsis. We used National Institutes of Health image analysis software and histologic assessment to analyze wound closure and found that EP increased expression of gWIZ luciferase vector up to 53-fold compared with transfection without EP (p < 0.001). EP-assisted plasmid transfection was found to be localized to skin. Septic rats had a 4.7 times larger average wound area on day 9 compared with control (p < 0.001). Rats that underwent PCDNA3.1/KGF-1 transfection with EP had 60% smaller wounds on day 12 compared with vector without EP (p < 0.009). Quality of healing with KGF-1 vector plus EP scored 3.0 +/- 0.3 and was significantly better than that of 1.8 +/- 0.3 for treatment with vector alone (p < 0.05). We conclude that both the rate and quality of healing were improved with DNA plasmid expression vector for growth factor delivered with EP to septic rats.

  10. Characterization of the minimal replicon of a cryptic Deinococcus radiodurans SARK plasmid and development of versatile Escherichia coli-D. radiodurans shuttle vectors.

    Science.gov (United States)

    Meima, R; Lidstrom, M E

    2000-09-01

    The nucleotide sequence of a 12-kb fragment of the cryptic Deinococcus radiodurans SARK plasmid pUE10 was determined, in order to direct the development of small, versatile cloning systems for Deinococcus. Annotation of the sequence revealed 12 possible open reading frames. Among these are the repU and resU genes, the predicted products of which share similarity with replication proteins and site-specific resolvases, respectively. The products of both genes were demonstrated using an overexpression system in Escherichia coli. RepU was found to be required for replication, and ResU was found to be required for stable maintenance of pUE10 derivatives. Gel shift analysis using purified His-tagged RepU identified putative binding sites and suggested that RepU may be involved in both replication initiation and autoregulation of repU expression. In addition, a gene encoding a possible antirestriction protein was found, which was shown to be required for high transformation frequencies. The arrangement of the replication region and putative replication genes for this plasmid from D. radiodurans strain SARK is similar to that for plasmids found in Thermus but not to that for the 45.7-kb plasmid found in D. radiodurans strain R1. The minimal region required for autonomous replication in D. radiodurans was determined by sequential deletion of segments from the 12-kb fragment. The resulting minimal replicon, which consists of approximately 2.6 kb, was used for the construction of a shuttle vector for E. coli and D. radiodurans. This vector, pRAD1, is a convenient general-purpose cloning vector. In addition, pRAD1 was used to generate a promoter probe vector, and a plasmid containing lacZ and a Deinococcus promoter was shown to efficiently express LacZ.

  11. Generation of integration-free human induced pluripotent stem cells from postnatal blood mononuclear cells by plasmid vector expression.

    Science.gov (United States)

    Dowey, Sarah N; Huang, Xiaosong; Chou, Bin-Kuan; Ye, Zhaohui; Cheng, Linzhao

    2012-11-01

    Several human postnatal somatic cell types have been successfully reprogrammed to induced pluripotent stem cells (iPSCs). Blood mononuclear cells (MNCs) offer several advantages compared with other cell types. They are easily isolated from umbilical cord blood (CB) or adult peripheral blood (PB), and can be used fresh or after freezing. A short culture allows for more efficient reprogramming, with iPSC colonies forming from blood MNCs in 14 d, compared with 28 d for age-matched fibroblastic cells. The advantages of briefly cultured blood MNCs may be due to favorable epigenetic profiles and gene expression patterns. Blood cells from adults, especially nonlymphoid cells that are replenished frequently from intermittently activated blood stem cells, are short-lived in vivo and may contain less somatic mutations than skin fibroblasts, which are more exposed to environmental mutagens over time. We describe here a detailed, validated protocol for effective generation of integration-free human iPSCs from blood MNCs by plasmid vectors.

  12. Agrobacterium-mediated plant transformation by novel mini-T vectors in conjunction with a high-copy vir region helper plasmid.

    Science.gov (United States)

    Zyprian, E; Kado, C I

    1990-08-01

    A new binary vector system for Agrobacterium-mediated plant transformation was developed. A set of four mini-T vectors comprised of T-DNA border sequences from nopaline-type Ti-plasmid pTiC58 flanking a chimaeric hygromycin-resistance gene for selection of transformants and up to eight unique restriction sites for cloning foreign DNA was constructed on a broad-host replicon containing the oriV of plasmid pSa. In two of the constructs these multiple cloning sites are flanked by a strong promoter to activate transcription of inserted DNA in planta. High-efficiency transformation was prompted by a high-copy, stable virulence helper plasmid pUCD2614, which contains a cloned virulence region of pTiC58 and tandem copies of the par locus of plasmid pTAR. Southern blot hybridization and genetic analyses of the progeny of transformed plants showed that the hygromycin resistance gene was stably inherited.

  13. Determination of protein expression and plasmid copy number from cloned genes in Escherichia coli by flow injection analysis using an enzyme indicator vector.

    Science.gov (United States)

    Schendel, F J; Baude, E J; Flickinger, M C

    1989-10-20

    On-line determination of expression rates from cloned genes in Escherichia coli and of plasmid copy number would be useful for monitoring accumulation of non-secreted proteins. As an initial model for monitoring gene expression in intact cells, a non-gene-fusion enzyme-based indicator plasmid has been constructed containing the phoA gene coding for alkaline phosphatase (AP) in pUCIS and pACYC184. The activity of AP can be rapidly determined in permeabilized cells. A flow injection analysis (FIA) assay has been developed which allows the direct real-time measurement of the AP activity during cell growth. A model target gene coding for E. coli cyanase (cynS) has been inserted in order to determine the ratio between the expression of the target and indicator, AP. A linear relationship has been found between plasmid copy number and AP activity for the high-copy pUC vector. To minimize indicator expression, transcription terminators have been inserted between the cynS and phoA genes, altering the target-to-indicator ratio by 10- to 40-fold. These vectors may be useful for the rapid continuous determination of plasmid copy number and target gene expression for nonsecreted proteins and would overcome the limitations of in situ probe biosensors for real-time determination of the accumulation of proteins from cloned genes in E. coli.

  14. Construction of a stable plasmid vector for industrial production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) by a recombinant Cupriavidus necator H16 strain.

    Science.gov (United States)

    Sato, Shunsuke; Fujiki, Tetsuya; Matsumoto, Keiji

    2013-12-01

    A new stable plasmid vector (pCUP3) was developed for high and stable production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBH) using Cupriavidus necator H16 as the host strain. In pCUP3, it was found that the plasmid partition and replication region of the megaplasmid pMOL28 in the Cupriavidus metallidurans CH34 strain plays an important role in plasmid stability in C. necator H16. Moreover, the partition locus (comprising parA28 and parB28 and the parS28 region) is essential for plasmid maintenance under high-PHBH-accumulation. PHBH productivity by the C. necator H16/ds strain (phaC1 deactivated mutant strain) harboring a phaCAc NSDG within pCUP3 was identical to the productivity of poly(3-hydroxybutyrate) by the C. necator H16 strain when palm kernel oil was used as the sole carbon source without any antibiotics. This new vector is important for industrial mass production of polyhydroxyalkanoates using the C. necator H16 strain as the host, dispensing the necessity of the application of selective pressure such as antibiotics. Copyright © 2013 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  15. A plasmid RK2-based broad-host-range cloning vector useful for transfer of metagenomic libraries to a variety of bacterial species.

    Science.gov (United States)

    Aakvik, Trine; Degnes, Kristin Fløgstad; Dahlsrud, Rannveig; Schmidt, Frank; Dam, Ragnar; Yu, Lihua; Völker, Uwe; Ellingsen, Trond Erling; Valla, Svein

    2009-06-01

    The majority of microorganisms in natural environments are difficult to cultivate, but their genes can be studied via metagenome libraries. To enhance the chances that these genes become expressed we here report the construction of a broad-host-range plasmid vector (pRS44) for fosmid and bacterial artificial chromosome (BAC) cloning. pRS44 can be efficiently transferred to numerous hosts by conjugation. It replicates in such hosts via the plasmid RK2 origin of replication, while in Escherichia coli it replicates via the plasmid F origin. The vector was found to be remarkably stable due to the insertion of an additional stability element (parDE). The copy number of pRS44 is adjustable, allowing for easy modifications of gene expression levels. A fosmid metagenomic library consisting of 20 000 clones and BAC clones with insert sizes up to 200 kb were constructed. The 16S rRNA gene analysis of the fosmid library DNA confirmed that it represents a variety of microbial species. The entire fosmid library and the selected BAC clones were transferred to Pseudomonas fluorescens and Xanthomonas campestris (fosmids only), and heterologous proteins from the fosmid library were confirmed to be expressed in P. fluorescens. To our knowledge no other reported vector system has a comparable potential for functional screening across species barriers.

  16. Construction and use of a broad-host-range plasmid expressing the lamB gene for utilization of bacteriophage lambda vectors in the marine bacterium Vibrio harveyi.

    Science.gov (United States)

    Jasiecki, J; Czy, A; Gabig, M; Wegrzyn, G

    2001-07-01

    The remarkable success of Escherichia coli as a model organism in molecular genetics was dependent, among other things, on its susceptibility to genetic manipulation. Many versatile and sophisticated genetic tools for molecular biology studies are derived from bacteriophage lambda. However, this bacteriophage is specific for E. coli, and thus lambda-based techniques have been restricted to this bacterium. Plasmids expressing the E. coli gene coding for bacteriophage lambda receptor were reported previously, and introduction of such plasmids into cells of some other bacteria made them sensitive to phage lambda infection. However, we found that these systems were not efficient for Vibrio harveyi, one of the most frequently investigated species of marine bacteria. Here we describe construction of a broad-host-range plasmid expressing the lamB gene. Introduction of this plasmid to V. harveyi cells and expression of lamB made this strain susceptible to bacteriophage lambda adsorption and lambda DNA injection. Foreign genetic material could be introduced into cells of this strain using a cosmid vector.

  17. Development of a vector and host system and characterization of replication of plasmid pSQ10 in moderately halophilic Nocardiopsis

    Institute of Scientific and Technical Information of China (English)

    Ana Zeng; Tao Wang; Haiyang Xia; Shiyuan Peng; Weihua Chen; Chenglin Jiang; Lihua Xu

    2011-01-01

    The genus of Nocardiopsis is a new source of antibiotics,chemicals,and enzymes.Here we reported the development of a vector and host system in moderately halophilic Nocardiopsis via an oriT-mediated conjugation.By screening about 80 Nocardiopsis strains,6 of them harbored 8 plasmids (18-80 kb).The complete nucleotide sequence of pSQ10 consisted of 18,219 bp,with 71.9% G + Ccontent,encoding 17 open reading frames,5 of them resembled those of Streptomyces plasmids.A rep locus (iteron within the gene) was identified for replication in Nocardiopsis sp.YIM 90083,and rep protein bound to its iteron sequence.This system may be useful for gene cloning and expression in Nocardiopsis.

  18. Prognostic impact of HER-2 Subclonal Amplification in breast cancer.

    Science.gov (United States)

    Di Oto, Enrico; Brandes, Alba A; Cucchi, Maria C; Foschini, Maria P

    2017-06-02

    The presence of a limited number of cells with HER-2 amplification (Subclonal Amplification) in breast carcinomas is occasionally encountered, but its prognostic impact is poorly known. The purpose of this study is to evaluate the prognostic impact of HER-2 Subclonal Amplification in a retrospective series of breast cancers. Accordingly, 81 consecutive breast carcinomas showing HER-2 Subclonal Amplification were obtained from the histology files (case series). These cases were subdivided into two groups: (a) those cases in which the HER-2 Subclonal Amplification was consonant to the accepted criteria for amplification, showing clusters of amplified cells, and (b) those cases with rare HER-2 Subclonal Amplification that did not reflect the accepted criteria for amplification, showing scattered amplified cells only. The incidence of metastases and late recurrences of the case series was compared with a series composed of 109 consecutive cases, being HER-2 homogeneous (comprising 14 Amplified and 95 Non-Amplified cases), matched for grade and stage (control series). It appeared that cases showing Subclonal Amplification had an incidence of metastases intermediate between the cases Amplified and Non-Amplified. Specifically, Subclonal Amplification with clustered cells had a lower incidence of metastases than Amplified cases (12.9 versus 21.4%). On the contrary, Subclonal Amplification with scattered cells showed an incidence of metastases higher than Non-Amplified cases (14 versus 9.47%). In addition, patients Subclonal Amplification with clustered cells, who were treated with the specific monoclonal antibody, had a lower incidence of metastases than patients showing Subclonal Amplification with scattered cells, who did not receive target therapy. These data, together with those recently published, indicate that Subclonal Amplification has an impact on prognosis and should be taken into consideration to correctly plan the treatment of breast cancer patients.

  19. Two-plasmid vector system for independently controlled expression of green and red fluorescent fusion proteins in Staphylococcus aureus.

    Science.gov (United States)

    Brzoska, Anthony J; Firth, Neville

    2013-05-01

    We have constructed a system for the regulated coexpression of green fluorescent protein (GFP) and red fluorescent protein (RFP) fusions in Staphylococcus aureus. It was validated by simultaneous localization of cell division proteins FtsZ and Noc and used to detect filament formation by an actin-like ParM plasmid partitioning protein in its native coccoid host.

  20. Dual-function vector for protein expression in both mammalian cells and Xenopus laevis oocytes

    DEFF Research Database (Denmark)

    Jespersen, Thomas; Grunnet, M; Angelo, K;

    2002-01-01

    will often engage both oocytes and mammalian cells. Efficient expression of a protein in both systems have thus far only been possible by subcloning the cDNA into two different vectors because several different molecular requirements should be fulfilled to obtain a high protein level in both mammalian cells...... and oocytes. To address this problem, we have constructed a plasmid vector, pXOOM, that can function as a template for expression in both oocytes and mammalian cells. By including all the necessary RNA stability elements for oocyte expression in a standard mammalian expression vector, we have obtained a dual......-function vector capable of supporting protein production in both Xenopus oocytes and CHO-K1 cells at an expression level equivalent to the levels obtained with vectors optimized for either oocyte or mammalian expression. Our functional studies have been performed with hERGI, KCNQ4, and Kv1.3 potassium channels....

  1. A bivalent typhoid live vector vaccine expressing both chromosome- and plasmid-encoded Yersinia pestis antigens fully protects against murine lethal pulmonary plague infection.

    Science.gov (United States)

    Galen, James E; Wang, Jin Yuan; Carrasco, Jose A; Lloyd, Scott A; Mellado-Sanchez, Gabriela; Diaz-McNair, Jovita; Franco, Olga; Buskirk, Amanda D; Nataro, James P; Pasetti, Marcela F

    2015-01-01

    Live attenuated bacteria hold great promise as multivalent mucosal vaccines against a variety of pathogens. A major challenge of this approach has been the successful delivery of sufficient amounts of vaccine antigens to adequately prime the immune system without overattenuating the live vaccine. Here we used a live attenuated Salmonella enterica serovar Typhi strain to create a bivalent mucosal plague vaccine that produces both the protective F1 capsular antigen of Yersinia pestis and the LcrV protein required for secretion of virulence effector proteins. To reduce the metabolic burden associated with the coexpression of F1 and LcrV within the live vector, we balanced expression of both antigens by combining plasmid-based expression of F1 with chromosomal expression of LcrV from three independent loci. The immunogenicity and protective efficacy of this novel vaccine were assessed in mice by using a heterologous prime-boost immunization strategy and compared to those of a conventional strain in which F1 and LcrV were expressed from a single low-copy-number plasmid. The serum antibody responses to lipopolysaccharide (LPS) induced by the optimized bivalent vaccine were indistinguishable from those elicited by the parent strain, suggesting an adequate immunogenic capacity maintained through preservation of bacterial fitness; in contrast, LPS titers were 10-fold lower in mice immunized with the conventional vaccine strain. Importantly, mice receiving the optimized bivalent vaccine were fully protected against lethal pulmonary challenge. These results demonstrate the feasibility of distributing foreign antigen expression across both chromosomal and plasmid locations within a single vaccine organism for induction of protective immunity. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  2. Vectores

    OpenAIRE

    2016-01-01

    Documento que contiene la explicación sobre las temáticas de Sistemas coordenados, Cantidades vectoriales y escalares, Algunas propiedades de los vectores, Componentes de un vector y vectores unitarios

  3. Dynamic properties of the Sulfolobus CRISPR/Cas and CRISPR/Cmr systems when challenged with vector-borne viral and plasmid genes and protospacers

    DEFF Research Database (Denmark)

    Guðbergsdóttir, Sóley Ruth; Deng, Ling; Chen, Zhengjun

    2011-01-01

    The adaptive immune CRISPR/Cas and CRISPR/Cmr systems of the crenarchaeal thermoacidophile Sulfolobus were challenged by a variety of viral and plasmid genes, and protospacers preceded by different dinucleotide motifs. The genes and protospacers were constructed to carry sequences matching...... individual spacers of CRISPR loci, and a range of mismatches were introduced. Constructs were cloned into vectors carrying pyrE/pyrF genes and transformed into uracil auxotrophic hosts derived from Sulfolobus solfataricus P2 or Sulfolobus islandicus REY15A. Most constructs, including those carrying different...... protospacer mismatches, yielded few viable transformants. These were shown to carry either partial deletions of CRISPR loci, covering a broad spectrum of sizes and including the matching spacer, or deletions of whole CRISPR/Cas modules. The deletions occurred independently of whether genes or protospacers...

  4. PLASMIDS FROM ANAEROCELLUM THERMOPHILUM AND USES THEREOF

    DEFF Research Database (Denmark)

    2003-01-01

    The present invention concerns the isolation of plasmids from extremely thermophilic anaerobic microorganisms and their use in genetic transformation of thermophilic and mesophilic microorganisms. More particular the invention concerns the use of thermostable plasmid vectors as tools for creating...

  5. Construction of plasmid vector pAFP-HSVtk-IRES2-EGFP and its effect on the cytotoxicity of ganciclovir to hepatocellular carcinoma

    Institute of Scientific and Technical Information of China (English)

    Lai Zhiyong; Qin Qin; Yu Baofeng; Xie Jun; Gao Ranpeng; Zhang Tiantian; Li Chunfeng

    2014-01-01

    Background Herpes simplex virus thymidine kinase phosphorylates ganciclovir to ganciclovir monophosphate,which is then converted to ganciclovir triphosphate by endogenous cellular nucleoside kinases.The ganciclovir triphosphate acts as a DNA chain terminator due to the lack of a functional 3'-OH group and terminates the process of DNA replication,hence leading to cell apoptosis.At present,HSVtk gene usually acts as suicide gene to kill tumor cells.The aim of this study was to investigate the selective cytotoxicity of the herpes simplex virus thymidine kinase/ganciclovir (HSVtK/GCV) suicide gene system controlled by the α-fetoprotein (AFP) promoter on hepatocellular carcinoma (HCC) cells in vitro.Methods pAFP-HSVtk-IRES2-EGFP recombinant plasmid vectors driven by the AFP promoter were constructed.HL-7702 liver cells,HUH-7 HCC,and HepG2 HCC were transfected with the recombinant plasmids.HSVtK gene expression was detected using Western blotting analysis.HepG2 cells line stably expressing HSVtk gene was selected by G418 reagent.The cytotoxicity of HSVtK/GCV suicide gene system on hepatoma cells was measured by CCK-8 reagents when different doses of ganciclovir were added.Results Plasmid pAFP-TK-IRES2-EGFP-expressed HSVtk gene was constructed successfully.HSVtk gene expression level was significantly higher in AFP-positive hepatoma cells than in AFP-negative liver cells.After G418 selection,a HepG2 cells line stably expressing HSVtk gene was acquired.With the increase of the dose of ganciclovir the optical density at 450 nm of HepG2 cells stably expressing HSVtk gene gradually decreased (P <0.05).Conclusion The HSVtK gene-specific expression in hepatoma cells as well as the cytotoxicity of the suicide gene system in HepG2 cells provided the basis for the targeted gene therapy of HCC.

  6. Construction and Expression of Eukaryotic Expression Vector and Plasmid Expressing siRNA of Human Protection of Telomeres 1

    Institute of Scientific and Technical Information of China (English)

    Di-Nan HUANG; Ying-Hua JIANG; Hou GAN

    2005-01-01

    @@ 1 Introduction The POT1 (protection of telomeres 1 ) protein binds the single-stranded overhang at the ends of chromosomes in diverse eukaryocytes. It is essential for chromosome end-protection in the fission yeast Schizosaccharomyces pombe, and it is involved in regulation of telomere lengthin human cells. Human POT1 had been identified in 2001year. Its amino terminal is highly conservative in eukaryocytes. Since Pot1 can bind internal loops and directly adjacent DNA-binding sites, it is likely to fully coat and protect both G-strand overhangs and the displaced G strand of a T-loop. It also participates in the regulation of telomere maintenance by telomerase or of chromosome by other enzymes. But its biological function and mechanism are still unknown. hPOT eukaryotic expression vector(pcDNA3- hPOT1 ) and 3 kinds of siRNA expression vectors(pmU6-shDNA1, pmU6-shDNA2 and pmU6-shDNA3)had been constructed. And there were overexpression and RNA silence in HeLa cells.

  7. Vectors

    DEFF Research Database (Denmark)

    Boeriis, Morten; van Leeuwen, Theo

    2017-01-01

    This article revisits the concept of vectors, which, in Kress and van Leeuwen’s Reading Images (2006), plays a crucial role in distinguishing between ‘narrative’, action-oriented processes and ‘conceptual’, state-oriented processes. The use of this concept in image analysis has usually focused...... on the most salient vectors, and this works well, but many images contain a plethora of vectors, which makes their structure quite different from the linguistic transitivity structures with which Kress and van Leeuwen have compared ‘narrative’ images. It can also be asked whether facial expression vectors...... should be taken into account in discussing ‘reactions’, which Kress and van Leeuwen link only to eyeline vectors. Finally, the question can be raised as to whether actions are always realized by vectors. Drawing on a re-reading of Rudolf Arnheim’s account of vectors, these issues are outlined...

  8. A new method for rapidly generating gene-targeting vectors by engineering BACs through homologous recombination in bacteria.

    Science.gov (United States)

    Cotta-de-Almeida, Vinicius; Schonhoff, Susan; Shibata, Tomoyuki; Leiter, Andrew; Snapper, Scott B

    2003-09-01

    Generating knockout mice is still an expensive and highly time-consuming process. Target construct generation, the first labor-intensive step in this process, requires the manipulation of large fragments of DNA and numerous, and often cumbersome, cloning steps. Here we show the development of a rapid approach for generating targeting constructs that capitalizes on efficient homologous recombination between linear DNA fragments and circular plasmids in Escherichia coli ("recombineering"), the availability of bacterial artificial chromosomes (BACs), and the accessibility of the sequence of the mouse genome. Employing recombineering, we demonstrate with only 1-2 template plasmids, short homologies (40-50bp) between donor and target DNA, and one subcloning step that we can efficiently manipulate BACs in situ to generate a complicated targeting vector. This procedure avoids the need to construct or screen genomic libraries and permits the generation of most standard, conditional, or knock-in targeting vectors, often within two weeks.

  9. Hybrid cytomegalovirus-U6 promoter-based plasmid vectors improve efficiency of RNA interference in zebrafish.

    Science.gov (United States)

    Su, Jianguo; Zhu, Zuoyan; Xiong, Feng; Wang, Yaping

    2008-01-01

    Short hairpin RNA (shRNA) directed by RNA polymerase III (Pol III) or Pol II promoter was shown to be capable of silencing gene expression, which should permit analyses of gene functions or as a potential therapeutic tool. However, the inhibitory effect of shRNA remains problematic in fish. We demonstrated that silencing efficiency by shRNA produced from the hybrid construct composed of the CMV enhancer or entire CMV promoter placed immediately upstream of a U6 promoter. When tested the exogenous gene, silencing of an enhanced green fluorescent protein (EGFP) target gene was 89.18 +/- 5.06% for CMVE-U6 promoter group and 88.26 +/- 6.46% for CMV-U6 promoter group. To test the hybrid promoters driving shRNA efficiency against an endogenous gene, we used shRNA against no tail (NTL) gene. When vectorized in the zebrafish, the hybrid constructs strongly repressed NTL gene expression. The NTL phenotype occupied 52.09 +/- 3.06% and 51.56 +/- 3.68% for CMVE-U6 promoter and CMV-U6 promoter groups, respectively. The NTL gene expression reduced 82.17 +/- 2.96% for CMVE-U6 promoter group and 83.06 +/- 2.38% for CMV-U6 promoter group. We concluded that the CMV enhancer or entire CMV promoter locating upstream of the U6-promoter could significantly improve inhibitory effect induced by the shRNA for both exogenous and endogenous genes compared with the CMV promoter or U6 promoter alone. In contrast, the two hybrid promoter constructs had similar effects on driving shRNA.

  10. Cloning of 1183 bp Fragment from Rhoptry Protein I (ROPI Gene of Toxoplasma gondii (RH in Expression Prokaryote Plasmid PET32a

    Directory of Open Access Journals (Sweden)

    Zahra Eslamirad

    2013-10-01

    Full Text Available Background: Toxoplasma gondii is an obligatory intracellular protozoan. Considering to high prevalence of this disease the best way to reduce the raised loses is prevention of human and animal infection, rapid diagnosis, differentiation between acute and chronic disease. Rhoptry protein 1 of Toxoplasma gondii is an excretory-secretory antigen that exists in the most stages of life cycle. According to specifications of excretory-secretory antigen that seems this antigen is a suitable candidate to produce recombinant vaccine and diagnostic kit. The main object of the present work was cloning rhoptry protein 1 (ROP1 gene of Toxoplasma gondii (RH in a cloning vector for further production of rhoptry proteins.Materials and Methods: Genomic DNA was extracted by phenol-chloroform method. The ROP1 fragment was amplified by PCR. This product was approved by sequencing and was cloned between the EcoR1 and Sal1 sites of the pTZ57R/T vector. Then transformed into Escherichia coli DH5α strain and screened by IPTG and X-Gal. After isolating of this gene from pTZ57R/T, it was subcloned into pET32a plasmid.Results: The plasmid was purified and approved by electrophoresis, enzyme restriction and PCR. After isolating of this gene from pTZ57R/T, it was subcloned into pET32a plasmid. After enzyme restriction and electrophoresis a fragment about 1183bp was separated from pET32a.Conclusion: Recombinant plasmid of ROP1 gene was constructed and ready for future study. That seems the antigen is a suitable candidate to produce recombinant vaccine and diagnostic kit.

  11. CONSTRUCTION OF EUKARYOTIC EXPRESSION VECTOR WITH GRANULOCYTE-MACROPHAGE COLONY-STIMULATING FACTOR GENE

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Objective: To construct the eukaryotic expression vector that express human granulocyte-macrophage colony-stimulating factor (hGM-CSF) gene for making highly express in mammalian cells. Methods: Extract totally RNA from the induced human fetal lung (HFL) cell line. HGM-CSF cDNA was obtained by reverse transcription-polymerase chain reaction (RT-PCR), and then directionally subcloned into the HindIII and EcoRI site on the pcDNA3.1 plasmid, which was controlled by the CMV promoter, to form the recombinant expressing vector pcDNA3.1-GM-CSF. Results: The PCR amplification was identified and the sequence was analyzed, the results showed that hGM-CSF was properly inserted into the vector and the sequence was correct.

  12. Parallel evolution of tumour subclones mimics diversity between tumours.

    Science.gov (United States)

    Martinez, Pierre; Birkbak, Nicolai Juul; Gerlinger, Marco; McGranahan, Nicholas; Burrell, Rebecca A; Rowan, Andrew J; Joshi, Tejal; Fisher, Rosalie; Larkin, James; Szallasi, Zoltan; Swanton, Charles

    2013-08-01

    Intratumour heterogeneity (ITH) may foster tumour adaptation and compromise the efficacy of personalized medicine approaches. The scale of heterogeneity within a tumour (intratumour heterogeneity) relative to genetic differences between tumours (intertumour heterogeneity) is unknown. To address this, we obtained 48 biopsies from eight stage III and IV clear cell renal cell carcinomas (ccRCCs) and used DNA copy-number analyses to compare biopsies from the same tumour with 440 single tumour biopsies from the Cancer Genome Atlas (TCGA). Unsupervised hierarchical clustering of TCGA and multi-region ccRCC samples revealed segregation of samples from the same tumour into unrelated clusters; 25% of multi-region samples appeared more similar to unrelated samples than to any other sample originating from the same tumour. We found that the majority of recurrent DNA copy number driver aberrations in single biopsies were not present ubiquitously in late-stage ccRCCs and were likely to represent subclonal events acquired during tumour progression. Such heterogeneous subclonal genetic alterations within individual tumours may impair the identification of robust ccRCC molecular subtypes classified by distinct copy number alterations and clinical outcomes. The co-existence of distinct subclonal copy number events in different regions of individual tumours reflects the diversification of individual ccRCCs through multiple evolutionary routes and may contribute to tumour sampling bias and impact upon tumour progression and clinical outcome.

  13. Versatile shuttle vectors and genomic libraries for use with Schizosaccharomyces pombe.

    Science.gov (United States)

    Barbet, N; Muriel, W J; Carr, A M

    1992-05-01

    We have constructed a variety of pUC-based vectors designed for maintenance in Schizosaccharomyces pombe. These can be used for both gene bank construction and subcloning. Plasmids pUR18 and pUR19 are modifications of pUC vectors containing the Sc. pombe ars1 and ura4 sequences and retaining the lacZ XGal blue-white selection system for screening for DNA inserts. These vectors have been used to construct representative Sc. pombe and Saccharomyces cerevisiae genomic libraries. To assist in the creation of gene deletions, we have constructed another two plasmids. Combined with the technique of partially filling-in 5' overhangs created with restriction enzymes, these plasmids simplify the replacement of all or part of an open reading frame by a functional ura4 gene. Furthermore, such constructs can be excised with SfiI as a linear fragment for use in Sc. pombe transformations. When integrated into the Sc. pombe genome, the site of integration can be easily mapped by pulsed-field gel electrophoresis using the presence of a novel NotI site.

  14. Constructing retroviral vector carrying green fluorescent protein (GFP) and investigating the expression of GFP in primary rat myoblast

    Institute of Scientific and Technical Information of China (English)

    Shuling Rong; Yongxin Lu; Yuhua Liao; Xiaolin Wang; Xiaoqing Li; Jiahua Zhang; Yanli He

    2006-01-01

    Objective: To construct green fluorescent protein (GFP) retroviral vector (pLgXSN), and to investigate the expression of GFP in primary rat myoblast. Methods: GFP cDNA was subcloned into the plasmid pLgXSN, and the recombinant vector was transfected into packaging cell PT67. G418 was used to select positive colony. Myoblasts were infected by a high-titer viral supernatant. The recombinant retroviral plasmid vector was identified by restriction endonuclease analysis and DNA sequence analysis. Confocal microscopy and flow cytometry were used to detect the expression of GFP. Results: The GFP cDNA sequence was identical to that of GenBank. Recombinant retroviral plasmid vector pLgGFPSN was constructed successfully. The titer of the packaged recombinant retrovirus was 1 × 106 cfu/ml. Bright green fluorescence of the transfected cells was observed under confocal microscope 48 h after transfection. The transfection rate was 33%. The effective expression of GFP in myoblast infected by recombinant retrovirus lasted for 6 weeks. Conclusion: GFP gene could be effectively and stably expressed in myoblast, which suggests that GFP could act as a marker for studies on myoblast.

  15. Construction and identification of plasmid-based replicon vector system derived from Alphavirus%质粒型甲病毒复制子载体系统的构建及其功能鉴定

    Institute of Scientific and Technical Information of China (English)

    张凤娟; 唐丽; 尉研; 王焕琴; 杨蕴芝; 吴萌; 张鹏; 梁国栋; 朱武洋

    2014-01-01

    目的 构建质粒型甲病毒复制子载体系统,并对其功能进行鉴定.方法 在XJ-160病毒感染性cDNA克隆的基础上,将病毒非结构基因序列分为三个片段扩增,分步克隆至真核表达载体pVAX1 CMV启动子下游,并用多克隆位点序列替代病毒的结构基因构建XJ-160病毒质粒型复制子载体.将病毒核蛋白基因及包膜糖蛋白基因分别克隆至pVAX1 CMV启动子下游构建载体的两个辅助质粒.通过绿色荧光蛋白报告基因及海肾荧光素酶报告基因的表达检验该质粒型载体系统的功能特性.结果 成功构建了包括复制子载体和辅助质粒的质粒型甲病毒复制子载体系统,且该复制子载体系统可成功表达绿色荧光蛋白报告基因及海肾荧光素酶报告基因.结论 本研究构建了能够表达外源基因的质粒型甲病毒复制子载体系统,为目标基因表达、重组病毒颗粒制备等奠定了基础.%Objective To construct a plasmid-based replicon vector system derived from Alphavirus,and its functions were identified.Methods based on XJ-160 viral infectious cDNA clone,the virus non-structural gene sequence were devided into three fragments to amplify and then cloned into the eukaryotic expression vector pVAX1 CMV promoter downstream by step clone.A multiple cloning site sequence replacing the virus structural gene,plasmid-based replicon vector derived from XJ-160 virus was constructed.Virus nucleoprotein gene and envelope glycoprotein gene were respectively cloned into pVAX1 CMV promoter downstream,constructing two helper plasmid vectors.Then by the expression of green fluorescent protein reporter gene and renilla luciferase reporter gene we test the function of the plasmid-based replicon vector system.Results The plasmid-based replicon vector system derived from XJ-160 virus was successfully constructed,and green fluorescent protein reporter gene and renilla luciferase reporter gene were successfully expressed

  16. Stathmin-specific siRNA expression plasmid inhibits the expression of stathmin%Stathmin特异性siRNA表达质粒抑制stathmin的表达

    Institute of Scientific and Technical Information of China (English)

    向秋; 王建红; 熊伟明; 田晶; 蒋伟; 肖胜军; 何晓松; 雷迅

    2012-01-01

    目的:构建微管解聚蛋白stathmin基因的特异性siRNA质粒表达栽体,以便研究stathmin在鼻咽癌中的生物学作用.方法:合成stathmin特异性DNA片段,退火形成的双链DNA片段克隆于质粒表达载体pGenesil-1.3;载体经扩增后,进行酶切和测序鉴定;采用QIAGEN公司脂质体(effectene transfection reagent)将鉴定后的重组质粒转入鼻咽癌CNE2细胞:RT-PCR与Western blot检测stathmin基因表达.结果:酶切和测序分析表明,插入siRNA质粒表达栽体中的DNA片段,其碱基序列和插入方向正确.表达分析证实特异性siRNA质粒表达载体能有效抑制鼻咽癌CNE2细胞中stathmin的表达.结论:构建的siRNA质粒表达栽体对stathmin的表达有很好的抑制作用.%Objective Construct microtubule depolymerizing protein stathmin specific siRNA expression plasmid vector and study the biological function of stathmin in nasopharyngeal carcinoma. Methods Synthesize stathmin specific DNA fragments and anneal them to form double-strand DNA fragment. Subclone the DNA fragment into plasmid expression vector pGenesil-1.3. After the vector was amplified , it was identified by enzyme digestion and sequencing.Then, the identified recombinant plasmid vector was transferred into nasopharyngeal carcinoma CNE2 cell line by effectene transfection reagent of QIAGEN Company. Expression of stathmin was analyzed by RT-PCR and western blot. Results Enzyme digestion and sequencing data showed that siRNA expression plasmid vectors had been inserted stathmin-specific DNA fragment with right direction. Expression analysis confirmed that stathmin specific siRNA expression plasmid vector could effectively inhibit stathmin expression in nasopharyngeal CNE2 cells. Conclusion Recombinant stathmin specific siRNA expression plasmid vector can silence stathmin.

  17. Plasmid Rolling-Circle Replication.

    Science.gov (United States)

    Ruiz-Masó, J A; MachóN, C; Bordanaba-Ruiseco, L; Espinosa, M; Coll, M; Del Solar, G

    2015-02-01

    Plasmids are DNA entities that undergo controlled replication independent of the chromosomal DNA, a crucial step that guarantees the prevalence of the plasmid in its host. DNA replication has to cope with the incapacity of the DNA polymerases to start de novo DNA synthesis, and different replication mechanisms offer diverse solutions to this problem. Rolling-circle replication (RCR) is a mechanism adopted by certain plasmids, among other genetic elements, that represents one of the simplest initiation strategies, that is, the nicking by a replication initiator protein on one parental strand to generate the primer for leading-strand initiation and a single priming site for lagging-strand synthesis. All RCR plasmid genomes consist of a number of basic elements: leading strand initiation and control, lagging strand origin, phenotypic determinants, and mobilization, generally in that order of frequency. RCR has been mainly characterized in Gram-positive bacterial plasmids, although it has also been described in Gram-negative bacterial or archaeal plasmids. Here we aim to provide an overview of the RCR plasmids' lifestyle, with emphasis on their characteristic traits, promiscuity, stability, utility as vectors, etc. While RCR is one of the best-characterized plasmid replication mechanisms, there are still many questions left unanswered, which will be pointed out along the way in this review.

  18. Construction and expression of the eukaryotic expressed plasmid of MIC3 gene from Toxoplasma gondii in IBRS-2 cells

    Institute of Scientific and Technical Information of China (English)

    Tao JIANG; Donglin ZHANG; Hao NIE; Baoan YAO; Junlong ZHAO

    2008-01-01

    The sequence encoding MIC3 was obtained by amplification from genomic DNA of Toxoplasma gondii RH strain and cloned into the vector pMD18-T. The tar-get gene was subcloned into the eukaryotic vector pcDNA3.1 after the identification of pMD18-T-MIC3 by enzyme digesting, PCR amplification and sequencing. Then the target recombinant plasmids pcMIC3 were transfected into IBRS-2 cells, and the positive cells con-taining pcMIC3 plasmids were obtained under the selec-tion of G418. The expressed proteins from the positive cells were detected by SDS-PAGE, Western blot and ELISA. The results showed that the DNA sequence iden-tity was 99.9% between amplified MIC3 and that from GenBank. The molecular weight of the recombinant MIC3 protein with good immuno-activity was about 39.2 ku. These available data would lay the foundation for further studies on DNA vaccine against Toxoplasma gondii.

  19. A high-copy T7 Escherichia coli expression vector for the production of recombinant proteins with a minimal N-terminal His-tagged fusion peptide

    Directory of Open Access Journals (Sweden)

    Ramos C.R.R.

    2004-01-01

    Full Text Available We report here the construction of a vector derived from pET3-His and pRSET plasmids for the expression and purification of recombinant proteins in Escherichia coli based on T7 phage RNA polymerase. The resulting pAE plasmid combined the advantages of both vectors: small size (pRSET, expression of a short 6XHis tag at N-terminus (pET3-His and a high copy number of plasmid (pRSET. The small size of the vector (2.8 kb and the high copy number/cell (200-250 copies facilitate the subcloning and sequencing procedures when compared to the pET system (pET3-His, 4.6 kb and 40-50 copies and also result in high level expression of recombinant proteins (20 mg purified protein/liter of culture. In addition, the vector pAE enables the expression of a fusion protein with a minimal amino-terminal hexa-histidine affinity tag (a tag of 9 amino acids using XhoI restriction enzyme for the 5'cloning site as in the case of pET3-His plasmid and in contrast to proteins expressed by pRSET plasmids (a tag of 36 amino acids using BamHI restriction enzyme for the 5'cloning site. Thus, although proteins expressed by pRSET plasmids also have a hexa-histidine tag, the fusion peptide is much longer and may represent a problem for some recombinant proteins.

  20. Construction of a shuttle vector consisting of the Escherichia coli plasmid pACYC177 inserted into the Streptomyces cattleya phage TG1.

    Science.gov (United States)

    Foor, F; Morin, N

    1990-09-28

    The Escherichia coli plasmid, pACYC177, was inserted into the single PstI site of a deletion derivative of the Streptomyces cattleya phage, TG1. The hybrid molecule can be propagated as a phage in S. cattleya and as a plasmid in E. coli and is readily transferred between the two species by transfection and transformation. The kanamycin-resistance-encoding gene derived from pACYC177 is not expressed in lysogens of the hybrid phage. Analysis of deletion mutants of the hybrid phage indicated that at least 7.5 kb of phage DNA is dispensable. Some of the deletion mutants fail to lysogenize S. cattleya (Lyg- phenotype). The locations of these deletions are consistent with the location of the phage att site as previously established by Southern hybridization analysis. The thiostrepton-resistance-encoding gene derived from Streptomyces azureus was inserted into Lyg+ and Lyg- deletion derivatives and is expressed in S. cattleya.

  1. Expression and humoral immune response to Hepatitis C virus using a plasmid DNA construct

    Directory of Open Access Journals (Sweden)

    Ray S

    2003-01-01

    Full Text Available PURPOSE: The objective of this study was to clone a c-DNA fragment of hepatitis C virus in a eukaryotic expression vector and to measure the efficacy of humoral immune responses in mice inoculated with this recombinant plasmid. This study was an attempt to lay a foundation for HCV nucleic acid vaccine development in the future. METHODS: A c-DNA fragment of BK146, a clone of HCV type 1b, was sub-cloned into an eukaryotic expression vector pMT3. HepG2 and COS cells were transfected with this construct, named pMT3-BK146. The expression of HCV mRNA and proteins was studied by reverse transcribed polymerase chain reaction, radio Immunoprecipitation (RIPA and immunofluorescence (IFA. The DNA of this construct was injected into the footpad of BALB/c mice and antibody response was tested by enzyme immunoassay and indirect immunofluorescence. RESULTS: COS and HepG2 cells transiently transfected with the recombinant plasmid pMT3-BK146 showed the expression of HCV proteins by RT-PCR, RIPA and immunofluorescence. This DNA clone when injected into Balb/c mice was able to generate specific antibody response to hepatitis C virus by ELISA and IFA. CONCLUSIONS: A c-DNA fragment of HCV cloned in an eukaryotic expression vector was able to express core protein. This DNA clone was also able to elicit antibody response in mice. This can be an initial step towards the development of a potential DNA vaccine for hepatitis C virus infection.

  2. Why close a bacterial genome? The plasmid of Alteromonas macleodii HOT1A3 is a vector for inter-specific transfer of a flexible genomic island

    Directory of Open Access Journals (Sweden)

    Eduard eFadeev

    2016-03-01

    Full Text Available Genome sequencing is rapidly becoming a staple technique in environmental and clinical microbiology, yet computational challenges still remain, leading to many draft genomes which are typically fragmented into many contigs. We sequenced and completely assembled the genome of a marine heterotrophic bacterium, Alteromonas macleodii HOT1A3, and compared its full genome to several draft genomes obtained using different reference-based and de-novo methods. In general, the de-novo assemblies clearly outperformed the reference-based or hybrid ones, covering>99% of the genes and representing essentially all of the gene functions. However, only the fully closed genome (~4.5Mbp allowed us to identify the presence of a large, 148 kbp plasmid, pAM1A3. While HOT1A3 belongs to Alteromonas macleodii, typically found in surface waters (surface ecotype, this plasmid consists of an almost complete flexible genomic island, containing many genes involved in metal resistance previously identified in the genomes of Alteromonas mediterranea (deep ecotype. Indeed, similar to A. mediterranea, A. macleodii HOT1A3 grows at concentrations of zinc, mercury and copper that are inhibitory for other A. macleodii strains. The presence of a plasmid encoding almost an entire flexible genomic island suggests that wholesale genomic exchange between heterotrophic marine bacteria belonging to related but ecologically different populations is not uncommon.

  3. Construction and expressing activity of microRNA-125a plasmid expression vectors%MicroRNA-125a质粒表达载体的构建及其表达活性

    Institute of Scientific and Technical Information of China (English)

    张涛; 李东; 李华; 高辉

    2012-01-01

    背景:pSuper是一种非编码RNA的真核表达载体,可在细胞质内表达miRNA前体,经过细胞自身的Dicer酶切后形成miRNA成熟体,其过程能够完全模仿细胞内源性miRNA形成过程.因此,利用pSuper表达载体构建一种肝癌抑制性miRNA:miR-125a的表达载体,为下一步研究其抗癌机制奠定了基础.目的:构建miR-125a的真核表达质粒pSuper-miR-125a,并验证其表达miR-125a的效率和特异性.方法:PCR扩增miR-125a前体(Pre-miR-125a)的目的片段,将其T-A克隆入pMD18-T过渡质粒载体,限制性内切酶切下目的片段,连接入pSuper质粒表达载体得到pSuper-miR-125a重组质粒;将重组质粒转染Hela细胞,miRNA定量PCR检测重组质粒表达miR-125a的效率和特异性.结果与结论:成功将miR-125a前体(Pre-miR-125a)片段克隆入真核表达质粒pSuper H1启动子下游,获得pSuper-miR-125a重组表达质粒,转染该质粒进入Hela细胞后能够在细胞内高效和特异的表达miR-125a,进一步证明 pSuper真核表达质粒适合miRNA的表达研究.%BACKGROUND: pSuper is a kind of eukaryotic expression vectors of non-coding RNA. It expresses the precursor of miRNA in plasma, and then the precursor is cut by Dicer enzyme to produce mature miRNA. This process completely imitates the formation process of endogenous miRNA. In this study, pSuper is utilized to construct an expression vector of miR-125a, which could inhibit liver cancer. It provides basis for the further investigation on antitumor mechanism. OBJECTIVE: To construct the eukaryotic plasmid pSuper-miR-125a expressing miR-125a and to confirm the expressing efficiency and specificity of miR-125a in the plasmid. METHODS: Target fragments in the precursor of miR-125a (Pre-miR-125a) were amplified by PCR. The fragments were cloned into pMD18-T transition plasmid using T-A cloning method. The target fragments were cut by restriction enzyme and then connected to pSuper plasmid to construct recombinant plasmid p

  4. Prevalence and characteristics of Escherichia coli sequence type 131 and its H30 and H30Rx subclones: a multicenter study from Korea.

    Science.gov (United States)

    Kim, Soo-Young; Park, Yeon-Joon; Johnson, James R; Yu, Jin Kyung; Kim, Yong-Kyun; Kim, Yeong Sic

    2016-02-01

    We investigated the prevalence and characteristics of Escherichia coli sequence type 131 (ST131) and its subclones among 268 E. coli isolates. The isolates were collected from 21 Korean hospitals without use of selection criteria and were screened for ST131 status by PCR. ST131 isolates were characterized for extended-spectrum β-lactamase variants, fluoroquinolone resistance genes, plasmid addiction systems, and replicon types. The collection's 57 identified ST131 isolates (21% of 268) were distributed disproportionately by clonal subset, as follows: 21 (37%) H30Rx, 27 (47%) H30 non-Rx, and 8 (14%) non-H30. Most (93%) ST131 isolates were ciprofloxacin resistant, and all H30 isolates had the same 5 nonsynonymous mutations in gyrA, parC, and parE. Twenty (95%) of H30Rx isolates harbored CTX-M-15, whereas only 14 (52%) of H30 non-Rx isolates harbored CTX-M-14 or CTX-M-27. Most (97%) ST131 isolates harbored IncF plasmids, but vagCD was confined exclusively to H30Rx. Our findings suggest that the distinctive characteristics of H30Rx isolates could have contributed to this subclone's recent epidemiologic success.

  5. Historical Events That Spawned the Field of Plasmid Biology.

    Science.gov (United States)

    Kado, Clarence I

    2014-10-01

    This chapter revisits the historical development and outcome of studies focused on the transmissible, extrachromosomal genetic elements called plasmids. Early work on plasmids involved structural and genetic mapping of these molecules, followed by the development of an understanding of how plasmids replicate and segregate during cell division. The intriguing property of plasmid transmission between bacteria and between bacteria and higher cells has received considerable attention. The utilitarian aspects of plasmids are described, including examples of various plasmid vector systems. This chapter also discusses the functional attributes of plasmids needed for their persistence and survival in nature and in man-made environments. The term plasmid biology was first conceived at the Fallen Leaf Lake Conference on Promiscuous Plasmids, 1990, Lake Tahoe, California. The International Society for Plasmid Biology was established in 2004 (www.ISPB.org).

  6. Cloning of Two Bacteriocin Genes from a Lactococcal Bacteriocin Plasmid

    NARCIS (Netherlands)

    Belkum, Marco J. van; Hayema, Bert Jan; Geis, Arnold; Kok, Jan; Venema, Gerard

    1989-01-01

    Lactococcus lactis subsp. cremoris 9B4 plasmid p9B4-6 (60 kilobases [kb]), which specifies bacteriocin production and immunity, was analyzed with restriction endonucleases, and fragments of this plasmid were cloned into shuttle vectors based on the broad-host-range plasmid pWVO1. Two regions on p9B4

  7. Combination of the somatic cell nuclear transfer method and RNAi technology for the production of a prion gene-knockdown calf using plasmid vectors harboring the U6 or tRNA promoter.

    Science.gov (United States)

    Wongsrikeao, Pimprapar; Sutou, Shizuyo; Kunishi, Miho; Dong, Ya Juan; Bai, Xuejin; Otoi, Takeshige

    2011-01-01

    By combining RNAi technology with SCNT method, we attempted to produce transgenic calves with knocked down bPRNP for technological assessments. The respective utilities of type II (tRNA) and type III (hU6) Pol III promoters in mediating plasmid vector-based RNAi for the production of a bPRNP-knockdown calf were compared. Plasmid harboring DNA for siRNA expression was introduced stably into the genome of primary cultured bovine cells. By inserting the transgenic cell into an enucleated bovine egg, SCNT embryos were produced. The ability for SCNT embryos to develop to blastocysts was higher in hU6 based vector groups (44-53%) than in a tRNA group (32%). In all, 30 hU6-embryos and 12 tRNA-embryos were transferred to 11 recipients. Only tRNA-embryos were able to impregnate recipients (6 out of 11 transfers), resulting in four aborted fetuses, one stillbirth, and one live-born calf. The expression of EGFP, a marker, was detected in all six. The bPRNP transcript levels in the nervous tissues (brain, cerebellum, spinal bulb, and spinal cord) from the calf, which was killed 20 days after birth, were reduced to 35% of those of the control calf on average, as determined by qRT-PCR. The PrPC levels, as estimated by western blot were reduced to 86% on average in the nervous tissues. These findings suggest that SCNT technology remains immature, that the tRNA promoter is useful, and that RNAi can significantly reduce PRNP mRNA levels, but insufficient reduction of PrPC levels exists in cattle under these conditions.

  8. A Plasmodium vivax plasmid DNA- and adenovirus-vectored malaria vaccine encoding blood stage antigens AMA1 and MSP142 in a prime/boost heterologous immunization regimen partially protects Aotus monkeys against blood stage challenge.

    Science.gov (United States)

    Obaldia, Nicanor; Stockelman, Michael G; Otero, William; Cockrill, Jennifer A; Ganeshan, Harini; Abot, Esteban N; Zhang, Jianfeng; Limbach, Keith; Charoenvit, Yupin; Doolan, Denise L; Tang, De-Chu C; Richie, Thomas L

    2017-02-08

    Malaria is caused by parasites of the genus Plasmodium that are transmitted to humans by the bites of Anopheles mosquitoes. After the elimination of P. falciparum it is predicted that Plasmodium vivax will remain an important cause of morbidity and mortality outside of Africa, stressing the importance of developing a vaccine against malaria. In this study we assess the immunogenicity and protective efficacy of two P. vivax antigens, AMA1 and MSP142 in a recombinant DNA plasmid prime/adenoviral vector (Ad) boost regimen in Aotus monkeys. Groups of 4 to 5 monkeys were immunized with DNA alone, Ad alone, prime/boost regimens of each antigen, prime/boost with both antigens, and empty vector controls, and then subjected to blood stage challenge. The heterologous immunization regimen with the antigen pair was more protective than either antigen alone or both antigens delivered with a single vaccine platform, based on their ability to induced the longest pre-patent period and time to peak parasitemia; the lowest peak and mean parasitemia; the smallest area under the parasitemia curve and the highest self-cured rate. Overall, pre-challenge MSP1 antibody titers strongly correlated with decreased parasite burden. Nevertheless, a significant proportion of immunized animals developed anemia. In conclusion, P. vivax plasmid DNA/Ad5 vaccine encoding blood stage parasite antigens AMA1 and MSP142 in a heterologous prime/boost immunization regimen, provided significant protection against blood-stage challenge in Aotus monkeys, indicating the suitability of these antigens and regimen for further development.

  9. BMP2基因重组慢病毒载体质粒的构建及鉴定%Construction and identification of lentiviral vector plasmid recombined by BMP2 gene

    Institute of Scientific and Technical Information of China (English)

    林昭伟; 李奇; 林荔军; 刘云龙; 帅明; 谢小波

    2013-01-01

    目的 构建重组慢病毒载体质粒pLV.EX2d.P/neo-EF1A>BMP-2/T2A/EGFP并进行鉴定.方法 从Genbank获得BMP2基因序列,结合载体上的酶切位点需要,设计上下游引物,通过PCR方法扩增目的 基因片段,利用Gateway技术 BP反应构建pDown-BMP2-T2A-EGFP,并进行阳性克隆测序,应用LR反应把pDown-BMP2-T2A-EGFP重组入慢病毒目的 载体质粒pLV.Des2d.P/neo,进行阳性克隆测序.结果 获得长度为1 191 bp的BMP2目的 基因片段,质粒pLV.EX2d.P/neo-EF1A>BMP2/T2A/EGFP经双酶切后凝胶电泳鉴定正确,测序结果与Genbank报道序列一致.结论 成功构建重组慢病毒载体质粒pLV.EX2d.P/neo-EF1A>BMP2/T2A/EGFP.%Objective To construct and identify a lentiviral vector carrying human BMP2 gene. Methods BMP2 gene was obtained from Genbank directly. Primers were designed according to the BMP2 gene sequences reported in Genbank and the restriction sites of the vector. The BMP2 gene was amplified by polymerase chain reaction(PCR). pDown-BMP2-T2A-EGFP was constructed with the BP reaction of Gateway technology, then positive cloning was sequenced. pDown-BMP2-T2A-EGFP was inserted into destination vector plasmid pLV. Des2d. P/neo with the LR reaction of Gateway technology. The positive cloning was sequenced. Results 1 191 bp BMP2 gene fragment was obtained,pLV. EX2d. P/neo-EFlA>BMP2/T2A/EGFP was identified with double digestion and sequencing, the result was completely in accordance with the BMP2 gene sequences reported in Genbank. Conclusion Recombination lentiviral vector plasmid pLV. EX2d. P/neo-EFlA>BMP2/T2A/EGFP is constructed successfully.

  10. Construction of a eukaryotic expression plasmid pcDNA3.1-HuR-FLAG and its transient expression in NIH3T3 cells

    Directory of Open Access Journals (Sweden)

    Tao LI

    2011-04-01

    Full Text Available Objective To construct a eukaryotic expression vector for HuR and analyze its expression and biological function in NIH3T3 cells.Methods The total RNA was extracted from NIH3T3 cells and reverse transcribed to cDNAs.The coding region sequence of mouse HuR was then amplified by PCR and subcloned into the pcDNA3.1-FLAG plasmid.The recombinant plasmid pcDNA3.1-HuR-FLAG was verified by PCR and restriction endonuclease analysis,confirmed by DNA sequence analysis,and then transiently transfected into NIH3T3 cells with Lipofectamine LTX.The expression of HuR protein was determined by Western blotting,and the mRNA level of HuR and DUSP1 were analyzed by using real-time PCR.Result The recombinant plasmid pcDNA3.1-HuR-FLAG was correctly constructed.Twenty-four hours after transfection of the recombinant plasmid into NIH3T3 cells,the fusion protein was found to have highly expressed in the cells as revealed by Western blotting.Real-time PCR results detected that the over-expression of HuR could up-regulate the expression of DUSP1.Conclusion The eukaryotic expression vector for HuR-FLAG fusion protein has been successfully constructed and transiently expressed in NIH3T3 cells.It can be used in further analysis of the posttranscriptional regulation of DUSP1 by HuR in cancer cells.

  11. Non-cell-autonomous driving of tumour growth supports sub-clonal heterogeneity.

    Science.gov (United States)

    Marusyk, Andriy; Tabassum, Doris P; Altrock, Philipp M; Almendro, Vanessa; Michor, Franziska; Polyak, Kornelia

    2014-10-02

    Cancers arise through a process of somatic evolution that can result in substantial sub-clonal heterogeneity within tumours. The mechanisms responsible for the coexistence of distinct sub-clones and the biological consequences of this coexistence remain poorly understood. Here we used a mouse xenograft model to investigate the impact of sub-clonal heterogeneity on tumour phenotypes and the competitive expansion of individual clones. We found that tumour growth can be driven by a minor cell subpopulation, which enhances the proliferation of all cells within a tumour by overcoming environmental constraints and yet can be outcompeted by faster proliferating competitors, resulting in tumour collapse. We developed a mathematical modelling framework to identify the rules underlying the generation of intra-tumour clonal heterogeneity. We found that non-cell-autonomous driving of tumour growth, together with clonal interference, stabilizes sub-clonal heterogeneity, thereby enabling inter-clonal interactions that can lead to new phenotypic traits.

  12. Plasmid DNA entry into postmitotic nuclei of primary rat myotubes.

    OpenAIRE

    Dowty, M E; Williams, P.; G. Zhang; Hagstrom, J E; Wolff, J A

    1995-01-01

    These studies were initiated to elucidate the mechanism of DNA nuclear transport in mammalian cells. Biotin- or gold-labeled plasmid and plasmid DNA expression vectors for Escherichia coli beta-galactosidase or firefly luciferase were microinjected into the cytoplasm of primary rat myotubes in culture. Plasmid DNA was expressed in up to 70% of the injected myotubes, which indicates that it entered intact, postmitotic nuclei. The nuclear transport of plasmid DNA occurred through the nuclear po...

  13. Construction of eukaryotic expression vector with brain-derived neurotrophic factor receptor trkB gene

    Institute of Scientific and Technical Information of China (English)

    HUANG Tao; JIANG Xiao-dan; XU Zhong; YUAN Jun; DING Lian-shu; ZOU Yu-xi; XU Ru-xiang

    2005-01-01

    Objective: To construct an eukaryotic expression vector carrying rat brain-derived neurotrophic factor receptor trkB gene. Methods: Using the total RNA isolated from rat brain as template, the trkB gene was amplified by reverse-transcription-polymerase chain reaction (RT-PCR) with a pair of specific primers which contained the restrictive sites of EcoR I and BamH I. The amplified fragment of trkB gene was digested with EcoR I and BamH I, and then subcloned into cloning vector pMD18-T and expression vector pEGFP-C2 respectively. The recombinant plasmids were identified by restriction endonuclease enzyme analysis and PCR. Results: The amplified DNA fragment was about 1461 bp in length. Enzyme digestion and PCR analysis showed that the gene of trkB had been successfully cloned into vector pMD18-T and pEGFP-C2. Conclusions: The trkB gene of rat has been amplified and cloned into the eukaryotic expression vector pEGFP-C2.

  14. Stress responses and replication of plasmids in bacterial cells

    Directory of Open Access Journals (Sweden)

    Wegrzyn Alicja

    2002-05-01

    Full Text Available Abstract Plasmids, DNA (or rarely RNA molecules which replicate in cells autonomously (independently of chromosomes as non-essential genetic elements, play important roles for microbes grown under specific environmental conditions as well as in scientific laboratories and in biotechnology. For example, bacterial plasmids are excellent models in studies on regulation of DNA replication, and their derivatives are the most commonly used vectors in genetic engineering. Detailed mechanisms of replication initiation, which is the crucial process for efficient maintenance of plasmids in cells, have been elucidated for several plasmids. However, to understand plasmid biology, it is necessary to understand regulation of plasmid DNA replication in response to different environmental conditions in which host cells exist. Knowledge of such regulatory processes is also very important for those who use plasmids as expression vectors to produce large amounts of recombinant proteins. Variable conditions in large-scale fermentations must influence replication of plasmid DNA in cells, thus affecting the efficiency of recombinant gene expression significantly. Contrary to extensively investigated biochemistry of plasmid replication, molecular mechanisms of regulation of plasmid DNA replication in response to various environmental stress conditions are relatively poorly understood. There are, however, recently published studies that add significant data to our knowledge on relations between cellular stress responses and control of plasmid DNA replication. In this review we focus on plasmids derived from bacteriophage λ that are among the best investigated replicons. Nevertheless, recent results of studies on other plasmids are also discussed shortly.

  15. Construction of eukaryotic expression vector encoding ATP synthase lipid-binding protein-like protein gene of Sj and its expression in HeLa cells

    Institute of Scientific and Technical Information of China (English)

    Ouyang Danming; Hu Yongxuan; Li Mulan; Zeng Xiaojun; He Zhixiong; Yuan Caijia

    2008-01-01

    Objective: To clone and construct the recombinant plasmid containing ATP synthase lipid-binding protein-like protein gene of Schistosoma japonicum,(SjAslp) and transfer it into mammalian cells to express the objective protein. Methods: By polymerase chain reaction (PCR) technique, SjAslp was amplified from the constructed recombinant plasmid pBCSK+/SjAslp, and inserted into cloning vector pUCm-T. Then, SjAslp was subcloned into an eukaryotic expression vector pcDNA3.1(+). After identifying it by PCR, restrictive enzymes digestion and DNA sequencing, the recombinant plasmid was transfected into HeLa cells using electroporation, and the expression of the recombinant protein was analyzed by immunocytochemical assay. Resnlts: The specific gene fragment of 558 bp was successfully amplified. The DNA vaccine of SjAslp was successfully constructed. Immunocytochemical assay showed that SjAslp was expressed in the cytoplasm of HeLa cells. Conclusion: SjAslp gene can be expressed in eukaryotic system, which lays the foundation for development of the SjAslp DNA vaccine against schitosomiasis.

  16. Construction and expression of SET gene and siRNA recombinant adenovirus vectors

    Institute of Scientific and Technical Information of China (English)

    Xu Bo-qun; Lu Pin-hong; Li Ying; Xue Kai; Li Mei; Ma Xiang; Diao Fei-yan; Cui Yu-gui; Liu Jia-yin

    2010-01-01

    Objective: To construct SET gene recombinant adenovirus vector and SET gene small interfering RNA (SiRNA) recombinant adenovirus vector for over-expression or knock-down of SET levels.Methods: The cDNA sequence of SET was cloned by reverse transcriptive polymerase chain reaction (RT-PCR) and the SET gene fragment was subcloned into adenovirus shuttle plasmid pAdTrack-CMV to construct the shuttle plasmid pAdTrack-SET. The shuttle plasmid pAdtrack-SET was transformed into BJ5183 cells with the adenoviral backbone pAdEasy-1 to obtain the homologous recombinant Ad-CMV-SET and the recombinant Ad-CMV-SET was packaged and amplified in the AD293 cells. The expression of SET in AD293 cells was detected by Western blot. In addition, we constructed SET gene SiRNA recombinant adenovirus vector (Ad-H1-SiRNA/SET) and its efficacy of knockdown of SET protein was detected in infected GC-2spd(ts) cells by Western blot. Results: The recombinant adenovirus vectors, both SET gene recombinant adenovirus vector Ad-CMV-SET and SET gene SiRNA recombinant adenovirus vector Ad-H1-SiRNA/SET, were proven to be constructed successfully by the evidence of endonulease digestion and sequencing. AD293 cells infected with either recombinant adenovirus vector of Ad-CMV-SET or Ad-H1-SiRNA/SET were observed to express GFP. The expression of SET protein was up-regulated significantly in AD293 cells infected with SET gene recombinant adenovirus vector. On the contrast, SET protein was significantly down-regulated in the GC-2spd(ts) cells infected with Ad-H1-SiRNA/SET (P<0.05) and the knockdown efficiency was approximately 50%-70%. Conclusion: The recombinant adenovirus vector Ad-CMV-SET and Ad-H1-SiRNA/SET were successfully constructed and effectively expressed in germ cells and somatic cells. It provides an experimental tool for further study of SET gene in the physiological and pathophysiological mechanism of reproduction-related diseases.

  17. Establishment of hepatitis E virus infection-permissive and -non-permissive human hepatoma PLC/PRF/5 subclones.

    Science.gov (United States)

    Shiota, Tomoyuki; Li, Tian-Cheng; Yoshizaki, Sayaka; Kato, Takanobu; Wakita, Takaji; Ishii, Koji

    2015-02-01

    PLC/PRF/5 cells show limited permissiveness, meaning that almost all subclones are permissive; however, some subclones do not exhibit permissiveness for hepatitis E virus (HEV) infection. In this study, the single-cell cloning of PLC/PRF/5 was performed and heterogeneous subclones characterized. Notably, the efficiency of intracellular virus replication did not correlate with the permissiveness for HEV infection. However, as well as binding permissive subclones, virus-like particles bound non-permissive subclones on various levels, suggesting that these subclones have some deficiencies in the attachment and entry steps of infection. Our data would be useful for investigating the HEV life cycle. © 2014 The Societies and Wiley Publishing Asia Pty Ltd.

  18. PEG-b-PPS-b-PEI micelles and PEG-b-PPS/PEG-b-PPS-b-PEI mixed micelles as non-viral vectors for plasmid DNA: tumor immunotoxicity in B16F10 melanoma.

    Science.gov (United States)

    Velluto, Diana; Thomas, Susan N; Simeoni, Eleonora; Swartz, Melody A; Hubbell, Jeffrey A

    2011-12-01

    Cationic micelles formed from poly(ethylene glycol)-bl-poly(propylene sulfide)-bl-poly(ethylene imine) (PEG-b-PPS-b-PEI) and from mixtures of poly(ethylene glycol)-bl-poly(propylene sulfide) (PEG-b-PPS) with PEG-b-PPS-b-PEI were explored as non-viral vectors for plasmid DNA (pDNA) transfection in a tumor immunotoxicity model. Complexes with pDNA were found to be templated exclusively by the size of the pDNA-free micelles and ranged from 240 nm (for PEG-b-PPS-b-PEI) to 30 nm (for mixed micelles of PEG-b-PPS/PEG-b-PPS-b-PEI). Both formulations transfected melanoma cells well in vitro. As a model with a functional read-out of tumor cell death, one with likely only small bystander effects, tumors were transfected with an antigen transgene, using an antigen to which the recipient animals had been previously vaccinated with a Th1-biasing adjuvant. Reduction in tumor growth, increase in intratumoral infiltration of cytotoxic T lymphocytes and accumulation of Th1-biasing cytokines indicated that both micelle formulations transfected efficiently compared with naked pDNA and with low cytotoxicity.

  19. Plasmid-encoded asp operon confers a proton motive metabolic cycle catalyzed by an aspartate-alanine exchange reaction.

    Science.gov (United States)

    Abe, Keietsu; Ohnishi, Fumito; Yagi, Kyoko; Nakajima, Tasuku; Higuchi, Takeshi; Sano, Motoaki; Machida, Masayuki; Sarker, Rafiquel I; Maloney, Peter C

    2002-06-01

    Tetragenococcus halophila D10 catalyzes the decarboxylation of L-aspartate with nearly stoichiometric release of L-alanine and CO(2). This trait is encoded on a 25-kb plasmid, pD1. We found in this plasmid a putative asp operon consisting of two genes, which we designated aspD and aspT, encoding an L-aspartate-beta-decarboxylase (AspD) and an aspartate-alanine antiporter (AspT), respectively, and determined the nucleotide sequences. The sequence analysis revealed that the genes of the asp operon in pD1 were in the following order: promoter --> aspD --> aspT. The deduced amino acid sequence of AspD showed similarity to the sequences of two known L-aspartate-beta-decarboxylases from Pseudomonas dacunhae and Alcaligenes faecalis. Hydropathy analyses suggested that the aspT gene product encodes a hydrophobic protein with multiple membrane-spanning regions. The operon was subcloned into the Escherichia coli expression vector pTrc99A, and the two genes were cotranscribed in the resulting plasmid, pTrcAsp. Expression of the asp operon in E. coli coincided with appearance of the capacity to catalyze the decarboxylation of aspartate to alanine. Histidine-tagged AspD (AspDHis) was also expressed in E. coli and purified from cell extracts. The purified AspDHis clearly exhibited activity of L-aspartate-beta-decarboxylase. Recombinant AspT was solubilized from E. coli membranes and reconstituted in proteoliposomes. The reconstituted AspT catalyzed self-exchange of aspartate and electrogenic heterologous exchange of aspartate with alanine. Thus, the asp operon confers a proton motive metabolic cycle consisting of the electrogenic aspartate-alanine antiporter and the aspartate decarboxylase, which keeps intracellular levels of alanine, the countersubstrate for aspartate, high.

  20. Constructing recombinant replication-defective adenoviral vectors that express glucose transporter-1 through in vitro ligation

    Institute of Scientific and Technical Information of China (English)

    Fangcheng Li; Junliang Li; Ranyi Liu; Xinke Xu; Kaichang Yuan; Zhonghua Wu

    2008-01-01

    BACKGROUND: We constructed a homologous recombination bacterial method based on the pAdEasy system, a widely used system, for generating recombinant adenoviral vectors that express glucose transporter-1 (GLUT1) in rats.OBJECTIVE: This study was designed to investigate the feasibility of generating recombinant replication-defective adenoviral vectors that express GLUT1 in rats by in vitro ligation based on the Adeno-XTM system. DESIGN: An in vitro cell-based experiment. SETTING: This study was performed at the Linbaixin Medical Research Center of the Second Hospital Affiliated to Sun Yat-sen University and Central Laboratory for Prevention and Treatment of Tumor, Sun Yat-sen University between January and August 2004. MATERIALS: Male, adult, Sprague Dawley rats were used to extract total RNA from brain tissue. E. coli DH5?and human embryonic kidney 293 cells (HEK293 cells) used in the present study were cryo-preserved by the Second Hospital Affiliated to Sun Yat-sen University. Rabbit anti-rat GLUT1 polyclonal antibody (Chemicon, U.S.A.) and primers (Shanghai Boya Bioengineering Co., Ltd) were also used. METHODS: E1/E3-deleted replication-defective adenoviral vectors were used. Using in vitro ligation, the target gene was first sub-cloned into a shuttle vector plasmid to obtain the fragment containing target gene expression cassettes by enzyme digestion. Subsequently, the fragment was co-transformed with linearized adenoviral backbone vector into the E. coli strain. The recombinant adenoviral plasmid was transfected into HEK293 cells to assembly recombinant adenoviral vectors with replication capabilities. The procedure was repeated several times for recombinant adenoviral vectors amplification. MAIN OUTCOME MEASURES: Efficiency of recombinant adenoviral vectors to express the target gene was measured by gene and protein expression through polymerase chain reaction and Western Blot assays, respectively.RESULTS: Results demonstrated that recombinant adenoviral

  1. Ultra-deep sequencing reveals the subclonal structure and genomic evolution of oral squamous cell carcinoma

    DEFF Research Database (Denmark)

    Tabatabaeifar, Siavosh; Thomassen, Mads; Larsen, Martin Jakob

    Background: Oral squamous cell carcinoma (OSCC), a subgroup of head and neck squamous cell carcinoma (HNSCC), is primarily caused by alcohol consumption and tobacco use. Recent DNA sequencing studies suggests that HNSCC are very heterogeneous between patients; however the intra-patient subclonal...... structure remains unexplored due to lack of sampling multiple tumor biopsies from each patient. Materials and methods: To examine the clonal structure and describe the genomic cancer evolution we applied whole-exome sequencing combined with targeted ultra-deep targeted sequencing on biopsies from 5stage IV...... complex subclonal architectures comprising distinct subclones only found in geographically distinct regions of the tumors. The metastatic potential of the tumor is acquired early in the tumor evolution, as indicated by the lymph node sharing the majority of the mutations with the tumor biopsies, while...

  2. Comparative Immunization in BALB/c Mice with Recombinant Replication-Defective Adenovirus Vector and DNA Plasmid Expressing a SARS-CoV Nucleocapsid Protein Gene

    Institute of Scientific and Technical Information of China (English)

    Chunling Ma; Kun Yao; Feng Zhou; Minsheng Zhu

    2006-01-01

    In order to investigate immunogenicity in the induction of humoral and cellular immune responses, severe acute respiratory syndrome associated coronavirus (SARS-CoV)-N gene recombinant replication-defective adenoviral vector, rAd-N, was generated and immunized BALB/c mice in a pcDNA3.1-N prime-rAd-N boost regimen. After humoral and cellular immune response detection, different levels of SARS-CoV N protein specific antibodies and interferon-γ (IFN-γ) secretion are shown compared to controls. The humoral immune response was induced more effectively by the DNA priming and recombinant adenovirus boosting regimen. There is a significant difference between heterogeneous and homologous vaccinations. The heterogeneous combinations were all higher than those of the homologous combinations in the induction of anti-N antibody response. Among the three heterogeneous combinations, pcDNA3.1-N/pcDNA3.1-N/pcDNA3.1-N/rAd-N induced the strongest antibody response. In the induction of IFN-γ production, the homologous combination of rAd-N/rAd-N/rAd-N/rAd- N was significantly stronger than that of pcDNA3.1-N/pcDNA3. 1-N/pcDNA3.1-N/pcDNA3.1-N, but was relatively weaker than the heterogeneous combination of pcDAN3.1-N/pcDAN3.1-N/pcDAN3.1-N/rAd-N. This combination was a most efficient immunization regimen in induction of SARS-CoV-N-specific (IFN-γ) secretion just as the antibody response. These results suggest that DNA immunization followed by recombinant adenovirus boosting could be used as a potential SARS-CoV vaccine.

  3. Conservation of plasmids among Escherichia coli K1 isolates of diverse origins.

    Science.gov (United States)

    Mercer, A A; Morelli, G; Heuzenroeder, M; Kamke, M; Achtman, M

    1984-12-01

    Escherichia coli K1 isolates of various O types were previously assigned to different clonal groups. Members of the two clones defined by membrane pattern 9 (MP9) and serotypes O18:K1 and O1:K1 had been found to be very similar to each other. The plasmid contents of these bacteria confirmed this conclusion. Both groups carried a self-transmissible plasmid of the FI incompatibility group that coded for colicin production and a major outer membrane protein called the plasmid-coded protein (PCP). The size of this plasmid varied from 76 to 96 megadaltons, but restriction endonuclease digestion and DNA heteroduplex analysis revealed that these plasmids were highly related. O18:K1 bacteria of MP6 had previously been determined to represent a subclone, related to but different from O18:K1 MP9 bacteria. These MP6 bacteria carried a different, smaller IncFI plasmid which did not code for colicin production or the PCP protein. This smaller plasmid was primarily related to the larger plasmid within the regions of DNA encoding incompatibility, replication, and conjugation. O1:K1 bacteria of MP5 contained other unrelated plasmids in agreement with the previous conclusion that they are unrelated to O1:K1 bacteria of MP9. The bacteria examined had been isolated from two continents over a time span of 38 years, and the results attest to conservative inheritance of plasmids within bacteria of common descent.

  4. Conservation of plasmids among Escherichia coli K1 isolates of diverse origins.

    Science.gov (United States)

    Mercer, A A; Morelli, G; Heuzenroeder, M; Kamke, M; Achtman, M

    1984-01-01

    Escherichia coli K1 isolates of various O types were previously assigned to different clonal groups. Members of the two clones defined by membrane pattern 9 (MP9) and serotypes O18:K1 and O1:K1 had been found to be very similar to each other. The plasmid contents of these bacteria confirmed this conclusion. Both groups carried a self-transmissible plasmid of the FI incompatibility group that coded for colicin production and a major outer membrane protein called the plasmid-coded protein (PCP). The size of this plasmid varied from 76 to 96 megadaltons, but restriction endonuclease digestion and DNA heteroduplex analysis revealed that these plasmids were highly related. O18:K1 bacteria of MP6 had previously been determined to represent a subclone, related to but different from O18:K1 MP9 bacteria. These MP6 bacteria carried a different, smaller IncFI plasmid which did not code for colicin production or the PCP protein. This smaller plasmid was primarily related to the larger plasmid within the regions of DNA encoding incompatibility, replication, and conjugation. O1:K1 bacteria of MP5 contained other unrelated plasmids in agreement with the previous conclusion that they are unrelated to O1:K1 bacteria of MP9. The bacteria examined had been isolated from two continents over a time span of 38 years, and the results attest to conservative inheritance of plasmids within bacteria of common descent. Images PMID:6094355

  5. Quantitative Analysis of Mutant Subclones in Chronic Myeloid Leukemia: Comparison of Different Methodological Approaches.

    Science.gov (United States)

    Preuner, Sandra; Barna, Agnes; Frommlet, Florian; Czurda, Stefan; Konstantin, Byrgazov; Alikian, Mary; Machova Polakova, Katerina; Sacha, Tomasz; Richter, Johan; Lion, Thomas; Gabriel, Christian

    2016-04-29

    Identification and quantitative monitoring of mutant BCR-ABL1 subclones displaying resistance to tyrosine kinase inhibitors (TKIs) have become important tasks in patients with Ph-positive leukemias. Different technologies have been established for patient screening. Various next-generation sequencing (NGS) platforms facilitating sensitive detection and quantitative monitoring of mutations in the ABL1-kinase domain (KD) have been introduced recently, and are expected to become the preferred technology in the future. However, broad clinical implementation of NGS methods has been hampered by the limited accessibility at different centers and the current costs of analysis which may not be regarded as readily affordable for routine diagnostic monitoring. It is therefore of interest to determine whether NGS platforms can be adequately substituted by other methodological approaches. We have tested three different techniques including pyrosequencing, LD (ligation-dependent)-PCR and NGS in a series of peripheral blood specimens from chronic myeloid leukemia (CML) patients carrying single or multiple mutations in the BCR-ABL1 KD. The proliferation kinetics of mutant subclones in serial specimens obtained during the course of TKI-treatment revealed similar profiles via all technical approaches, but individual specimens showed statistically significant differences between NGS and the other methods tested. The observations indicate that different approaches to detection and quantification of mutant subclones may be applicable for the monitoring of clonal kinetics, but careful calibration of each method is required for accurate size assessment of mutant subclones at individual time points.

  6. Safety and tolerability of conserved region vaccines vectored by plasmid DNA, simian adenovirus and modified vaccinia virus ankara administered to human immunodeficiency virus type 1-uninfected adults in a randomized, single-blind phase I trial.

    Directory of Open Access Journals (Sweden)

    Emma-Jo Hayton

    Full Text Available TRIAL DESIGN: HIV-1 vaccine development has advanced slowly due to viral antigenic diversity, poor immunogenicity and recently, safety concerns associated with human adenovirus serotype-5 vectors. To tackle HIV-1 variation, we designed a unique T-cell immunogen HIVconsv from functionally conserved regions of the HIV-1 proteome, which were presented to the immune system using a heterologous prime-boost combination of plasmid DNA, a non-replicating simian (chimpanzee adenovirus ChAdV-63 and a non-replicating poxvirus, modified vaccinia virus Ankara. A block-randomized, single-blind, placebo-controlled phase I trial HIV-CORE 002 administered for the first time candidate HIV-1- vaccines or placebo to 32 healthy HIV-1/2-uninfected adults in Oxford, UK and elicited high frequencies of HIV-1-specific T cells capable of inhibiting HIV-1 replication in vitro. Here, detail safety and tolerability of these vaccines are reported. METHODS: Local and systemic reactogenicity data were collected using structured interviews and study-specific diary cards. Data on all other adverse events were collected using open questions. Serum neutralizing antibody titres to ChAdV-63 were determined before and after vaccination. RESULTS: Two volunteers withdrew for vaccine-unrelated reasons. No vaccine-related serious adverse events or reactions occurred during 190 person-months of follow-up. Local and systemic events after vaccination occurred in 27/32 individuals and most were mild (severity grade 1 and predominantly transient (<48 hours. Myalgia and flu-like symptoms were more strongly associated with MVA than ChAdV63 or DNA vectors and more common in vaccine recipients than in placebo. There were no intercurrent HIV-1 infections during follow-up. 2/24 volunteers had low ChAdV-63-neutralizing titres at baseline and 7 increased their titres to over 200 with a median (range of 633 (231-1533 post-vaccination, which is of no safety concern. CONCLUSIONS: These data demonstrate

  7. Chemotherapy of Bacterial Plasmids

    Science.gov (United States)

    1979-01-29

    render them non-susceptible to K: z plasmid-encoded enzymes. (3) Development of drugs which are selective inhibitor! 1 4, of plasmid DNA replication. (4... Development of drugs which inhibit phenotypic as expression of plasmid genes, and (5) Development of drugs which are inhibitors o, drug-inactivating...Barnes [2] them non-susceptible to plasmid-encoded enzymes, tabulated data on the incidence of Gram-negative 3) development of drugs which are

  8. Computational Model Reveals Limited Correlation between Germinal Center B-Cell Subclone Abundancy and Affinity: Implications for Repertoire Sequencing

    Science.gov (United States)

    Reshetova, Polina; van Schaik, Barbera D. C.; Klarenbeek, Paul L.; Doorenspleet, Marieke E.; Esveldt, Rebecca E. E.; Tak, Paul-Peter; Guikema, Jeroen E. J.; de Vries, Niek; van Kampen, Antoine H. C.

    2017-01-01

    Immunoglobulin repertoire sequencing has successfully been applied to identify expanded antigen-activated B-cell clones that play a role in the pathogenesis of immune disorders. One challenge is the selection of the Ag-specific B cells from the measured repertoire for downstream analyses. A general feature of an immune response is the expansion of specific clones resulting in a set of subclones with common ancestry varying in abundance and in the number of acquired somatic mutations. The expanded subclones are expected to have BCR affinities for the Ag higher than the affinities of the naive B cells in the background population. For these reasons, several groups successfully proceeded or suggested selecting highly abundant subclones from the repertoire to obtain the Ag-specific B cells. Given the nature of affinity maturation one would expect that abundant subclones are of high affinity but since repertoire sequencing only provides information about abundancies, this can only be verified with additional experiments, which are very labor intensive. Moreover, this would also require knowledge of the Ag, which is often not available for clinical samples. Consequently, in general we do not know if the selected highly abundant subclone(s) are also the high(est) affinity subclones. Such knowledge would likely improve the selection of relevant subclones for further characterization and Ag screening. Therefore, to gain insight in the relation between subclone abundancy and affinity, we developed a computational model that simulates affinity maturation in a single GC while tracking individual subclones in terms of abundancy and affinity. We show that the model correctly captures the overall GC dynamics, and that the amount of expansion is qualitatively comparable to expansion observed from B cells isolated from human lymph nodes. Analysis of the fraction of high- and low-affinity subclones among the unexpanded and expanded subclones reveals a limited correlation between

  9. Plasmid segregation mechanisms

    DEFF Research Database (Denmark)

    Ebersbach, Gitte; Gerdes, Kenn; Charbon, Gitte Ebersbach

    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 ...

  10. Ion pair-reversed phase HPLC approach facilitates subcloning of PCR products and screening of recombinant colonies.

    Science.gov (United States)

    Shaw-Bruha, C M; Lamb, K A

    2000-04-01

    The isolation of a single DNA molecule for cloning is technically difficult, and the subsequent screening of colonies for recombinant DNA clones is time consuming. Ion pair-reversed phase HPLC (IP-RP-HPLC) analysis of nucleic acids improves the resolution and isolation of PCR products to be cloned. We demonstrate that PCR products analyzed and collected using the IP-RP-HPLC approach (WAVE DNA Fragment Analysis System) can be cloned directly into a plasmid vector. In addition, we demonstrate that when IP-RP-HPLC analysis is extended to the colony screening process, the time required for these procedures is reduced.

  11. 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...

  12. Constructionof the recombinant adenovirus vectors of CALB2 gene and small interfering RNA, and application in testicular Leydig cells

    Institute of Scientific and Technical Information of China (English)

    Luo Jian; Wang Jing; Liu Shan; Sun Xue-ping; Gao Chao; Gao Li; Yang Xiao-yu; Liu Jia-yin; Cui Yu-gui

    2011-01-01

    Objective:To construct the recombinant adenovirus vectors of calretinin (CALB2) gene and small interfering RNA (siRNA),for over-expression or knock-down of CALB2,as the basis of functional investigation of CALB2 in testicular Leydig cells.Methods:The cDNA sequence of CALB2 was cloned by the reverse transcriptive polymerase chain reaction (RT-PCR).A CALB2 gene fragment was sub-cloned into adenovirus shuttle plasmid pAdTrack-CMV to construct the shuttle plasmid pAdTrack-CALB2.Then it was transformed into BJ5183 cells with the adenoviral backbone pAdEasy-1 to obtain the homologous recombinant AdCMV-CALB2.The recombinant AdCMV-CALB2 was further packaged and amplificated in AD293 cells.The expression of CALB2 protein in AD293 cells was detected by Western blotting.CALB2 protein was over-expressed in mouse Leydig cell line (MLTC-1 cells) by the constructed AdCMVCALB2.CALB2 gene siRNA recombinant adenovirus vector (Ad-H1-siRNA/CALB2 was also constructed simultaneously.Its efficacy was detected in AD293 cells by Western blotting.Results:The CALB2 gene recombinant adenovirus vector AdCMV-CALB2 and the CALB2 gene siRNA recombinant adenovirus vector Ad-H1-siRNA/CALB2 were constructed successfully by endonulease digestion and sequencing.AD293 cells infected with AdCMV-CALB2 or Ad-H1-SiRNA/CALB2 significantly expressed GFP protein.The expression of CALB2 protein was significantly up-regulated in AD293 cells infected with AdCMV-CALB2 plasmids,while the expression of CALB2 protein was down-regulated by 60% in the CALB2 cells infected with Ad-H1SiRNA/CALB2.MLTC-1 cells did not markedly express CALLB2 protein,while MLTC-1 cells infected with AdCMV-CALB2 expressed CALB2 protein at a high level.Conclusions:The recombinant adenovirus vectors of AdCMV-CALB2 and Ad-H1-SiRNA/CALB2 were successfully constructed.Both vectors effectively expressed in AD293.CALB2 protein was over-expressed in the cultured MLTC-1 cells by AdCMV-CALB2.These vectors of CALB2 gene and Leydig cell line are

  13. A simplified approach to construct infectious cDNA clones of a tobamovirus in a binary vector.

    Science.gov (United States)

    Junqueira, Bruna Rayane Teodoro; Nicolini, Cícero; Lucinda, Natalia; Orílio, Anelise Franco; Nagata, Tatsuya

    2014-03-01

    Infectious cDNA clones of RNA viruses are important tools to study molecular processes such as replication and host-virus interactions. However, the cloning steps necessary for construction of cDNAs of viral RNA genomes in binary vectors are generally laborious. In this study, a simplified method of producing an agro-infectious Pepper mild mottle virus (PMMoV) clone is described in detail. Initially, the complete genome of PMMoV was amplified by a single-step RT-PCR, cloned, and subcloned into a small plasmid vector under the T7 RNA polymerase promoter to confirm the infectivity of the cDNA clone through transcript inoculation. The complete genome was then transferred to a binary vector using a single-step, overlap-extension PCR. The selected clones were agro-infiltrated to Nicotiana benthamiana plants and showed to be infectious, causing typical PMMoV symptoms. No differences in host responses were observed when the wild-type PMMoV isolate, the T7 RNA polymerase-derived transcripts and the agroinfiltration-derived viruses were inoculated to N. benthamiana, Capsicum chinense PI 159236 and Capsicum annuum plants.

  14. Plasmid DNA Manufacturing for Indirect and Direct Clinical Applications.

    Science.gov (United States)

    Schmeer, Marco; Buchholz, Tatjana; Schleef, Martin

    2017-10-01

    Plasmid DNA is currently gaining increasing importance for clinical research applications in gene therapy and genetic vaccination. For direct gene transfer into humans, good manufacturing practice (GMP)-grade plasmid DNA is mandatory. The same holds true if the drug substance contains a genetically modified cell, for example chimeric antigen receptor (CAR) T cells, where these cells as well as the contained plasmids are used. According to the responsible regulatory agencies, they have to be produced under full GMP. On the other hand, for GMP production of, for example, mRNA or viral vectors (lentiviral vectors, adeno-associated virus vectors, etc.), in many cases, High Quality Grade plasmid DNA is accepted as a starting material. The manufacturing process passes through different production steps. To ensure the right conditions are used for the plasmid, a pilot run must be conducted at the beginning. In this step, a followed upscaling with respect to reproducibility and influences on product quality is performed. Subsequently, a cell bank of the transformed productions strain is established and characterized. This cell bank is used for the cultivation process. After cell harvesting and lysis, several chromatography steps are conducted to receive a pure plasmid product. Depending on the respective required quality grade, the plasmid product is subject to several quality controls. The last step consists of formulation and filling of the product.

  15. Genomic libraries: II. Subcloning, sequencing, and assembling large-insert genomic DNA clones.

    Science.gov (United States)

    Quail, Mike A; Matthews, Lucy; Sims, Sarah; Lloyd, Christine; Beasley, Helen; Baxter, Simon W

    2011-01-01

    Sequencing large insert clones to completion is useful for characterizing specific genomic regions, identifying haplotypes, and closing gaps in whole genome sequencing projects. Despite being a standard technique in molecular laboratories, DNA sequencing using the Sanger method can be highly problematic when complex secondary structures or sequence repeats are encountered in genomic clones. Here, we describe methods to isolate DNA from a large insert clone (fosmid or BAC), subclone the sample, and sequence the region to the highest industry standard. Troubleshooting solutions for sequencing difficult templates are discussed.

  16. Construction of polyoma virus middle T gene vector and its expression in eucaryotic cells%多瘤病毒MT基因真核表达载体的构建

    Institute of Scientific and Technical Information of China (English)

    何三纲; 赵怡芳; 贾俊

    2001-01-01

    Objective:To construct a plasmid for exploring the role ofpolyoma virus middle T (PyMT) protein in the development of hemangioma. Methods:The open reading frame of PyMT gene from Polyoma virus (Py) genome deleted replication starting sites was digested with restricted enzyme and subcloned into pUC19 plasmid.The resulting plasmid pPyMT was digested with HindⅢ and EcoRI.The PyMT fragment was inserted to plasmid pEGFP1. The recombinant plasmids with proper orientation were identified with analysis of restriction enzymes and PCR.The recombinant vector pPyMT-GFP and the vector-alone pEGFP1 were lined and transfected into mouse skin fibroblasts with electroporation technique.After selected with G418, resistant colonies were obtained. Results:The results showed that the recombinant plasmid could express PyMT efficiently under the control of polyoma virus promoter and enhancer. Conclusions:Because the eukaryotic expression vector pPyMT is derived from the polyoma virus, the PyMT gene could express steadily in mammalian cells.The recombinant vector pPyMT-GFP could be used further to study the effect of PyMT protein on the development of hemangioma.%目的:构建携带多瘤病毒MT癌基因的真核细胞表达载体。方法:从野生型多瘤病毒上取其nt4632至nt1560片段克隆到pUC19上,再将该片段定点插入pEGFP1的HindⅢ和EcoRI位点间,构建携带多瘤病毒MT癌基因的真核表达载体pPyMT-GFP。经线性化,将重组体导入鼠皮肤成纤维细胞中,G418选择培养,得到抗性细胞克隆。结果:构建的pPyMT-GFP质粒在鼠成纤维细胞中有稳定表达。结论:来源于野生型多瘤病毒的PyMT基因在哺乳动物细胞中有稳定表达,其真核表达载体pPyMT-GFP可被进一步用于研究PyMT蛋白在血管瘤发生中的作用。

  17. SubClones (Alan Parsons) visit CERN – and the subatomic world

    CERN Multimedia

    Jordan Juras

    2011-01-01

    While the LHC has been creating subatomic particles, Alan Parsons has re-entered the studio with his new project, SubClones. The three-piece electronic rock group has joined the legendary Alan Parsons Live Project on tour, and their common friend Patrick Geeraert gave them the chance to drop into CERN for a visit.   Alan Parsons during his visit to CERN. The Alan Parsons Live Project is back on tour, with dates scheduled for the end of the summer across Europe and the Americas. A stop at CERN recently complemented a day off between two cities and allowed the band to move from the frontier of the music industry to the frontier of science. “We saw everything there was to see at CERN,” explains Alan Parsons. “It was all fascinating stuff but unfortunately we couldn’t see what was underground, though, so I think we will have to come back.” Parsons’ new project, SubClones, features three members whose identities remain a secret. Althoug...

  18. Development of prophylactic recombinant HPV58-attenuated Shigeila live vector vaccine and evaluation of its protective efficacy and immunogenicity in the guinea pig keratoconjunctivitis model

    Institute of Scientific and Technical Information of China (English)

    Wensheng Li; Hongli Liu; Xiaofeng Yang; Jin Zheng; Yili Wang; Lusheng Si

    2009-01-01

    To develop a prophylactic recombinant HPV58L1-attenuated Shigella live vector vaccine and evaluate its protective efficacy and immunogenicity in the guinea pig keratoconjunctivitis model, the HPV58L1 gene was cloned into vector pUCmt, and then subcloned into the suicide vector pCVD442. The recombinant plasmid pCVD442-HPV58L1 was introduced into attenuated Shigella (sf301:△virG) with the helper plasmid PRK2013 by filter mating. The positive colonies were harvested and confirmed by polymerase chain reaction. The expression of the HPV58L1 protein with a molecu-lar weight of 60 kDa was confirmed by western blot. The ability of the interested protein to self-assemble into virus-like particles was identified by transmission electron microscope, and murine erythrocyte hemagglu-tination assay. The guinea pig keratoconjunctivitis model was used to evaluate the protective efficacy and immunogenicity of the vaccine. Animal experiments showed that there was no keratoconjunctivitis occurred in the immunized group (HPV58-attenuated Shigella), and the serum levels of anti-HPV58L1-IgG and -IgA were obviously increased (P0.05). Enzyme-linked immunosorbent spot assay showed that HPV58L1-specific IgA-antibody-secreting cells (ASC) and IgG-ASC of spleen and lymph nodes were also obviously increased (P<0.01). In this study, a recombi-nant HPV58L1-attenuated Shigella live vector vaccine was successfully constructed, and it could induce strong humoral immune responses in the immunized animals, and induce protective antibody production.

  19. 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.

  20. Characterization of pMC11, a plasmid with dual origins of replication isolated from Lactobacillus casei MCJ and construction of shuttle vectors with each replicon

    DEFF Research Database (Denmark)

    Chen, Zhengjun; Lin, Jinzhong; Ma, Chengjie

    2014-01-01

    %. These vectors were employed to express a green fluorescent protein (GFP) using the promoter of S-layer protein SlpA from Lactobacillus acidophilus. And a growth-phase regulated expression of GFP was observed in different strains. In conclusion, these shuttle vectors provide efficient genetic tools for DNA...

  1. Detection and characterization of pCT-like plasmid vectors for blaCTX-M-14 in Escherichia coli isolates from humans, turkeys and cattle in England and Wales

    NARCIS (Netherlands)

    Stokes, M.O.; Cottel, J.L.; Piddock, L.J.; Wu, G.; Wootton, M.; Mevius, D.J.; Randall, L.P.; Teale, C.J.; Fielder, M.D.; Coldham, N.G.

    2012-01-01

    Objectives - To detect and characterize Escherichia coli strains and pCT-like plasmids implicated in the dissemination of the CTX-M-14 gene in animals and humans, in England and Wales. Methods UK CTX-M-14-producing E. coli (n¿=¿70) from cattle (n¿=¿33), turkeys (n¿=¿9), sheep (n¿=¿2) and humans (n¿=

  2. Cloning of HBsAg-encoded genes in different vectors and their expression in eukaryotic cells

    Institute of Scientific and Technical Information of China (English)

    Shan Qin; Hong Tang; Lian-San Zhao; Fang He; Yong Lin; Li Liu; Xiao-Mei He

    2003-01-01

    AIM: To compare the efficiency of different plasmids as DNA vectors by cloning three HBsAg-encoded genes into two eukaryotic expression vectors, pRc/CMV and pSG5UTPL/Flag, and to express HBsAg S, MS, and LS proteins in SP2/0 cells, and to establish monoclone SP2/0 cell strains that are capable of expressing S or S2S proteins stably.METHODS: Segments of S, preS2-S, preS1-preS2-S genes of Hepatitis B virus were amplified by routine PCR and preS1S fragment was amplified by Over-Lap Extension PCR. The amplified segments were cleaved with restricted endonuclease Hind Ⅲ/Not Ⅰ followed by ligation with pRc/CMV, or BamHI/EcoR Ⅰ followed by ligation with pSG5UTPL/Flag. After the plasmid vectors were cleaved with the correspond enzymes, the amplified segments were inserted into pRc/CMV or pSGSUTPL/Flag plasmid vectors with T4DNA ligase. KOZAK sequence was added before the initial ATG code of each fragment using specific primer. The inserted segments in the recombinant plasmids were sequenced after subcloning. BALB/c mice myeloma cells (SP2/0 cell line) were transfected with the recombinant plasmids. The expressions of the different recombinants were compared by Western-blot, using a monoclonal anti-HBs antibody as the primary antibody and peroxidase-labeled multi-linker as the secondary. Stable SP2/0-pRc/CMV-S or SP2/0- pRc/CMV-MS clones were established through clone screening with G418.RESULTS: Fragments with anticipated size were harvested after PCR. After recombination and screening, the sequences of the inserted segments in the recombinants were confirmed to be S, preS2S, preSl-preS2S and preSlS encoding genes,determined by sequencing. The results of Western-blot hybridization were positive for the anticipated proteins.Among them, pRc/CMV-S or pRc/CMV-MS demonstrated the highest expressing their respective antigen.CONCLUSION: Eight recombinant plasmids expressing S,M, L or preSlS proteins are obtained. For hepatitis surface antigen expression in eukaryotic cells

  3. [A novel Salmonella Typhimurium plasmid, pAnkS: an example for plasmid evolution in antibiotic resistance].

    Science.gov (United States)

    Sahin, Fikret; Karasartova, Djursun; Gerçeker, Devran; Aysev, A Derya; Erdem, Birsel

    2008-07-01

    In this study, a plasmid, carrying ampicillin resistance (ampR) gene, isolated from a clinical isolate of Salmonella enterica serotype Typhimurium presenting ACSSuT (ampicilin, chloramphenicol, streptomycin, sulphonamide, tetracycline) resistance phenotype, was defined. The length of complete sequence of this plasmid was 8271 base pairs (bp), and it was named as pAnkS owing to its isolation place (plasmid-Ankara- Salmonella). The plasmid was analyzed for potential reading frames and structural features indicative of transposons and transposon relics. The Xmnl enzyme restriction fragments of pAnkS were cloned into E. coli plasmid vectors (pBSK), sequenced and analyzed with the BLAST programs. Plasmid pAnkS has contained a previously defined enterohemorrhagic E. coli (EHEC) plasmid p4821 as a core region and also contained a complete Tn3-like transposon of 4950 bp consisting of the left terminal repeat, Tn3-related tnpR and tnpA genes for transposition functions, ampicillin resistance gene bla(TEM), and the right terminal repeats, pAnkS showed strong homology with another Salmonella plasmid, pNTP16, for sequences that belong to p4821 and partial Tn3 segments. It was found that pNTP16 also carries kanamycin resistance gene (kanR) in addition to ampR gene. Plasmid pAnkS is one of the few completely sequenced plasmids from Salmonella Typhimurium and is in the middle of the pathway of evolution of plasmid from p4821 to pNTP16. The identification of pAnkS might help better understanding of plasmid evolution.

  4. The Subclonal Structure and Genomic Evolution of Oral Squamous Cell Carcinoma Revealed by Ultra-deep Sequencing

    DEFF Research Database (Denmark)

    Tabatabaeifar, Siavosh; Thomassen, Mads; Larsen, Martin Jakob

    Background: Oral squamous cell carcinoma (OSCC), a subgroup of head and neck squamous cell carcinoma (HNSCC), is primarily caused by alcohol consumption and tobacco use. Recent DNA sequencing studies suggests that HNSCC are very heterogeneous between patients; however the intra-patient subclonal...... structure remains unexplored due to lack of sampling multiple tumor biopsies from each patient. Materials and methods: To examine the clonal structure and describe the genomic cancer evolution we applied whole-exome sequencing combined with targeted ultra-deep targeted sequencing on biopsies from 5stage IV...... complex subclonal architectures comprising distinct subclones only found in geographically distinct regions of the tumors. The metastatic potential of the tumor is acquired early in the tumor evolution, as indicated by the lymph node sharing the majority of the mutations with the tumor biopsies, while...

  5. An Improved Method for Including Upper Size Range Plasmids in Metamobilomes

    DEFF Research Database (Denmark)

    Norman, Anders; Riber, Leise; Luo, Wenting

    2014-01-01

    cloning vector (pBR322), and a 56 Kbp conjugative plasmid (pKJK10), to represent lower- and upper plasmid size ranges, respectively. Subjecting a mixture of these plasmids to the overall isolation protocol revealed a 34-fold over-amplification of pBR322 after MDA. To address this bias, we propose......, as gene functions associated with these plasmids, such as conjugation, was exclusively encoded in the data output generated through the modified protocol. Thus, with the suggested modification, access to a large uncharacterized pool of accessory elements that reside on medium-to-large plasmids has been...

  6. The MEC1 and MEC2 lines represent two CLL subclones in different stages of progression towards prolymphocytic leukemia.

    Directory of Open Access Journals (Sweden)

    Eahsan Rasul

    Full Text Available The EBV carrying lines MEC1 and MEC2 were established earlier from explants of blood derived cells of a chronic lymphocytic leukemia (CLL patient at different stages of progression to prolymphocytoid transformation (PLL. This pair of lines is unique in several respects. Their common clonal origin was proven by the rearrangement of the immunoglobulin genes. The cells were driven to proliferation in vitro by the same indigenous EBV strain. They are phenotypically different and represent subsequent subclones emerging in the CLL population. Furthermore they reflect the clinical progression of the disease. We emphasize that the support for the expression of the EBV encoded growth program is an important differentiation marker of the CLL cells of origin that was shared by the two subclones. It can be surmised that proliferation of EBV carrying cells in vitro, but not in vivo, reflects the efficient surveillance that functions even in the severe leukemic condition. The MEC1 line arose before the aggressive clinical stage from an EBV carrying cell within the subclone that was in the early prolymphocytic transformation stage while the MEC2 line originated one year later, from the subsequent subclone with overt PLL characteristics. At this time the disease was disseminated and the blood lymphocyte count was considerably elevated. The EBV induced proliferation of the MEC cells belonging to the subclones with markers of PLL agrees with earlier reports in which cells of PLL disease were infected in vitro and immortalized to LCL. They prove also that the expression of EBV encoded set of proteins can be determined at the event of infection. This pair of lines is particularly important as they provide in vitro cells that represent the subclonal evolution of the CLL disease. Furthermore, the phenotype of the MEC1 cells shares several characteristics of ex vivo CLL cells.

  7. Construction of targeted plasmid vector pcDNA3.1-Egr.1p-p16 and its expression in pancreatic cancer JF305 cells induced by radiation in vitro

    Institute of Scientific and Technical Information of China (English)

    Hong-Bing Ma; Ming-Hua Bai; Xi-Jing Wang; Zheng-Li Di; Hui Xia; Zheng Li; Jie Liu; Jie Ma; Hua-Fen Kang; Cong-Mei Wu

    2007-01-01

    AIM: To construct pcDNA3.1-Egr.1p-p16 recombinant plasmid and investigate the expression of p16 in pancreatic cancer JF305 cells induced by radiation and the feasibility of gene radiotherapy for pancreatic carcinoma.METHODS: Human p16 cDNA was ligated to th edownstream of Egr-1 promotor to construct pcDNA3.1-Egr.1p-p16 plasmid by restriction enzyme digested. The recombined plasmids were transfected into pancreatic cancer JF305 cells with lipofectamine. p16 mRNA level was detected by RT-PCR. The expression of p16 after different doses of X-ray radiation was detected by Western blot technique. Cell survival was assessed by clonogenic assays and cell viability was analysed by trypen blue exclusion. Flow cytometry was performed to study the apoptosis of JF305 cells.RESULTS: Restriction enzyme digestion showed the correctly constructed pcDNA3.1-Egr.1p-p16. The p16expression in cells transfected with pcDNA3.1-Egr.1p-p16induced by different doses of radiation was higher than that in the control group (P < 0.05). Eight hours after 2 Gy X-ray radiation, the expression reached its peak(87.00 ng/L), and was significantly higher than that in the control group (P < 0.0.5). Clonogenic analysis and trypan blue extraction test showed that the pcDNA3.1-Egr.1p-p16 transfer enhanced radiation-induced cell killing in p16-null JF305 cell lines. The induction of apoptosis was lower in combined transfection and irradiation group than that in irradiation alone.CONCLUSION: X-ray can induce the recombinant plasmid pcDNA3.1-Egr.1p-p16 expression in JF305 cells.The detection of dose and time provides an experimental basis for in vivo study in future.

  8. Plasmid Transfer into the Homoacetogen Acetobacterium woodii by Electroporation and Conjugation

    OpenAIRE

    Strätz, Michael; Sauer, Uwe; Kuhn, Anita; Dürre, Peter

    1994-01-01

    Shuttle vectors (pMS3 and pMS4) which replicated in Escherichia coli and in gram-positive Acetobacterium woodii were constructed by ligating the replication origin of plasmid pAMβ1 with the E. coli cloning vector pUC19 and the tetM gene of streptococcal transposon Tn916. Electrotransformation of A. woodii was achieved at frequencies of 4.5 × 103 transformants per μg of plasmid DNA. For conjugal plasmid transfer, the mobilizable shuttle vector pKV12 was constructed by cloning the tetM gene int...

  9. The subclonal structure and genomic evolution of oral squamous cell carcinoma revealed by ultra-deep sequencing

    DEFF Research Database (Denmark)

    Tabatabaeifar, Siavosh; Thomassen, Mads; Larsen, Martin J

    2017-01-01

    Recent studies suggest that head and neck squamous cell carcinomas are very heterogeneous between patients; however the subclonal structure remains unexplored mainly due to studies using only a single biopsy per patient. To deconvolutethe clonal structure and describe the genomic cancer evolution...

  10. Mechanisms of plasmid segregation: have multicopy plasmids been overlooked?

    Science.gov (United States)

    Million-Weaver, Samuel; Camps, Manel

    2014-09-01

    Plasmids are self-replicating pieces of DNA typically bearing non-essential genes. Given that plasmids represent a metabolic burden to the host, mechanisms ensuring plasmid transmission to daughter cells are critical for their stable maintenance in the population. Here we review these mechanisms, focusing on two active partition strategies common to low-copy plasmids: par systems type I and type II. Both involve three components: an adaptor protein, a motor protein, and a centromere, which is a sequence area in the plasmid that is recognized by the adaptor protein. The centromere-bound adaptor nucleates polymerization of the motor, leading to filament formation, which can pull plasmids apart (par I) or push them towards opposite poles of the cell (par II). No such active partition mechanisms are known to occur in high copy number plasmids. In this case, vertical transmission is generally considered stochastic, due to the random distribution of plasmids in the cytoplasm. We discuss conceptual and experimental lines of evidence questioning the random distribution model and posit the existence of a mechanism for segregation in high copy number plasmids that moves plasmids to cell poles to facilitate transmission to daughter cells. This mechanism would involve chromosomally-encoded proteins and the plasmid origin of replication. Modulation of this proposed mechanism of segregation could provide new ways to enhance plasmid stability in the context of recombinant gene expression, which is limiting for large-scale protein production and for bioremediation.

  11. Plasmid-to-plasmid recombination in Haemophilus influenzae

    Energy Technology Data Exchange (ETDEWEB)

    Balganesh, M.; Setlow, J.K.

    1986-01-01

    No recombination between plasmids was observed after conjugal transfer of a plasmid into a cell carrying another plasmid. Two types of such recombination took place after transformation, one type being Rec/sup +/ dependent and suggesting a preferred site of recombination. The other much rarer type was at least partially Rec/sup +/ independent.

  12. Plasmid interference for curing antibiotic resistance plasmids in vivo.

    Science.gov (United States)

    Kamruzzaman, Muhammad; Shoma, Shereen; Thomas, Christopher M; Partridge, Sally R; Iredell, Jonathan R

    2017-01-01

    Antibiotic resistance increases the likelihood of death from infection by common pathogens such as Escherichia coli and Klebsiella pneumoniae in developed and developing countries alike. Most important modern antibiotic resistance genes spread between such species on self-transmissible (conjugative) plasmids. These plasmids are traditionally grouped on the basis of replicon incompatibility (Inc), which prevents coexistence of related plasmids in the same cell. These plasmids also use post-segregational killing ('addiction') systems, which poison any bacterial cells that lose the addictive plasmid, to guarantee their own survival. This study demonstrates that plasmid incompatibilities and addiction systems can be exploited to achieve the safe and complete eradication of antibiotic resistance from bacteria in vitro and in the mouse gut. Conjugative 'interference plasmids' were constructed by specifically deleting toxin and antibiotic resistance genes from target plasmids. These interference plasmids efficiently cured the corresponding antibiotic resistant target plasmid from different Enterobacteriaceae in vitro and restored antibiotic susceptibility in vivo to all bacterial populations into which plasmid-mediated resistance had spread. This approach might allow eradication of emergent or established populations of resistance plasmids in individuals at risk of severe sepsis, enabling subsequent use of less toxic and/or more effective antibiotics than would otherwise be possible, if sepsis develops. The generalisability of this approach and its potential applications in bioremediation of animal and environmental microbiomes should now be systematically explored.

  13. 细粒棘球绦虫转基因植物载体重组pBI-Eg95质粒构建及鉴定%Construction and identification of the transgenic plant vector recombinant pBI-Eg95 plasmid of Echinococcus granulosus

    Institute of Scientific and Technical Information of China (English)

    周辉; 李文桂

    2008-01-01

    Objective To construct and identify the transgenic plant vector recombinant pBI-Eg95 plasmid of Echinococcus granulosus. Methods Total RNA was extracted from hydatid cyst protoscoleces of Echinococcus granulosus after sonication. A couple of specific primers were designed on the basis of known sequences of Eg95 gene. The desired gene was amplified by PCR technique from the cDNA, and then was cloned into the plant expression vector pBI121 to construct the recombinant pBI-Eg95 plasmid. The recombinant plasmid was electroporated into Agrobocterium tumefaciens (At) LBA4404 strain. The positive recombinant clones were confirmed by restriction endonuclease digestion and characterized by PCR. Results For RT-PCR, a specific band around 471 bp was amplified. The result of DNA sequencing of Eg95 showed the identity with the published sequence. The same band was obtained by restriction endonuclease digestion and PER from the plasmids of positive recombinant At(rAt). Conclusions The recombinant pBI-Eg95 plasmid was successfully constructed, and it provides the basis to further research of the transgenic plant vaccine of Echinococcus granulosus.%目的 构建并鉴定细粒棘球绦虫(Eg)转基因植物载体重组pBI-Eg95质粒.方法 从细粒棘球蚴包囊中分离原头节.经超声粉碎后抽提总RNA,反转录成eDNA,设计合成引物,以eDNA为模板.通过PCR从cDNA中扩增出目的 基因Eg95,经电泳及测序鉴定后,将该基因定向克隆到植物表达载体pBI121中构建pBI-Eg95重组质粒,电穿孔转化根癌农杆菌(At)LBA4404株;从转化的At阳性株中抽提质粒进行双酶切和以抽提的质粒为模板进行PCR鉴定.结果 RT-PCR扩增出1条约471 bp的特异性条带,DNA序列分析与GenBank知的序列同源性为100%.从转化的At中抽提的质粒,双酶切及PCR测定的结果与预期相符.结论 成功构建了细粒棘球绦虫转基因植物载体重组pBI-Eg95质粒,为进一步构建细粒棘球绦虫转基因植物疫苗奠定了基础.

  14. Safety and immunogenicity of recombinant low-dosage HIV-1 A vaccine candidates vectored by plasmid pTHr DNA or modified vaccinia virus Ankara (MVA) in humans in East Africa.

    Science.gov (United States)

    Jaoko, Walter; Nakwagala, Frederick N; Anzala, Omu; Manyonyi, Gloria Omosa; Birungi, Josephine; Nanvubya, Annet; Bashir, Farah; Bhatt, Kirana; Ogutu, Hilda; Wakasiaka, Sabina; Matu, Lucy; Waruingi, Wambui; Odada, Jane; Oyaro, Micah; Indangasi, Jackton; Ndinya-Achola, Jeckonia; Konde, Carol; Mugisha, Emmanuel; Fast, Patricia; Schmidt, Claudia; Gilmour, Jill; Tarragona, Tony; Smith, Carol; Barin, Burc; Dally, Len; Johnson, Bruce; Muluubya, Andrew; Nielsen, Leslie; Hayes, Peter; Boaz, Mark; Hughes, Peter; Hanke, Tomás; McMichael, Andrew; Bwayo, Job; Kaleebu, Pontiano

    2008-05-23

    The safety and immunogenicity of plasmid pTHr DNA, modified vaccinia virus Ankara (MVA) human immunodeficiency virus type 1 (HIV-1) vaccine candidates were evaluated in four Phase I clinical trials in Kenya and Uganda. Both vaccines, expressing HIV-1 subtype A gag p24/p17 and a string of CD8 T-cell epitopes (HIVA), were generally safe and well-tolerated. At the dosage levels and intervals tested, the percentage of vaccine recipients with HIV-1-specific cell-mediated immune responses, assessed by a validated ex vivo interferon gamma (IFN-gamma) ELISPOT assay and Cytokine Flow Cytometry (CFC), did not significantly differ from placebo recipients. These trials demonstrated the feasibility of conducting high-quality Phase 1 trials in Africa.

  15. 人胰岛素样生长因子1基因质粒载体的构建及其在人脐血源性神经干细胞中的表达%Construction of Plasmid Vector Containing Human Insulin-Like Growth Factor 1 and Its Expression in Human Umbilical Cord Blood-Derived Neural Stem Cells

    Institute of Scientific and Technical Information of China (English)

    王军; 吴值荣; 朱登纳; 高峰; 侯艳艳; 陈海; 王留霞

    2011-01-01

    Objective To construct the plasmid expression vector containing human insulin - like growth factor 1 ( IGF - 1 ), and examine the expression of IGF - 1 gene in human umbilical cord blood - derived neural stem cells(NSCs).Methods The IGF - 1 gene was extracted from the fetal liver via reverse tranacription polymerase chain reaction( RT - PCR) ,then the product of PCR and plasmid pcDNA3.1 were purified by gel extraction.After enzyme digestion of DNA restriction enzyme - BamH Ⅰ and Hind Ⅲ, purified IGF - 1 gene was cloned into expression plasmid vector pcDNA3.1 by T4 DNA Ligase.Recombinant plasmid was identified by DNA sequencing method and enzyme digestion of DNA restriction enzyme - BamH Ⅰ and Hind Ⅲ.Recombinant pcDNA3.1 - IGF - 1 and empty plasmid were transfected into human umbilical cord blood - derived NSCs through lipofectin transfection.After transfection, transfected umbilical cord blood - derived NSCs were filtered with neomycin( G418 ).The expression of IGF - 1 gene in the gene transfected umbilical cord blood - derived was examined by immunocytochemical method and RT - PCR.Results IGF - 1 gene was successfully extracted from the fetal liver.Recombinants peDNA3.1 -IGF - 1 was proved accurate by restriction enzyme digestion and sequencing.Recombinant was transfected into human umbilical cord blood -derived NSCs by liposome for 24 hours,then selection cell clones appeared after 2 weeks for G418 filtering.In umbilical cord blood -derived NSCs transfected by recombinant plasmid vector, the expression of IGF- 1 gene was successful, which was detected by immtmocyto-chemical method and the expression of IGF - 1 mRNA was also positive while the other was negative compared with controla,which was examined by RT - PCR.Conclusion IGF - 1 gene can expressed in umbilical cord blood - derived NSCs transfected by recombinant plasmid vector.%目的 构建人胰岛素样生长因子1(IGF-1)质粒表达载体,并观察重组体pcDNA3.1-IGF-1转染后的脐血

  16. Plasmid interference for curing antibiotic resistance plasmids in vivo

    Science.gov (United States)

    Kamruzzaman, Muhammad; Shoma, Shereen; Thomas, Christopher M.; Partridge, Sally R.

    2017-01-01

    Antibiotic resistance increases the likelihood of death from infection by common pathogens such as Escherichia coli and Klebsiella pneumoniae in developed and developing countries alike. Most important modern antibiotic resistance genes spread between such species on self-transmissible (conjugative) plasmids. These plasmids are traditionally grouped on the basis of replicon incompatibility (Inc), which prevents coexistence of related plasmids in the same cell. These plasmids also use post-segregational killing (‘addiction’) systems, which poison any bacterial cells that lose the addictive plasmid, to guarantee their own survival. This study demonstrates that plasmid incompatibilities and addiction systems can be exploited to achieve the safe and complete eradication of antibiotic resistance from bacteria in vitro and in the mouse gut. Conjugative ‘interference plasmids’ were constructed by specifically deleting toxin and antibiotic resistance genes from target plasmids. These interference plasmids efficiently cured the corresponding antibiotic resistant target plasmid from different Enterobacteriaceae in vitro and restored antibiotic susceptibility in vivo to all bacterial populations into which plasmid-mediated resistance had spread. This approach might allow eradication of emergent or established populations of resistance plasmids in individuals at risk of severe sepsis, enabling subsequent use of less toxic and/or more effective antibiotics than would otherwise be possible, if sepsis develops. The generalisability of this approach and its potential applications in bioremediation of animal and environmental microbiomes should now be systematically explored. PMID:28245276

  17. New Plasmid Tools for Genetic Analysis of Actinobacilus pleuropneumoniae and Other Pasteurellaceae

    National Research Council Canada - National Science Library

    Janine T Bossé; Andrew L Durham; Andrew N Rycroft; J Simon Kroll; Paul R Langford

    2009-01-01

      We have generated a set of plasmids, based on the mobilizable shuttle vector pMIDG100, which can be used as tools for genetic manipulation of Actinobacillus pleuropneumoniae and other members of the Pasteurellaceae...

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

    Energy Technology Data Exchange (ETDEWEB)

    Benoit, T.G.; Wilson, G.R.; Bull, D.L.; Aronson, A.I. (Department of Agriculture, College Station, TX (USA))

    1990-08-01

    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.

  19. Molecular cloning of virB12 gene ofBrucella melitensis 16M strain in pET28a vector

    Institute of Scientific and Technical Information of China (English)

    Shiva Mirkalantari; Nour Amirmozafari; Bahram Kazemi; Gholamreza Irajian

    2012-01-01

    ABSTRACT Objective:To clone the virB12 gene in pET28a expression vector for production of recombinant protein to be used as antigenic component for future serological test development.Methods:Brucella melitensis (B. melitensis)16M strain was cultured and bacterialDNA was extracted by BioneerAccuPrep®GenomicDNAExtractionKit.Oligonucleotide primer pair was designed based onBrucella virB12 gene sequence withBamHI andHindIII restriction site at5´ end ofthe forward and reverse primers, respectively.DNA amplification was performed usingPrimSTAR®HSDNA polymerase and thePCR product was purified byDNAAccuPrep®GelPurificationKit.Purified DNA was cloned into pJET1.2 cloning vector.VirB12 gene fragment was excised from pJET1.2 usingBamHI/HindIII and subsequently subcloned into pET28a(+).Results: Brucella virB12 gene was successfully cloned in pJET1.2 and then in pET28a(+) plasmids.PCR and restriction enzyme digestion confirms the procedure.Conclusion:We cloned and expressed theBrucella virB12 gene which could be used as antigenic component for specific serological assay development.

  20. Plasmid containing a DNA ligase gene from Haemophilus influenzae

    Energy Technology Data Exchange (ETDEWEB)

    McCarthy, D.; Griffin, K.; Setlow, J.K.

    1984-05-01

    A ligase gene from Haemophilus influenzae was cloned into the shuttle vector pDM2. Although the plasmid did not affect X-ray sensitivity, it caused an increase in UV sensitivity of the wild-type but not excision-defective H. influenzae and a decrease in UV sensitivity of the rec-1 mutant. 14 references, 2 figures.

  1. MICROARRAY ANALYSIS OF DIFFERENT GENE EXPRESSION OF HUMAN CERVICAL CANCER SUBCLONE CELL LINES

    Institute of Scientific and Technical Information of China (English)

    Chen Wei; Li Xu; Wang Xiang

    2006-01-01

    Objective To examine the differentially expressed invasion-related genes in two anchorage-independent uterine cervical carcinoma cell lines derived from the same patient using a cDNA array. Methods Two human uterine cervical carcinoma subclonal cell lines CS03 and CS07 derived from a single donor line CS1213 were established by limited dilution procedure. The two cDNA samples retro-transcribed from total RNA derived from CS03 and CS07 cells were screened by a cDNA microarray carrying 234 human cell-cycle related genes and 1011 human signal transduction and membrane receptor -associated genes, scanned with a ScanArray 3000 laser scanner. Results The cDNA microarray analysis showed that 12 genes in CS03 were up-regulated compared to CS07, and 24 genes in CS07 were up-regulated. The function of a number of differentially expressed genes was consistently associated with cell-cycle, cell proliferation, migration, apoptosis, signal transduction and tumor metastasis, including p34cdc2, TSC22, plasminogen activator inhibitor I (PAI-1)and desmosome associated protein(Pinin). Conclusion Multiple genes are differentially expressed in uterine cervical carcinoma cell lines even came from the same patient. It is suggested that these genes are involved in the different phenotypic characteristics and development of cervical carcinoma.

  2. Tracking sub-clonal TP53 mutated tumor cells in human metastatic renal cell carcinoma.

    Science.gov (United States)

    Bousquet, Guilhem; El Bouchtaoui, Morad; Leboeuf, Christophe; Battistella, Maxime; Varna, Mariana; Ferreira, Irmine; Plassa, Louis-François; Hamdan, Diaddin; Bertheau, Philippe; Feugeas, Jean-Paul; Damotte, Diane; Janin, Anne

    2015-08-07

    Renal Cell Carcinomas (RCCs) are heterogeneous tumors with late acquisition of TP53 abnormalities during their evolution. They harbor TP53 abnormalities in their metastases. We aimed to study TP53 gene alterations in tissue samples from primary and metastatic RCCs in 36 patients followed up over a median of 4.2 years, and in xenografted issued from primary RCCs. In 36 primary RCCs systematically xenografted in mice, and in biopsies of metastases performed whenever possible during patient follow-up, we studied p53-expressing tumor cells and TP53 gene abnormalities.We identified TP53 gene alterations in primary tumors, metastases and xenografts. Quantification of tumors cells with TP53 gene alterations showed a significant increase in the metastases compared to the primary RCCs, and, strikingly, the xenografts were similar to the metastases and not to the primary RCCs from which they were derived.Using laser-microdissection of p53-expressing tumor cells, we identified TP53-mutated tumor cells in the xenografts derived from the primary RCC, and in a lung metastasis later developed in one patient. The mutation enabled us to track back their origin to a minority sub-clone in the primary heterogeneous RCC. Combining in situ and molecular analyses, we demonstrated a clonal expansion in a living patient with metastatic RCC.

  3. Characterization of the Lactobacillus plantarum plasmid pCD033 and generation of the plasmid free strain L. plantarum 3NSH.

    Science.gov (United States)

    Heiss, Silvia; Grabherr, Reingard; Heinl, Stefan

    2015-09-01

    Lactobacillus plantarum CD033, a strain isolated from grass silage in Austria, harbors a 7.9 kb plasmid designated pCD033. Sequence analysis identified 14 open reading frames and 8 of these were supposed to be putative coding sequences. Gene annotation revealed no putative essential genes being plasmid encoded, but a plasmid addiction system based on a PemI/PemK-like toxin-antitoxin system, able to stabilize plasmid maintenance. Absence of a replication initiation protein, a double strand origin as well as a single strand origin on plasmid pCD033 suggests replication via a new type of theta mechanism, whereby plasmid replication is potentially initiated and regulated by non-coding RNA. Detailed examination of segregational stability of plasmid vectors consisting of pCD033-fragments, combined with a selection marker, resulted in definition of a stably maintained minimal replicon. A gene encoding a RepB/OrfX-like protein was found to be not essential for plasmid replication. Alignment of the amino acid sequence of this protein with related proteins unveiled a highly conserved amino acid motif (LLDQQQ). L. plantarum CD033 was cured of pCD033 resulting in the novel plasmid free strain L. plantarum 3NSH. Plasmid curing demonstrated that no essential features are provided by pCD033 under laboratory conditions.

  4. Gene therapy for hepatocellular carcinoma using non-viral vectors composed of bis guanidinium-tren-cholesterol and plasmids encoding the tissue inhibitors of metalloproteinases TIMP-2 and TIMP-3.

    Science.gov (United States)

    Tran, Phuong-Lan; Vigneron, Jean-Pierre; Pericat, David; Dubois, Sylvie; Cazals, Dominique; Hervy, Martial; DeClerck, Yves A; Degott, Claude; Auclair, Christian

    2003-06-01

    Metalloproteinases (MMPs) and their natural inhibitors (TIMPs) contribute to the regulation of tumor microenvironment. Their expressions are deregulated in almost all human cancers. We report a novel approach to gene therapy of hepatocellular carcinoma (HCC), using repeated injections of DNA plasmids encoding the tissue inhibitors of metalloproteinases (TIMPs) TIMP-2 or TIMP-3, and a novel competent formulation of gene transfer based on nontoxic cationic cholesterol derivatives. The new gene delivery system was efficient in demonstrating the antitumor efficiency of TIMP-2 or TIMP-3 in inhibiting tumor growth of human HuH7 HCC cells xenografted into nude mice. We show, for the first time, an in vivo effect of TIMP-3 in delaying HCC tumor growth. No treatment-related toxicity was noted. An inhibition of angiogenesis and tumor necrosis accompanied the inhibitory effects of TIMP-2 or TIMP-3 on tumor expansion and invasion. We also report a bystander effect produced by transfected HuH7 tumor cells mixed with untransfected cells in 1:1 ratio in culture that resulted in killing 98% of cells within 96 h. In addition, the soluble forms of TIMP-2 and TIMP-3 expressed by transfected cells exerted a cytotoxic effect on untransfected HuH7 cell cultures. Taken together, these results demonstrate the potential efficacy of repeated treatment of secreted TIMP-2 and TIMP-3 for the design of nonviral gene therapy for hepatocarcinoma.

  5. Use of FabV-Triclosan Plasmid Selection System for Efficient Expression and Production of Recombinant Proteins in Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Syed A Ali

    Full Text Available Maintenance of recombinant plasmid vectors in host bacteria relies on the presence of selection antibiotics in the growth media to suppress plasmid -free segregants. However, presence of antibiotic resistance genes and antibiotics themselves is not acceptable in several applications of biotechnology. Previously, we have shown that FabV-Triclosan selection system can be used to select high and medium copy number plasmid vectors in E. coli. Here, we have extended our previous work and demonstrated that expression vectors containing FabV can be used efficiently to express heterologous recombinant proteins in similar or better amounts in E. coli host when compared with expression vectors containing β-lactamase. Use of small amount of non-antibiotic Triclosan as selection agent in growth medium, enhanced plasmid stability, applicability in various culture media, and compatibility with other selection systems for multiple plasmid maintenance are noteworthy features of FabV-Triclosan selection system.

  6. Characterization of two novel plasmids from Geobacillus sp. 610 and 1121 strains.

    Science.gov (United States)

    Kananavičiūtė, Rūta; Butaitė, Elena; Citavičius, Donaldas

    2014-01-01

    We describe two cryptic low molecular weight plasmids, pGTD7 (3279bp) and pGTG5 (1540bp), isolated from Geobacillus sp. 610 and 1121 strains, respectively. Homology analysis of the replication protein (Rep) sequences and detection of ssDNA indicate that both of them replicate via rolling circle mechanism. As revealed by sequence similarities of dso region and Rep protein, plasmid pGTD7 belongs to pC194/pUB110 plasmid family. The replicon of pGTD7 was proved to be functional in another Geobacillus host. For this purpose, a construct pUCK7, containing a replicon of the analyzed plasmid, was created and transferred to G. stearothermophilus NUB3621R strain by electroporation. Plasmid pGTG5, based on Rep protein sequence similarity, was found to be related mostly to some poorly characterized bacterial plasmids. Rep proteins encoded by these plasmids contain conservative motifs that are most similar to those of Microviridae phages. This feature suggests that pGTG5, together with other plasmids containing the same motifs, could constitute a new family of bacterial plasmids. To date, pGTG5 is the smallest plasmid identified in bacteria belonging to the genus Geobacillus. The two plasmids described in this study can be used for the construction of new vectors suitable for biotechnologically important bacteria of the genus Geobacillus.

  7. Bacillus stearothermophilus contains a plasmid-borne gene for alpha-amylase.

    Science.gov (United States)

    Mielenz, J R

    1983-01-01

    The gene for thermostable alpha-amylase from the thermophilic bacterium Bacillus stearothermophilus has been cloned and expressed in Escherichia coli. Each alpha-amylase-producing colony contained at least a 9.7-kilobase-pair (kb) chimeric plasmid composed of the vector pBR322 and a common 5.4-kb HindIII fragment of DNA. B. stearothermophilus contains four plasmids with sizes from 12 kb to over 108 kb. Restriction endonuclease analysis of these naturally occurring plasmids showed they also contain a 5.4-kb HindIII fragment of DNA. Cloning experiments with the four plasmids yielded alpha-amylase-producing E. coli that contained the same 9.7-kb chimeric plasmid. Restriction endonuclease analysis and further recombinant DNA experiments identified a 26-kb plasmid that contains the gene for alpha-amylase. A spontaneous mutant of B. stearothermophilus unable to produce alpha-amylase was missing the 26-kb plasmid but contained a 20-kb plasmid. A 6-kb deletion within the region of the 5.4-kb HindIII fragment yielded the 20-kb plasmid unable to code for alpha-amylase. A nick-translated probe for the alpha-amylase coding region did not hybridize to either plasmid or total cellular DNA from this mutant strain of B. stearothermophilus. These results demonstrate the gene for alpha-amylase is located exclusively on a 26-kb plasmid in B. stearothermophilus with no genetic counterpart present on the chromosome. Images PMID:6193526

  8. Construction of Recombinant Plasmid Containing S. Mutans F-ATPase β Subunit Gene

    Institute of Scientific and Technical Information of China (English)

    YU Dan-ni; JIANG Li

    2005-01-01

    objective: construct a homologous recombinant plasmid which was expected to be transformed into S. mutans Methods: a region at the 5' terminus of the S. mutans F-ATPase β subunit gene was amplified by PCR, the PCR product was inserted into vector pVA891, yielding recombinant plasmid. Results: the DNA sequence of the recombinant plasmid was identified correct in whole by restriction endonuclease and DNA sequence techniques. Conclusion: the recombinant plasmid of S. mutans DNA was cloned in effect ,it may assist in construction of homologues recombinant mutant.

  9. Inhibition of Corneal Neovascularization with the Combination of Bevacizumab and Plasmid Pigment Epithelium-Derived Factor-Synthetic Amphiphile INTeraction-18 (p-PEDF-SAINT-18 Vector in a Rat Corneal Experimental Angiogenesis Model

    Directory of Open Access Journals (Sweden)

    Ching-Hsein Chen

    2013-04-01

    Full Text Available Bevacizumab, a 149-kDa protein, is a recombinant humanized monoclonal antibody to VEGF. PEDF, a 50-kDa glycoprotein, has demonstrated anti-vasopermeability properties. In this study, we demonstrated that the combination of bevacizumab and plasmid pigment epithelium-derived factor-synthetic amphiphile INTeraction-18 (p-PEDF-SAINT-18 has a favorable antiangiogenic effect on corneal NV. Four groups (Group A: 0 μg + 0 μg, B: 0.1 μg + 0.1 μg, C: 1 μg + 1 μg, and D: 10 μg + 10 μg of bevacizumab + p-PEDF-SAINT-18 were prepared and implanted into the rat subconjunctival substantia propria 1.5 mm from the limbus on the temporal side. Then, 1 μg of p-bFGF-SAINT-18 was prepared and implanted into the rat corneal stroma 1.5 mm from the limbus on the same side. The inhibition of NV was observed and quantified from days 1 to 60. Biomicroscopic examination, western blot analysis and immunohistochemistry were used to analyze the 18-kDa bFGF, 50-kDa PEDF and VEGF protein expression. No inhibition activity for normal limbal vessels was noted. Subconjunctival injection with the combination of bevacizumab and p-PEDF-SAINT-18 successfully inhibited corneal NV. The bFGF and PEDF genes were successfully expressed as shown by western blot analysis, and a mild immune response to HLA-DR was shown by immunohistochemistry. We concluded that the combination of bevacizumab and p-PEDF-SAINT-18 may have more potent and prolonged antiangiogenic effects, making it possible to reduce the frequency of subconjunctival.Bevacizumab, a 149-kDa protein, is a recombinant humanized monoclonalantibody to VEGF. PEDF, a 50-kDa glycoprotein, has demonstrated anti-vasopermeabilityproperties. In this study, we demonstrated that the combination of bevacizumaband plasmid pigment epithelium-derived factor-synthetic amphiphile INTeraction-18(p-PEDF-SAINT-18 has a favorable antiangiogenic effect on corneal NV. Four groups(Group A: 0 μg + 0 μg, B: 0.1 μg + 0.1 μg, C: 1 μg + 1 μg, and

  10. 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 successful propagation and long-term continued existence of these extra-chromosomal elements is extensive. Apart from the accessory genetic elements that may provide plasmid-harboring cells a selective advantage, special focus is placed on the mechanisms conjugative plasmids employ to ensure their stable...... maintenance in the host cell. These importantly include the ability to self-mobilize in a process termed conjugative transfer, which may occur across species barriers. Other plasmid stabilizing mechanisms include the multimer resolution system, active partitioning, and post-segregational-killing of plasmid...

  11. Toxin Plasmids of Clostridium perfringens

    Science.gov (United States)

    Li, Jihong; Adams, Vicki; Bannam, Trudi L.; Miyamoto, Kazuaki; Garcia, Jorge P.; Uzal, Francisco A.; Rood, Julian I.

    2013-01-01

    SUMMARY In both humans and animals, Clostridium perfringens is an important cause of histotoxic infections and diseases originating in the intestines, such as enteritis and enterotoxemia. The virulence of this Gram-positive, anaerobic bacterium is heavily dependent upon its prolific toxin-producing ability. Many of the ∼16 toxins produced by C. perfringens are encoded by large plasmids that range in size from ∼45 kb to ∼140 kb. These plasmid-encoded toxins are often closely associated with mobile elements. A C. perfringens strain can carry up to three different toxin plasmids, with a single plasmid carrying up to three distinct toxin genes. Molecular Koch's postulate analyses have established the importance of several plasmid-encoded toxins when C. perfringens disease strains cause enteritis or enterotoxemias. Many toxin plasmids are closely related, suggesting a common evolutionary origin. In particular, most toxin plasmids and some antibiotic resistance plasmids of C. perfringens share an ∼35-kb region containing a Tn916-related conjugation locus named tcp (transfer of clostridial plasmids). This tcp locus can mediate highly efficient conjugative transfer of these toxin or resistance plasmids. For example, conjugative transfer of a toxin plasmid from an infecting strain to C. perfringens normal intestinal flora strains may help to amplify and prolong an infection. Therefore, the presence of toxin genes on conjugative plasmids, particularly in association with insertion sequences that may mobilize these toxin genes, likely provides C. perfringens with considerable virulence plasticity and adaptability when it causes diseases originating in the gastrointestinal tract. PMID:23699255

  12. Conjugative plasmids of Neisseria gonorrhoeae.

    Directory of Open Access Journals (Sweden)

    Emilia Pachulec

    Full Text Available Many clinical isolates of the human pathogen Neisseria gonorrhoeae contain conjugative plasmids. The host range of these plasmids is limited to Neisseria species, but presence of a tetracycline (tetM determinant inserted in several of these plasmids is an important cause of the rapid spread of tetracycline resistance. Previously plasmids with different backbones (Dutch and American type backbones and with and without different tetM determinants (Dutch and American type tetM determinants have been identified. Within the isolates tested, all plasmids with American or Dutch type tetM determinants contained a Dutch type plasmid backbone. This demonstrated that tetM determinants should not be used to differentiate between conjugal plasmid backbones. The nucleotide sequences of conjugative plasmids with Dutch type plasmid backbones either not containing the tetM determinant (pEP5233 or containing Dutch (pEP5289 or American (pEP5050 type tetM determinants were determined. Analysis of the backbone sequences showed that they belong to a novel IncP1 subfamily divergent from the IncP1alpha, beta, gamma, delta and epsilon subfamilies. The tetM determinants were inserted in a genetic load region found in all these plasmids. Insertion was accompanied by the insertion of a gene with an unknown function, and rearrangement of a toxin/antitoxin gene cluster. The genetic load region contains two toxin/antitoxins of the Zeta/Epsilon toxin/antitoxin family previously only found in Gram positive organisms and the virulence associated protein D of the VapD/VapX toxin/antitoxin family. Remarkably, presence of VapX of pJD1, a small cryptic neisserial plasmid, in the acceptor strain strongly increased the conjugation efficiency, suggesting that it functions as an antitoxin for the conjugative plasmid. The presence of the toxin and antitoxin on different plasmids might explain why the host range of this IncP1 plasmid is limited to Neisseria species. The isolated plasmids

  13. Introducing Vectors.

    Science.gov (United States)

    Roche, John

    1997-01-01

    Suggests an approach to teaching vectors that promotes active learning through challenging questions addressed to the class, as opposed to subtle explanations. Promotes introducing vector graphics with concrete examples, beginning with an explanation of the displacement vector. Also discusses artificial vectors, vector algebra, and unit vectors.…

  14. Shuttle cloning vectors for the cyanobacterium Anacystis nidulans.

    OpenAIRE

    Gendel, S; Straus, N; Pulleyblank, D; Williams, J

    1983-01-01

    Hybrid plasmids capable of acting as shuttle cloning vectors in Escherichia coli and the cyanobacterium Anacystis nidulans R2 were constructed by in vitro ligation. DNA from the small endogenous plasmid of A. nidulans was combined with two E. coli vectors, pBR325 and pDPL13, to create vectors containing either two selectable antibiotic resistance markers or a single marker linked to a flexible multisite polylinker. Nonessential DNA was deleted from the polylinker containing plasmid pPLAN B2 t...

  15. Phenotypic plasticity in bacterial plasmids.

    Science.gov (United States)

    Turner, Paul E

    2004-01-01

    Plasmid pB15 was previously shown to evolve increased horizontal (infectious) transfer at the expense of reduced vertical (intergenerational) transfer and vice versa, a key trade-off assumed in theories of parasite virulence. Whereas the models predict that susceptible host abundance should determine which mode of transfer is selectively favored, host density failed to mediate the trade-off in pB15. One possibility is that the plasmid's transfer deviates from the assumption that horizontal spread (conjugation) occurs in direct proportion to cell density. I tested this hypothesis using Escherichia coli/pB15 associations in laboratory serial culture. Contrary to most models of plasmid transfer kinetics, my data show that pB15 invades static (nonshaking) bacterial cultures only at intermediate densities. The results can be explained by phenotypic plasticity in traits governing plasmid transfer. As cells become more numerous, the plasmid's conjugative transfer unexpectedly declines, while the trade-off between transmission routes causes vertical transfer to increase. Thus, at intermediate densities the plasmid's horizontal transfer can offset selection against plasmid-bearing cells, but at high densities pB15 conjugates so poorly that it cannot invade. I discuss adaptive vs. nonadaptive causes for the phenotypic plasticity, as well as potential mechanisms that may lead to complex transfer dynamics of plasmids in liquid environments. PMID:15166133

  16. Automated Solid-Phase Subcloning Based on Beads Brought into Proximity by Magnetic Force

    DEFF Research Database (Denmark)

    Hudson, Elton P.; Nikoshkov, Andrej; Uhlén, Mathias;

    2012-01-01

    and the gene are immobilized to separate paramagnetic beads and brought into proximity by magnetic force. Ligation events were directly evaluated using fluorescent-based microscopy and flow cytometry. The highest ligation efficiencies were obtained when gene- and vector-coated beads were brought into close...... contact by application of a magnet during the ligation step. An automated procedure was developed using a laboratory workstation to transfer genes into various expression vectors and more than 95% correct clones were obtained in a number of various applications. The method presented here is suitable...

  17. Conjugation efficiency depends on intra and intercellular interactions between distinct plasmids: Plasmids promote the immigration of other plasmids but repress co-colonizing plasmids.

    Science.gov (United States)

    Gama, João Alves; Zilhão, Rita; Dionisio, Francisco

    2017-08-24

    Conjugative plasmids encode the genes responsible for the synthesis of conjugative pili and plasmid transfer. Expression of the conjugative machinery (including conjugative pili) may be costly to bacteria, not only due to the energetic/metabolic cost associated with their expression but also because they serve as receptors for certain viruses. Consequently, the presence of two plasmids in the same cell may be disadvantageous to each plasmid, because they may impose a higher fitness cost on the host. Therefore, plasmids may encode mechanisms to cope with co-resident plasmids. Moreover, it is possible that the transfer rate of a plasmid is affected by the presence of a distinct plasmid in the recipient cell. In this work, we measured transfer rates of twelve natural plasmids belonging to seven incompatibility groups in three situations, namely when: (i) donor cells contain a plasmid and recipient cells are plasmid-free; (ii) donor cells contain two unrelated plasmids and recipient cells are plasmid-free; and (iii) half of the cells contain a given plasmid and the other half contain another, unrelated, plasmid. In the third situation, recipient cells of a plasmid are the donor cells of the other plasmid. We show that there are more negative interactions (reduction of a plasmid's conjugative efficiency) between plasmids if they reside in the same cell than if they reside in different cells. However, if plasmids interacted intercellularly, the transfer rate of one of the plasmids was often higher (when the unrelated conjugative plasmid was present in the recipient cell) than if the recipient cell was plasmid-free - a positive effect. Experimental data retrieved from the study of mutant plasmids not expressing conjugative pili on the cell surface suggest that positive effects result from a higher efficiency of mating pair formation. Overall, our results suggest that negative interactions are significantly more frequent when plasmids occupy the same cell. Such

  18. Construction and expression of recombined human AFP eukaryotic expression vector

    Institute of Scientific and Technical Information of China (English)

    Li-Wang Zhang; Yang-Lin Pan; Stephen M Festein; Jun Ren; Liang Zhang; Hong-Mei Zhang; Bin Jin; Bo-Rong Pan; Xiao-Ming Si; Yan-Jun Zhang; Zhong-Hua Wang

    2003-01-01

    AIM: To construct a recombined human AFP eukaryotic expression vector for the purpose of gene therapy and target therapy of hepatocellular carcinoma (HCC).METHODS: The full length AFP-cDNA of prokaryotic vector was digested, and subcloned to the multi-clony sites of the eukaryotic vector. The constructed vector was confirmed by enzymes digestion and electrophoresis, and the product expressed was detected by electrochemiluminescence and immunofluorescence methods.RESULTS: The full length AFP-cDNA successfully cloned to the eukaryotic vector through electrophoresis, 0.9723 IU/ml AFP antigen was detected in the supernatant of AFPCHO by electrochemiluminescence method. Compared with the control groups, the differences were significant (P<0.05).AFP antigen molecule was observed in the plasma of AFPCHO by immunofluorescence staining.CONCLUSION: The recombined human AFP eukaryotic expression vector can express in CHO cell line. It provides experimental data for gene therapy and target therapy of hepatocellular carcinoma.

  19. Plasmid recombination in Haemophilus influenzae

    Energy Technology Data Exchange (ETDEWEB)

    McCarthy, D.

    1982-01-01

    DNA recombination in exponential phase and competent Haemophilus influenzae was measured by an electron microscopic assay that relies on the conversion of plasmid RSF0885 monomers into multimeric forms. Dimer circles were present at a frequency of 2% in plasmid preparations from competent Rd (wild-type) cells; multimers were present at a frequency of 0.2% in preparations from exponential phase cells. Thus, plasmid recombination was stimulated in competent cells. Multimer formation occurred efficiently in cells of the transformation defective mutant rec2, implying that the rec2 gene product is not required for plasmid recombination. However, the absence of multimer plasmids in preparations from competent cells of the transformation defective mutant rec1 suggests that the rec1 gene product is required. Digestion of purified plasmids with restriction endonuclease PvuII, which makes a single cut in the monomer, revealed the presence of recombination intermediates composed of two linear plasmids joined to form two pairs of arms resembling the Greek letter chi. Length measurements of these arms taken from a population of recombination intermediates gave evidence that the plasmids were joined at sites of homology. The distributions of individual DNA strands, at the intersections of the four arms, could be resolved in some recombination intermediates and were of two types. The first type of junction appeared as a single-stranded arm appended to each corner. The second type of junction consisted of a single strand of DNA linking the two linear plasmids at a site of homology. The single-stranded linker was frequently situated at the edge of a short gap on one of the plasmids in the pair. The fine structures of the recombinational joints have been interpreted in terms of previously proposed models of recombination.

  20. Selection of BW 5147 subclones devoid of non-specific suppressive activity for use in cell hybridization.

    Science.gov (United States)

    Chaouat, G; Liacopoulos-Briot, M; Couderc, J; Parlebas, J; Perrodon, Y; Briffaut, O; Liacopoulos, P

    1985-02-01

    Culture supernatants of BW 5147 cells widely used for T-cell hybridization often manifest non-MHC-restricted, non-antigen-specific regulatory activities on the mixed lymphocyte reaction (MLR) of mouse cells. This report demonstrates that, whereas supernatants of BW 5147 cells grown at low concentrations (2 X 10(5)/ml) enhanced MLR, high cell concentration (2 X 10(6)/ml) supernatants markedly inhibited this reaction. BW 5147 cell-free extracts significantly inhibited MLR and in vitro antibody production (PFC), as well as the mitogenic response to lipopolysaccharide E. coli (LPS) of mouse spleen cells, but did not affect the response to an optimal dose of phytohaemagglutinin (PHA). Both supernatant and cell-free extract inhibitory activities were located in 60,000 MW fraction. Inhibitory material of low MW (less than 12,000) was also found in high cell concentration supernatants. A similar suppressive activity was exerted by cell-free extracts of P3 X 63 NS cells used for B-cell hybridization. The suppressive activity seemed to stem from some kind of interaction between BW 5147 cells and the fetal calf serum (FCS) of the culture medium. Supernatants from subclones of BW 5147 cells obtained in selected batches of FCS and maintained in the same serum, even at high cell concentrations, did not affect MLR, whereas the supernatants from the same subclones maintained in other batches definitely suppressed this reaction. Thus, provided that culture conditions are chosen carefully, subclones of BW 5147 devoid of effect on in vitro immune reactions can be obtained.

  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. Genome Sequence of Listeria monocytogenes Plasmid pLM-C-273 Carrying Genes Related to Stress Resistance.

    Science.gov (United States)

    Liang, Lindsay; Gnaneshan, Saravanamuttu; Garduño, Rafael A; Mallo, Gustavo V

    2016-10-13

    Mobile genetic elements in bacteria, such as plasmids, act as important vectors for the transfer of antibiotic resistance, virulence, and metal resistance genes. Here, we report the genome sequence of a new plasmid pLM-C-273, identified in a Listeria monocytogenes strain isolated from a clinical sample in Ontario, Canada.

  3. Genome Sequence of Listeria monocytogenes Plasmid pLM-C-273 Carrying Genes Related to Stress Resistance

    Science.gov (United States)

    Liang, Lindsay; Gnaneshan, Saravanamuttu; Garduño, Rafael A.

    2016-01-01

    Mobile genetic elements in bacteria, such as plasmids, act as important vectors for the transfer of antibiotic resistance, virulence, and metal resistance genes. Here, we report the genome sequence of a new plasmid pLM-C-273, identified in a Listeria monocytogenes strain isolated from a clinical sample in Ontario, Canada. PMID:27738039

  4. DNASU plasmid and PSI:Biology-Materials repositories: resources to accelerate biological research.

    Science.gov (United States)

    Seiler, Catherine Y; Park, Jin G; Sharma, Amit; Hunter, Preston; Surapaneni, Padmini; Sedillo, Casey; Field, James; Algar, Rhys; Price, Andrea; Steel, Jason; Throop, Andrea; Fiacco, Michael; LaBaer, Joshua

    2014-01-01

    The mission of the DNASU Plasmid Repository is to accelerate research by providing high-quality, annotated plasmid samples and online plasmid resources to the research community through the curated DNASU database, website and repository (http://dnasu.asu.edu or http://dnasu.org). The collection includes plasmids from grant-funded, high-throughput cloning projects performed in our laboratory, plasmids from external researchers, and large collections from consortia such as the ORFeome Collaboration and the NIGMS-funded Protein Structure Initiative: Biology (PSI:Biology). Through DNASU, researchers can search for and access detailed information about each plasmid such as the full length gene insert sequence, vector information, associated publications, and links to external resources that provide additional protein annotations and experimental protocols. Plasmids can be requested directly through the DNASU website. DNASU and the PSI:Biology-Materials Repositories were previously described in the 2010 NAR Database Issue (Cormier, C.Y., Mohr, S.E., Zuo, D., Hu, Y., Rolfs, A., Kramer, J., Taycher, E., Kelley, F., Fiacco, M., Turnbull, G. et al. (2010) Protein Structure Initiative Material Repository: an open shared public resource of structural genomics plasmids for the biological community. Nucleic Acids Res., 38, D743-D749.). In this update we will describe the plasmid collection and highlight the new features in the website redesign, including new browse/search options, plasmid annotations and a dynamic vector mapping feature that was developed in collaboration with LabGenius. Overall, these plasmid resources continue to enable research with the goal of elucidating the role of proteins in both normal biological processes and disease.

  5. Structural similarity and distribution of small cryptic plasmids of Lactobacillus curvatus and L. sake.

    Science.gov (United States)

    Vogel, R F; Lohmann, M; Weller, A N; Hugas, M; Hammes, W P

    1991-11-15

    Plasmid profiles of strains of Lactobacillus curvatus and L. sake isolated from meat or sauerkraut were analysed to investigate plasmid homology and distribution in relation to the ecology of these organisms in fermenting foods. A hybridisation probe was constructed by cloning of pLc2, a cryptic, 2.6-kbp plasmid from L. curvatus LTH683, into the Escherichia coli plasmid pRV50. In Southern hybridisations with the digoxygenine labeled pLc2 probe, pLc2-related small plasmids were frequently detected in meat-borne strains of L. casei subsp. pseudoplantarum, L. curvatus, L. sake, L. alimentarius, L. farciminis and L. halotolerans and in L. curvatus and L. sake isolated from sauerkraut. Among 27 Lactobacillus type strains originally isolated from habitats other than meat this type of homology was detected only with plasmids of L. buchneri and L. mali. Restriction-enzyme mapping of six small cryptic plasmids from L. curvatus and L. sake revealed strong structural homology but no similarity to previously characterized plasmids of lactobacilli. The presence of a variable region in addition to a conserved one and the occurrence of deletions during cloning of pLc2 suggest that vectors derived from these plasmids are likely to be structurally unstable.

  6. Enhanced Replication of Hepatitis E Virus Strain 47832c in an A549-Derived Subclonal Cell Line

    Science.gov (United States)

    Schemmerer, Mathias; Apelt, Silke; Trojnar, Eva; Ulrich, Rainer G.; Wenzel, Jürgen J.; Johne, Reimar

    2016-01-01

    Hepatitis E virus (HEV) is a human pathogen with increasing importance. The lack of efficient cell culture systems hampers systematic studies on its replication cycle, virus neutralization and inactivation. Here, several cell lines were inoculated with the HEV genotype 3c strain 47832c, previously isolated from a chronically infected transplant patient. At 14 days after inoculation the highest HEV genome copy numbers were found in A549 cells, followed by PLC/PRF/5 cells, whereas HepG2/C3A, Huh-7 Lunet BLR and MRC-5 cells only weakly supported virus replication. Inoculation of A549-derived subclone cell lines resulted in most cases in reduced HEV replication. However, the subclone A549/D3 was susceptible to lower virus concentrations and resulted in higher virus yields as compared to parental A549 cells. Transcriptome analysis indicated a downregulation of genes for carcinoembryonic antigen-related cell adhesion molecules (CEACAM) 5 and 6, and an upregulation of the syndecan 2 (SDC2) gene in A549/D3 cells compared to A549 cells. However, treatment of A549/D3 cells or A549 cells with CEACAM- or syndecan 2-specific antisera did not influence HEV replication. The results show that cells supporting more efficient HEV replication can be selected from the A549 cell line. The specific mechanisms responsible for the enhanced replication remain unknown. PMID:27690085

  7. Effects of the presence of ColE1 plasmid DNA in Escherichia coli on the host cell metabolism

    Directory of Open Access Journals (Sweden)

    Węgrzyn Alicja

    2006-11-01

    Full Text Available Abstract Background Although understanding of physiological interactions between plasmid DNA and its host is important for vector design and host optimization in many biotechnological applications, to our knowledge, global studies on plasmid-host interactions have not been performed to date even for well-characterized plasmids. Results Escherichia coli cells, either devoid of plasmid DNA or bearing plasmid pOri1 (with a single ColE1 replication origin or plasmid pOri2 (with double ColE1 replication origins, were cultured in a chemostat. We used a combination of metabolic flux analysis, DNA microarray and enzyme activity analysis methods to explore differences in the metabolism between these strains. We found that the presence of plasmids significantly influenced various metabolic pathways in the host cells, e.g. glycolysis, the tricarboxylic acid (TCA cycle and the pentose phosphate (PP pathway. Expression of rpiA, a gene coding for ribose-5-phosphate isomerase A, was considerably decreased in E. coli carrying a high copy number plasmid relative to E. coli carrying a low copy number plasmid and plasmid-free E. coli. The rpiA gene was cloned into an expression vector to construct plasmid pETrpiA. Following induction of pETrpiA-bearing E. coli, which harbored either pOri1 or pOri2, with isopropyl-β-D-thiogalactopyranoside (IPTG, the copy number of pOri1 and pOri2 was sigificantly higher than that measured in a host devoid of pETrpiA. Conclusion The presence of plasmids can significantly influence some metabolic pathways in the host cell. We believe that the results of detailed metabolic analysis may be useful in optimizing host strains, vectors and cultivation conditions for various biotechnological purposes.

  8. Characterization of subpopulations (clones and subclones of the 21 SF strain of Trypanosoma cruzi after long lasting maintenance in the laboratory

    Directory of Open Access Journals (Sweden)

    Rozalia MF Campos

    1996-12-01

    Full Text Available Several studies have shown a clonal structure of Trypanosoma cruzi and its possible correlation with the behavioral heterogeneity of the parasite strains. In the present study, the 21 SF strain, that have been maintained in laboratory by successive passages in mice, for more than 15 years, showing a stability of biological and isoenzymic characteristics has been cloned, with the objective of establishing the characters of its clones and subclones. With the technique of isolation of a single parasite from the blood of infected mice, 5 clones and 14 subclones have been obtained. After four passages into mice, inoculum of 10(5 was obtained for each clone and subclone and inoculated into mice weighing 10 to 12 g. These were used for the study of the biological behavior of the clones: evolution of parasitemia, morphology of blood forms and host mortality. For isoenzymic characterization, the clones and subclones were analyzed for ALAT, ASAT, GPI and PGM enzymes. Results have shown that the 5 clones and the 14 subclones disclosed a biological behavior similar to the parental strain, with minor variability of the parasitemic profiles and also the same isoenzymic patterns. These results confirm the stability of the 21 SF strain and indicate a clonal homogeneity of its populations. This is compatible with the hypothesis that the T. cruzi strains represent an equilibrium of either homogenous or heterogeneous populations.

  9. 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...

  10. Novel cloning vectors for Bacillus thuringiensis.

    OpenAIRE

    Baum, J A; Coyle, D M; Gilbert, M P; Jany, C S; Gawron-Burke, C

    1990-01-01

    Seven replication origins from resident plasmids of Bacillus thuringienis subsp. kurstaki HD263 and HD73 were cloned in Escherichia coli. Three of these replication origins, originating from plasmids of 43, 44, and 60 MDa, were used to construct a set of compatible shuttle vectors that exhibit structural and segregational stability in the Cry- strain B. thuringiensis HD73-26. These shuttle vectors, pEG597, pEG853, and pEG854, were designed with rare restriction sites that permit various adapt...

  11. Low-dose plasmid DNA treatment increases plasma vasopressin and regulates blood pressure in experimental endotoxemia

    Directory of Open Access Journals (Sweden)

    Malardo Thiago

    2012-11-01

    Full Text Available Abstract Background Although plasmid DNA encoding an antigen from pathogens or tumor cells has been widely studied as vaccine, the use of plasmid vector (without insert as therapeutic agent requires further investigation. Results Here, we showed that plasmid DNA (pcDNA3 at low doses inhibits the production of IL-6 and TNF-α by lipopolysaccharide (LPS-stimulated macrophage cell line J774. These findings led us to evaluate whether plasmid DNA could act as an anti-inflammatory agent in a Wistar rat endotoxemia model. Rats injected simultaneously with 1.5 mg/kg of LPS and 10 or 20 μg of plasmid DNA had a remarkable attenuation of mean arterial blood pressure (MAP drop at 2 hours after treatment when compared with rats injected with LPS only. The beneficial effect of the plasmid DNA on MAP was associated with decreased expression of IL-6 in liver and increased concentration of plasma vasopressin (AVP, a known vasoconstrictor that has been investigated in hemorrhagic shock management. No difference was observed in relation to nitric oxide (NO production. Conclusion Our results demonstrate for the first time that plasmid DNA vector at low doses presents anti-inflammatory property and constitutes a novel approach with therapeutic potential in inflammatory diseases.

  12. Identification of resistance mechanisms in erlotinib-resistant subclones of the non-small cell lung cancer cell line HCC827 by exome sequencing

    DEFF Research Database (Denmark)

    Jacobsen, Kirstine; Alcaraz, Nicolas; Lund, Rikke Raaen

    Background: Erlotinib (Tarceva®, Roche) has significantly changed the treatment of non-small cell lung cancer (NSCLC) as 70% of patients show significant tumor regression upon treatment (Santarpia et. al., 2013). However, all patients relapse due to development of acquired resistance, which...... mutations in erlotinib-resistant subclones of the NSCLC cell line, HCC827. Materials & Methods: We established 3 erlotinib-resistant subclones (resistant to 10, 20, 30 µM erlotinib, respectively). DNA libraries of each subclone and the parental HCC827 cell line were prepared in biological duplicates using...... exhibited a significant difference in viability over a time course of 25 days when treated with erlotinib. Importantly, the resistant clones did not acquire the T790M or other EGFR or KRAS mutations, potentiating the identification of novel resistance mechanisms in these clones. For the sensitive and the 3...

  13. Anion exchange purification of plasmid DNA using expanded bed adsorption.

    Science.gov (United States)

    Ferreira, G N; Cabral, J M; Prazeres, D M

    2000-01-01

    Recent developments in gene therapy with non-viral vectors and DNA vaccination have increased the demand for large amounts of pharmaceutical-grade plasmid DNA. The high viscosity of process streams is of major concern in the purification of plasmids, since it can cause high back pressures in column operations, thus limiting the throughput. In order to avoid these high back pressures, expanded bed anion exchange chromatography was evaluated as an alternative to fixed bed chromatography. A Streamline 25 column filled with 100 ml of Streamline QXL media, was equilibrated with 0.5 M NaCl in TE (10 mM Tris, 1 mM EDTA, pH = 8.0) buffer at an upward flow of 300 cmh-1, E. coli lysates (obtained from up to 3 liters of fermentation broth) were injected in the column. After washing out the unbound material, the media was allowed to sediment and the plasmid was eluted with 1 M NaCl in TE buffer at a downward flow of 120 cmh-1. Purification factors of 36 +/- 1 fold, 26 +/- 0.4 plasmid purity, and close to 100% yields were obtained when less than one settled column volume of plasmid feed was injected. However, both recovery yield and purity abruptly decreased when larger amounts were processed-values of 35 +/- 2 and 5 +/- 0.7 were obtained for the recovery yield and purity, respectively, when 250 ml of feedstock were processed. In these cases, gel clogging and expansion collapse were observed. The processing of larger volumes, thus larger plasmid quantities, was only possible by performing an isopropanol precipitation step prior to the chromatographic step. This step led to an enhancement of the purification step.

  14. Plasmids of Carotenoid-Producing Paracoccus spp. (Alphaproteobacteria) - Structure, Diversity and Evolution

    Science.gov (United States)

    Maj, Anna; Dziewit, Lukasz; Czarnecki, Jakub; Wlodarczyk, Miroslawa; Baj, Jadwiga; Skrzypczyk, Grazyna; Giersz, Dorota; Bartosik, Dariusz

    2013-01-01

    Plasmids are components of many bacterial genomes. They enable the spread of a large pool of genetic information via lateral gene transfer. Many bacterial strains contain mega-sized replicons and these are particularly common in Alphaproteobacteria. Considerably less is known about smaller alphaproteobacterial plasmids. We analyzed the genomes of 14 such plasmids residing in 4 multireplicon carotenoid-producing strains of the genus Paracoccus (Alphaproteobacteria): P. aestuarii DSM 19484, P. haeundaensis LG P-21903, P. marcusii DSM 11574 and P. marcusii OS22. Comparative analyses revealed mosaic structures of the plasmids and recombinational shuffling of diverse genetic modules involved in (i) plasmid replication, (ii) stabilization (including toxin-antitoxin systems of the relBE/parDE, tad-ata, higBA, mazEF and toxBA families) and (iii) mobilization for conjugal transfer (encoding relaxases of the MobQ, MobP or MobV families). A common feature of the majority of the plasmids is the presence of AT-rich sequence islets (located downstream of exc1-like genes) containing genes, whose homologs are conserved in the chromosomes of many bacteria (encoding e.g. RelA/SpoT, SMC-like proteins and a retron-type reverse transcriptase). The results of this study have provided insight into the diversity and plasticity of plasmids of Paracoccus spp., and of the entire Alphaproteobacteria. Some of the identified plasmids contain replication systems not described previously in this class of bacteria. The composition of the plasmid genomes revealed frequent transfer of chromosomal genes into plasmids, which significantly enriches the pool of mobile DNA that can participate in lateral transfer. Many strains of Paracoccus spp. have great biotechnological potential, and the plasmid vectors constructed in this study will facilitate genetic studies of these bacteria. PMID:24260361

  15. Subcloning and Sequencing of DNA Fragment Related to Salt Tolerance in Sinorhizobium fredii RT19%费氏中华根瘤菌与耐盐有关的DNA片段的亚克隆和测序

    Institute of Scientific and Technical Information of China (English)

    卞学琳; 葛世超; 杨苏声

    2000-01-01

    将费氏中华根瘤菌(Sinorhizobium fredii) RT19与耐盐有关的23kb DNA片段用BamH酶切成大小不同的长度,分别与质粒pML122连接,然后转化大肠杆菌(Escherichia coli) S17-1,筛选出3个转化子。以这些转化子为供体, RT19的盐敏感突变株RC3-3为受体,分别 进行二亲本杂交,筛选到接合子BR2,得到4.4kb与耐盐有关的DNA片段。根据其物理图谱,酶切成6个DNA片段,并分别连接到质粒pUC18进行测序。测序分析表明,该4.4kb DNA片段含有fixO、fixN基因和3个开放阅读框(ORF)。%A 23kb DNA fragment related to salt tolerance was obtained from the gene library of S. fredii strain RT19. In this study, BamH was selected to digest 23kb DNA fragment into different length of DNA fragments. The resulting fragments were ligated with plasmid pML122, then the recombinant plasmids were transformed to competent cells of E. coli S17-1 on selective medium and three transformants TR were obtained. Two-Parental mating experiments were carried out with these transformants as donor and salt sensitive S. fredii strain RC3-3 as recipient, and the transconjugant BR2 was selected on FY plates containing gentamycin and 0.4mol/L NaCl. Thus, a 4.4kb DNA fragment related to salt tolerance was obtained. Based on its physical map, six restriction fragments were subcloned into plasmid pUC18 for DNA sequencing. Subsequently, sequencing and analysis of 4.4kb DNA fragment showed that fixO, fixN genes and three ORFs were obtained.

  16. The replication origin of a repABC plasmid

    Directory of Open Access Journals (Sweden)

    Cevallos Miguel A

    2011-06-01

    Full Text Available Abstract Background repABC operons are present on large, low copy-number plasmids and on some secondary chromosomes in at least 19 α-proteobacterial genera, and are responsible for the replication and segregation properties of these replicons. These operons consist, with some variations, of three genes: repA, repB, and repC. RepA and RepB are involved in plasmid partitioning and in the negative regulation of their own transcription, and RepC is the limiting factor for replication. An antisense RNA encoded between the repB-repC genes modulates repC expression. Results To identify the minimal region of the Rhizobium etli p42d plasmid that is capable of autonomous replication, we amplified different regions of the repABC operon using PCR and cloned the regions into a suicide vector. The resulting vectors were then introduced into R. etli strains that did or did not contain p42d. The minimal replicon consisted of a repC open reading frame under the control of a constitutive promoter with a Shine-Dalgarno sequence that we designed. A sequence analysis of repC revealed the presence of a large A+T-rich region but no iterons or DnaA boxes. Silent mutations that modified the A+T content of this region eliminated the replication capability of the plasmid. The minimal replicon could not be introduced into R. etli strain containing p42d, but similar constructs that carried repC from Sinorhizobium meliloti pSymA or the linear chromosome of Agrobacterium tumefaciens replicated in the presence or absence of p42d, indicating that RepC is an incompatibility factor. A hybrid gene construct expressing a RepC protein with the first 362 amino acid residues from p42d RepC and the last 39 amino acid residues of RepC from SymA was able to replicate in the presence of p42d. Conclusions RepC is the only element encoded in the repABC operon of the R. etli p42d plasmid that is necessary and sufficient for plasmid replication and is probably the initiator protein. The ori

  17. 大鼠生精相关基因TSARG1原核表达载体的构建和表达%Construction and Expression of pGEX-KG/TSARG1 Recombinant Vector in Rats

    Institute of Scientific and Technical Information of China (English)

    张懿; 刘刚

    2011-01-01

    Objective To construct pGEX- KG/TSARG1 recombinant vector in rats. Methods The open reading frame (ORF) of TSARG1 was amplified from rats' testis RNA by RT - PCR. The products were cloned into pUCm - T vectors and then sequenced. Then the recombinant plasmid was digested and subcloned into pGEX - KG vector. The recombiant plasmids were identified by DNA sequence analysis end transformed into component E. coli BL21 cells, the GST/TSARG1 fusion protein was expressed with IPTG induction. Results The pGEX - KG/TSARG1 recombinant vector was successfully constructed. The GST/TSARG1 fusion protein was expressed abundantly after IPTG induction. Conclusions pGEX- KG/TSARG1 recombinant vector is constructed successfully, which may facilitate further investigation of the role of TSARG1 in spermatogenesis.%目的 构建大鼠生精相关基因pGEX-KG/TSARG1重组载体并进行原核表达.方法 应用RT-PCR技术从大鼠睾丸组织mRNA中扩增TSARG1的开放阅读框(ORF),T-A克隆后将TSARGI插入到原核表达载体丙EX-KG中,经测序鉴定后将重组质粒转入宿主菌E.coli BL21,IPTG诱导表达GST/TSARG1融合蛋白.结果 成功构建pGEX一KG/TSARG1重组质粒;转化重组质粒的E.coli BL21经IPTG 37℃诱导高效表达GST/TSARG1融合蛋白.结论 pGEX一KG/TSARG1原核表达载体的成功构建为进一步研究TSARG1的生物学功能奠定了基础.

  18. Production of lentiviral vectors

    Directory of Open Access Journals (Sweden)

    Otto-Wilhelm Merten

    2016-01-01

    Full Text Available Lentiviral vectors (LV have seen considerably increase in use as gene therapy vectors for the treatment of acquired and inherited diseases. This review presents the state of the art of the production of these vectors with particular emphasis on their large-scale production for clinical purposes. In contrast to oncoretroviral vectors, which are produced using stable producer cell lines, clinical-grade LV are in most of the cases produced by transient transfection of 293 or 293T cells grown in cell factories. However, more recent developments, also, tend to use hollow fiber reactor, suspension culture processes, and the implementation of stable producer cell lines. As is customary for the biotech industry, rather sophisticated downstream processing protocols have been established to remove any undesirable process-derived contaminant, such as plasmid or host cell DNA or host cell proteins. This review compares published large-scale production and purification processes of LV and presents their process performances. Furthermore, developments in the domain of stable cell lines and their way to the use of production vehicles of clinical material will be presented.

  19. Transformation of Azotobacter vinelandii OP with a broad host range plasmid containing a cloned chromosomal nif-DNA marker.

    Science.gov (United States)

    Bingle, W H

    1988-05-01

    The non-nitrogen-fixing (Nif-) strain UW10 of Azotobacter vinelandii OP (UW) was naturally induced to competence and transformed with broad host range plasmid pKT210 containing the cloned wild-type nif-10 locus from A. vinelandii UW (Nif+); this marker was unable to complement the nif-10 mutation in trans, but could through recombination with the chromosome. The most frequent type of transformation event observed was recombination between the homologous regions of the plasmid and chromosome (producing Nif+ transformants) with loss of the plasmid vector. At a substantially lower frequency, transformants expressing the plasmid-encoded antibiotic resistance determinants were isolated which were phenotypically Nif-. Agarose gel electrophoresis showed that these transformants contained a plasmid migrating with the same mobility as the original donor plasmid. During culture these transformants acquired a Nif+ phenotype without the loss of the plasmid, as judged by the use of a hybridization probe specific for the cloned nif-DNA fragment. These data indicate that plasmids carrying sequences homologous to chromosomal sequences could be maintained in recombination-proficient A. vinelandii UW. The introduction of plasmids containing sequences homologous to chromosomal sequences was facilitated by prelinearization of the plasmid using a restriction endonuclease generating cohesive ends. Because the site of linearization could be chosen outside the region of shared homology, it was unlikely that the route of plasmid establishment occurred via a homology-facilitated transformation mechanism. The data also indicated that A. vinelandii UW could harbor broad host range cloning vectors based on plasmid RSF1010 without significant impairment of its nitrogen-fixation ability.

  20. [Chromatographic separation of plasmid DNA by anion-exchange cryogel].

    Science.gov (United States)

    Guo, Yantao; Shen, Shaochuan; Yun, Junxian; Yao, Kejian

    2012-08-01

    Plasmid DNA (pDNA) is used as an important vector for gene therapy, and its wide application is restricted by the purity and yield. To obtain high-purity pDNA, a chromatographic method based on anion-exchange supermacroporous cryogel was explored. The anion-exchange cryogel was prepared by grafting diethylaminoethyl-dextran to the epoxide groups of polyacrylamide-based matrix and pUC19 plasmid was used as a target to test the method. The plasmid was transferred into Escherichia coli DH5alpha, cultivated, harvested and lysed. The obtained culture was centrifuged and the supernatant was used as the plasmid feedstock, which was loaded into the anion-exchange cryogel bed for chromatographic separation. By optimizing the pH of running buffer and the elution conditions, high-purity pDNA was obtained by elution with 0.5 mol/L sodium chloride solution at pH 6.6. Compared to the traditional methods for purification of pDNA, animal source enzymes and toxic reagents were not involved in the present separation process, ensuring the safety of both the purification operations and the obtained pDNA.

  1. About vectors

    CERN Document Server

    Hoffmann, Banesh

    1975-01-01

    From his unusual beginning in ""Defining a vector"" to his final comments on ""What then is a vector?"" author Banesh Hoffmann has written a book that is provocative and unconventional. In his emphasis on the unresolved issue of defining a vector, Hoffmann mixes pure and applied mathematics without using calculus. The result is a treatment that can serve as a supplement and corrective to textbooks, as well as collateral reading in all courses that deal with vectors. Major topics include vectors and the parallelogram law; algebraic notation and basic ideas; vector algebra; scalars and scalar p

  2. Vector analysis

    CERN Document Server

    Newell, Homer E

    2006-01-01

    When employed with skill and understanding, vector analysis can be a practical and powerful tool. This text develops the algebra and calculus of vectors in a manner useful to physicists and engineers. Numerous exercises (with answers) not only provide practice in manipulation but also help establish students' physical and geometric intuition in regard to vectors and vector concepts.Part I, the basic portion of the text, consists of a thorough treatment of vector algebra and the vector calculus. Part II presents the illustrative matter, demonstrating applications to kinematics, mechanics, and e

  3. Co-resident plasmids travel together.

    Science.gov (United States)

    Gama, João Alves; Zilhão, Rita; Dionisio, Francisco

    2017-08-24

    Conjugative plasmids encode genes that enable them to transfer, by conjugation, from a given host cell to another cell. Conjugative transfer, despite being an important feature of conjugative plasmids, is not constitutive for most plasmids, the reason being that genes involved in horizontal transfer are mostly repressed. Only upon their transient de-repression are plasmids able to transfer horizontally. If host cells harbour multiple plasmids, their simultaneous transfer depends on simultaneous transient de-repression of all plasmids. If de-repression of different plasmids was random and independent events, simultaneous de-repression should be a rare event because the probability of simultaneous de-repression would be the product of the probabilities of de-repression of each plasmid. Some previous observations support this hypothesis, while others show that co-transfer of plasmids is more frequent than this reasoning indicates. Here, we show that co-transfer of multiple plasmids mainly results from non-independent events: the probability that all plasmids within a cell become de-repressed is much higher than if de-repression of plasmids genes were independent. We found a simple model for the probability of co-transfer: the plasmid having the lowest conjugation rates is the one who limits co-transfer. In this sense, cells receiving the plasmid with the lower transfer rate also receive the other plasmid. If de-repression happens simultaneously on co-resident plasmids, common cues may stimulate de-repression of distinct plasmids. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. The Chlamydophila felis plasmid is highly conserved.

    Science.gov (United States)

    Harley, Ross; Day, Sarinder; Di Rocco, Camillo; Helps, Chris

    2010-11-20

    The presence of a plasmid in the Chlamydiaceae is both species and strain specific. Knowledge of the prevalence of the plasmid in different Chlamydia species is important for future studies aiming to investigate the role of the plasmid in chlamydial biology and disease. Although strains of Chlamydophila felis with or without the plasmid have been identified, only a small number of laboratory-adapted strains have been analysed and the prevalence of the plasmid in field isolates has not been determined. This study aimed to determine the prevalence of the plasmid in C. felis-positive conjunctival and oropharyngeal clinical samples submitted for routine diagnosis of C. felis by real-time (Q)PCR. DNA extracts from four laboratory-adapted strains were also analysed. QPCR assays targeting regions of C. felis plasmid genes pCF01, pCF02 and pCF03 were developed for the detection of plasmid DNA. QPCR analysis of DNA extracts from C. felis-positive clinical samples found evidence of plasmid DNA in 591 of 595 samples representing 561 of 564 (99.5%) clinical cases. Plasmid DNA was also detected by QPCR in laboratory-adapted strains 1497V, K2487 and K2490, but not strain 905. We conclude that the plasmid is highly conserved in C. felis, and plasmid-deficient strains represent a rare but important population for future studies of chlamydial plasmid function.

  5. Quantification of Plasmid Copy Number with Single Colour Droplet Digital PCR.

    Science.gov (United States)

    Plotka, Magdalena; Wozniak, Mateusz; Kaczorowski, Tadeusz

    2017-01-01

    Bacteria can be considered as biological nanofactories that manufacture a cornucopia of bioproducts most notably recombinant proteins. As such, they must perfectly match with appropriate plasmid vectors to ensure successful overexpression of target genes. Among many parameters that correlate positively with protein productivity plasmid copy number plays pivotal role. Therefore, development of new and more accurate methods to assess this critical parameter will result in optimization of expression of plasmid-encoded genes. In this study, we present a simple and highly accurate method for quantifying plasmid copy number utilizing an EvaGreen single colour, droplet digital PCR. We demonstrate the effectiveness of this method by examining the copy number of the pBR322 vector within Escherichia coli DH5α cells. The obtained results were successfully validated by real-time PCR. However, we observed a strong dependency of the plasmid copy number on the method chosen for isolation of the total DNA. We found that application of silica-membrane-based columns for DNA purification or DNA isolation with use of bead-beating, a mechanical cell disruption lead to determination of an average of 20.5 or 7.3 plasmid copies per chromosome, respectively. We found that recovery of the chromosomal DNA from purification columns was less efficient than plasmid DNA (46.5 ± 1.9% and 87.4 ± 5.5%, respectively) which may lead to observed differences in plasmid copy number. Besides, the plasmid copy number variations dependent on DNA template isolation method, we found that droplet digital PCR is a very convenient method for measuring bacterial plasmid content. Careful determination of plasmid copy number is essential for better understanding and optimization of recombinant proteins production process. Droplet digital PCR is a very precise method that allows performing thousands of individual PCR reactions in a single tube. The ddPCR does not depend on running standard curves and is a

  6. Sample displacement chromatography of plasmid DNA isoforms.

    Science.gov (United States)

    Černigoj, Urh; Martinuč, Urška; Cardoso, Sara; Sekirnik, Rok; Krajnc, Nika Lendero; Štrancar, Aleš

    2015-10-02

    Sample displacement chromatography (SDC) is a chromatographic technique that utilises different relative binding affinities of components in a sample mixture and has been widely studied in the context of peptide and protein purification. Here, we report a use of SDC to separate plasmid DNA (pDNA) isoforms under overloading conditions, where supercoiled (sc) isoform acts as a displacer of open circular (oc) or linear isoform. Since displacement is more efficient when mass transfer between stationary and mobile chromatographic phases is not limited by diffusion, we investigated convective interaction media (CIM) monoliths as stationary phases for pDNA isoform separation. CIM monoliths with different hydrophobicities and thus different binding affinities for pDNA (CIM C4 HLD, CIM-histamine and CIM-pyridine) were tested under hydrophobic interaction chromatography (HIC) conditions. SD efficiency for pDNA isoform separation was shown to be dependent on column selectivity for individual isoform, column efficiency and on ammonium sulfate (AS) concentration in loading buffer (binding strength). SD and negative mode elution often operate in parallel, therefore negative mode elution additionally influences the efficiency of the overall purification process. Optimisation of chromatographic conditions achieved 98% sc pDNA homogeneity and a dynamic binding capacity of over 1mg/mL at a relatively low concentration of AS. SDC was successfully implemented for the enrichment of sc pDNA for plasmid vectors of different sizes, and for separation of linear and and sc isoforms, independently of oc:sc isoform ratio, and flow-rate used. This study therefore identifies SDC as a promising new approach to large-scale pDNA purification, which is compatible with continuous, multicolumn chromatography systems, and could therefore be used to increase productivity of pDNA production in the future. Copyright © 2015 Elsevier B.V. All rights reserved.

  7. Sequence type 131 fimH30 and fimH41 subclones amongst Escherichia coli isolates in Australia and New Zealand.

    Science.gov (United States)

    Rogers, Benjamin A; Ingram, Paul R; Runnegar, Naomi; Pitman, Matthew C; Freeman, Joshua T; Athan, Eugene; Havers, Sally; Sidjabat, Hanna E; Gunning, Earlleen; De Almeida, Mary; Styles, Kaylene; Paterson, David L

    2015-04-01

    The clonal composition of Escherichia coli causing extra-intestinal infections includes ST131 and other common uropathogenic clones. Drivers for the spread of these clones and risks for their acquisition have been difficult to define. In this study, molecular epidemiology was combined with clinical data from 182 patients enrolled in a case-control study of community-onset expanded-spectrum cephalosporin-resistant E. coli (ESC-R-EC) in Australia and New Zealand. Genetic analysis included antimicrobial resistance mechanisms, clonality by DiversiLab (rep-PCR) and multilocus sequence typing (MLST), and subtyping of ST131 by identification of polymorphisms in the fimH gene. The clonal composition of expanded-spectrum cephalosporin-susceptible E. coli and ESC-R-EC isolates differed, with six MLST clusters amongst susceptible isolates (median 7 isolates/cluster) and three clusters amongst resistant isolates, including 40 (45%) ST131 isolates. Population estimates indicate that ST131 comprises 8% of all E. coli within our population; the fluoroquinolone-susceptible H41 subclone comprised 4.5% and the H30 subclone comprised 3.5%. The H30 subclone comprised 39% of all ESC-R-EC and 41% of all fluoroquinolone-resistant E. coli within our population. Patients with ST131 were also more likely than those with non-ST131 isolates to present with an upper than lower urinary tract infection (RR=1.8, 95% CI 1.01-3.1). ST131 and the H30 subclone were predominant amongst ESC-R-EC but were infrequent amongst susceptible isolates where the H41 subclone was more prevalent. Within our population, the proportional contribution of ST131 to fluoroquinolone resistance is comparable with that of other regions. In contrast, the overall burden of ST131 is low by global standards.

  8. Complete genetic analysis of plasmids carrying mcr-1 and other resistance genes in an Escherichia coli isolate of animal origin.

    Science.gov (United States)

    Li, Ruichao; Xie, Miaomiao; Lv, Jingzhang; Wai-Chi Chan, Edward; Chen, Sheng

    2017-03-01

    To investigate the genetic features of three plasmids recovered from an MCR-1 and ESBL-producing Escherichia coli strain, HYEC7, and characterize the transmission mechanism of mcr-1 . The genetic profiles of three plasmids were determined by PCR, S1-PFGE, Southern hybridization and WGS analysis. The ability of the mcr-1 -bearing plasmid to undergo conjugation was also assessed. The mcr-1 -bearing transposon Tn 6330 was characterized by PCR and DNA sequencing. Complete sequences of three plasmids were obtained. A non-conjugative phage P7-like plasmid, pHYEC7- mcr1 , was found to harbour the mcr-1 -bearing transposon Tn 6330 , which could be excised from the plasmid by generating a circular intermediate harbouring mcr-1 and the IS Apl1 element. The insertion of the circular intermediate into another plasmid, pHYEC7-IncHI2, could form pHNSHP45-2, the original IncHI2-type mcr-1 -carrying plasmid that was reported. The third plasmid, pHYEC7-110, harboured two replicons, IncX1 and IncFIB, and comprised multiple antimicrobial resistance mobile elements, some of which were shared by pHYEC7-IncHI2. The Tn 6330 element located in the phage-like plasmid pHYEC7- mcr1 could be excised from the plasmid and formed a circular intermediate that could be integrated into plasmids containing the IS Apl1 element. This phenomenon indicated that Tn 6330 is a key element responsible for widespread dissemination of mcr-1 among various types of plasmids and bacterial chromosomes. The dissemination rate of such an element may be further enhanced upon translocation into phage-like vectors, which may also be transmitted via transduction events.

  9. Differential behavior of plasmids containing chromosomal DNA insertions of various sizes during transformation and conjugation in Haemophilus influenzae

    Energy Technology Data Exchange (ETDEWEB)

    Balganesh, M.; Setlow, J.K.

    1985-01-01

    Plasmids with chromosomal insertions were constructed by removal of a 1.1-kilobase-pair piece from the 9.8-kilobase-pair vector plasmid pDM2 by EcoRI digestion and inserting in its place various lengths of chromosomal DNA (1.7, 3.4, and 9.0 kilobase pairs) coding for resistance to novobiocin. A fourth plasmid was constructed by insertion of the largest piece of chromosomal DNA into the SmaI site of pDM2. The plasmids without inserts were taken up poorly by competent cells and thus were considered not to contain specific DNA uptake sites. The presence of even the smallest insert of chromosomal DNA caused a large increase in transformation of Rec/sup +/ and Rec/sup -/ strains. The frequency of plasmid establishment in Rec/sup +/ cells by transformation increased exponentially with increasing insert size, but in Rec/sup -/ cells there was less transformation by the larger plasmids. Conjugal transfer of these plasmids was carried out with the 35-kilobase-pair mobilizing plasmid pHD147. The frequency of establishment of plasmids by this method not only was not markedly affected by the presence of the insertions, but also decreased somewhat with increase in insert size and was independent of rec-1 and rec-2 genes. Recombination between plasmid and chromosome was readily detected after transformation, but could not be detected after transconjugation even when the recipient cells were Rec/sup +/ and made competent. These data suggested that there is a special processing of plasmid DNA that enters the competent cells in transformation that makes possible recombination of homologous regions of the plasmid with the chromosome and pairing with the chromosome that aids plasmid establishment.

  10. Plasmid required for virulence of Agrobacterium tumefaciens

    Energy Technology Data Exchange (ETDEWEB)

    Watson, B.; Currier, T.C.; Gordon, M.P.; Chilton, M.D.; Nester, E.W.

    1975-07-01

    The irreversible loss of crown gall-inducing ability of Agrobacterium tumefaciens strain C-58 during growth at 37/sup 0/C is shown to be due to loss of a large plasmid (1.2 x 10/sup 8/ daltons). The gene responsible for this high rate of plasmid loss at elevated temperatures seems to be located on the plasmid. In addition, another spontaneous avirulent variant, A. tumefaciens strain IIBNV6, is shown to lack the virulence plasmid which its virulent sibling strain, IIBV7, possesses. Deoxyribonucleic acid reassociation measurements prove that the plasmid is eliminated, not integrated into the chromosome, in both of the avirulent derivatives. Transfer of virulence from donor strain C-58 to avirulent recipient strain A136 results from the transfer of a plasmid, which appears identical to the donor plasmid by deoxyribonucleic acid reassociation measurements. The transfer of virulence in another cross, K27 x A136, was also shown to result from the transfer of a large plasmid. These findings establish unequivocally that the large plasmid determines virulence. Two additional genetic determinants have been located on the virulence plasmid of A. tumefaciens strain C-58: the ability to utilize nopaline and sensitivity to a bacteriocin produced by strain 84. The latter trait can be exploited for selection of avirulent plasmid-free derivatives of strain C-58. The trait of nopaline utilization appears to be on the virulence plasmid also in strains IIBV7 and K27.

  11. Elementary vectors

    CERN Document Server

    Wolstenholme, E Œ

    1978-01-01

    Elementary Vectors, Third Edition serves as an introductory course in vector analysis and is intended to present the theoretical and application aspects of vectors. The book covers topics that rigorously explain and provide definitions, principles, equations, and methods in vector analysis. Applications of vector methods to simple kinematical and dynamical problems; central forces and orbits; and solutions to geometrical problems are discussed as well. This edition of the text also provides an appendix, intended for students, which the author hopes to bridge the gap between theory and appl

  12. Isolation and characterization of yeast DNA repair genes. II. Isolation of plasmids that complement the mutations rad50-1, rad51-1, rad54-3, and rad55-3

    Energy Technology Data Exchange (ETDEWEB)

    Calderon, I.L.; Contopoulou, C.R.; Mortimer, R.K.

    1983-01-01

    Plasmids that complement the yeast mutations rad50-1, rad51-1, rad54-3, and rad55-3 were obtained by transforming strains that carried a leu2 marker and the particular rad mutation, with YEp 13 plasmids containing near random yeast DNA inserts. Integration of these plasmids or of fragments of these plasmids was accomplished. Genetic studies using the integrants established the presence of the genes RAD50, RAD54 and RAD55 in the respective plasmids. However, a BamHI subclone of the rad50-1 complementing plasmid failed to integrate at the RAD50 locus, indicating that no homology exists between this fragment and the RAD50 gene. A BamHI fragment for the RAD54 plasmid was shown to be internal to the RAD54 gene: its integration within a wild type copy of RAD54 causes the cell to become Rad/sup -/; its excision is X-ray inducible and restores the Rad/sup -/ phenotype. Since cells bearing a disrupted copy of RAD54 are able to survive, the author concludes that this is not essential.

  13. Origin and Evolution of Rickettsial Plasmids.

    Science.gov (United States)

    El Karkouri, Khalid; Pontarotti, Pierre; Raoult, Didier; Fournier, Pierre-Edouard

    2016-01-01

    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 transfer as well as

  14. 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

  15. Transformation of Rhizobia with Broad-Host-Range Plasmids by Using a Freeze-Thaw Method

    OpenAIRE

    Vincze, Eva; Bowra, Steve

    2006-01-01

    Several species of rhizobia were successfully transformed with broad-host-range plasmids of different replicons by using a modified freeze-thaw method. A generic binary vector (pPZP211) was maintained in Mesorhizobium loti without selection and stably inherited during nodulation. The method could extend the potential of rhizobia as a vehicle for plant transformation.

  16. Chlamydophila felis: plasmid detection in Italian isolates.

    Science.gov (United States)

    Di Francesco, Antonietta; Donati, Manuela; Salvatore, Daniela; Cevenini, Roberto; Di Paolo, Maria; Baldelli, Raffaella

    2010-04-01

    Plasmids have been detected in the majority of strains in the genus Chlamydia and in many Chlamydophila species. Previous studies showed that FP Pring and FP Cello Chlamydophila felis strains have an extrachromosomial plasmid, whereas the FP Baker strain does not. Azuma et al. recently sequenced the entire genomic DNA sequence of the Japanese Cp. felis strain Fe/C-56 and described a 7,552 base pair circular plasmid. In the present study a highly conserved plasmid gene was detected in 11 Italian Cp. felis isolates, showing 100% nucleotide identity with the plasmid gene of Fe/C-56 Cp. felis strain.

  17. FabV/Triclosan Is an Antibiotic-Free and Cost-Effective Selection System for Efficient Maintenance of High and Medium -Copy Number Plasmids in Escherichia coli

    OpenAIRE

    Ali, Syed A.; Yik Wei Chew

    2015-01-01

    Antibiotic resistance genes and antibiotics are frequently used to maintain plasmid vectors in bacterial hosts such as Escherichia coli. Due to the risk of spread of antibiotic resistance, the regulatory authorities discourage the use of antibiotic resistance genes/antibiotics for the maintenance of plasmid vectors in certain biotechnology applications. Overexpression of E. coli endogenous fabI gene and subsequent selection on Triclosan has been proposed as a practical alternative to traditio...

  18. 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

  19. Study on the construction of recombinant plasmid coexpressing newcastle disease virus F protein and chicken IL-2

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    This study investigated the protection against the ND in chickens by a recombinant DNA vaccine. A plasmid vector encoding NDV F protein, which is reqired for virus cell fusion and is important for vaccine induced immunity, was used as a model to study how DNA vaccines may be modulated by the simulaneous expression of chicken IL-2. The NDV D26 strain F gene with CMV promotor and BGH polyA signal sequence was amplified by PCR from eukaryotic plasmid pcDNA-F, which contains the full-length NDV F gene, and clond into reconstructed eukaryotic plasmid pcDNA-IL2, which contains chicken IL-2 gene. Restriction endonuclease cleavage and PCR amplification showed that a bicistronic plasmid encoding NDV F gene and chicken IL-2 separately was successfully constructed. Two-week-old SPF chickens were intramuscularly innoculated the recombinant plasmid. Antibody and lymphocyte proliferative assay showed that the humoral and cellular immunity of chickens vaccinated the recombinant plasmid greatly increased compared with those innoculated only plasmid expressing NDV F protein. Challenged with the lethal dose of NDV F48E9 strain, 72% chickens vaccinated recombinant plasmid were survived, and 30% chickens vaccinated plasmid expressing F protein were survived. These results proved the adjuvant effect of chicken IL-2, and further showed that the efficacy of a DNA vaccine can be greatly improved by simultaneous expression of IL-2.

  20. The establishment of 20 different human embryonic stem cell lines and subclones; a report on derivation, culture, characterisation and banking.

    Science.gov (United States)

    Englund, Mikael C O; Caisander, Gunilla; Noaksson, Karin; Emanuelsson, Katarina; Lundin, Kersti; Bergh, Christina; Hansson, Charles; Semb, Henrik; Strehl, Raimund; Hyllner, Johan

    2010-04-01

    This report summarises our efforts in deriving, characterising and banking of 20 different human embryonic stem cell lines. We have derived a large number of human embryonic stem cell lines between 2001 and 2005. One of these cell lines was established under totally xeno-free culture conditions. In addition, several subclones have been established, including a karyoptypical normal clone from a trisomic mother line. A master cell banking system has been utilised in concert with an extensive characterisation programme, ensuring a supply of high quality pluripotent stem cells for further research and development. In this report we also present the first data on a proprietary novel antibody, hES-Cellect, that exhibits high specificity for undifferentiated hES cells. In addition to the traditional manual dissection approach of propagating hES cells, we here also report on the successful approaches of feeder-free cultures as well as single cell cultures based on enzymatic digestion. All culture systems used as reported here have maintained the hES cells in a karyotypical normal and pluripotent state. These systems also have the advantage of being the principal springboards for further scale up of cultures for industrial or clinical applications that would require vastly more cells that can be produced by mechanical means.

  1. Recombinogenic engineering of conjugative plasmids with fluorescent marker cassettes

    DEFF Research Database (Denmark)

    Reisner, A.; Molin, Søren; Zechner, E.L.

    2002-01-01

    functions using very short regions of homology. Initial manipulation of the IncFII target plasmids R1 and R1drd19 indicated that the linear targeting DNA should be devoid of all extraneous homologies to. the target molecule for optimal insertion specificity. Indeed, a simple recombination assay proved...... that in the presence of additional homologous regions in the targeting DNA, strand exchanges occurred exclusively within the longest regions of homology. A versatile panel of vectors was created to facilitate convenient PCR amplification of targeting DNAs containing various combinations of different antibiotic...

  2. Improving DNA vaccine performance through vector design.

    Science.gov (United States)

    Williams, James A

    2014-01-01

    DNA vaccines are a rapidly deployed next generation vaccination platform for treatment of human and animal disease. DNA delivery devices, such as electroporation and needle free jet injectors, are used to increase gene transfer. This results in higher antigen expression which correlates with improved humoral and cellular immunity in humans and animals. This review highlights recent vector and transgene design innovations that improve DNA vaccine performance. These new vectors improve antigen expression, increase plasmid manufacturing yield and quality in bioreactors, and eliminate antibiotic selection and other potential safety issues. A flowchart for designing synthetic antigen transgenes, combining antigen targeting, codon-optimization and bioinformatics, is presented. Application of improved vectors, of antibiotic free plasmid production, and cost effective manufacturing technologies will be critical to ensure safety, efficacy, and economically viable manufacturing of DNA vaccines currently under development for infectious disease, cancer, autoimmunity, immunotolerance and allergy indications.

  3. Vector analysis

    CERN Document Server

    Brand, Louis

    2006-01-01

    The use of vectors not only simplifies treatments of differential geometry, mechanics, hydrodynamics, and electrodynamics, but also makes mathematical and physical concepts more tangible and easy to grasp. This text for undergraduates was designed as a short introductory course to give students the tools of vector algebra and calculus, as well as a brief glimpse into these subjects' manifold applications. The applications are developed to the extent that the uses of the potential function, both scalar and vector, are fully illustrated. Moreover, the basic postulates of vector analysis are brou

  4. Factors affecting plasmid production in Escherichia coli from a resource allocation standpoint

    Directory of Open Access Journals (Sweden)

    Cunningham Drew S

    2009-05-01

    Full Text Available Abstract Background Plasmids are being reconsidered as viable vector alternatives to viruses for gene therapies and vaccines because they are safer, non-toxic, and simpler to produce. Accordingly, there has been renewed interest in the production of plasmid DNA itself as the therapeutic end-product of a bioprocess. Improvement to the best current yields and productivities of such emerging processes would help ensure economic feasibility on the industrial scale. Our goal, therefore, was to develop a stoichiometric model of Escherichia coli metabolism in order to (1 determine its maximum theoretical plasmid-producing capacity, and to (2 identify factors that significantly impact plasmid production. Results Such a model was developed for the production of a high copy plasmid under conditions of batch aerobic growth on glucose minimal medium. The objective of the model was to maximize plasmid production. By employing certain constraints and examining the resulting flux distributions, several factors were determined that significantly impact plasmid yield. Acetate production and constitutive expression of the plasmid's antibiotic resistance marker exert negative effects, while low pyruvate kinase (Pyk flux and the generation of NADPH by transhydrogenase activity offer positive effects. The highest theoretical yield (592 mg/g resulted under conditions of no marker or acetate production, nil Pyk flux, and the maximum allowable transhydrogenase activity. For comparison, when these four fluxes were constrained to wild-type values, yields on the order of tens of mg/g resulted, which are on par with the best experimental yields reported to date. Conclusion These results suggest that specific plasmid yields can theoretically reach 12 times their current experimental maximum (51 mg/g. Moreover, they imply that abolishing Pyk activity and/or transhydrogenase up-regulation would be useful strategies to implement when designing host strains for plasmid

  5. 人CCR5基因真核表达质粒的构建及其鉴定%Construction and characterization of plasmid expressing human CCR5 gene in eukaryotes

    Institute of Scientific and Technical Information of China (English)

    程林; 宋红勇; 吴喜林; 吴稚伟

    2012-01-01

    目的:构建人CCR5基因的真核表达质粒并对其进行功能鉴定.方法:PCR扩增人CCR5基因,将其克隆入真核表达载体pcDNA3.1内,构建含人CCR5基因的真核表达质粒pcDNA3.1-CCR5.使用RT-PCR、流式细胞术和HIV假病毒感染实验的方法,鉴定CCR5在真核细胞中的表达和功能.结果:克隆的人CCR5基因与GenBank中已登记的基因序列100%同源.瞬时转染真核细胞后,RT-PCR在预期的位置检测出目的条带,流式细胞术检测到约25.6%的细胞表达CCR5蛋白,且该蛋白能介导HIV假病毒的感染.结论:成功构建了含人CCR5基因的真核表达质粒.%Objective:To construct and characterize a eukaryotic system for expressing human CCR5 gene. Methods; Human CCR5 gene was amplified by PCR, and subcloned into pcDNA3.1 vector to construct a recombinant plasmid pcDNA3. 1-CCR5. The expression of human CCR5 gene in eukaryotic cells was verified by RT-PCR and flow cytometry. HIV-1 env pseudotyped virus infection assay was used to detect the function of CCR5 gene in eukaryotic cells. Results:The sequence of inserted CCR5 gene fragment was 100% homology compared to human CCR5 gene registered in GenBank. After transfection of eukaryotic cells with pcDNA3. 1-CCR5, the target band was identified by RT-PCR and about 25. 6% of the CCR5 protein was detected by flow cytometry. Furthermore, the protein could mediate HIV pseudotype virus infection. Conclusion:A functional eukaryotic expression plasmid pcDNA3. 1-CCR5 has been established successfully.

  6. Generation of human induced pluripotent stem cells by simple transient transfection of plasmid DNA encoding reprogramming factors

    Directory of Open Access Journals (Sweden)

    Lough John W

    2010-08-01

    Full Text Available Abstract Background The use of lentiviruses to reprogram human somatic cells into induced pluripotent stem (iPS cells could limit their therapeutic usefulness due to the integration of viral DNA sequences into the genome of the recipient cell. Recent work has demonstrated that human iPS cells can be generated using episomal plasmids, excisable transposons, adeno or sendai viruses, mRNA, or recombinant proteins. While these approaches offer an advance, the protocols have some drawbacks. Commonly the procedures require either subcloning to identify human iPS cells that are free of exogenous DNA, a knowledge of virology and safe handling procedures, or a detailed understanding of protein biochemistry. Results Here we report a simple approach that facilitates the reprogramming of human somatic cells using standard techniques to transfect expression plasmids that encode OCT4, NANOG, SOX2, and LIN28 without the need for episomal stability or selection. The resulting human iPS cells are free of DNA integration, express pluripotent markers, and form teratomas in immunodeficient animals. These iPS cells were also able to undergo directed differentiation into hepatocyte-like and cardiac myocyte-like cells in culture. Conclusions Simple transient transfection of plasmid DNA encoding reprogramming factors is sufficient to generate human iPS cells from primary fibroblasts that are free of exogenous DNA integrations. This approach is highly accessible and could expand the use of iPS cells in the study of human disease and development.

  7. Handling S/MAR vectors.

    Science.gov (United States)

    Hagedorn, Claudia; Baiker, Armin; Postberg, Jan; Ehrhardt, Anja; Lipps, Hans J

    2012-06-01

    Nonviral episomal vectors represent attractive alternatives to currently used virus-based expression systems. In the late 1990s, it was shown that a plasmid containing an expression cassette linked to a scaffold/matrix attached region (S/MAR) replicates as a low copy number episome in all cell lines tested, as well as primary cells, and can be used for the genetic modification of higher animals. Once established in the cell, the S/MAR vector replicates early during S-phase and, in the absence of selection, is stably retained in the cells for an unlimited period of time. This vector can therefore be regarded as a minimal model system for studying the epigenetic regulation of replication and functional nuclear architecture. In theory, this construct represents an almost "ideal" expression system for gene therapy. In practice, S/MAR-based vectors stably modify mammalian cells with efficiencies far below those of virus-based constructs. Consequently, they have not yet found application in gene therapy trials. Furthermore, S/MAR vector systems are not trivial to handle and several critical technical issues have to be considered when modifying these vectors for various applications.

  8. An Autonomously Replicating Transforming Vector for Sulfolobus solfataricus

    Science.gov (United States)

    Cannio, Raffaele; Contursi, Patrizia; Rossi, Mosè; Bartolucci, Simonetta

    1998-01-01

    A plasmid able to transform and to be stably maintained both in Sulfolobus solfataricus and in Escherichia coli was constructed by insertion into an E. coli plasmid of the autonomously replicating sequence of the virus particle SSV1 and a suitable mutant of the hph (hygromycin phosphotransferase) gene as the transformation marker. The vector suffered no rearrangement and/or chromosome integration, and its copy number in Sulfolobus was increased by exposure of the cells to mitomycin C. PMID:9620978

  9. Enhanced gene disruption by programmable nucleases delivered by a minicircle vector.

    Science.gov (United States)

    Dad, A-B K; Ramakrishna, S; Song, M; Kim, H

    2014-11-01

    Targeted genetic modification using programmable nucleases such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) is of great value in biomedical research, medicine and biotechnology. Minicircle vectors, which lack extraneous bacterial sequences, have several advantages over conventional plasmids for transgene delivery. Here, for the first time, we delivered programmable nucleases into human cells using transient transfection of a minicircle vector and compared the results with those obtained using a conventional plasmid. Surrogate reporter assays and T7 endonuclease analyses revealed that cells in the minicircle vector group displayed significantly higher mutation frequencies at the target sites than those in the conventional plasmid group. Quantitative PCR and reverse transcription-PCR showed higher vector copy number and programmable nuclease transcript levels, respectively, in 293T cells after minicircle versus conventional plasmid vector transfection. In addition, tryphan blue staining and flow cytometry after annexin V and propidium iodide staining showed that cell viability was also significantly higher in the minicircle group than in the conventional plasmid group. Taken together, our results show that gene disruption using minicircle vector-mediated delivery of ZFNs and TALENs is a more efficient, safer and less toxic method than using a conventional plasmid, and indicate that the minicircle vector could serve as an advanced delivery method for programmable nucleases.

  10. Mutations in the regulatory network underlie the recent clonal expansion of a dominant subclone of the Mycobacterium tuberculosis Beijing genotype.

    Science.gov (United States)

    Schürch, Anita C; Kremer, Kristin; Warren, Robin M; Hung, Nguyen V; Zhao, Yanlin; Wan, Kanglin; Boeree, Martin J; Siezen, Roland J; Smith, Noel H; van Soolingen, Dick

    2011-04-01

    The Beijing genotype family is an epidemiologically important sub-group of Mycobacterium tuberculosis. It has been suggested that the high frequency of the Beijing isolates in some areas could be explained by selective advantages. Some evidence suggests that the emerging and most frequently isolated "Typical Beijing" lineage has the ability to circumvent BCG-induced immunity. To investigate the phylogeny of the Beijing genotype of M. tuberculosis, the genome of six Beijing strains from three different countries was sequenced with next-generation sequencing. The phylogeny of these strains was established using single nucleotide polymorphisms (SNPs). The three Typical Beijing strains clustered very tightly in the Beijing phylogeny suggesting that Typical Beijing strains represent a monophyletic lineage and resulted from recent diversification. Typing of 150 M. tuberculosis strains with a subset of the SNPs and comparison of the IS6110 restriction-fragment length polymorphism (RFLP) patterns of these strains to a database of 1522 Beijing RFLP patterns revealed that about 80% of all Beijing strains belong to the Typical Beijing subclone, which indicates clonal expansion. To identify the genomic changes that are characteristic for all Typical Beijing strains and to reconstruct their most recent common ancestor, the presence of SNPs was assayed in other Beijing strains. We identified 51 SNPs that define the minimal set of polymorphisms for all Typical Beijing strains. Nonsynonymous polymorphisms in genes coding for the regulatory network were over-represented in this set of mutations. We suggest that alterations in the response to environmental signals may have enabled Typical Beijing strains to develop the emerging phenotype.

  11. 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.

  12. Shuttle vectors for cloning recombinant DNA in Escherichia coli and Streptomyces griseofuscus C581.

    OpenAIRE

    Larson, J L; Hershberger, C L

    1984-01-01

    The replicon of the Streptomyces plasmid SCP2 was located on a 5.9-kilobase EcoRI-SalI restriction fragment. The SCP2 replicon was combined with Escherichia coli plasmid pBR322 and genes specifying neomycin resistance and thiostrepton resistance in streptomycetes to construct shuttle vectors that are useful for cloning in E. coli and streptomycetes.

  13. Persistence of Antibiotic Resistance Plasmids in Biofilms

    Science.gov (United States)

    2014-10-01

    plasmids* in*populations*of* Gram > negative *bacteria*grown*in*biofilms*and*well>mixed*liquid*cultures.** * Task2:*Characterize*the*evolution*of*plasmid...R.! Edwards.! 2005.! Overview! of! nosocomial! infections! caused! by! gramP negative ! bacilli .!Clin.!Infect.!Dis.!41:848P854.! LoftiePEaton,!W.,!A... negative ! interaction!between!one!of! its!chromosomal!segments!and!the!plasmid! by!simply!deleting!the!appropriate!chromosomal!segment.!! 7. None

  14. Cyanobacterium sp. host cell and vector for production of chemical compounds in Cyanobacterial cultures

    Energy Technology Data Exchange (ETDEWEB)

    Piven, Irina; Friedrich, Alexandra; Duhring, Ulf; Uliczka, Frank; Baier, Kerstin; Inaba, Masami; Shi, Tuo; Wang, Kui; Enke, Heike; Kramer, Dan

    2016-04-19

    A cyanobacterial host cell, Cyanobacterium sp., that harbors at least one recombinant gene for the production of a chemical compounds is provided, as well as vectors derived from an endogenous plasmid isolated from the cell.

  15. Cyanobacterium sp. host cell and vector for production of chemical compounds in cyanobacterial cultures

    Science.gov (United States)

    Piven, Irina; Friedrich, Alexandra; Duhring, Ulf; Uliczka, Frank; Baier, Kerstin; Inaba, Masami; Shi, Tuo; Wang, Kui; Enke, Heike; Kramer, Dan

    2014-09-30

    A cyanobacterial host cell, Cyanobacterium sp., that harbors at least one recombinant gene for the production of a chemical compounds is provided, as well as vectors derived from an endogenous plasmid isolated from the cell.

  16. Novel cloning vectors for Bacillus thuringiensis.

    Science.gov (United States)

    Baum, J A; Coyle, D M; Gilbert, M P; Jany, C S; Gawron-Burke, C

    1990-11-01

    Seven replication origins from resident plasmids of Bacillus thuringienis subsp. kurstaki HD263 and HD73 were cloned in Escherichia coli. Three of these replication origins, originating from plasmids of 43, 44, and 60 MDa, were used to construct a set of compatible shuttle vectors that exhibit structural and segregational stability in the Cry- strain B. thuringiensis HD73-26. These shuttle vectors, pEG597, pEG853, and pEG854, were designed with rare restriction sites that permit various adaptations, including the construction of small recombinant plasmids lacking antibiotic resistance genes. The cryIA(c) and cryIIA insecticidal crystal protein genes were inserted into these vectors to demonstrate crystal protein production in B. thuringiensis. Introduction of a cloned cryIA(c) gene from strain HD263 into a B. thuringiensis subsp. aizawai strain exhibiting good insecticidal activity against Spodoptera exigua resulted in a recombinant strain with an improved spectrum of insecticidal activity. Shuttle vectors of this sort should be valuable in future genetic studies of B. thuringiensis as well as in the development of B. thuringiensis strains for use as microbial pesticides.

  17. DNA Ministrings: Highly Safe and Effective Gene Delivery Vectors

    Directory of Open Access Journals (Sweden)

    Nafiseh Nafissi

    2014-01-01

    Full Text Available Conventional plasmid DNA vectors play a significant role in gene therapy, but they also have considerable limitations: they can elicit adverse immune responses because of bacterial sequences they contain for maintenance and amplification in prokaryotes, their bioavailability is compromised because of their large molecular size, and they may be genotoxic. We constructed an in vivo platform to produce ministring DNA—mini linear covalently closed DNA vectors—that are devoid of unwanted bacterial sequences and encode only the gene(s of interest and necessary eukaryotic expression elements. Transfection of rapidly and slowly dividing human cells with ministring DNA coding for enhanced green fluorescent protein resulted in significantly improved transfection, bioavailability, and cytoplasmic kinetics compared with parental plasmid precursors and isogenic circular covalently closed DNA counterparts. Ministring DNA that integrated into the genome of human cells caused chromosomal disruption and apoptotic death of possibly oncogenic vector integrants; thus, they may be safer than plasmid and circular DNA vectors.

  18. Mechanism of acquisition of chromosomal markers by plasmids in Haemophilus influenzae

    Energy Technology Data Exchange (ETDEWEB)

    Setlow, J.K.; Cabrera-Juarez, E.; Griffin, K.

    1984-11-01

    The hybrid plasmid pNov1 readily acquired genetic information from the chromosome of wild-type, but not rec-2, cells. Most of the recombination had taken place 1 h after entrance of the plasmid into the cell, as judged by transformation of rec-2 by lysates made from wild-type cells exposed to pNova. Measurement of physical transfer from radioactively labeled cellular DNA to plasmids recombining in wild-type cells failed, since there was little more radioactivity in plasmids from such cells than from labeled rec-2 recipients, in which no recombination took place. EcoRI digestion of pNov1 divided the DNA into a 1.7-kilobase-pair fragment containing the novobiocin resistance marker and a 13-kilobase-pair fragment containing all of the original vector and considerable portions homologous to the chromosome. Transformation by the large fragment alone resulted in a plasmid the size of the original pNov1. The hypothesis to explain the data is that genetic transfer from chromosome to plasmid took place by a copy choice mechanism.

  19. Integration host factor is required for replication of pYGK-derived plasmids in Aggregatibacter actinomycetemcomitans.

    Science.gov (United States)

    Torres-Escobar, Ascención; Juárez-Rodríguez, María D; Demuth, Donald R

    2014-08-01

    In this study, we show that integration host factor protein (IHF) is required for replication of pYGK plasmids in Aggregatibacter actinomycetemcomitans. YGK plasmids were not replicated in A. actinomycetemcomitans strains lacking either the α- or β- subunit of IHF. However, the deletion mutants were complemented, and plasmid replication was restored when the promoter region and gene for either ihfA or ihfB was cloned into pYGK. We also identified two motifs that resemble the consensus IHF-binding site in a 813-bp fragment containing the pYGK origin of replication. Using electrophoretic mobility shift assays, purified IHFα-IHFβ protein complex was shown to bind to probes containing either of these motifs. To our knowledge, this is the first report showing that plasmid replication is IHF-dependent in the family Pasteurellaceae. In addition, using site-direct mutagenesis, the XbaI and KpnI restriction sites in the suicide vector pJT1 were modified to generate plasmid pJT10. The introduction of these new unique sites in pJT10 facilitates the transfer of transcriptional or translational lacZ fusion constructs for the generation of single-copy chromosomal insertion of the reporter construct. Plasmid pJT10 and its derivatives will be useful for genetic studies in Aggregatibacter (Actinobacillus) and probably other genera of Pasteurellaceae, including Haemophilus, Pasteurella, and Mannheimia.

  20. Construction of Txt5 Adenovirus Vector and Analysis of Its Expression in Cadiomyocytes%Txt5腺病毒载体构建及其在心肌细胞中的表达

    Institute of Scientific and Technical Information of China (English)

    彭浩; 陈宇; 李佑锋; 朱旋; 周作琼; 吴秀山; 范雄伟; 李永青

    2015-01-01

    设计小鼠 Txt5基因的特异性引物,将小鼠 cDNA 作为模板,用 PCR 扩增出 mTxt5编码区,并加入 HA标签序列.将这一片段插入 pMD -18T 载体,再亚克隆至 pAdtrack - cmv 穿梭载体上.线性化后,转化 BJ5183感受态细胞,在 BJ5183中发生同源重组获得 pAd - Txt5质粒.pAd - Txt5线性化后转染293A 细胞,包装得到含 Txt5基因的病毒.将病毒溶液感染大鼠原代心肌细胞,一段时间后观察绿色荧光,然后通过 RT - PCR 和免疫印迹法检测 HA 标签蛋白的表达.结果表明成功构建小鼠 Txt5腺病毒表达载体并实现在大鼠原代心肌细胞中的表达.%Specific primers for the mouse Txt5 gene were designed and employed to amplify the coding sequence using mouse cDNAs as the template .HA - tagged sequence was induced into the PCR primers .The PCR product was inserted into the pMD -18T vector and subcloned into pAd - track - cmv shuttle vector .The recombinant plasmid was linearized and transformed into bacteria BJ5183 .After homologous recombination in the bacteria ,plasmid pAd - Txt5 was obtained .Linearized pAd - Txt5 was transfected into 293 A cells .Then , the recombinant virus containing Txt5 was obtained after packaging . Cardiomyocytes were infected with the collected virus fluid .To detect the expression of Txt5 ,green fluorescence protein (GFP) was observed by green fluorescence and RT - PCR , immunoblotting was performed . The results showed that mouse Txt5 adenovirus vector was constructed successfully ,and it could induce the sequence coding Txt5 - HA into cardiomyocytes .

  1. Use of human MAR elements to improve retroviral vector production.

    Science.gov (United States)

    Buceta, M; Galbete, J L; Kostic, C; Arsenijevic, Y; Mermod, N

    2011-01-01

    Retroviral vectors have many favorable properties for gene therapies, but their use remains limited by safety concerns and/or by relatively lower titers for some of the safer self-inactivating (SIN) derivatives. In this study, we evaluated whether increased production of SIN retroviral vectors can be achieved from the use of matrix attachment region (MAR) epigenetic regulators. Two MAR elements of human origin were found to increase and to stabilize the expression of the green fluorescent protein transgene in stably transfected HEK-293 packaging cells. Introduction of one of these MAR elements in retroviral vector-producing plasmids yielded higher expression of the viral vector RNA. Consistently, viral titers obtained from transient transfection of MAR-containing plasmids were increased up to sixfold as compared with the parental construct, when evaluated in different packaging cell systems and transfection conditions. Thus, use of MAR elements opens new perspectives for the efficient generation of gene therapy vectors.

  2. Plasmid profiles of Moraxella bovis isolates.

    Science.gov (United States)

    McDonald, T J; Pugh, G W

    1986-04-01

    Two-hundred isolates of Moraxella bovis were selected at random and examined for the presence of plasmid DNA by a rapid alkaline-detergent lysis method. All isolates contained from 1 to 6 plasmids, with varying agarose-gel electrophoretic migration patterns. Most (80%) isolates carried 2 to 4 plasmids, which ranged in molecular weight from 2.6 to 80 megadaltons. Seemingly, plasmid profiles can be used as a simple, reliable epizootiologic tool to establish a strain identification scheme for M bovis.

  3. Plasmid transfer systems in the rhizobia.

    Science.gov (United States)

    Ding, Hao; Hynes, Michael F

    2009-08-01

    Rhizobia are agriculturally important bacteria that can form nitrogen-fixing nodules on the roots of leguminous plants. Agricultural application of rhizobial inoculants can play an important role in increasing leguminous crop yields. In temperate rhizobia, genes involved in nodulation and nitrogen fixation are usually located on one or more large plasmids (pSyms) or on symbiotic islands. In addition, other large plasmids of rhizobia carry genes that are beneficial for survival and competition of rhizobia in the rhizosphere. Conjugative transfer of these large plasmids thus plays an important role in the evolution of rhizobia. Therefore, understanding the mechanism of conjugative transfer of large rhizobial plasmids provides foundations for maintaining, monitoring, and predicting the behaviour of these plasmids during field release events. In this minireview, we summarize two types of known rhizobial conjugative plasmids, including quorum sensing regulated plasmids and RctA-repressed plasmids. We provide evidence for the existence of a third type of conjugative plasmid, including pRleVF39c in Rhizobium leguminosarum bv. viciae strain VF39SM, and we provide a comparison of the different types of conjugation genes found in members of the rhizobia that have had their genomes sequenced so far.

  4. Construction of plant expression plasmid p2355-gtfB containing glucosyltransferase B of Streptococcus mutans%变异链球菌葡糖基转移酶植物表达质粒p2355-gtfB的构建

    Institute of Scientific and Technical Information of China (English)

    陈筑; 刘建国; 马欣荣; 张燕; 唐琳; 吴家媛; 张剑

    2012-01-01

    Objective To construct plant plasmid p2355-gtfB which contains multiple catalytic sites and antigen epitopes of glucosyltransferase of Streptococcus mutans. Methods Plasmid pcDNA3-gtfB was used as template to obtain the gtfB by PCR amplification. Then gtfB was inserted into inter-mediate vector pMD18-T, subcloned from the recombi-nant plasmid T-gtfB to plant expression vector p2355, then p2355-gtfB was transformed into Agrobacterium tumefaciens EHA105 by electroporation. Results Genetic segments 2. 9 kb and 3. 7 kb were obtained through restriction enzyme mapping analysis and DNA sequencing, and inserted gene 3 675 bp was shown in the construction recombinant plasmid p2355-gtfB through series analysis and detection. Conclusion The recombinant plasmid was constructed successfully. This research provided basis for further study on plants vaccine against dental caries.%目的 构建含变异链球菌葡糖基转移酶多个免疫显性抗原表位的植物表达质粒p2355-gtfB,为其转化植物研究奠定物质基础,为可食防龋疫苗研究提供条件.方法 以真核表达质粒pcDNA3-gtfB为模板,通过聚合酶链反应(polymerase chain reaction,PCR)扩增含编码变异链球菌葡糖基转移酶抗原表位的目的基因gtfB.将回收纯化的PCR产物经T-A克隆技术克隆于中间载体pMD18-T,双酶切鉴定插入方向后,将目的基因从中间载体释放,再克隆至高效的植物表达载体p2355,用电转化法转化根癌农杆菌EHA105,对重组质粒阳性克隆株进行筛选与鉴定.结果 通过对重组质粒T-gtfB进行酶切图谱分析,获得2.9kb和3.7 kb的2个片断,将重组质粒p2355-gtfB进行酶切、PCR及测序分析,显示p2355-gtfB中插入基因长度为3 675 bp,基因序列与双脱氧链终止法的测定结果相同,证明植物表达质粒p2355-gtfB构建成功,开放阅读框架正确.结论 本研究成功构建了含变异链球菌多个葡糖基转移酶免疫显性抗原表位编码

  5. Cloning vector

    Science.gov (United States)

    Guilfoyle, R.A.; Smith, L.M.

    1994-12-27

    A vector comprising a filamentous phage sequence containing a first copy of filamentous phage gene X and other sequences necessary for the phage to propagate is disclosed. The vector also contains a second copy of filamentous phage gene X downstream from a promoter capable of promoting transcription in a bacterial host. In a preferred form of the present invention, the filamentous phage is M13 and the vector additionally includes a restriction endonuclease site located in such a manner as to substantially inactivate the second gene X when a DNA sequence is inserted into the restriction site. 2 figures.

  6. Cloning vector

    Science.gov (United States)

    Guilfoyle, Richard A.; Smith, Lloyd M.

    1994-01-01

    A vector comprising a filamentous phage sequence containing a first copy of filamentous phage gene X and other sequences necessary for the phage to propagate is disclosed. The vector also contains a second copy of filamentous phage gene X downstream from a promoter capable of promoting transcription in a bacterial host. In a preferred form of the present invention, the filamentous phage is M13 and the vector additionally includes a restriction endonuclease site located in such a manner as to substantially inactivate the second gene X when a DNA sequence is inserted into the restriction site.

  7. The interplay between relatedness and horizontal gene transfer drives the evolution of plasmid-carried public goods.

    Science.gov (United States)

    Mc Ginty, Sorcha É; Lehmann, Laurent; Brown, Sam P; Rankin, Daniel J

    2013-06-22

    Plasmids carry a wide range of genes that are often involved in bacterial social behaviour. The question of why such genes are frequently mobile has received increasing attention. Here, we use an explicit population genetic approach to model the evolution of plasmid-borne bacterial public goods production. Our findings highlight the importance of both transmission and relatedness as factors driving the evolution of plasmid-borne public goods production. We partition the effects of plasmid transfer of social traits into those of infectivity and the effect of increased relatedness. Our results demonstrate that, owing to its effect on relatedness, plasmid mobility increases the invasion and stability of public goods, in a way not seen in individually beneficial traits. In addition, we show that plasmid transfer increases relatedness when public goods production is rare but this effect declines when production is common, with both scenarios leading to an increase in the frequency of plasmid-borne public goods. Plasmids remain important vectors for the spread of social genes involved in bacterial virulence thus an understanding of their dynamics is highly relevant from a public health perspective.

  8. Plasmids from Food Lactic Acid Bacteria: Diversity, Similarity, and New Developments

    Directory of Open Access Journals (Sweden)

    Yanhua Cui

    2015-06-01

    Full Text Available 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.

  9. Effect of chromosome homology an plasmid transformation and plasmid conjugal transfer in Haemophilus influenzae

    Energy Technology Data Exchange (ETDEWEB)

    Balganesh, M.; Setlow, J.K.

    1984-05-14

    The pairing between plasmid and the homologous part of the chromosome associated with plasmid establishment may differ from the pairing which results from integration of a homologous region of the plasmid into the chromosome. Thus the rate of novobiocin transformation decreases with duplication of the chromosomal portion in pMB2, but the rate of establishment of the plasmid increases with this duplication. A model to explain these data is given. 17 references, 5 figures, 4 tables.

  10. The expression of a plasmid-specified exported protein causes structural plasmid instability in Bacillus subtilis

    NARCIS (Netherlands)

    Cordes, C.; Meima, R; Twiest, B; Kazemier, B; Venema, G; vanDijl, JM; Bron, S

    The rolling-circle plasmid pGP1 was used to study the effects of the expression of a plasmid-specified exported protein on structural plasmid stability in Bacillus subtilis. pGP1 contains a fusion between the Bacillus licheniformis penP gene, encoding a C-terminally truncated penicillinase, and the

  11. hLMO3基因真核表达载体的构建及蛋白的表达和定位%Construction of eucaryotic plasmid of human LMO3 Gene and the expression and localization of fusion protein

    Institute of Scientific and Technical Information of China (English)

    顾卉; 佟宇鑫; 刘彤; 李妍; 李丹妮; 袁正伟

    2011-01-01

    Objective To construct the expression plasmid of human LIM domain only 3 (hLMO3) gene and identify the expression and localization of its fusion protein in human HEK293 cells. Methods The hLMO3 coding sequence was amplified by polymerase chain reaction by using cDNA library derived from human fetal brain as the template and subcloned into pEGFP vector. After the target region was identified by enzyme digestion and sequencing, the plasmid was transfected into HEK293 cells. The expression of the recombinant plasmid in HEK293 cells was detected by Western blot. The localization of pEGFP-LMO3 in HEK293 cells was observed with laser scanning confocal microscopy. Results hLMO3 was constructed into the expressing vector pEGFP successfully, the length of the fragment identified by restriction enzyme digestion was 440bp. The expression of pEGFP-LMO3 fusion protein with a molecular weight of 42 kDa was detected by Western blot in human HEK293 cells. The pEGFP-LMO3 fusion protein was mostly localized in the nucleus of HEK293 cells. Conclusion The recombinant plasmid of hLMO3 gene was successfully cloned into eukaryotic expressing vector, and the pEGFP-LMO3 fusion protein was mostly localized in the nucleus of HEK293 cells.%目的 构建hLMO3真核表达载体并证实融合蛋白在细胞内的表达及定位.方法 以人胎脑文库cDNA为模板,PCR扩增hLMO3基因cDNA全长,亚克隆至pEGFP表达载体中.将构建的重组质粒进行酶切测序鉴定,并转染到人上皮细胞HEK293细胞中,提取细胞蛋白进行Western blot检测.利用激光扫描共聚焦显微镜观察pEGFP-hLMO3在HEK293细胞内的定位.结果 hLMO3基因cDNA全长克隆到了真核表达载体pEGFP中,酶切鉴定片段为440 bp;Western blot检测到融合蛋白在HEK293细胞表达,分子量约为42kDa,pEGFP-LMO3在人HEK293细胞中主要定位于细胞核内.结论 成功构建了hLMO3基因cDNA全长的真核表达载体,pEGFP-LMO3蛋白在HEK293细胞中主要定位于细胞核内.

  12. The heat-shock DnaK protein is required for plasmid R1 replication and it is dispensable for plasmid ColE1 replication.

    Science.gov (United States)

    Giraldo-Suárez, R; Fernández-Tresguerres, E; Díaz-Orejas, R; Malki, A; Kohiyama, M

    1993-01-01

    Plasmid R1 replication in vitro is inactive in extracts prepared from a dnaK756 strain but is restored to normal levels upon addition of purified DnaK protein. Replication of R1 in extracts of a dnaKwt strain can be specifically inhibited with polyclonal antibodies against DnaK. RepA-dependent replication of R1 in dnaK756 extracts supplemented with DnaKwt protein at maximum concentration is partially inhibited by rifampicin and it is severely inhibited at sub-optimal concentrations of DnaK protein. The copy number of a run-away R1 vector is reduced in a dnaK756 background at 30 degrees C and at 42 degrees C the amplification of the run-away R1 vector is prevented. However a runaway R1 vector containing dnaK gene allows the amplification of the plasmid at high temperature. These data indicate that DnaK is required for both in vitro and in vivo replication of plasmid R1 and show a partial compensation for the low level of DnaK by RNA polymerase. In contrast ColE1 replication is not affected by DnaK as indicated by the fact that ColE1 replicates with the same efficiency in extracts from dnaKwt and dnaK756 strains. Images PMID:8265367

  13. Construction of recombinant eukaryotic expression plasmid containing murine CD40 ligand gene and its expression in H22 cells

    Institute of Scientific and Technical Information of China (English)

    Yong-Fang Jiang; Yan He; Guo-Zhong Gong; Jun Chen; Chun-Yan Yang; Yun Xu

    2005-01-01

    AIM: To construct a recombinant murine CD40 ligand (mCD40L) eukaryotic expression vector for gene therapy and target therapy of hepatocellular carcinoma (HCC).METHODS: mCD40L cDNA was synthesized by RT-PCR with the specific primers and directly cloned into T vector to generate middle recombinant. After digestion with restriction endonuclease, the target fragment was subcloned into the multi-clone sites of the eukaryotic vector. The constructed vector was verified by enzyme digestion and sequencing,and the product expressed was detected by RT-PCR and immunofluorescence methods.RESULTS: The full-length mCD40L-cDNA was successfully cloned into the eukaryotic vector through electrophoresis,and mCD40L gene was integrated into the genome of infected H22 cells by RT-PCR. Murine CD40L antigen molecule was observed in the plasma of mCD40L-H22 by indirect immuno-fluorescence staining.CONCLUSION: The recombined mCD40L eukaryotic expression vector can be expressed in H22 cell line. It providesexperimental data for gene therapy and target therapy ofhepatocellular carcinoma.

  14. A Series of Medium and High Copy Number Arabinose-Inducible Escherichia coli Expression Vectors Compatible with pBR322 and pACYC184

    OpenAIRE

    Chakravartty, Vandana; Cronan, John E.

    2015-01-01

    The original pBAD24 plasmid and the derived lower copy number (the pBAD322 series) expression vectors have been widely used in Escherichia coli, Salmonella enterica, and related bacteria. However, a flexible pBAD expression system has been available only in pMB1 (ColE1) vectors. We report a series of pBAD vectors that replicate using the origin of plasmid RSF1030 that are compatible with pMB1 (ColE1) and p15A (pACYC) vectors. Both high (≥pBAD24) and medium (~pBAD322) copy number plasmids enco...

  15. 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.

  16. A low-copy-number plasmid for retrieval of toxic genes from BACs and generation of conditional targeting constructs.

    Science.gov (United States)

    Na, Giyoun; Wolfe, Andrew; Ko, Chemyong; Youn, Hyesook; Lee, Young-Min; Byun, Sung June; Jeon, Iksoo; Koo, Yongbum

    2013-06-01

    Bacterial Artificial Chromosome (BAC) clones are widely used for retrieving genomic DNA sequences for gene targeting. In this study, low-copy-number plasmids pBAC-FB, pBAC-FC, and pBAC-DE, which carry the F plasmid replicon, were generated from pBACe3.6. pBAC-FB was successfully used to retrieve a sequence of a BAC that was resistant to retrieval by a high-copy-number plasmid via λ Red-mediated recombineering (gap-repair cloning). This plasmid was also used to retrieve two other genes from BAC, indicating its general usability retrieving genes from BAC. The retrieved genes were manipulated in generating targeting vectors for gene knockouts by recombineering. The functionality of the targeting vector was further validated in a targeting experiment with C57BL/6 embryonic stem cells. The low-copy-number plasmid pBAC-FB is a plasmid of choice to retrieve toxic DNA sequences from BACs and to manipulate them to generate gene-targeting constructs by recombineering.

  17. Shuttle vectors for the archaebacterium Halobacterium volcanii.

    OpenAIRE

    Lam, W. L.; Doolittle, W F

    1989-01-01

    Progress in archaebacterial molecular biology requires tools for genetic analysis. We describe vectors that can be selected and maintained in either Halobacterium volcanii or Escherichia coli. A genetic determinant for resistance to the 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor mevinolin was isolated by "shotgun cloning" into a derivative of the endogenous H. volcanii plasmid pHV2, to form pWL2, which transforms sensitive H. volcanii to mevinolin resistance at high frequency. ...

  18. High-throughput Binary Vectors for Plant Gene Function Analysis

    Institute of Scientific and Technical Information of China (English)

    Zhi-Yong Lei; Ping Zhao; Min-Jie Cao; Rong Cui; Xi Chen; Li-Zhong Xiong; Qi-Fa Zhang; David J. Oliver; Cheng-Bin Xiang

    2007-01-01

    A series of high-throughput binary cloning vectors were constructed to facilitate gene function analysis in higher plants. This vector series consists of plasmids designed for plant expression, promoter analysis, gene silencing,and green fluorescent protein fusions for protein localization. These vectors provide for high-throughput and efficient cloning utilizing sites for λ phage integrase/excisionase. In addition, unique restriction sites are incorporated in a multiple cloning site and enable promoter replacement. The entire vector series are available with complete sequence information and detailed annotations and are freely distributed to the scientific community for non-commercial uses.

  19. Vector geometry

    CERN Document Server

    Robinson, Gilbert de B

    2011-01-01

    This brief undergraduate-level text by a prominent Cambridge-educated mathematician explores the relationship between algebra and geometry. An elementary course in plane geometry is the sole requirement for Gilbert de B. Robinson's text, which is the result of several years of teaching and learning the most effective methods from discussions with students. Topics include lines and planes, determinants and linear equations, matrices, groups and linear transformations, and vectors and vector spaces. Additional subjects range from conics and quadrics to homogeneous coordinates and projective geom

  20. SubClonal Hierarchy Inference from Somatic Mutations: Automatic Reconstruction of Cancer Evolutionary Trees from Multi-region Next Generation Sequencing.

    Directory of Open Access Journals (Sweden)

    Noushin Niknafs

    2015-10-01

    Full Text Available Recent improvements in next-generation sequencing of tumor samples and the ability to identify somatic mutations at low allelic fractions have opened the way for new approaches to model the evolution of individual cancers. The power and utility of these models is increased when tumor samples from multiple sites are sequenced. Temporal ordering of the samples may provide insight into the etiology of both primary and metastatic lesions and rationalizations for tumor recurrence and therapeutic failures. Additional insights may be provided by temporal ordering of evolving subclones--cellular subpopulations with unique mutational profiles. Current methods for subclone hierarchy inference tightly couple the problem of temporal ordering with that of estimating the fraction of cancer cells harboring each mutation. We present a new framework that includes a rigorous statistical hypothesis test and a collection of tools that make it possible to decouple these problems, which we believe will enable substantial progress in the field of subclone hierarchy inference. The methods presented here can be flexibly combined with methods developed by others addressing either of these problems. We provide tools to interpret hypothesis test results, which inform phylogenetic tree construction, and we introduce the first genetic algorithm designed for this purpose. The utility of our framework is systematically demonstrated in simulations. For most tested combinations of tumor purity, sequencing coverage, and tree complexity, good power (≥ 0.8 can be achieved and Type 1 error is well controlled when at least three tumor samples are available from a patient. Using data from three published multi-region tumor sequencing studies of (murine small cell lung cancer, acute myeloid leukemia, and chronic lymphocytic leukemia, in which the authors reconstructed subclonal phylogenetic trees by manual expert curation, we show how different configurations of our tools can

  1. Detection of Clonal and Subclonal Copy-Number Variants in Cell-Free DNA from Patients with Breast Cancer Using a Massively Multiplexed PCR Methodology

    OpenAIRE

    Eser Kirkizlar; Bernhard Zimmermann; Tudor Constantin; Ryan Swenerton; Bin Hoang; Nicholas Wayham; Babiarz, Joshua E; Zachary Demko; Pelham, Robert J.; Stephanie Kareht; Simon, Alexander L.; Kristine N. Jinnett; Matthew Rabinowitz; Styrmir Sigurjonsson; Matthew Hill

    2015-01-01

    We demonstrate proof-of-concept for the use of massively multiplexed PCR and next-generation sequencing (mmPCR-NGS) to identify both clonal and subclonal copy-number variants (CNVs) in circulating tumor DNA. This is the first report of a targeted methodology for detection of CNVs in plasma. Using an in vitro model of cell-free DNA, we show that mmPCR-NGS can accurately detect CNVs with average allelic imbalances as low as 0.5%, an improvement over previously reported whole-genome sequencin...

  2. STUDY REGARDING EFFICIENCY OF INDUCED GENETIC TRANSFORMATION IN BACILLUS LICHENIFORMIS WITH PLASMID DNA

    Directory of Open Access Journals (Sweden)

    VINTILĂ T.

    2007-01-01

    Full Text Available A strain of Bacillus licheniformis was subject to genetic transformation with plasmidvectors (pLC1 and pNC61, using electroporation technique, protoplasttransformation and bivalent cations (CaCl2 mediated transformation. In the case oftransformation by electroporation of Bacillus licheniformis B40, the highest numberof transformed colonies (3 were obtained only after a 1,79 KV electric shock, for 2,2milliseconds. Using this transformation technique we have obtained six kanamycinresistant transformants. The frequency of Bacillus licheniformis B40 protoplaststransformation 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, whichconfers trimethoprim resistance, does not integrate in receiver cells by protoplasttransformation. The direct genetic transformation in the presence of bivalent cations(CaCl2, mediated by pLC1 and pNC61 plasmid vectors, produce a lowtransformation frequency. Using this technique, we have obtained three trimethoprimresistant colonies and four kanamycin resistant colonies. The chemical way oftransformation is the only technique, which realizes the integration of pNC61 in B.licheniformis B40 cells.

  3. Advances in Non-Viral DNA Vectors for Gene Therapy

    Directory of Open Access Journals (Sweden)

    Cinnamon L. Hardee

    2017-02-01

    Full Text Available Uses of viral vectors have thus far eclipsed uses of non-viral vectors for gene therapy delivery in the clinic. Viral vectors, however, have certain issues involving genome integration, the inability to be delivered repeatedly, and possible host rejection. Fortunately, development of non-viral DNA vectors has progressed steadily, especially in plasmid vector length reduction, now allowing these tools to fill in specifically where viral or other non-viral vectors may not be the best options. In this review, we examine the improvements made to non-viral DNA gene therapy vectors, highlight opportunities for their further development, address therapeutic needs for which their use is the logical choice, and discuss their future expansion into the clinic

  4. Advances in Non-Viral DNA Vectors for Gene Therapy

    Science.gov (United States)

    Hardee, Cinnamon L.; Arévalo-Soliz, Lirio Milenka; Hornstein, Benjamin D.; Zechiedrich, Lynn

    2017-01-01

    Uses of viral vectors have thus far eclipsed uses of non-viral vectors for gene therapy delivery in the clinic. Viral vectors, however, have certain issues involving genome integration, the inability to be delivered repeatedly, and possible host rejection. Fortunately, development of non-viral DNA vectors has progressed steadily, especially in plasmid vector length reduction, now allowing these tools to fill in specifically where viral or other non-viral vectors may not be the best options. In this review, we examine the improvements made to non-viral DNA gene therapy vectors, highlight opportunities for their further development, address therapeutic needs for which their use is the logical choice, and discuss their future expansion into the clinic. PMID:28208635

  5. BglBrick vectors and datasheets: A synthetic biology platform for gene expression

    Directory of Open Access Journals (Sweden)

    Lee Taek

    2011-09-01

    Full Text Available Abstract Background As engineered biological systems become more complex, it is increasingly common to express multiple operons from different plasmids and inducible expression systems within a single host cell. Optimizing such systems often requires screening combinations of origins of replication, expression systems, and antibiotic markers. This procedure is hampered by a lack of quantitative data on how these components behave when more than one origin of replication or expression system are used simultaneously. Additionally, this process can be time consuming as it often requires the creation of new vectors or cloning into existing but disparate vectors. Results Here, we report the development and characterization of a library of expression vectors compatible with the BglBrick standard (BBF RFC 21. We have designed and constructed 96 BglBrick-compatible plasmids with a combination of replication origins, antibiotic resistance genes, and inducible promoters. These plasmids were characterized over a range of inducer concentrations, in the presence of non-cognate inducer molecules, and with several growth media, and their characteristics were documented in a standard format datasheet. A three plasmid system was used to investigate the impact of multiple origins of replication on plasmid copy number. Conclusions The standardized collection of vectors presented here allows the user to rapidly construct and test the expression of genes with various combinations of promoter strength, inducible expression system, copy number, and antibiotic resistance. The quantitative datasheets created for these vectors will increase the predictability of gene expression, especially when multiple plasmids and inducers are utilized.

  6. Transposon insertion reveals pRM, a plasmid of Rickettsia monacensis.

    Science.gov (United States)

    Baldridge, Gerald D; Burkhardt, Nicole Y; Felsheim, Roderick F; Kurtti, Timothy J; Munderloh, Ulrike G

    2007-08-01

    Until the recent discovery of pRF in Rickettsia felis, the obligate intracellular bacteria of the genus Rickettsia (Rickettsiales: Rickettsiaceae) were thought not to possess plasmids. We describe pRM, a plasmid from Rickettsia monacensis, which was detected by pulsed-field gel electrophoresis and Southern blot analyses of DNA from two independent R. monacensis populations transformed by transposon-mediated insertion of coupled green fluorescent protein and chloramphenicol acetyltransferase marker genes into pRM. Two-dimensional electrophoresis showed that pRM was present in rickettsial cells as circular and linear isomers. The 23,486-nucleotide (31.8% G/C) pRM plasmid was cloned from the transformant populations by chloramphenicol marker rescue of restriction enzyme-digested transformant DNA fragments and PCR using primers derived from sequences of overlapping restriction fragments. The plasmid was sequenced. Based on BLAST searches of the GenBank database, pRM contained 23 predicted genes or pseudogenes and was remarkably similar to the larger pRF plasmid. Two of the 23 genes were unique to pRM and pRF among sequenced rickettsial genomes, and 4 of the genes shared by pRM and pRF were otherwise found only on chromosomes of R. felis or the ancestral group rickettsiae R. bellii and R. canadensis. We obtained pulsed-field gel electrophoresis and Southern blot evidence for a plasmid in R. amblyommii isolate WB-8-2 that contained genes conserved between pRM and pRF. The pRM plasmid may provide a basis for the development of a rickettsial transformation vector.

  7. The immunogenicity of viral haemorragic septicaemia rhabdovirus (VHSV) DNA vaccines can depend on plasmid regulatory sequences.

    Science.gov (United States)

    Chico, V; Ortega-Villaizan, M; Falco, A; Tafalla, C; Perez, L; Coll, J M; Estepa, A

    2009-03-18

    A plasmid DNA encoding the viral hemorrhagic septicaemia virus (VHSV)-G glycoprotein under the control of 5' sequences (enhancer/promoter sequence plus both non-coding 1st exon and 1st intron sequences) from carp beta-actin gene (pAE6-G(VHSV)) was compared to the vaccine plasmid usually described the gene expression is regulated by the human cytomegalovirus (CMV) immediate-early promoter (pMCV1.4-G(VHSV)). We observed that these two plasmids produced a markedly different profile in the level and time of expression of the encoded-antigen, and this may have a direct effect upon the intensity and suitability of the in vivo immune response. Thus, fish genetic immunisation assays were carried out to study the immune response of both plasmids. A significantly enhanced specific-antibody response against the viral glycoprotein was found in the fish immunised with pAE6-G(VHSV). However, the protective efficacy against VHSV challenge conferred by both plasmids was similar. Later analysis of the transcription profile of a set of representative immune-related genes in the DNA immunized fish suggested that depending on the plasmid-related regulatory sequences controlling its expression, the plasmid might activate distinct patterns of the immune system. All together, the results from this study mainly point out that the selection of a determinate encoded-antigen/vector combination for genetic immunisation is of extraordinary importance in designing optimised DNA vaccines that, when required for inducing protective immune response, could elicit responses biased to antigen-specific antibodies or cytotoxic T cells generation.

  8. Construction of disarmed Ti plasmids transferable between Escherichia coli and Agrobacterium species.

    Science.gov (United States)

    Kiyokawa, Kazuya; Yamamoto, Shinji; Sakuma, Kei; Tanaka, Katsuyuki; Moriguchi, Kazuki; Suzuki, Katsunori

    2009-04-01

    Agrobacterium-mediated plant transformation has been used widely, but there are plants that are recalcitrant to this type of transformation. This transformation method uses bacterial strains harboring a modified tumor-inducing (Ti) plasmid that lacks the transfer DNA (T-DNA) region (disarmed Ti plasmid). It is desirable to develop strains that can broaden the host range. A large number of Agrobacterium strains have not been tested yet to determine whether they can be used in transformation. In order to improve the disarming method and to obtain strains disarmed and ready for the plant transformation test, we developed a simple scheme to make certain Ti plasmids disarmed and simultaneously maintainable in Escherichia coli and mobilizable between E. coli and Agrobacterium. To establish the scheme in nopaline-type Ti plasmids, a neighboring segment to the left of the left border sequence, a neighboring segment to the right of the right border sequence of pTi-SAKURA, a cassette harboring the pSC101 replication gene between these two segments, the broad-host-range IncP-type oriT, and the gentamicin resistance gene were inserted into a suicide-type sacB-containing vector. Replacement of T-DNA with the cassette in pTiC58 and pTi-SAKURA occurred at a high frequency and with high accuracy when the tool plasmid was used. We confirmed that there was stable maintenance of the modified Ti plasmids in E. coli strain S17-1lambdapir and conjugal transfer from E. coli to Ti-less Agrobacterium strains and that the reconstituted Agrobacterium strains were competent to transfer DNA into plant cells. As the modified plasmid delivery system was simple and efficient, conversion of strains to the disarmed type was easy and should be applicable in studies to screen for useful strains.

  9. Recursive directional ligation by plasmid reconstruction allows rapid and seamless cloning of oligomeric genes.

    Science.gov (United States)

    McDaniel, Jonathan R; Mackay, J Andrew; Quiroz, Felipe García; Chilkoti, Ashutosh

    2010-04-12

    This paper reports a new strategy, recursive directional ligation by plasmid reconstruction (PRe-RDL), to rapidly clone highly repetitive polypeptides of any sequence and specified length over a large range of molecular weights. In a single cycle of PRe-RDL, two halves of a parent plasmid, each containing a copy of an oligomer, are ligated together, thereby dimerizing the oligomer and reconstituting a functional plasmid. This process is carried out recursively to assemble an oligomeric gene with the desired number of repeats. PRe-RDL has several unique features that stem from the use of type IIs restriction endonucleases: first, PRe-RDL is a seamless cloning method that leaves no extraneous nucleotides at the ligation junction. Because it uses type IIs endonucleases to ligate the two halves of the plasmid, PRe-RDL also addresses the major limitation of RDL in that it abolishes any restriction on the gene sequence that can be oligomerized. The reconstitution of a functional plasmid only upon successful ligation in PRe-RDL also addresses two other limitations of RDL: the significant background from self-ligation of the vector observed in RDL, and the decreased efficiency of ligation due to nonproductive circularization of the insert. PRe-RDL can also be used to assemble genes that encode different sequences in a predetermined order to encode block copolymers or append leader and trailer peptide sequences to the oligomerized gene.

  10. Plasmid selection in Escherichia coli using an endogenous essential gene marker

    Directory of Open Access Journals (Sweden)

    Good Liam

    2008-08-01

    Full Text Available Abstract Background Antibiotic resistance genes are widely used for selection of recombinant bacteria, but their use risks contributing to the spread of antibiotic resistance. In particular, the practice is inappropriate for some intrinsically resistant bacteria and in vaccine production, and costly for industrial scale production. Non-antibiotic systems are available, but require mutant host strains, defined media or expensive reagents. An unexplored concept is over-expression of a host essential gene to enable selection in the presence of a chemical inhibitor of the gene product. To test this idea in E. coli, we used the growth essential target gene fabI as the plasmid-borne marker and the biocide triclosan as the selective agent. Results The new cloning vector, pFab, enabled selection by triclosan at 1 μM. Interestingly, pFab out-performed the parent pUC19-ampicillin system in cell growth, plasmid stability and plasmid yield. Also, pFab was toxic to host cells in a way that was reversed by triclosan. Therefore, pFab and triclosan are toxic when used alone but in combination they enhance growth and plasmid production through a gene-inhibitor interaction. Conclusion The fabI-triclosan model system provides an alternative plasmid selection method based on essential gene over-expression, without the use of antibiotic-resistance genes and conventional antibiotics.

  11. Construction of a novel kind of expression plasmid by homologous recombination in Saccharomyces cerevisiae

    Institute of Scientific and Technical Information of China (English)

    CHEN Xiangling; YUAN Hanying; HE Wei; HU Xianghua; LU Hong; LI Yuyang

    2005-01-01

    Based on a previously used plasmid pHC11, a new plasmid pHC11R was constructed. Cutting plasmid pHC11R with proper restriction enzymes, the resulting larger DNA fragment pHC11R' was co-transformed with a PCR amplified expression cassette of human IFNα2b into yeast. By means of the homologous sequences at both ends of two DNA fragments, a novel expression plasmid pHC11R-IFNα2b was formed via homologous recombination in the yeast. Compared with pHC11-IFNα2b, the expression plasmid pHC11R-IFNα2b was smaller in size and in absence of antibiotic resistant gene. The stability and copy number of pHC11R- IFNα2b were greatly increased and the expression level of heterologous protein was improved. As the derivatives of pHC11R, a series of recombination expression vectors pHRs containing different combination of expression elements were developed. This led to a rapid and powerful method for cloning and expressing of different genes in yeast.

  12. Construction of Prokaryotic Expression Vector for pbv220/NT4-ADNF-9

    Institute of Scientific and Technical Information of China (English)

    ZHENG Guo-xi; ZHU Kang; JING Yang; WEI Jun-rong; ZHU Hong-liang

    2008-01-01

    Objective To construct a prokaryotic expression vector bearing fusion gene NT4-ADNF-9 for future studies on genetic therapies for sensorineural deafness. Methods Double strand ADNF-9 Edna was synthesized using asymmetrical primer/templates and ligated to the 3' terminal of signal and leader peptides of neurotrophin 4 (NT4). The fusion gene NT4-ADNF-9, was subeloned into prokaryotic expression vector Pbv220, and named Pbv220/NT4-ADNF-9. DNA sequence of the fusion gene was analyzed. The fusion protein was isolated by SDS-PAGE and its bioactivity was evaluated using primary culture of day 8 chicken embryonic DRGcells. Results The correct sequence of fusion gene NT4-ADNF-9 was successfully subcloned into the Pbv220 vector. The expressed ADNF-9 protein showed its effects in promoting cell survival and neurite growth. Conclusion Prokaryotic expression vector Pbv220/NT4-ADNF-9 was constructed successfully and the expressed fusion protein demonstrated satisfactory bioactivity.

  13. 人源核不均一核糖核蛋白E1真核表达载体在神经细胞中的表达%Expression of recombinant plasmid of homo heterogeneous nuclear ribonucleoprotein E1 in SH-SY5Y cells

    Institute of Scientific and Technical Information of China (English)

    霍丽蓉; 王兰英; 邹俊华; 钟南

    2014-01-01

    背景:人源核不均一核糖核蛋白E1功能广泛,可参与神经系统骨架蛋白的表达。目的:为深入研究其在神经细胞中的作用,构建其真核表达载体,观察其在神经细胞中的表达。方法:利用真核表达载体pcDNATM4/His C,通过亚克隆构建核不均一核糖核蛋白E1-pcDNATM 4/His C重组质粒,经酶切、测序鉴定,通过转染神经细胞SH-SY5Y,采用western-blot,RT-PCR鉴定核不均一核糖核蛋白E1重组质粒的表达,并观察转染细胞的生长现象。结果与结论:成功构建了核不均一核糖核蛋白 E1的真核表达载体,mRNA 和蛋白水平上均证实了该质粒可在神经细胞SH-SY5Y中正确表达。SH-SY5Y细胞在转染核不均一核糖核蛋白E1后表现为加速生长。提示该蛋白对神经细胞的生长发育具有重要的作用。该载体为进一步研究核不均一核糖核蛋白E1在神经系统中的功能提供了前提条件。%BACKGROUND:The functions of homo heterogeneous ribonucleoprotein E1 are very wide. It can participate in the expression of skeleton proteins in the nervous system. OBJECTIVE:To construct the recombinant plasmid of homo heterogeneous ribonucleoprotein E1 and observe its expression in nerve cells for further studying the functions of it in neurocytes. METHODS:Using pcDNATM4/His C, the homo heterogeneous ribonucleoprotein E1 was subcloned into recombinant plasmid E1-pcDNATM 4/His C, fol owed by enzyming and sequencing. After SH-SY5Y cells were transfected with the recombinant plasmid, western blot analysis and real time RT-PCR were used to detect the expression of homo heterogeneous ribonucleoprotein E1 in SH-SY5Y cells. And the growth of SH-SY5Y cells was observed. RESULTS AND CONCLUSION:We successful y constructed the eukaryotic expressed vector of homo heterogeneous ribonucleoprotein E1. The recombinant plasmids were verified to express in SH-SY5Y cells correctly at mRNA and protein levels. And SH-SY5Y cells

  14. ColE1-Plasmid Production in Escherichia coli: Mathematical Simulation and Experimental Validation

    Science.gov (United States)

    Freudenau, Inga; Lutter, Petra; Baier, Ruth; Schleef, Martin; Bednarz, Hanna; Lara, Alvaro R.; Niehaus, Karsten

    2015-01-01

    Plasmids have become very important as pharmaceutical gene vectors in the fields of gene therapy and genetic vaccination in the past years. In this study, we present a dynamic model to simulate the ColE1-like plasmid replication control, once for a DH5α-strain carrying a low copy plasmid (DH5α-pSUP 201-3) and once for a DH5α-strain carrying a high copy plasmid (DH5α-pCMV-lacZ) by using ordinary differential equations and the MATLAB software. The model includes the plasmid replication control by two regulatory RNA molecules (RNAI and RNAII) as well as the replication control by uncharged tRNA molecules. To validate the model, experimental data like RNAI- and RNAII concentration, plasmid copy number (PCN), and growth rate for three different time points in the exponential phase were determined. Depending on the sampled time point, the measured RNAI- and RNAII concentrations for DH5α-pSUP 201-3 reside between 6 ± 0.7 and 34 ± 7 RNAI molecules per cell and 0.44 ± 0.1 and 3 ± 0.9 RNAII molecules per cell. The determined PCNs averaged between 46 ± 26 and 48 ± 30 plasmids per cell. The experimentally determined data for DH5α-pCMV-lacZ reside between 345 ± 203 and 1086 ± 298 RNAI molecules per cell and 22 ± 2 and 75 ± 10 RNAII molecules per cell with an averaged PCN of 1514 ± 1301 and 5806 ± 4828 depending on the measured time point. As the model was shown to be consistent with the experimentally determined data, measured at three different time points within the growth of the same strain, we performed predictive simulations concerning the effect of uncharged tRNA molecules on the ColE1-like plasmid replication control. The hypothesis is that these tRNA molecules would have an enhancing effect on the plasmid production. The in silico analysis predicts that uncharged tRNA molecules would indeed increase the plasmid DNA production. PMID:26389114

  15. ColE1-plasmid production in Escherichia coli: Mathematical Simulation and Experimental Validation

    Directory of Open Access Journals (Sweden)

    Inga eFreudenau

    2015-09-01

    Full Text Available Plasmids have become very important as pharmaceutical gene vectors in the fields of gene therapy and genetic vaccination in the last years. In this study, we present a dynamic model to simulate the ColE1-like plasmid replication control, once for a DH5α-strain carrying a low copy plasmid (DH5α-pSUP 201-3 and once for a DH5α-strain carrying a high copy plasmid (DH5α-pCMV-lacZ by using ordinary differential equations (ODE and the MATLAB software. The model includes the plasmid replication control by two regulatory RNA molecules (RNAI and RNAII as well as the replication control by uncharged tRNA molecules. To validate the model, experimental data like RNAI- and RNAII concentration, plasmid copy number (PCN, and growth rate for three different time points in the exponential phase were determined. Depending on the sampled time point, the measured RNAI and RNAII concentrations for DH5α-pSUP 201-3 reside between 6 ±0.7 to 34 ±7 RNAI molecules per cell and 0.44 ±0.1 to 3 ±0.9 RNAII molecules per cell. The determined plasmid copy numbers (PCN averaged between 46 ±26 to 48 ±30 plasmids per cell. The experimentally determined data for DH5α-pCMV-lacZ reside between 345 ±203 to 1086 ±298 RNAI molecules per cell and 22 ±2 to 75 ±10 RNAII molecules per cell with an averaged PCN of 1514 ±1301 to 5806 ±4828 depending on the measured time point. As the model was shown to be consistent with the experimentally determined data, measured at three different time points within the growth of the same strain, we performed predictive simulations concerning the effect of uncharged tRNA molecules on the ColE1-like plasmid replication control. The hypothesis is that these tRNA molecules would have an enhancing effect on the plasmid production. The in silico analysis predicts that uncharged tRNA molecules would indeed increase the pDNA production.

  16. Detection of Clonal and Subclonal Copy-Number Variants in Cell-Free DNA from Patients with Breast Cancer Using a Massively Multiplexed PCR Methodology

    Science.gov (United States)

    Kirkizlar, Eser; Zimmermann, Bernhard; Constantin, Tudor; Swenerton, Ryan; Hoang, Bin; Wayham, Nicholas; Babiarz, Joshua E.; Demko, Zachary; Pelham, Robert J.; Kareht, Stephanie; Simon, Alexander L.; Jinnett, Kristine N.; Rabinowitz, Matthew; Sigurjonsson, Styrmir; Hill, Matthew

    2015-01-01

    We demonstrate proof-of-concept for the use of massively multiplexed PCR and next-generation sequencing (mmPCR-NGS) to identify both clonal and subclonal copy-number variants (CNVs) in circulating tumor DNA. This is the first report of a targeted methodology for detection of CNVs in plasma. Using an in vitro model of cell-free DNA, we show that mmPCR-NGS can accurately detect CNVs with average allelic imbalances as low as 0.5%, an improvement over previously reported whole-genome sequencing approaches. Our method revealed differences in the spectrum of CNVs detected in tumor tissue subsections and matching plasma samples from 11 patients with stage II breast cancer. Moreover, we showed that liquid biopsies are able to detect subclonal mutations that may be missed in tumor tissue biopsies. We anticipate that this mmPCR-NGS methodology will have broad applicability for the characterization, diagnosis, and therapeutic monitoring of CNV-enriched cancers, such as breast, ovarian, and lung cancer. PMID:26500031

  17. Plasmid typing of Shigella sonnei epidemic strains and molecular relationship of their R-plasmids.

    Science.gov (United States)

    Mendoza, M C; Gonzalez, A J; Mendez, F J; Hardisson, C

    1988-06-01

    We conducted a surveillance program on epidemic and/or endemic Shigella strains in Asturias (Spain), their frequency and dispersion in our community, and their R-plasmids. We analyzed initial isolates of Shigella sonnei from two epidemic outbreaks using antibiotic resistance patterns and plasmid profile analysis as epidemiological markers. We found that the 2 outbreaks were caused by different S. sonnei strains, which respectively carried one and two R-plasmids together with other plasmids. The molecular relationship among these and three other R-plasmids from two S. sonnei strains isolated during a previous outbreak, were studied by restriction enzyme analysis and DNA-DNA hybridizations. We were able to establish different levels of relationship among the six R-plasmids.

  18. Electrotransformation of Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis with various plasmids.

    Science.gov (United States)

    Serror, Pascale; Sasaki, Takashi; Ehrlich, S Dusko; Maguin, Emmanuelle

    2002-01-01

    We describe, for the first time, a detailed electroporation procedure for Lactobacillus delbrueckii. Three L. delbrueckii strains were successfully transformed. Under optimal conditions, the transformation efficiency was 10(4) transformants per microg of DNA. Using this procedure, we identified several plasmids able to replicate in L. delbrueckii and integrated an integrative vector based on phage integrative elements into the L. delbrueckii subsp. bulgaricus chromosome. These vectors provide a good basis for developing molecular tools for L. delbrueckii and open the field of genetic studies in L. delbrueckii.

  19. Using Plasmids as DNA Vaccines for Infectious Diseases.

    Science.gov (United States)

    Tregoning, John S; Kinnear, Ekaterina

    2014-12-01

    DNA plasmids can be used to induce a protective (or therapeutic) immune response by delivering genes encoding vaccine antigens. That naked DNA (without the refinement of coat proteins or host evasion systems) can cross from outside the cell into the nucleus and be expressed is particularly remarkable given the sophistication of the immune system in preventing infection by pathogens. As a result of the ease, low cost, and speed of custom gene synthesis, DNA vaccines dangle a tantalizing prospect of the next wave of vaccine technology, promising individual designer vaccines for cancer or mass vaccines with a rapid response time to emerging pandemics. There is considerable enthusiasm for the use of DNA vaccination as an approach, but this enthusiasm should be tempered by the successive failures in clinical trials to induce a potent immune response. The technology is evolving with the development of improved delivery systems that increase expression levels, particularly electroporation and the incorporation of genetically encoded adjuvants. This review will introduce some key concepts in the use of DNA plasmids as vaccines, including how the DNA enters the cell and is expressed, how it induces an immune response, and a summary of clinical trials with DNA vaccines. The review also explores the advances being made in vector design, delivery, formulation, and adjuvants to try to realize the promise of this technology for new vaccines. If the immunogenicity and expression barriers can be cracked, then DNA vaccines may offer a step change in mass vaccination.

  20. Construction the hairpin RNA recombinant plasmids targeting human Pokemon gene%靶向人Pokemon基因的shRNA重组质粒的构建

    Institute of Scientific and Technical Information of China (English)

    Wenmiao Cao; Tao Zhang; Buhai Wang; Xizhi Zhang; Jinghua Ren; Jie Yao

    2011-01-01

    Objective:The aim of this study was to establish the foundation for studying the role of pokemon gene in tumorigenesis and development by constructing recombinant plasmids that can express small interfering RNA (siRNA) targeting human Pokemon gene. Methods: Hairpin siRNA templates targeting Pokemon gene were synthesized and cloned into plasmid vector psiRNA-H1neo. Three vectors derived siRNAs (psiRNA1, 2, 3) and one mocking psiRNAc (as control) were constructed.The recombinant Pokemon siRNA plasmids were constructed and identified using restrictive enzyme analysis and DNA sequencing. Results: Restrictive enzyme analysis and DNA sequencing revealed the successful construction of siRNA expression plasmids. Conclusion: Constructing siRNA templates targeting Pokemon gene may provide us with practical tools for further study the role of Pokemon gene in the development of some diseases and gene therapy of tumor.

  1. Prevalence and molecular characterization of plasmid- mediated ...

    African Journals Online (AJOL)

    lactamase genes among nosocomial Staphylococcus aureus drug resistance isolates in Taiwan. .... Table 2: Plasmid profiles of the clinical antibiotic-resistant pathogens. Strain. Profile .... Madec J. Characterization of clinical canine methicillin-.

  2. antimicrobial susceptibility and plasmids from escherichia coli ...

    African Journals Online (AJOL)

    2001-10-10

    Oct 10, 2001 ... transmission to humans of E. coli containing antibiotic resistance plasmids ... resistant micro-organisms, which may in turn transfer resistance to .... cells were washed with sterile normal saline to remove leached. Я-lactamase ...

  3. Protein diversity confers specificity in plasmid segregation.

    Science.gov (United States)

    Fothergill, Timothy J G; Barillà, Daniela; Hayes, Finbarr

    2005-04-01

    The ParG segregation protein (8.6 kDa) of multidrug resistance plasmid TP228 is a homodimeric DNA-binding factor. The ParG dimer consists of intertwined C-terminal domains that adopt a ribbon-helix-helix architecture and a pair of flexible, unstructured N-terminal tails. A variety of plasmids possess partition loci with similar organizations to that of TP228, but instead of ParG homologs, these plasmids specify a diversity of unrelated, but similarly sized, partition proteins. These include the proteobacterial pTAR, pVT745, and pB171 plasmids. The ParG analogs of these plasmids were characterized in parallel with the ParG homolog encoded by the pseudomonal plasmid pVS1. Like ParG, the four proteins are dimeric. No heterodimerization was detectable in vivo among the proteins nor with the prototypical ParG protein, suggesting that monomer-monomer interactions are specific among the five proteins. Nevertheless, as with ParG, the ParG analogs all possess significant amounts of unordered amino acid residues, potentially highlighting a common structural link among the proteins. Furthermore, the ParG analogs bind specifically to the DNA regions located upstream of their homologous parF-like genes. These nucleoprotein interactions are largely restricted to cognate protein-DNA pairs. The results reveal that the partition complexes of these and related plasmids have recruited disparate DNA-binding factors that provide a layer of specificity to the macromolecular interactions that mediate plasmid segregation.

  4. 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......-like apparatus in prokaryotes. The identification of chromosomal homologues of the well-characterized plasmid partitioning genes indicates that there could be a general mechanism of bacterial DNA partitioning. Udgivelsesdato: July 1...

  5. Vector velocimeter

    DEFF Research Database (Denmark)

    2012-01-01

    The present invention relates to a compact, reliable and low-cost vector velocimeter for example for determining velocities of particles suspended in a gas or fluid flow, or for determining velocity, displacement, rotation, or vibration of a solid surface, the vector velocimeter comprising a laser...... assembly for emission of a measurement beam for illumination of an object in a measurement volume with coherent light whereby a signal beam emanating from the object in the measurement volume is formed in response to illumination of the object by the measurement beam, a reference beam generator...... detector element signal when the fringe pattern formed by the interfering signal beam and reference beam moves across the first detector array; and a signal processor that is adapted for generation of a velocity signal corresponding to a first velocity component of movement of the object in the measurement...

  6. Vector velocimeter

    DEFF Research Database (Denmark)

    2012-01-01

    for generation of a reference beam, a detector system comprising a first detector arrangement arranged in such a way that the signal beam and the reference beam are incident upon the first detector arrangement with the reference beam propagating at an angle relative to a signal beam, and wherein the first......The present invention relates to a compact, reliable and low-cost vector velocimeter for example for determining velocities of particles suspended in a gas or fluid flow, or for determining velocity, displacement, rotation, or vibration of a solid surface, the vector velocimeter comprising a laser...... assembly for emission of a measurement beam for illumination of an object in a measurement volume with coherent light whereby a signal beam emanating from the object in the measurement volume is formed in response to illumination of the object by the measurement beam, a reference beam generator...

  7. Curing of plasmid pXO1 from Bacillus anthracis using plasmid incompatibility.

    Directory of Open Access Journals (Sweden)

    Xiankai Liu

    Full Text Available The large plasmid pXO1 encoding the anthrax toxin is important for the virulence of Bacillus anthracis. It is essential to cure pXO1 from B. anthracis to evaluate its role in the pathogenesis of anthrax infection. Because conventional methods for curing plasmids (e.g., curing agents or growth at elevated temperatures can induce mutations in the host chromosomal DNA, we developed a specific and reliable method to eliminate pXO1 from B. anthracis using plasmid incompatibility. Three putative replication origins of pXO1 were inserted into a temperature-sensitive plasmid to generate three incompatible plasmids. One of the three plasmids successfully eliminated the large plasmid pXO1 from B. anthracis vaccine strain A16R and wild type strain A16. These findings provided additional information about the replication/partitioning of pXO1 and demonstrated that introducing a small incompatible plasmid can generate plasmid-cured strains of B. anthracis without inducing spontaneous mutations in the host chromosome.

  8. Multiple plasmid interference - Pledging allegiance to my enemy's enemy.

    Science.gov (United States)

    Gama, João Alves; Zilhão, Rita; Dionisio, Francisco

    2017-08-24

    As shown in the previous article, two distinct conjugative plasmids sometimes interact within bacterial cells, implicating changes of transfer rates. In most cases of interactions within bacteria, the transfer of one of the plasmids decreases. Less frequently, the transfer rate of one of the plasmids increases. Here we analyse what happens if three distinct conjugative plasmids colonize the same bacterial cell. Our aim is to understand how interactions between two plasmids affect the transfer rate of the third plasmid. After showing that plasmids interact in 59 out of 84 possible interactions we show that, with some exceptions, if the transfer rate of a plasmid decreases in the presence of a second plasmid, a decrease is also observed in the presence of a third plasmid. Moreover, if the conjugation rate of a plasmid increases in the presence of another, an increase is also observed if there is a third plasmid in the cell. Both types of interactions are mostly independent of the third plasmid's identity, even if sometimes the third plasmid quantitatively distorts the interaction of the other two plasmids. There is a bias towards negative intensifying interactions, which provide good news concerning the spread conjugative plasmids encoding antibiotic-resistance genes and virulence factors. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. Insect cell transformation vectors that support high level expression and promoter assessment in insect cell culture

    Science.gov (United States)

    A somatic transformation vector, pDP9, was constructed that provides a simplified means of producing permanently transformed cultured insect cells that support high levels of protein expression of foreign genes. The pDP9 plasmid vector incorporates DNA sequences from the Junonia coenia densovirus th...

  10. Cloning of C-Terminal of Opioid μ-Receptor and Construction of Its Expression Plasmid for Yeast Two Hybrid System

    Institute of Scientific and Technical Information of China (English)

    YANHui; GONGZe-hui

    2004-01-01

    Aim: To obtain the C-terminal DNA and construct the expression plasmid in yeast two-hybrid. Methods: About 177bp DNA fragment was amplified from the complete sequence of ( receptor by PCR. After being sequenced, the C-terminal fragment was ligased into EcoR I-BamH I site of pGBKT7 vector to form recombinants. The recombinant plasmid

  11. Isolation of T—DNA flanking plant DNA from T—DNA insertional embryo—lethal mutants of Arabidopsis thaliana by plasmid rescue technique

    Institute of Scientific and Technical Information of China (English)

    YAOXIAOLI; JIANGESUN; 等

    1996-01-01

    Three T-DNA insertional embryonic lethal mutants from NASC(The Nottingham Arabidopsis Stock Center) were first checked with their segregation ratio of abortive and normal seeds and the copy number of T-DNA insertion.The N4081 mutant has a segregation ratio of 1:3.04 in average and one T-DNA insertion site according to our assay.It was therefore chosen for further analysis.To isolate the joint fragment of T-DNA and plant DNA,the plasmid rescue technique was used.pEL-7,one of plasmids from left border of T-DNA,which contained pBR322 was selected from ampicillin plate.The T-DNA fragment of pEL-7 was checked by restriction enzyme analysis and Southern Blot.Restriction analysis confirmed the presence of known sites of EcoRI,PstI and PvuII on it.For confirming the presence of flanking plant DNA in this plasmid,pEL-7 DNA was labeled and hybridized with wild type and mutant plant DNA.The Southern Blot indicated the hybridization band in both of them.Furthermore,the junction of T-DNA/plant DNA was subcloned into bluescript SK+ and sequenced by Applied Biosystem 373A sequencer.The results showed the 822 bp fragment contained a 274 bp sequence,which is 99.6%homolog(273bp/274bp) to Ti plasmid pTi 15955,DNA.The bp of left 25 bp border repeat were also found in the juction of T-DNA and Plant DNA. Taken together,pEL-7 should coutain a joint fragment of T-DNA and flanking plant DNA.This plasmid DNA could be used for the isolation of plant gene,which will be helpful to elucidate the relationship between gene function and plant embryo development.

  12. Clostridium perfringens type A–E toxin plasmids

    Science.gov (United States)

    Freedman, John C.; Theoret, James R.; Wisniewski, Jessica A.; Uzal, Francisco A.; Rood, Julian I.; McClane, Bruce A.

    2014-01-01

    Clostridium perfringens relies upon plasmid-encoded toxin genes to cause intestinal infections. These toxin genes are associated with insertion sequences that may facilitate their mobilization and transfer, giving rise to new toxin plasmids with common backbones. Most toxin plasmids carry a transfer of clostridial plasmids locus mediating conjugation, which likely explains the presence of similar toxin plasmids in otherwise unrelated C. perfringens strains. The association of many toxin genes with insertion sequences and conjugative plasmids provides virulence flexibility when causing intestinal infections. However, incompatibility issues apparently limit the number of toxin plasmids maintained by a single cell. PMID:25283728

  13. 人Nanog基因重组腺病毒载体的构建及其在人脐带间充质干细胞中的表达%Construction of recombinant adenovirus vector carrying human Nanog gene and its expression in human umbilical cord mesenchymal stem cells

    Institute of Scientific and Technical Information of China (English)

    孙靖; 徐哲; 钱茜; 严家来; 陈圆; 张徐; 高硕; 钱晖; 许文荣

    2011-01-01

    Objective To construct recombinant adenovirus vector carrying human Nanog gene and transfect human umbilical cord mesenchymal stem cells (hucMSCs). Methods The amplification products of Nanog in polymernse chain reaction (PCR) by using a pair of primers containing the sites of restriction endonuclease Kpn I and Xho I were subcloned into shuttle plasmid pAdtrack-CMV.After analysis of restriction endonuclease and confirmation by sequencing, the recombinant shuttle plasmid pAdTrack-CMV-Nanog was linearized by Pme I , and then transformed into E. coli. BJ5183 which was transformed by adenoviral backbone plasmid pAdEasy-1.The recombinant plasmid pAd-Nanog obtained from screening was confirmed by PCR and restriction endonuclease analysis. The pAdNanog plasmid was linearized by Pac Ⅰ and transfected into human embryonal kidney cell 293A via liposome. The recombinant adenovirus was packaged and amplified in 293A cells following three amplification. The prepared highly expresed Ad-Nanog was transfected into hucMSCs. Results PCR and restriction endonuclease analysis confirmed that Nanog gene was inserted into the recombinant adenovirus vector successfully. The efficiently expressed green fluorescent protein (GFP) in transfected hucMSCs was visualized by fluorescent microscopy. Conclusion Recombinant adenovirus vector containing Nanog was constructed successfully and efficiently transfected hucMSCs which should be useful in the successive investigation on transgenic mesenchymal stem cells.%目的 构建重组人Nanog基因腺病毒栽体(Ad-Nanog),转染人脐带间充质干细胞(hucMSCs),用于后续研究.方法 设计含有Kpn Ⅰ及Xho Ⅰ酶切位点的引物,PCR扩增Nanog基因,将扩增产物亚克隆到pAdTrack-CMV穿梭质粒上,经双酶切和基因测序鉴定,重组穿梭质粒经Pine Ⅰ线性化后,在含有腺病毒骨架质粒pAdEasy-1的BJ5183中同源重组,筛选获得Ad-Nanog重组腺病毒质粒.经Pac Ⅰ酶切线性化,脂质体转染293A细胞,包

  14. Nanopolymers Delivery of the Bone Morphogenetic Protein-4 Plasmid to Mesenchymal Stem Cells Promotes Articular Cartilage Repair In Vitro and In Vivo

    Directory of Open Access Journals (Sweden)

    Junjun Shi

    2012-01-01

    Full Text Available The clinical application of viral vectors for gene therapy is limited for biosafety consideration. In this study, to promote articular cartilage repair, poly (lactic-co glycolic acid (PLGA nanopolymers were used as non-viral vectors to transfect rabbit mesenchymal stem cells (MSCs with the pDC316-BMP4-EGFP plasmid. The cytotoxicity and transfection efficiency in vitro were acceptable measuring by CCK-8 and flow cytometry. After transfection, Chondrogenic markers (mRNA of Col2a1, Sox9, Bmp4, and Agg of experimental cells (MSCs being transfected with BMP-4 plasmid by PLGA nanopolymers were increased more than those of control cells (MSCs being transfected with naked BMP-4 plasmid alone. In vivo study, twelve rabbits (24 knees with large full thickness articular cartilage defects were randomly divided into the experimental group (MSCs being transfected with BMP-4 plasmid by PLGA nanopolymers and the control group (MSCs being transfected with naked BMP-4 plasmid. The experimental group showed better regeneration than the control group 6 and 12 weeks postoperatively. Hyaline-like cartilage formed at week 12 in the experimental group, indicating the local delivery of BMP-4 plasmid to MSCs by PLGA nanopolymers improved articular cartilage repair significantly. PLGA nanopolymers could be a promising and effective non-viral vector for gene therapy in cartilage repair.

  15. Coupling of importin beta binding peptide on plasmid DNA: transfection efficiency is increased by modification of lipoplex's physico-chemical properties

    Directory of Open Access Journals (Sweden)

    Escriou Virginie

    2003-09-01

    Full Text Available Abstract Background Non-viral vectors for gene transfer are less immunogenic than viral vectors but also less efficient. Significant effort has focused on enhancing non-viral gene transfer efficiency by increasing nuclear import of plasmid DNA, particularly by coupling nuclear localization peptidic sequences to plasmid DNA. Results We have coupled a 62-aminoacid peptide derived from hSRP1α importin beta binding domain, called the IBB peptide to plasmid DNA by using the heterobifunctional linker N-(4-azido-2,3,5,6 tetrafluorobenzyl-6-maleimidyl hexanamide (TFPAM-6. When covalently coupled to plasmid DNA, IBB peptide did not increase the efficiency of cationic lipid mediated transfection. The IBB peptide was still able to interact with its nuclear import receptor, importin β, but non-specifically. However, we observed a 20-fold increase in reporter gene expression with plasmid DNA / IBB peptide complexes under conditions of inefficient transfection. In which case, IBB was associated with plasmid DNA through self assembling ionic interaction. Conclusions The improvement of transfection activity was not due to an improved nuclear import of DNA, but rather by the modification of physicochemical properties of IBB peptide / plasmid complexes. IBB peptide increased lipoplex size and these larger complexes were more efficient for gene transfer.

  16. Convenient broad-host-range unstable vectors for studying stabilization cassettes in diverse bacteria.

    Science.gov (United States)

    Bartosik, Aneta A; Glabski, Krzysztof; Kulinska, Anna; Lewicka, Ewa; Godziszewska, Jolanta; Markowska, Aleksandra; Jagura-Burdzy, Grazyna

    2016-04-05

    Low-copy-number vectors of potential wide application in biotechnology need to encode stabilization modules ensuring their stable inheritance. The efficiency of stabilization may vary depending on the plasmid host so a thorough analysis of stabilization functions is required before use. To facilitate such analysis highly unstable, mobilizable, broad-host-range (BHR) vectors based on RK2 replicon were constructed. The vectors are suitable for testing of various stabilization functions, including plasmid and chromosomal partitioning cassettes encoding ParB homologues capable of spreading on DNA. The xylE or lacZ reporter systems facilitate easy monitoring of plasmid segregation. The range of BHR vectors with different reporter cassettes and alternative mobilization systems expands their application in diverse bacterial species.

  17. Development of expression vectors for Escherichia coli based on the pCR2 replicon

    Directory of Open Access Journals (Sweden)

    Deb J K

    2007-05-01

    Full Text Available Abstract Background Recent developments in metabolic engineering and the need for expanded compatibility required for co-expression studies, underscore the importance of developing new plasmid vectors with properties such as stability and compatibility. Results We utilized the pCR2 replicon of Corynebacterium renale, which harbours multiple plasmids, for constructing a range of expression vectors. Different antibiotic-resistance markers were introduced and the vectors were found to be 100% stable over a large number of generations in the absence of selection pressure. Compatibility of this plasmid was studied with different Escherichia coli plasmid replicons viz. pMB1 and p15A. It was observed that pCR2 was able to coexist with these E.coli plasmids for 60 generations in the absence of selection pressure. Soluble intracellular production was checked by expressing GFP under the lac promoter in an expression plasmid pCR2GFP. Also high level production of human IFNγ was obtained by cloning the h-IFNγ under a T7 promoter in the expression plasmid pCR2-IFNγ and using a dual plasmid heat shock system for expression. Repeated sub-culturing in the absence of selection pressure for six days did not lead to any fall in the production levels post induction, for both GFP and h-IFNγ, demonstrating that pCR2 is a useful plasmid in terms of stability and compatibility. Conclusion We have constructed a series of expression vectors based on the pCR2 replicon and demonstrated its high stability and sustained expression capacity, in the absence of selection pressure which will make it an efficient tool for metabolic engineering and co-expression studies, as well as for scale up of expression.

  18. 猪Mx1基因真核表达载体的构建、鉴定及其表达%Construction,Identification and Expression of Eukaryotic Expression Vector of Pig Mx1 Gene

    Institute of Scientific and Technical Information of China (English)

    覃兆鲜; 潘天彪; 黄光云; 谢炳坤

    2011-01-01

    The purpose of this study was to obtain pig fibroblast cells that transferred Mx1 gene.Total RNA was extracted from pig fibroblast cells that induced with IFN and the eDNA encoding pig Mx1 protein was cloned using reverse transcription polymerase chain reaction (RT-PCR).The Mx1 gene was subcloned into pMSCV-IRES-GFP vector and then the recombinant plasmids were transferred into Luchuan pig fibroblast cells by the method of Lipofectaming 2000.The results showed that Mx1 gene had been integrated into the genome of Luchuan pig fibroblast cells based on the analysis of fluoroscopy and PCR identification.%为获得转Mx1基因的阳性陆川猪成纤维细胞,本研究以干扰素诱导猪成纤维细胞Mx1基因表达,提取细胞总RNA,RT-PCR获得编码猪Mx1蛋白的cDNA;以pMSCV-IRES-GFP为骨架构建猪Mx1基因表达载体pMSCV-IRES-GFP-Mx1,并利用脂质体2000介导重组质粒转染陆川猪胎儿成纤维细胞,通过荧光观察和PCR检测分析结果表明Mx1蛋白基因整合进入陆川猪胎儿成纤维细胞.

  19. Isolation of a minireplicon of the plasmid pG6303 of Lactobacillus plantarum G63 and characterization of the plasmid-encoded Rep replication protein

    Indian Academy of Sciences (India)

    Jing Fan; Xuedong Xi; Yan Huang; Zhongli Cui

    2015-06-01

    A cryptic 10.0-kb plasmid pG6303 from a multiplasmid-containing Lactobacillus plantarum G63 was studied. The analysis of replicon was facilitated by the construction of shuttle vectors and electrotransformation into L. plantarum. The pG6303 replicon included (i) an open reading frame encoding the putative Rep replication initiation protein; and (ii) the putative origin of replication. The Rep protein was expressed as a fusion with the hexa-histidine (His) at its C-terminal end and purified by Ni-affinity chromatography. The electrophoretic mobility shift assays in pG6303 showed that the purified Rep protein specifically bound from 5582 to 5945 bp, differing from the putative origin of replication of pG6303. We speculate that pG6303 replication is a new mode of plasmid replication.

  20. Small, stable shuttle vectors for use in Xanthomonas.

    Science.gov (United States)

    DeFeyter, R; Kado, C I; Gabriel, D W

    1990-03-30

    Plasmids from three broad-host-range (bhr) incompatibility groups (Inc) were evaluated for use as cloning vectors in Xanthomonas campestris pv. malvacearum (Xcm), the causal agent of bacterial blight of cotton. The IncP vectors pLAFR3 and pVK102 could not be introduced into Xcm at a significant frequency (less than 1 x 10(-10] and IncQ vectors such as pKT210 were unstable in their maintenance and tended to delete cloned inserts. IncW vectors such as pSa747 also were lost readily from Xcm in the absence of selection pressure. We constructed two plasmids, pUFR027 and a cosmid derivative, pUFR034, which have proven useful as cloning vectors in Xcm and other xanthomonads. They contain the pSa origin of DNA replication, the partition locus parA from the Agrobacterium plasmid pTAR, a neomycin-resistance selection marker, and alacZ alpha cassette with cloning sites. pUFR027 is 9.3 kb, and pUFR034 is 8.7 kb in size. They can be mobilized by conjugation into Xcm at a frequency of approx. 1 x 10(-6) per recipient and are maintained stably (greater than 95% retention over 36 generations without selection pressure) in both broth culture and in planta. The plasmids were introduced and maintained stably in X. citri, and in X. campestris pathovars campestris, citrumelo, vesicatoria and translucens, and were moderately stable in X. phaseoli. No effects of the plasmids on pathogenicity have been observed.

  1. Development of shuttle vectors for transformation of diverse Rickettsia species.

    Directory of Open Access Journals (Sweden)

    Nicole Y Burkhardt

    Full Text Available Plasmids have been identified in most species of Rickettsia examined, with some species maintaining multiple different plasmids. Three distinct plasmids were demonstrated in Rickettsia amblyommii AaR/SC by Southern analysis using plasmid specific probes. Copy numbers of pRAM18, pRAM23 and pRAM32 per chromosome in AaR/SC were estimated by real-time PCR to be 2.0, 1.9 and 1.3 respectively. Cloning and sequencing of R. amblyommii AaR/SC plasmids provided an opportunity to develop shuttle vectors for transformation of rickettsiae. A selection cassette encoding rifampin resistance and a fluorescent marker was inserted into pRAM18 yielding a 27.6 kbp recombinant plasmid, pRAM18/Rif/GFPuv. Electroporation of Rickettsia parkeri and Rickettsia bellii with pRAM18/Rif/GFPuv yielded GFPuv-expressing rickettsiae within 2 weeks. Smaller vectors, pRAM18dRG, pRAM18dRGA and pRAM32dRGA each bearing the same selection cassette, were made by moving the parA and dnaA-like genes from pRAM18 or pRAM32 into a vector backbone. R. bellii maintained the highest numbers of pRAM18dRGA (13.3 - 28.1 copies, and R. parkeri, Rickettsia monacensis and Rickettsia montanensis contained 9.9, 5.5 and 7.5 copies respectively. The same species transformed with pRAM32dRGA maintained 2.6, 2.5, 3.2 and 3.6 copies. pRM, the plasmid native to R. monacensis, was still present in shuttle vector transformed R. monacensis at a level similar to that found in wild type R. monacensis after 15 subcultures. Stable transformation of diverse rickettsiae was achieved with a shuttle vector system based on R. amblyommii plasmids pRAM18 and pRAM32, providing a new research tool that will greatly facilitate genetic and biological studies of rickettsiae.

  2. Construction of Brain-derived Neurotrophic Factor Eukaryotic Expression Vector and its Ex-pression%pTracer-CMV/Bsd-BDNF真核表达载体的构建及表达

    Institute of Scientific and Technical Information of China (English)

    吴雄辉; 赵斯君; 李赟

    2015-01-01

    [Objective] To construct an eukaryotic expression vector of human brain‐derived neurotrophic factor (BDNF) and explore its expression in spiral ganglion cells (SGCs) of cochlear in mice .[Methods] The open reading frame (ORF) of BDNF was amplified from human peripheral blood by reverse transcription‐poly‐merase chain reaction (RT‐PCR) .TA cloning strategy was employed to insert target fragments into pUCm‐T vector .The recombinant plasmid was identified and noted as pUCm‐T‐BDNF .Then BDNF was subcloned into pTracer‐CMV/Bsd ,an eukaryotic expression vector .The plasmid of pTracer‐CMV/Bsd‐BDNF was sequenced and introduced into SCGs of cochlear by LipofectamineTM 2000 .The expression of green fluorescence protein (GFP) was observed by fluorescence microscope .The expression of BDNF was detected by Western blot after screening by blasticidin for 4 weeks .[Results]The eukaryotic expression plasmid pTracer‐CMV/Bsd‐BDNF was successfully constructed .GFP was found in transfected cells by fluorescence microscope .And the expres‐sion of BDNF was detected in transfected cells by Western blot .[Conclusion] The recombinant eukaryotic ex‐pression vector of pTracer‐CMV/Bsd‐BDNF has been constructed successfully .It can co‐express GFP and BD‐NF so as to serve as a tool for further gene therapy study .%【目的】构建人脑源性神经营养因子(BDNF)基因真核表达载体,并在小鼠耳蜗毛细胞与螺旋神经节细胞(SGCs)表达。【方法】应用RT‐PCR从人外周血中扩增BDNF基因开放阅读框(ORF),采用 TA克隆技术,将目的片段插入到pUCm‐T载体中进行鉴定,重组的质粒命名为pUCm‐T‐BDNF。随后,将BDNF进一步克隆到真核表达载体pT racer‐CM V/Bsd中。用脂质体将经过测序、验证的重组pT racer‐CM V/Bsd‐BD‐NF质粒转染小鼠耳蜗SGCs细胞,荧光显微镜下观察绿色荧光蛋白的表达,转染SGCs细胞,

  3. Bacteriophage selection against a plasmid-encoded sex apparatus leads to the loss of antibiotic-resistance plasmids

    OpenAIRE

    Jalasvuori, Matti; Friman, Ville-Petri; Nieminen, Anne; Jaana K.H. Bamford; Buckling, Angus

    2011-01-01

    Antibiotic-resistance genes are often carried by conjugative plasmids, which spread within and between bacterial species. It has long been recognized that some viruses of bacteria (bacteriophage; phage) have evolved to infect and kill plasmid-harbouring cells. This raises a question: can phages cause the loss of plasmid-associated antibiotic resistance by selecting for plasmid-free bacteria, or can bacteria or plasmids evolve resistance to phages in other ways? Here, we show that multiple ant...

  4. 基质金属蛋白酶组织抑制剂1基因重组腺病毒载体的构建%Construction of Recombinant Adenovirus Vector Carrying the Human Tissue Inhibitor of Metalloproteinase 1 cDNA

    Institute of Scientific and Technical Information of China (English)

    周毅; 屠冠军

    2012-01-01

    Objective To construct recombinant adenovirus vector carrying the human tissue inhibitor of metalloproteinase 1 (T1MP1) cD-NA and explore the feasibility of reversing or delaying intervertebral disc degeneration by gene therapy. Methods The hTIMPl cDNA was amplified from plasmid containing human TIMP1 ORF sequence by PCR. hTIMPl cDNA was subcloned into the adenovirus shuttle plasmid pDC316 of Ad5MaxTM adenovirus vector system,and the products were co-transfected into HEK293 cells with the helper plasmid pBH- Glox_El ,3Cre by lipofectin transfection method. The recombinant adenovirus Ad5-TIMP1 was generated by homologous recombination of the two plasmids in HEK 293 cells. After identification by PCR and enzyme digestion, Ad5-TTMP1 was amplified in HEK 293 adenoviral packaging cells, purified by column chromatography, and virus activity was measured by TCID50 assay. Results Recombinant adenoviral vector carrying hTIMPl(Ad5-hTIMPl) was successfully constructed with virus activity of l×l010 IU/ml,which was confirmed by PCR,restriction enzyme digestion and DNA sequencing. Conclusion The Recombinant adenoviral vector carrying hTIMPl (Ad5-hTlMPl) was successfully constructed, which provided a foundation for further research on reversing or delaying intervertebral disc degeneration by gene therapy.%目的 为探讨基因治疗逆转或延缓椎间盘退变的可行性,构建及制备人基质金属蛋白酶组织抑制剂1 (tissue inhibitor of metalloproteinase 1,TIMP1) cDNA重组腺病毒载体.方法 以含TIMP1 cDNA序列的质粒为模板,通过PCR方法扩增TIMP1cDNA片段,将TIMP1 cDNA全长定向克隆到Ad5MaxTM腺病毒系统的穿梭质粒pDC316上,构建pDC316-TIMP1穿梭质粒;使用Ad5MaxTM腺病毒包装系统,脂质体介导的穿梭质粒及骨架质粒pBHGlox-E1,3Cre共转染HEK293细胞,同源重组构建含TIMP1 cDNA的重组腺病毒Ad5-TIMP1,通过PCR方法鉴定重组腺病毒Ad5-TIM P1的正确性.通过阴离子柱层析

  5. Replicon RNA Viral Vectors as Vaccines

    Science.gov (United States)

    Lundstrom, Kenneth

    2016-01-01

    Single-stranded RNA viruses of both positive and negative polarity have been used as vectors for vaccine development. In this context, alphaviruses, flaviviruses, measles virus and rhabdoviruses have been engineered for expression of surface protein genes and antigens. Administration of replicon RNA vectors has resulted in strong immune responses and generation of neutralizing antibodies in various animal models. Immunization of mice, chicken, pigs and primates with virus-like particles, naked RNA or layered DNA/RNA plasmids has provided protection against challenges with lethal doses of infectious agents and administered tumor cells. Both prophylactic and therapeutic efficacy has been achieved in cancer immunotherapy. Moreover, recombinant particles and replicon RNAs have been encapsulated by liposomes to improve delivery and targeting. Replicon RNA vectors have also been subjected to clinical trials. Overall, immunization with self-replicating RNA viruses provides high transient expression levels of antigens resulting in generation of neutralizing antibody responses and protection against lethal challenges under safe conditions. PMID:27827980

  6. Plasmid-based generation of induced neural stem cells from adult human fibroblasts

    Directory of Open Access Journals (Sweden)

    Philipp Capetian

    2016-10-01

    Full Text Available Direct reprogramming from somatic to neural cell types has become an alternative to induced pluripotent stem cells. Most protocols employ viral expression systems, posing the risk of random genomic integration. Recent developments led to plasmid-based protocols, lowering this risk. However, these protocols either relied on continuous presence of a variety of small molecules or were only able to reprogram murine cells. We therefore established a reprogramming protocol based on vectors containing the Epstein-Barr virus (EBV-derived oriP/EBNA1 as well as the defined expression factors Oct3/4, Sox2, Klf4, L-myc, Lin28, and a small hairpin directed against p53. We employed a defined neural medium in combination with the neurotrophins bFGF, EGF and FGF4 for cultivation without the addition of small molecules. After reprogramming, cells demonstrated a temporary increase in the expression of endogenous Oct3/4. We obtained induced neural stem cells (iNSC 30 days after transfection. In contrast to previous results, plasmid vectors as well as a residual expression of reprogramming factors remained detectable in all cell lines. Cells showed a robust differentiation into neuronal (72% and glial cells (9% astrocytes, 6% oligodendrocytes. Despite the temporary increase of pluripotency-associated Oct3/4 expression during reprogramming, we did not detect pluripotent stem cells or non-neural cells in culture (except occasional residual fibroblasts. Neurons showed electrical activity and functional glutamatergic synapses. Our results demonstrate that reprogramming adult human fibroblasts to iNSC by plasmid vectors and basic neural medium without small molecules is possible and feasible. However, a full set of pluripotency-associated transcription factors may indeed result in the acquisition of a transient (at least partial pluripotent intermediate during reprogramming. In contrast to previous reports, the EBV-based plasmid system remained present and active inside

  7. 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.

  8. A new and improved host-independent plasmid system for RK2-based conjugal transfer.

    Directory of Open Access Journals (Sweden)

    Trine Aakvik Strand

    Full Text Available Bacterial conjugation is a process that is mediated either by a direct cell-to-cell junction or by formation of a bridge between the cells. It is often used to transfer DNA constructs designed in Escherichia coli to recipient bacteria, yeast, plants and mammalian cells. Plasmids bearing the RK2/RP4 origin of transfer (oriT are mostly mobilized using the E. coli S17-1/SM10 donor strains, in which transfer helper functions are provided from a chromosomally integrated RP4::Mu. We have observed that large plasmids were occasionally modified after conjugal transfer when using E. coli S17-1 as a donor. All modified plasmids had increased in size, which most probably was a result of co-transfer of DNA from the chromosomally located oriT. It has earlier also been demonstrated that the bacteriophage Mu is silently transferred to recipient cells by these donor strains, and both occurrences are very likely to lead to mutations within the recipient DNA. Here we report the construction of a new biological system addressing both the above mentioned problems in which the transfer helper functions are provided by a plasmid lacking a functional oriT. This system is compatible with all other replicons commonly used in conjugation experiments and further enables the use of diverse bacterial strains as donors. Plasmids containing large inserts were successfully conjugated and the plasmid modifications observed when E. coli S17-1 was used as donor were eliminated by the use of the new host-independent vector system.

  9. Detection of Clonal and Subclonal Copy-Number Variants in Cell-Free DNA from Patients with Breast Cancer Using a Massively Multiplexed PCR Methodology

    Directory of Open Access Journals (Sweden)

    Eser Kirkizlar

    2015-10-01

    Using an in vitro model of cell-free DNA, we show that mmPCR-NGS can accurately detect CNVs with average allelic imbalances as low as 0.5%, an improvement over previously reported whole-genome sequencing approaches. Our method revealed differences in the spectrum of CNVs detected in tumor tissue subsections and matching plasma samples from 11 patients with stage II breast cancer. Moreover, we showed that liquid biopsies are able to detect subclonal mutations that may be missed in tumor tissue biopsies. We anticipate that this mmPCR-NGS methodology will have broad applicability for the characterization, diagnosis, and therapeutic monitoring of CNV-enriched cancers, such as breast, ovarian, and lung cancer.

  10. Quantitative proteomics as a tool to identify resistance mechanisms in erlotinib-resistant subclones of the non-small cell lung cancer cell line HCC827

    DEFF Research Database (Denmark)

    Jacobsen, Kirstine

    Background: Erlotinib (Tarceva®, Roche) has significantly changed the treatment of non-small cell lung cancer (NSCLC) as approximately 70% of patients show significant tumor regression when treated (Santarpia et. al., 2013). However, all patients relapse due to development of acquired resistance......, which in 43-50% of cases are caused by a secondary mutation (T790M) in EGFR. Importantly, a majority of resistance cases are still unexplained (Lin & Bivona, 2012). Our aim is to identify novel resistance mechanisms – and potentially new drug targets - in erlotinib-resistant subclones of the NSCLC cell...... of erlotinib, and in biological triplicates on a Q-Exactive mass spectrometer. Only proteins identified with minimum 2 unique peptides and in minimum 2 of 3 replicates were accepted. Results: Importantly, the resistant clones did not acquire the T790M or other EGFR or KRAS mutations, potentiating...

  11. Selection vector for direct cloning of proof reading polymerase chain reaction products based on the lethal ccdB gene in Escherichia Coli

    OpenAIRE

    Weibel, Pascal; Ender, Miriam; Madon, Jerzy; Zinkernagel, Annelies; Schuepbach, Reto

    2013-01-01

    Introducing PCR products into plasmids vectors is key for molecular techniques. Ideally cloning vectors are easy to construct, modify and propagate, neither require advanced techniques nor special equipment or reagents and efficiently incorporate PCR products at close to zero empty vector background. We provide an easy to engineer self-made cloning vector, neither requiring sophisticated tools or techniques nor advanced cloning knowledge. Through recombination we obtained the pUC18ccdB vector...

  12. Most multifocal papillary thyroid carcinomas acquire genetic and morphotype diversity through subclonal evolution following the intra-glandular spread of the initial neoplastic clone.

    Science.gov (United States)

    Jovanovic, L; Delahunt, B; McIver, B; Eberhardt, N L; Grebe, S K G

    2008-06-01

    Papillary thyroid carcinoma (PTC) is frequently multifocal (mPTC), with synchronous tumour foci often showing varied morphology. The genetic mechanisms underlying the development of multiple and histologically diverse tumour foci remain uncertain. Different tumour foci might develop either through intrathyroidal dissemination of a single malignant clone, with morphotype differentiation occurring as a result of subclonal progression, or they may stem from independent transformational events involving multiple progenitor clones. To determine the clonal derivation of multiple tumour foci and to map their clonal relationships and genetic progression in mPTC, we evaluated genome-wide allelic imbalances (AI) and BRAF V600E mutation status in 55 synchronous tumour foci from 18 mPTC patients. For apparently monoclonal tumours, we calculated the probabilities of monoclonal derivation and used phylogenetic analysis to model clonal evolution. Genome-wide allelotyping and BRAF mutation analysis showed genetic alterations consistent with monoclonal origin in 83% of cases, mostly with evidence of subclonal evolution. BRAF V600E mutations were early events during clonal evolution of most, but not all cases. MPTC with morphologically diverse tumour foci also arose through monoclonal derivation in 75% of cases, demonstrating that morphotype-determining genetic changes can be acquired during clonal diversification, subsequent to the spread of the original malignant progenitor clone. In 17% of patients, discordant AI or BRAF V600E profiles implied that mPTCs can occasionally develop from distinct transformation events. This study suggests that mPTC originates usually from neoplastic transformation and subsequent intrathyroidal spread of a single malignant progenitor clone. Clonal progression and morphotype differentiation occur through progressive acquisition of genetic alterations subsequent to the initial intra-glandular spread. In monoclonal BRAF V600E-positive mPTCs, BRAF V600E

  13. Poly(ADP-ribose) polymerase activity in various U937 cell subclones with different susceptibility to HIV-1 infection: its dramatic decrease following persistent virus infection.

    Science.gov (United States)

    Tanaka, Y; Yoshihara, K; Kojima, K; Itaya, A; Kameoka, M; Ikuta, K; Kamiya, T

    1995-08-04

    Poly(ADP-ribose) polymerase, a nuclear enzyme, is suggested to be involved in apoptotic cell death. It is also known that apoptotic cell death following HIV-1 infection is the most important feature of AIDS pathogenesis. Thus, to evaluate the relations between the enzyme and HIV-1 infection, we examined the enzyme activity of several subclones of human promonocytic cell line U937, which showed different susceptibility to HIV-1 infection. The nuclear extracts of two "high type clones" (possessing high susceptibility to HIV-1 infection) contained approximately 4 to 7-fold less enzyme than two low type clones when assayed under a full activation of enzyme. Parent clone, possessing an intermediate susceptibility to HIV-1, showed an intermediate enzyme level, suggesting that low level of this enzyme in cells is important for an effective infection of HIV-1. Furthermore, when these U937 subclones persistently infected with HIV-1 were examined, a dramatic decrease of the enzyme activity, reaching 2 to 16% of uninfected cells, was observed in all of these clones. The levels of poly(ADP-ribose) glycohydrolase in these clones were relativity unchanged. Activity gel analysis and immunoblotting of the enzyme in the clones revealed that the low enzyme activities observed in uninfected "high type clones" and all HIV-1-infected clones were due to a marked decrease of the enzyme protein itself. All of these results suggest that HIV-1 infection involves some mechanism to downregulate cellular poly(ADP-ribose) polymerase and that a lower level of the enzyme may be essential for an effective production of the virus and/or for a stable virus/host interaction.

  14. Vector nematicons

    CERN Document Server

    Horikis, Theodoros P

    2016-01-01

    Families of soliton pairs, namely vector solitons, are found within the context of a coupled nonlocal nonlinear Schrodinger system of equations, as appropriate for modeling beam propagation in nematic liquid crystals. In the focusing case, bright soliton pairs have been found to exist provided their amplitudes satisfy a specific condition. In our analytical approach, focused on the defocusing regime, we rely on a multiscale expansion methods, which reveals the existence of dark-dark and antidark-antidark solitons, obeying an effective Korteweg-de Vries equation, as well as dark-bright solitons, obeying an effective Mel'nikov system. These pairs are discriminated by the sign of a constant that links all physical parameters of the system to the amplitude of the stable continuous wave solutions, and, much like the focusing case, the solitons' amplitudes are linked leading to mutual guiding.

  15. Distribution of small native plasmids in Streptococcus pyogenes in India.

    Science.gov (United States)

    Bergmann, René; Nerlich, Andreas; Chhatwal, Gursharan S; Nitsche-Schmitz, D Patric

    2014-05-01

    Complete characterization of a Streptococcus pyogenes population from a defined geographic region comprises information on the plasmids that circulate in these bacteria. Therefore, we determined the distribution of small plasmids (pyogenes isolates from India, where diversity of strains and incidence rates of S. pyogenes infections are high. The collection comprised 77 emm-types. For plasmid detection and discrimination, we developed PCRs for different plasmid replication initiation protein genes, the putative repressor gene copG and bacteriocin genes dysA and scnM57. Plasmid distribution was limited to 13 emm-types. Co-detection analysis using aforementioned PCRs revealed four distinct plasmid sub-types, two of which were previously unknown. Representative plasmids pA852 and pA996 of the two uncharacterized plasmid sub-types were sequenced. These two plasmids could be assigned to the pMV158 and the pC194/pUB110 family of rolling-circle plasmids, respectively. The majority of small plasmids found in India belonged to the two newly characterized sub-types, with pA852- and pA996-like plasmids amounting to 42% and 22% of all detected plasmids, respectively. None of the detected plasmids coded for a known antibiotic resistance gene. Instead, all of the four plasmid sub-types carried known or potential bacteriocin genes. These genes may have influence on the evolutionary success of certain S. pyogenes genotypes. Notably, pA852-like plasmids were found in all isolates of the most prevalent emm-type 11.0. Together, a priori fitness of this genotype and increased fitness due to the acquired plasmids may have rendered type emm11.0 successful and caused the prevalence of pA852-like plasmids in India.

  16. 牛TLR2全长基因表达质粒的构建及其在HEK293细胞中的表达(英文)%Construction of an Expression Plasmid pEGFP-N1-boTLR2 for Full-length Bovine TLR2 and Its Expression in HEK293 Cells

    Institute of Scientific and Technical Information of China (English)

    王玉明; 王静萱

    2012-01-01

    [Objective] This study aimed to construct a full-length bovine TLR2 expression plasmid pEGFP-N1-boTLR2 and express it in HEK293 cells. [Method] A fulllength coding sequence of bovine TLR2 was cloned by RT-PCR, and ligated into the pMD18-T simple vector and then subcloned into the pEGFP-N1 vector. A recombinant eukaryotic expression plasmid containing the full-length CDS region of bovine TLR2 was constructed and transiently transfected into HEK293 cells. The transfection efficiency and the location of recombinant protein were examined by FCM and confocal microscopy. Then the bovine TLR2 mRNA expression in HEK293/boTLR2 was detected by qRT-PCR. Finally, we analyzed the biological activity through the response that lipoteichoic acid stimulates HEK293/boTLR2 cells. [Result] The full-length TLR2 gene was successfully cloned and ligated into eukaryotic expression vector. The recombinant expression vector expressed bovine TLR2 in HEK293 cells. HEK293/boTLR2 cells produced higher levels of IL-8 secretion than nontransfected HEK293 cells when stimulated with LTA from Staphylococcus aureus. [Conclusion] The established cell model can provide a fast, flexible and convenient means for screening TLR agonists and antagonists, and may also be useful for investigating the interaction between TLR agonists and TLRs.%[目的]构建牛TLR2全长基因表达质粒,并在HEK293细胞中表达。[方法]利用RT-PCR技术克隆TLR2基因的全长编码区,连接到pMD18-Tsimplevector,再亚克隆到pEGFP-N1载体,得到包含TLR2基因全长的重组真核表达质粒。将重组质粒瞬时转染到HEK293细胞。流式细胞计数法和共聚焦显微镜法检测转染效率和表达蛋白在细胞中的定位;qRT-PCR法检测TLR2 mRNA在HEK293/boTLR2中的表达。最后,通过脂膜酸刺激HEK293/boTLR2细胞试验来分析TLR2蛋白的生物活性。[结果]成功克隆TLR2基因全长并连接到真核表达载体,并在HEK293细胞中表达。在LTA刺激的条件下,转染重

  17. Transfer of a gonococcal beta-lactamase plasmid to conjugation-deficient Neisseria cinerea strains by transformation.

    Science.gov (United States)

    Genco, C A; Clark, V L

    1988-12-01

    We have previously shown that some strains of Neisseria cinerea can serve as recipients in conjugation (Con+) with Neisseria gonorrhoeae while others cannot (Con-). To determine if a replication defect contributes to the inability of certain strains of N. cinerea to serve as recipients in conjugation, we attempted to introduce a naturally occurring gonococcal beta-lactamase plasmid into N. cinerea by transformation. Various methods were employed, and all proved unsuccessful. Since specific sequences are required for DNA uptake in transformation of N. gonorrhoeae, we constructed a number of hybrid plasmids containing N. cinerea chromosomal DNA inserted into the N. gonorrhoeae/Escherichia coli beta-lactamase shuttle vector, pLES2. When nine randomly selected plasmids with inserts were used to transform an N. cinerea strain which did not accept the gonococcal beta-lactamase plasmid by conjugation, transformants were observed with four of the hybrid plasmids. The presence of one of the hybrid plasmids, pCAG9, in transformants was confirmed by agarose gel electrophoresis, Southern hybridization, and beta-lactamase production. When an N. gonorrhoeae donor strain containing pCAG9 was used in conjugation with several N. cinerea strains, only those strains that were previously shown to act as recipients could accept and maintain pCAG9. The ability of pCAG9 and the other three hybrid plasmids to transform Con- strains demonstrates that the beta-lactamase plasmid can replicate in Con- strains, and, therefore, the Con- phenotype is due to a block in some other stage of the conjugation process.

  18. Plasmid-borne prokaryotic gene expression: Sources of variability and quantitative system characterization

    Science.gov (United States)

    Bagh, Sangram; Mazumder, Mostafizur; Velauthapillai, Tharsan; Sardana, Vandit; Dong, Guang Qiang; Movva, Ashok B.; Lim, Len H.; McMillen, David R.

    2008-02-01

    One aim of synthetic biology is to exert systematic control over cellular behavior, either for medical purposes or to “program” microorganisms. An engineering approach to the design of biological controllers demands a quantitative understanding of the dynamics of both the system to be controlled and the controllers themselves. Here we focus on a widely used method of exerting control in bacterial cells: plasmid vectors bearing gene-promoter pairs. We study two variants of the simplest such element, an unregulated promoter constitutively expressing its gene, against the varying genomic background of four Escherichia coli cell strains. Absolute protein numbers and rates of expression vary with both cell strain and plasmid type, as does the variability of expression across the population. Total variability is most strongly coupled to the cell division process, and after cell size is scaled away, plasmid copy number regulation emerges as a significant effect. We present simple models that capture the main features of the system behavior. Our results confirm that complex interactions between plasmids and their hosts can have significant effects on both expression and variability, even in deliberately simplified systems.

  19. Construction of Eukaryotic Coexpression Vector Containing Mycobacterium Tuberculosis Heat Shock Protein 70 and Herpes Simplex Virus Thymidine Kinase Genes%mtHSP70/HSV-TK双基因真核共表达质粒的构建

    Institute of Scientific and Technical Information of China (English)

    曾曙光; 刘启才; 王素文; 徐平平; 章锦才; 张积仁

    2011-01-01

    were obtained. Collected DNA was then ligated onto the T vector pMD18-T. The vector was used to transform Escherichia coli TG1, PCR was performed on the clones with specific primers of HSV-TK and mtHSP70 gene. Upstream and downstream sequencing were performed on positive pMD18T-TK recombinant plasmids and positive pMD18T-mtHSP70 recombinant plasmids. Results of sequencing was compared to the known gene sequences. One of these plasmids of each type was selected for sub-cloning of HSV-TK and mtHSP70 genes.PCMV-mtHSP70-IRES-TK was constructed. Resulted positive clones were shaken and a small amount of plasmids were extracted. Restriction enzyme verification was performed on the plasmids with single restriction enzyme of Not Ⅰ and double restriction enzymes of EcoR Ⅰ and Xho Ⅰ , respectively. Results Constructed plasmids pCMV-mtHSP70-IRESTK underwent restriction enzyme verification by single restriction enzyme of Not Ⅰ and double restriction enzymes of EcoR Ⅰ and Xho Ⅰ, respectively. Electrophoresis of cleavaged products showed specific restriction enzyme map, which confirmed that it was indeed recombinant plasmid pCMV-nuHSP70-IRES-TK. Conclusion This research work has made a base for further investigation of the synergistic effect of mycobacterium tuberculosis heat shock protein 70 gene combined with HSV-TK immunotherapy in rumor.

  20. Single primer-mediated circular polymerase chain reaction for hairpin DNA cloning and plasmid editing.

    Science.gov (United States)

    Huang, Jiansheng; Khan, Inamullah; Liu, Rui; Yang, Yan; Zhu, Naishuo

    2016-05-01

    We developed and validated a universal polymerase chain reaction (PCR) method, single primer circular (SPC)-PCR, using single primer to simultaneously insert and amplify a short hairpin sequence into a vector with a high success rate. In this method, the hairpin structure is divided into two parts and fused into a vector by PCR. Then, a single primer is used to cyclize the chimera into a mature short hairpin RNA (shRNA) expression vector. It is not biased by loop length or palindromic structures. Six hairpin DNAs with short 4-nucleotide loops were successfully cloned. Moreover, SPC-PCR was also applied to plasmid editing within 3 h with a success rate higher than 95%.

  1. Temporal Trends in Antimicrobial Resistance and Virulence-Associated Traits within the Escherichia coli Sequence Type 131 Clonal Group and Its H30 and H30-Rx Subclones, 1968 to 2012

    DEFF Research Database (Denmark)

    Olesen, Bente; Frimodt-Møller, Jakob; Leihof, Rikke Fleron;

    2014-01-01

    (2005) ST131 subclones. In contrast, neither the total virulence gene content nor the prevalence of biofilm production increased temporally, although non-H30 isolates increasingly qualified as extraintestinal pathogenic E. coli (ExPEC). Whereas virotype D occurred from 1968 forward, virotypes A and C...

  2. Construction and expression of porcine interleukin-15 cDNA eukaryotic expression vector and its enhancing of immunity as immunoadjuvants%猪IL-15 cDNA真核表达载体的构建、表达及其免疫佐剂效应

    Institute of Scientific and Technical Information of China (English)

    何谦; 陈钜豪; 房红莹; 卢俊鹏; 张欣; 崔敏; 曾小娜; 刘健; 罗满林

    2011-01-01

    According to the sequences of porcine interleukin-15 messenger ribonucleic acid(pIL-15mRNA) published in GenBank,the primers were designed to amplify IL-15 cDNA,which was cloned to into the pMD18-T vector.The product was proved correctly by PCR,enzyme digestion and sequencing,and subcloned into the eukaryotic expression vector pcDNA-3.1(+),then the recombinant plasmid pcDNA-pIL-15 was transfected to AD-293 cells by the liposome-mediated.With mouse anti-pig IL-15 as the first antibody,indirect immunofluorescence analysis showed that the porcine IL-15 gene were successfully carried out and transiently expressed in AD-293 cells,The immunization test to Balb/c mice showed that as an immunologic adjuvants,the pcDNA-pIL-15 could promote the proliferation of T lymphocyte cells and enhanced the specific antibody level for pcDNA-ORF2(PCV2) plasmids,which establish the foundation for further development of pig IL-15 gene adjuvant vaccine and conduct clinical trials.%参考GenBank发表猪IL-15mRNA序列设计引物,用RT-PCR方法扩增猪IL-15cDNA,并克隆到pMD18-T载体中,通过PCR、酶切和测序验证克隆正确,再亚克隆到真核表达载体pcDNA-3.1(+)上,得到重组质粒pcD-NA-pIL-15。在脂质体介导下,重组质粒pcDNA-pIL-15转染AD-293细胞。以鼠抗猪IL-15为一抗,用间接免疫荧光分析表明猪IL-15基因均在AD-293细胞中成功进行了瞬时表达。小鼠免疫试验表明,pcDNA-pIL-15作为免疫佐剂,能够有效提高小鼠的脾T

  3. 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

  4. 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 veter

  5. 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 veter

  6. The reversed terminator of octopine synthase gene on the Agrobacterium Ti plasmid has a weak promoter activity in prokaryotes.

    Science.gov (United States)

    Shao, Jun-Li; Long, Yue-Sheng; Chen, Gu; Xie, Jun; Xu, Zeng-Fu

    2010-06-01

    Agrobacterium tumefaciens transfers DNA from its Ti plasmid to plant host cells. The genes located within the transferred DNA of Ti plasmid including the octopine synthase gene (OCS) are expressed in plant host cells. The 3'-flanking region of OCS gene, known as OCS terminator, is widely used as a transcriptional terminator of the transgenes in plant expression vectors. In this study, we found the reversed OCS terminator (3'-OCS-r) could drive expression of hygromycin phosphotransferase II gene (hpt II) and beta-glucuronidase gene in Escherichia coli, and expression of hpt II in A. tumefaciens. Furthermore, reverse transcription-polymerase chain reaction analysis revealed that an open reading frame (ORF12) that is located downstream to the 3'-OCS-r was transcribed in A. tumefaciens, which overlaps in reverse with the coding region of the OCS gene in octopine Ti plasmid.

  7. Plasmids spread very fast in heterogeneous bacterial communities.

    Science.gov (United States)

    Dionisio, Francisco; Matic, Ivan; Radman, Miroslav; Rodrigues, Olivia R; Taddei, François

    2002-01-01

    Conjugative plasmids can mediate gene transfer between bacterial taxa in diverse environments. The ability to donate the F-type conjugative plasmid R1 greatly varies among enteric bacteria due to the interaction of the system that represses sex-pili formations (products of finOP) of plasmids already harbored by a bacterial strain with those of the R1 plasmid. The presence of efficient donors in heterogeneous bacterial populations can accelerate plasmid transfer and can spread by several orders of magnitude. Such donors allow millions of other bacteria to acquire the plasmid in a matter of days whereas, in the absence of such strains, plasmid dissemination would take years. This "amplification effect" could have an impact on the evolution of bacterial pathogens that exist in heterogeneous bacterial communities because conjugative plasmids can carry virulence or antibiotic-resistance genes. PMID:12524329

  8. CONSTRUCTION AND IDENTIFICATION OF THE RECOMBINANT OF THE AAV VECTOR AND HUMAN INTERFERON-GAMMA

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    To construct and identify further a recombinant of Adeno-associated virus and interferon-gamma for gene therapy, the full-length IFN-γcDNA containing signal peptide was amplified by PCR, and then cloned into the pUC18. After screening, the fragment from the positive clone was then subcloned into pwpl9. After the correct recombinant was identified by digestion with SacI and BamHI, it was transfected into lympho- cyte cell line H9 mediated by calcium phosphate, and the expression of IFN-γ was detected by RT-PCR and ELISA. The result showed that the IFN-y were expressed in the H9 cells transfected with pwp/IFN-y. The so constructed recombinant plasmid pwpl9/IFN-y containing the full-length IFN-y gene was expressed in mam- malian cells.

  9. PolI-driven integrative expression vectors for yeast.

    Science.gov (United States)

    Blancafort, P; Ferbeyre, G; Sariol, C; Cedergren, R

    1997-07-23

    A novel expression vector for yeast has been constructed from the regulatory elements present in the polI promoter and the enhancer/termination region (E/T) of rDNA. Under some conditions, this promoter/vector combination produces small RNAs such as the hammerhead RNA sequence at levels comparable to polII- and polIII-dependent systems. No stable transcription product can be demonstrated with this vector when the enhancer/termination sequence is less than 100 nucleotides downstream from the promoter. On the other hand, high expression of a stable, hammerhead RNA molecule can be obtained from this vector by inserting a 400-bp fragment containing the ADH1 transcription termination region upstream of the E/T. RNAs produced by this vector are polyadenylated and multiple copies of this plasmid can be stably integrated into the yeast chromosome.

  10. Endogenous mutagenesis in recombinant sulfolobus plasmids.

    Science.gov (United States)

    Sakofsky, Cynthia J; Grogan, Dennis W

    2013-06-01

    Low rates of replication errors in chromosomal genes of Sulfolobus spp. demonstrate that these extreme thermoacidophiles can maintain genome integrity in environments with high temperature and low pH. In contrast to this genetic stability, we observed unusually frequent mutation of the β-D-glycosidase gene (lacS) of a shuttle plasmid (pJlacS) propagated in Sulfolobus acidocaldarius. The resulting Lac(-) mutants also grew faster than the Lac(+) parent, thereby amplifying the impact of the frequent lacS mutations on the population. We developed a mutant accumulation assay and corrections for the effects of copy number and differential growth for this system; the resulting measurements and calculations yielded a corrected rate of 5.1 × 10(-4) mutational events at the lacS gene per plasmid replication. Analysis of independent lacS mutants revealed three types of mutations: (i) G · C-to-A · T transitions, (ii) slipped-strand events, and (iii) deletions. These mutations were frequent in plasmid-borne lacS expressed at a high level but not in single-copy lacS in the chromosome or at lower levels of expression in a plasmid. Substitution mutations arose at only two of 12 potential priming sites of the DNA primase of the pRN1 replicon, but nearly all these mutations created nonsense (chain termination) codons. The spontaneous mutation rate of plasmid-borne lacS was 175-fold higher under high-expression than under low-expression conditions. The results suggest that important DNA repair or replication fidelity functions are impaired or overwhelmed in pJlacS, with results analogous to those of the "transcription-associated mutagenesis" seen in bacteria and eukaryotes.

  11. Plasmid-mediated tetracycline resistance in Haemophilus ducreyi.

    OpenAIRE

    Albritton, W L; Maclean, I W; Slaney, L A; Ronald, A. R.; Deneer, H G

    1984-01-01

    Clinical isolates of Haemophilus ducreyi were shown to be resistant to tetracycline. Resistance was associated in some strains with a 30-megadalton plasmid capable of transferring resistance in conjugative matings with other strains of H. ducreyi and other species of Haemophilus. Restriction endonuclease digestion patterns suggest a relationship between H. ducreyi plasmids and other tetracycline resistance plasmids in Haemophilus. The presence of plasmid-mediated resistance to the tetracyclin...

  12. Replication of plasmids in gram-negative bacteria.

    OpenAIRE

    1989-01-01

    Replication of plasmid deoxyribonucleic acid (DNA) is dependent on three stages: initiation, elongation, and termination. The first stage, initiation, depends on plasmid-encoded properties such as the replication origin and, in most cases, the replication initiation protein (Rep protein). In recent years the understanding of initiation and regulation of plasmid replication in Escherichia coli has increased considerably, but it is only for the ColE1-type plasmids that significant biochemical d...

  13. Transient gene expression mediated by integrase-defective retroviral vectors.

    Science.gov (United States)

    Yu, Seung Shin; Dan, Kazuyuki; Chono, Hideto; Chatani, Emi; Mineno, Junichi; Kato, Ikunoshin

    2008-04-18

    Nonintegrating retroviral vectors were produced from a Moloney murine leukemia virus (MoMLV)-based retroviral vector system by introducing a point mutation into the integrase (IN) gene of the packaging plasmid. The efficacy of IN-defective retroviral vectors was measured through the transient expression of ZsGreen or luciferase in human cell lines. The IN-defective retroviral vectors could transduce target cells efficiently, but their gene expression was transient and lower than that seen with the integrating vectors. IN-defective retroviral vector gene expression decreased to background levels in fewer than 10 days. Southern blot analysis of transduced K562 cells confirmed the loss of a detectable vector sequence by 15 days. The residual integration activity of the IN-defective vector was 1000- to 10,000-fold lower than that of the integrating vector. These results demonstrate that the IN-defective retroviral vectors can provide a useful tool for efficient transient gene expression targeting of primary hematopoietic stem cells and lymphoid cells.

  14. Helper plasmid cloning in Streptococcus sanguis: cloning of a tetracycline resistance determinant from the Streptococcus mutans chromosome.

    Science.gov (United States)

    Tobian, J A; Macrina, F L

    1982-10-01

    A model system for testing the helper plasmid cloning system of Gryczan et al. (Mol. Gen. Genet. 177:459-467, 1980) was devised for the Streptococcus sanguis (Challis) host-vector system. In this system, linearized pVA736 plasmid efficiently transformed an S. sanguis (Challis) host containing a homologous plasmid, pVA380-1, but did not transform a plasmidless host or a host containing a nonhomologous plasmid, pVA380. In addition, whereas monomeric circular pVA736 transformed a plasmidless host with two-hit kinetics, it transformed a pVA380-1-containing host with one-hit kinetics. This helper plasmid cloning system was used to isolate two HindIII fragments (5.0 megadaltons [Mdal] and 1.9 Mdal in size) from the chromosome of Streptococcus mutans V825 which conferred high-level tetracycline resistance. One tetracycline-resistant clone was examined and found to contain three plasmids which were sized and designated pVA868 (9.0 Mdal), pVA869 (9.5 Mdal), and pVA870 (9.8 Mdal). Results of Southern blot hybridization and restriction endonuclease digestion confirmed that all three chimeras were composed of two HindIII fragments of the S. mutans V825 chromosome, as well as a large portion, varying in size for each chimera, of the 2.8 Mdal cloning vector, pVA380-1. Incompatibility observed between pVA380-1 and each of the chimeras indicated that replication of the chimeras was governed by the pVA380-1 replicative origin. Southern blotting experiments revealed that the chimeras hybridized to Tn916, providing the first evidence that transposon-related genes of enteric streptococcal origin are disseminated among oral streptococci.

  15. Plasmid Segregation: Spatial Awareness at the Molecular Level

    DEFF Research Database (Denmark)

    Møller-Jensen, Jakob; Gerdes, Kenn

    2007-01-01

    In bacteria, low-copy number plasmids ensure their stable inheritance by partition loci (par), which actively distribute plasmid replicates to each side of the cell division plane. Using time-lapse fluorescence microscopic tracking of segregating plasmid molecules, a new study provides novel insi...

  16. Multilocus sequence typing of IncN plasmids

    DEFF Research Database (Denmark)

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

    2011-01-01

    categorization of IncN plasmids. METHODS: Twelve fully sequenced IncN plasmids available at GenBank were analysed in silico for selecting the loci for the IncN-specific pMLST. A total of 58 plasmids originating from different reservoirs (human, pig, poultry, cattle and horses) and geographic regions (Italy...

  17. Boundaries of the origin of replication: creation of a pET-28a-derived vector with p15A copy control allowing compatible coexistence with pET vectors.

    Directory of Open Access Journals (Sweden)

    Sarmitha Sathiamoorthy

    Full Text Available During our studies involving protein-DNA interactions, we constructed plasmid pSAM to fulfill two requirements: 1 to facilitate transfer of cloned sequences from widely used expression vector pET-28a(+, and 2 to provide a vector compatible with pBR322-derived plasmids for use in cells harboring two different plasmids. Vector pSAM is a pET-28a(+-derived plasmid with the p15A origin of replication (ori; pET-28a(+ contains the pBR322 replicon that is incompatible with other pBR322-derived plasmids. By replacing the original pET-28a(+ replicon-comprising the ori, RNAI, RNAII, and Rom-with the p15A replicon, we generated pSAM, which contains the pET-28a(+ multiple cloning site and is now compatible with pBR322-derived vectors. Plasmid copy number was assessed using quantitative PCR: pSAM copy number was maintained at 18±4 copies per cell, consistent with that of other p15A-type vectors. Compatibility with pBR322-derived vectors was tested with pGEX-6p-1 and pSAM, which maintained their copy numbers of 49±10 and 14±4, respectively, when both were present within the same cell. Swapping of the ori is a common practice; however, it is vital that all regions of the original replicon be removed. Additional vector pSAMRNAI illustrated that incompatibility remains when portions of the replicon, such as RNAI and/or Rom, are retained; pSAMRNAI, which contains the intact RNAI but not ROM, lowered the copy number of pGEX-6p-1 to 18±2 in doubly transformed cells due to retention of the pET-28a(+-derived RNAI. Thus, pSAMRNAI is incompatible with vectors controlled by the pBR322 replicon and further demonstrates the need to remove all portions of the original replicon and to quantitatively assess copy number, both individually and in combination, to ensure vector compatibility. To our knowledge, this is the first instance where the nascent vector has been quantitatively assessed for both plasmid copy number and compatibility. New vector pSAM provides ease of

  18. One-oligonucleotide method for constructing vectors for RNA interference

    Institute of Scientific and Technical Information of China (English)

    Carlos Fabian FLORES-JASSO; Ines VELAZQUEZ-QUESADA; Carlos LANDA-SOLIS; Andres A GUTIERREZ; Luis VACA

    2005-01-01

    Aim: To develop an easy, fast, automated, and inexpensive method for constructing short-hairpin-RNA cassettes for RNAi studies. Methods: Using single oligonucleotides, a variety of DNA cassettes for RNAi vectors were constructed in only few minutes in an automated manner. The cassettes, targeting the eGFP,were cloned into plasmids driven by RNA polymerase Ⅲ promoter H 1. Then, the plasmids were transfected into HeLa cells that were later infected with a recombinant adenovirus encoding the eGFP gene. The level of eGFP fluorescence was evaluated by confocal imaging and flow cytometry. Results: The plasmids constructed with the DNA cassettes made by the one-oligonucleotide method inhibited eGFP with different potencies, ranging from 55% to 75%. Conclusion: By using the method reported here, it is possible to simultaneously construct hundreds of different DNA cassettes for RNAi experiments in an inexpensive, automated way. This method will facilitate functional genomics studies on mammalian cells.

  19. Conjugative Plasmid Transfer in Xylella fastidiosa Is Dependent on tra and trb Operon Functions

    Science.gov (United States)

    Van Horn, Christopher R.

    2017-01-01

    ABSTRACT The insect-transmitted plant pathogen Xylella fastidiosa is capable of efficient horizontal gene transfer (HGT) and recombination. Natural transformation occurs at high rates in X. fastidiosa, but there also is evidence that certain strains of X. fastidiosa carry native plasmids equipped with transfer and mobilization genes, suggesting conjugation as an additional mechanism of HGT in some instances. Two operons, tra and trb, putatively encoding a conjugative type IV secretion system, are found in some but not all X. fastidiosa isolates, often on native plasmids. X. fastidiosa strains that carry the conjugative transfer genes can belong to different subspecies and frequently differ in host ranges. Using X. fastidiosa strain M23 (X. fastidiosa subsp. fastidiosa) or Dixon (X. fastidiosa subsp. multiplex) as the donor strain and Temecula (X. fastidiosa subsp. fastidiosa) as the recipient strain, plasmid transfer was characterized using the mobilizable broad-host-range vector pBBR5pemIK. Transfer of plasmid pBBR5pemIK was observed under in vitro conditions with both donor strains and was dependent on both tra and trb operon functions. A conjugative mechanism likely contributes to gene transfer between diverse strains of X. fastidiosa, possibly facilitating adaptation to new environments or different hosts. IMPORTANCE Xylella fastidiosa is an important plant pathogen worldwide, infecting a wide range of different plant species. The emergence of new diseases caused by X. fastidiosa, or host switching of existing strains, is thought to be primarily due to the high frequency of HGT and recombination in this pathogen. Transfer of plasmids by a conjugative mechanism enables movement of larger amounts of genetic material at one time, compared with other routes of gene transfer such as natural transformation. Establishing the prevalence and functionality of this mechanism in X. fastidiosa contributes to a better understanding of HGT, adaptation, and disease emergence

  20. Conjugative plasmid transfer in Xylella fastidiosa is dependent on tra and trb operon functions.

    Science.gov (United States)

    Burbank, Lindsey P; Van Horn, Christopher R

    2017-08-14

    The insect-transmitted plant pathogen Xylella fastidiosa is capable of efficient horizontal gene transfer (HGT) and recombination. Natural transformation occurs at high rates in X. fastidiosa, but there also is evidence that certain strains of X. fastidiosa carry native plasmids equipped with transfer and mobilization genes, suggesting conjugation as an additional mechanism of HGT in some instances. Two operons, tra and trb, putatively encoding a conjugative Type IV secretion system are found in some but not all X. fastidiosa isolates, often on native plasmids. X. fastidiosa strains that carry the conjugative transfer genes can belong to different subspecies, and frequently differ in host ranges. Using X. fastidiosa strains M23 (subspecies fastidiosa) or Dixon (subspecies multiplex) as the donor strain and Temecula (subspecies fastidiosa) as the recipient strain, plasmid transfer was characterized using the mobilizable broad host range vector pBBR5pemIK. Transfer of plasmid pBBR5pemIK was observed under in vitro conditions with both donor strains, and was dependent on both tra and trb operon functions. A conjugative mechanism likely contributes to gene transfer between diverse strains of X. fastidiosa, possibly facilitating adaptation to new environments or different hosts.IMPORTANCEXylella fastidiosa is an important plant pathogen world-wide, infecting a wide range of different plant species. Emergence of new diseases caused by X. fastidiosa, or host-switching of existing strains is thought to be primarily due to the high frequency of HGT and recombination in this pathogen. Transfer of plasmids by a conjugative mechanism enables movement of larger amounts of genetic material at one time compared with other routes of gene transfer such as natural transformation. Establishing the prevalence and functionality of this mechanism in X. fastidiosa contributes to a better understanding of HGT and adaptation, and disease emergence in this diverse pathogen. This is a work

  1. Kloning Gen Melanoma Antigen 1 (Mage-1 dari Jaringan Testis untuk mendapatkan Plasmid Rekombinan Mage-1

    Directory of Open Access Journals (Sweden)

    Gondo Mastutik

    2015-12-01

    Full Text Available Melanoma antigen-1 (Mage-1 is expressed by spermatogonia cells of normal testicular tissue and 60−80% is expressed by the liver of hepatocellular carcinoma (HC patients. Mage-1 expression is a marker for diagnosing HC and predicting gastric and colorectal cancers. Isolation of messenger ribonucleid acid (mRNA Mage-1 from the liver tissue of HC patients is difficult; therefore, Mage-1 mRNA isolates can be obtained from tissues that express Mage-1 such as normal testicular tissues . This is an explorative research that was conducted at the Institute of Tropical Diseases of Airlangga University during August 2006–August 2008. The aim was to clone the coding sequence of Mage-1 gene from testicular tissues into a vector and to get recombinant plasmid Mage-1. Isolation of the full-length Mage-1 was performed using semi-nested polymerase chain reaction (PCR which was then cloned into plasmid pET101/D-TOPO and transformed into Escherichia coli (E. coli Top10 to get recombinant plasmid Mage-1. The length of pET101/D-TOPO was 5,753 bp and Mage-1 was 927 bp. The length of recombinant plasmid was 6,680 bp (5,753+927. Restriction analysis using EcoRV showed 4,230 and 2,450 bp bands (4,230+2,450=6,680. Sequence analyses showed that Mage-1 was 100% homologous with M77481 and NM_004988, 99% homologous with BC01755. In conclusion, according to the results of the restriction and sequences analysis, the recombinant plasmid pETGM/MAGE1-Testis contains the full length coding region of Mage-1 and is useful for developing the hepatocellular carcinoma diagnostic kits.

  2. Canine adenovirus type 2 vector generation via I-Sce1-mediated intracellular genome release.

    Directory of Open Access Journals (Sweden)

    Sandy Ibanes

    Full Text Available When canine adenovirus type 2 (CAdV-2, or also commonly referred to as CAV-2 vectors are injected into the brain parenchyma they preferentially transduce neurons, are capable of efficient axonal transport to afferent regions, and allow transgene expression for at last >1 yr. Yet, translating these data into a user-friendly vector platform has been limited because CAV-2 vector generation is challenging. Generation of E1-deleted adenovirus vectors often requires transfection of linear DNA fragments of >30 kb containing the vector genome into an E1-transcomplementing cell line. In contrast to human adenovirus type 5 vector generation, CAV-2 vector generation is less efficient due, in part, to a reduced ability to initiate replication and poor transfectibility of canine cells with large, linear DNA fragments. To improve CAV-2 vector generation, we generated an E1-transcomplementing cell line expressing the estrogen receptor (ER fused to I-SceI, a yeast meganuclease, and plasmids containing the I-SceI recognition sites flanking the CAV-2 vector genome. Using transfection of supercoiled plasmid and intracellular genome release via 4-OH-tamoxifen-induced nuclear translocation of I-SceI, we improved CAV-2 vector titers 1,000 fold, and in turn increased the efficacy of CAV-2 vector generation.

  3. Canine adenovirus type 2 vector generation via I-Sce1-mediated intracellular genome release.

    Science.gov (United States)

    Ibanes, Sandy; Kremer, Eric J

    2013-01-01

    When canine adenovirus type 2 (CAdV-2, or also commonly referred to as CAV-2) vectors are injected into the brain parenchyma they preferentially transduce neurons, are capable of efficient axonal transport to afferent regions, and allow transgene expression for at last >1 yr. Yet, translating these data into a user-friendly vector platform has been limited because CAV-2 vector generation is challenging. Generation of E1-deleted adenovirus vectors often requires transfection of linear DNA fragments of >30 kb containing the vector genome into an E1-transcomplementing cell line. In contrast to human adenovirus type 5 vector generation, CAV-2 vector generation is less efficient due, in part, to a reduced ability to initiate replication and poor transfectibility of canine cells with large, linear DNA fragments. To improve CAV-2 vector generation, we generated an E1-transcomplementing cell line expressing the estrogen receptor (ER) fused to I-SceI, a yeast meganuclease, and plasmids containing the I-SceI recognition sites flanking the CAV-2 vector genome. Using transfection of supercoiled plasmid and intracellular genome release via 4-OH-tamoxifen-induced nuclear translocation of I-SceI, we improved CAV-2 vector titers 1,000 fold, and in turn increased the efficacy of CAV-2 vector generation.

  4. Networking in microbes: conjugative elements and plasmids in the genus Alteromonas.

    Science.gov (United States)

    López-Pérez, Mario; Ramon-Marco, Nieves; Rodriguez-Valera, Francisco

    2017-01-05

    To develop evolutionary models for the free living bacterium Alteromonas the genome sequences of isolates of the genus have been extensively analyzed. However, the main genetic exchange drivers in these microbes, conjugative elements (CEs), have not been considered in detail thus far. In this work, CEs have been searched in several complete Alteromonas genomes and their sequence studied to understand their role in the evolution of this genus. Six genomes are reported here for the first time. We have found nine different plasmids of sizes ranging from 85 to 600 Kb, most of them were found in a single strain. Networks of gene similarity could be established among six of the plasmids that were also connected with another cluster of plasmids found in Shewanella strains. The cargo genes found in these plasmids included cassettes found before in chromosome flexible genomic islands of Alteromonas strains. We describe also the plasmids pAMCP48-600 and pAMCP49-600, the largest found in Alteromonas thus far (ca. 600 Kb) and containing all the hallmarks to be classified as chromids. We found in them some housekeeping genes and a cluster that code for an exocellular polysaccharide. They could represent the transport vectors for the previously described replacement flexible genomic islands. Integrative and conjugative elements (ICEs) were more common than plasmids and showed similar patterns of variation with cargo genes coding for components of additive flexible genomic islands. A nearly identical ICE was found in A. mediterranea MED64 and Vibrio cholera AHV1003 isolated from a human pathogen, indicating the potential exchange of these genes across phylogenetic distances exceeding the family threshold. We have seen evidence of how CEs can be vectors to transfer gene cassettes acquired in the chromosomal flexible genomic islands, both of the additive and replacement kind. These CEs showed evidence of how genetic material is exchanged among members of the same species but also

  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. Monitoring of prostate cancer growth and metastasis using a PSA luciferase report plasmid in a mouse model

    Institute of Scientific and Technical Information of China (English)

    Qi-Qi Mao; Yi-Wei Lin; Hong Chen; Kai Yang; De-Bo Kong; Hai Jiang

    2014-01-01

    Objective:To construct a PSA luciferase report plasmid and monitor the growth and metastasis of prostate cancer after emasculation in SCID mice. Methods: PSA promoter sequence and luciferase gene were amplified by PCR and subsequently inserted into pZsGreen1-1 vector to construct pPSA-FL-Luc vector. LNCaP cells that were stably transfected with pPSA-FL-Luc were used to establish a SCID mouse xenograft model. Then, the growth and metastasis of prostate cancer were monitored via living imaging. Results:We successfully constructed a PSA luciferase plasmid, pPSA-FL-Luc. DHT enhanced luciferase activity in a concentration-dependent manner in 293T cells with pPSA-FL-Luc transfection. Prostate cancer SCID mouse model was established with pPSA-FL-Luc transfected LNCaP cells. In tumor bearing mice with or without emasculation, pPSA-FL-Luc plasmid was applied to monitored tumor growth and metastasis based on bioluminescence imaging. Conclusions: We construct a pPSA-FL-Luc plasmid, which stably expresses luciferase and can be applied to monitor tumor development in a prostate SCID mouse model.

  7. Bacteriophages limit the existence conditions for conjugative plasmids.

    Science.gov (United States)

    Harrison, Ellie; Wood, A Jamie; Dytham, Calvin; Pitchford, Jonathan W; Truman, Julie; Spiers, Andrew; Paterson, Steve; Brockhurst, Michael A

    2015-06-02

    Bacteriophages are a major cause of bacterial mortality and impose strong selection on natural bacterial populations, yet their effects on the dynamics of conjugative plasmids have rarely been tested. We combined experimental evolution, mathematical modeling, and individual-based simulations to explain how the ecological and population genetics effects of bacteriophages upon bacteria interact to determine the dynamics of conjugative plasmids and their persistence. The ecological effects of bacteriophages on bacteria are predicted to limit the existence conditions for conjugative plasmids, preventing persistence under weak selection for plasmid accessory traits. Experiments showed that phages drove faster extinction of plasmids in environments where the plasmid conferred no benefit, but they also revealed more complex effects of phages on plasmid dynamics under these conditions, specifically, the temporary maintenance of plasmids at fixation followed by rapid loss. We hypothesized that the population genetic effects of bacteriophages, specifically, selection for phage resistance mutations, may have caused this. Further mathematical modeling and individual-based simulations supported our hypothesis, showing that conjugative plasmids may hitchhike with phage resistance mutations in the bacterial chromosome. Conjugative plasmids are infectious loops of DNA capable of transmitting DNA between bacterial cells and between species. Because plasmids often carry extra genes that allow bacteria to live in otherwise-inhospitable environments, their dynamics are central to understanding bacterial adaptive evolution. The plasmid-bacterium interaction has typically been studied in isolation, but in natural bacterial communities, bacteriophages, viruses that infect bacteria, are ubiquitous. Using experiments, mathematical models, and computer simulations we show that bacteriophages drive plasmid dynamics through their ecological and evolutionary effects on bacteria and ultimately

  8. Multiple Antibiotic Resistance Plasmids Allow Scalable, PCR-Mediated DNA Manipulation and Near-Zero Background Cloning

    Directory of Open Access Journals (Sweden)

    Remigiusz Arnak

    2016-01-01

    Full Text Available We have constructed two plasmids that can be used for cloning as templates for PCR-based gene disruption, mutagenesis and the construction of DNA chromosome translocation cassettes. To our knowledge, these plasmids are the first vectors that confer resistance to ampicillin, kanamycin and hygromycin B in bacteria, and to geneticin (G418 and hygromycin B in Saccharomyces cerevisiae simultaneously. The option of simultaneously using up to three resistance markers provides a highly stringent control of recombinant selection and the almost complete elimination of background resistance, while unique restriction sites allow easy cloning of chosen genetic material. Moreover, we successfully used these new vectors as PCR templates for the induction of chromosome translocation in budding yeast by the bridge-induced translocation system. Cells in which translocation was induced carried chromosomal rearrangements as expected and exhibited resistance to both, G418 and hygromycin B. These features make our constructs very handy tools for many molecular biology applications.

  9. Bacteriophage selection against a plasmid-encoded sex apparatus leads to the loss of antibiotic-resistance plasmids.

    Science.gov (United States)

    Jalasvuori, Matti; Friman, Ville-Petri; Nieminen, Anne; Bamford, Jaana K H; Buckling, Angus

    2011-12-23

    Antibiotic-resistance genes are often carried by conjugative plasmids, which spread within and between bacterial species. It has long been recognized that some viruses of bacteria (bacteriophage; phage) have evolved to infect and kill plasmid-harbouring cells. This raises a question: can phages cause the loss of plasmid-associated antibiotic resistance by selecting for plasmid-free bacteria, or can bacteria or plasmids evolve resistance to phages in other ways? Here, we show that multiple antibiotic-resistance genes containing plasmids are stably maintained in both Escherichia coli and Salmonella enterica in the absence of phages, while plasmid-dependent phage PRD1 causes a dramatic reduction in the frequency of antibiotic-resistant bacteria. The loss of antibiotic resistance in cells initially harbouring RP4 plasmid was shown to result from evolution of phage resistance where bacterial cells expelled their plasmid (and hence the suitable receptor for phages). Phages also selected for a low frequency of plasmid-containing, phage-resistant bacteria, presumably as a result of modification of the plasmid-encoded receptor. However, these double-resistant mutants had a growth cost compared with phage-resistant but antibiotic-susceptible mutants and were unable to conjugate. These results suggest that bacteriophages could play a significant role in restricting the spread of plasmid-encoded antibiotic resistance.

  10. Construction of New Campylobacter Cloning Vectors and a New Mutational Cat Cassette

    Science.gov (United States)

    1993-01-01

    mutational cat cassette PE - 61102A PR - 3M161102 6. AUTHOR(S) TA - BS13AK Yao R, Aim RA, Trust TJ, Guerry P WU- 1291 7. PERFORMING ORGANIZATION NAME(S) AND...mutational cat cassette %~ccesion For (Site-specific mutagenesis; recombinant DNA; multiple cloning site; PCR; shuttle vectors) NTIS CRA&I OTIC TAB E...incompatibility plasmids can mobilize shuttle vectors containing E. coi and C. coi plasmid Systems of experimental genetics are in the early stages replicons

  11. A rapid and safe plasmid isolation method for efficient engineering of recombinant lactobacilli expressing immunogenic or tolerogenic epitopes for oral administration

    NARCIS (Netherlands)

    Maassen, C.B.M.

    1999-01-01

    Recombinant lactobacilli are being developed which can be used as expression and delivery vectors of heterologous antigens in oral vaccination and other therapeutic applications. Because most Lactobacillus strains do not accept ligation mixtures, sufficiently pure plasmid DNA needs to be isolated fr

  12. A rapid and safe plasmid isolation method for efficient engineering of recombinant lactobacilli expressing immunogenic or tolerogenic epitopes for oral administration

    NARCIS (Netherlands)

    Maassen, C.B.M.

    1999-01-01

    Recombinant lactobacilli are being developed which can be used as expression and delivery vectors of heterologous antigens in oral vaccination and other therapeutic applications. Because most Lactobacillus strains do not accept ligation mixtures, sufficiently pure plasmid DNA needs to be isolated

  13. Utilization of an unstable plasmid and the I-SceI endonuclease to generate routine markerless deletion mutants in Francisella tularensis

    Science.gov (United States)

    Horzempa, Joseph; Shanks, Robert M.Q.; Brown, Matthew J.; Russo, Brian C.; O’Dee, Dawn M.; Nau, Gerard J.

    2011-01-01

    We engineered an efficient system to make Francisella tularensis deletion mutations using an unstable, poorly maintained plasmid to enhance the likelihood of homologous recombination. For counterselection, we adapted a strategy using I-SceI, which causes a double-stranded break in the integrated suicide vector, forcing a second recombination to mediate allelic replacement. PMID:19879904

  14. An ultra-deep sequencing strategy to detect sub-clonal TP53 mutations in presentation chronic lymphocytic leukaemia cases using multiple polymerases.

    Science.gov (United States)

    Worrillow, L; Baskaran, P; Care, M A; Varghese, A; Munir, T; Evans, P A; O'Connor, S J; Rawstron, A; Hazelwood, L; Tooze, R M; Hillmen, P; Newton, D J

    2016-10-06

    Chronic lymphocytic leukaemia (CLL) is the most common clonal B-cell disorder characterized by clonal diversity, a relapsing and remitting course, and in its aggressive forms remains largely incurable. Current front-line regimes include agents such as fludarabine, which act primarily via the DNA damage response pathway. Key to this is the transcription factor p53. Mutations in the TP53 gene, altering p53 functionality, are associated with genetic instability, and are present in aggressive CLL. Furthermore, the emergence of clonal TP53 mutations in relapsed CLL, refractory to DNA-damaging therapy, suggests that accurate detection of sub-clonal TP53 mutations prior to and during treatment may be indicative of early relapse. In this study, we describe a novel deep sequencing workflow using multiple polymerases to generate sequencing libraries (MuPol-Seq), facilitating accurate detection of TP53 mutations at a frequency as low as 0.3%, in presentation CLL cases tested. As these mutations were mostly clustered within the regions of TP53 encoding DNA-binding domains, essential for DNA contact and structural architecture, they are likely to be of prognostic relevance in disease progression. The workflow described here has the potential to be implemented routinely to identify rare mutations across a range of diseases.

  15. 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.

  16. 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.

  17. Genetically engineering adenoviral vectors for gene therapy.

    Science.gov (United States)

    Coughlan, Lynda

    2014-01-01

    Adenoviral (Ad) vectors are commonly used for various gene therapy applications. Significant advances in the genetic engineering of Ad vectors in recent years has highlighted their potential for the treatment of metastatic disease. There are several methods to genetically modify the Ad genome to incorporate retargeting peptides which will redirect the natural tropism of the viruses, including homologous recombination in bacteria or yeast. However, homologous recombination in yeast is highly efficient and can be achieved without the need for extensive cloning strategies. In addition, the method does not rely on the presence of unique restriction sites within the Ad genome and the reagents required for this method are widely available and inexpensive. Large plasmids containing the entire adenoviral genome (~36 kbp) can be modified within Saccharomyces cerevisiae yeast and genomes easily rescued in Escherichia coli hosts for analysis or amplification. A method for two-step homologous recombination in yeast is described in this chapter.

  18. 抗p185erbB2人鼠嵌合抗体慢病毒表达载体的构建%Construction of the Lentiviral Expression Vector for Anti-p185erbB2 Mouse/Human Chimeric Antibody

    Institute of Scientific and Technical Information of China (English)

    刘芳; 李力; 张玮; 王琪

    2013-01-01

    本研究构建抗p185erbB2人鼠嵌合抗体慢病毒表达载体,并将其转染293T细胞,明确转染后嵌合抗体基因的表达情况.采用PCR法扩增抗p185erbB2鼠单抗轻、重链可变区基因(vL和vH)和人IgG1的轻、重链恒定区基因(κ和γ1),再利用三引物PCR法将vL和κ,vH和γ1进行拼接,得到嵌合轻链基因(L)和嵌合重链基因(H),分别插入质粒pVAX1/IRES的IRES元件的下、上游.用内切酶将H-IRES-L从pVAX1/H-IRES-L上切下,插入慢病毒载体pWPI中,经相应酶切和测序鉴定,正确构建了慢病毒表达载体pWPI/H-IRES-L.将其转染293T细胞,48 h后通过荧光显微镜检测绿色荧光蛋白(GFP)的表达,RT-PCR和直接ELISA法检测嵌合抗体的表达.结果显示,转染pWPI/H-IRES-L的293T细胞中有嵌合轻链和嵌合重链基因的共同表达,而且表达的嵌合抗体能够与p185erbB2分子特异性结合,为今后抗p185erbB2工程抗体的研究奠定了基础.%This research was to construct the lentiviral expression vector for anti- pl85erbB2 mouse/human chimeric antibody and to determine the expression of the chimeric antibody gene in 293T cells transfected with this vector. The genes (vL and vH) coding light and heavy chain of variable regions of anti-pl85erbB2 mAb and the constant regions of human IgG1 (Κ and γ1) were cloned with PCR method. The target genes were assembled by three-primers PCR method to obtain the chimeric light chain (L) and the chimeric heavy chain (H). Both chains inserted into the down stream and upper stream of IRES gene of the plasmid pVAX1/IRES respectively. We digested the plasmid pVAX1/ H-IRES-L with endoenzyme and subcloned H-IRES-L into the lentiviral vector pWPI. The enzyme digestion and sequence analysis showed that the lentiviral expression vector pWPI/H-IRES-L was constructed correctly. Then, it was transfected into 293T cells and after 48h, GFP protein expression in 293T cells were detected by fluorescent microscope and the chimeric antibody

  19. Sequence and organization of pMAC, an Acinetobacter baumannii plasmid harboring genes involved in organic peroxide resistance.

    Science.gov (United States)

    Dorsey, Caleb W; Tomaras, Andrew P; Actis, Luis A

    2006-09-01

    Acinetobacter baumannii 19606 harbors pMAC, a 9540-bp plasmid that contains 11 predicted open-reading frames (ORFs). Cloning and transformation experiments using Acinetobacter calcoaceticus BD413 mapped replication functions within a region containing four 21-bp direct repeats (ori) and ORF 1, which codes for a predicted replication protein. Subcloning and tri-parental mating experiments mapped mobilization functions to the product of ORF 11 and an adjacent predicted oriT. Three ORFs code for proteins that share similarity to hypothetical proteins encoded by plasmid genes found in other bacteria, while the predicted products of three others do not match any known sequence. The product of ORF 8 is similar to Ohr, a hydroperoxide reductase responsible for organic peroxide detoxification and resistance in bacteria. This ORF is immediately upstream of a coding region whose product is related to the MarR family of transcriptional regulators. Disk diffusion assays showed that A. baumannii 19606 is resistant to the organic peroxide-generating compounds cumene hydroperoxide (CHP) and tert-butyl hydroperoxide (t-BHP), although to levels lower than those detected in Pseudomonas aeruginosa PAO1. Cloning and introduction of the ohr and marR ORFs into Escherichia coli was associated with an increase in resistance to CHP and t-BHP. This appears to be the first case in which the genetic determinants involved in organic peroxide resistance are located in an extrachromosomal element, a situation that can facilitate the horizontal transfer of genetic elements coding for a function that protects bacterial cells from oxidative damage.

  20. Role of Plasmid in Production of Acetobacter Xylinum Biofilms

    Directory of Open Access Journals (Sweden)

    Abbas Rezaee

    2005-01-01

    Full Text Available Acetobacter xylinum has the ability to produce cellulotic biofilms. Bacterial cellulose is expected to be used in many industrial or biomedical materials for its unique characteristics. A. xylinum contains a complex system of plasmid DNA molecules. A 44 kilobases (kb plasmid was isolated in wild type of A. xylinum. To improve the cellulose producing ability of A. xylinum, role of the plasmid in production of cellulose was studied. The comparisons between wild type and cured cells of A. xylinum showed that there is considerably difference in cellulose production. In order to study the relationship between plasmid and the rate of cellulose production, bacteria were screened for plasmid profile by a modified method for preparation of plasmid. This method yields high levels of pure plasmid DNA that can be used for common molecular techniques, such as digestion and transformation, with high efficiency.

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

    DEFF Research Database (Denmark)

    Klümper, Uli

    at high frequencies from diverse donors, I showed plasmid or donor dependence of plasmid transfer to other species. Additionally, environmental factors like stress also impact the permissiveness of phylogenetic groups towards plasmids. The developed method and results increase our ability to predict......Horizontal transfer of mobile genetic elements facilitates adaptive and evolutionary processes in bacteria. Among the known mobile genetic elements, plasmids can confer their hosts with accessory adaptive traits, such as antibiotic or heavy metal resistances, or additional metabolic pathways...... and the extent of bacterial phyla permissive towards plasmid receipt are largely unknown. Historically, methods exploring the underlying genetic and environmental factors of plasmid transfer have been heavily reliant on cultivation and expression of plasmid encoded phenotypes. This has provided an incomplete...

  2. A versatile one-step CRISPR-Cas9 based approach to plasmid-curing

    DEFF Research Database (Denmark)

    Lauritsen, Ida; Porse, Andreas; Sommer, Morten Otto Alexander

    2017-01-01

    tool enabling rapid removal of plasmids from bacterial cells is lacking. Results Based on replicon abundance and sequence conservation analysis, we show that the vast majority of bacterial cloning and expression vectors share sequence similarities that allow for broad CRISPR-Cas9 targeting. We have...... widely used for expression and engineering purposes. By virtue of the CRISPR-Cas9 targeting, our platform is highly expandable and can be applied in a broad host context. We exemplify the wide applicability of our system in Gram-negative bacteria by demonstrating the successful application in both...

  3. Constructing the recombinant plasmid-pLXSN-SjYF-of Yolk Ferritin gene of Schistosoma japonicum

    Institute of Scientific and Technical Information of China (English)

    ZhouJunmei; YuXinbing; WuZhongdao; ZhengYinan; LiYan

    1999-01-01

    TO construct the recotabinant plasmid —— pLXSN-SjYF to prepare for expression and DNA vaccine of Schistosoma japonicum gene. Methods :Mnpliffing DNA fragment coding yolk fereltin from a female adult Sehistosoma japonicum DNAs by PCR. The fragment was inseted into pLXSN retrovirus vector by digesting with restrictive enzymes and linking reactions. The positive clone was screened on LB plates contmaining amplcillin asld identified by restrictive enzymes digestion and PCR amplification, Resttlts The specific DNA fragmem SjYF was amplified from the female adult SjDNAs. IaLXSN-SjYF was constructed successfully and the further research will be carried out.

  4. Modeling sRNA-Regulated Plasmid Maintenance

    Science.gov (United States)

    Klumpp, Stefan

    2017-01-01

    We study a theoretical model for the toxin-antitoxin (hok/sok) mechanism for plasmid maintenance in bacteria. Toxin-antitoxin systems enforce the maintenance of a plasmid through post-segregational killing of cells that have lost the plasmid. Key to their function is the tight regulation of expression of a protein toxin by an sRNA antitoxin. Here, we focus on the nonlinear nature of the regulatory circuit dynamics of the toxin-antitoxin mechanism. The mechanism relies on a transient increase in protein concentration rather than on the steady state of the genetic circuit. Through a systematic analysis of the parameter dependence of this transient increase, we confirm some known design features of this system and identify new ones: for an efficient toxin-antitoxin mechanism, the synthesis rate of the toxin’s mRNA template should be lower that of the sRNA antitoxin, the mRNA template should be more stable than the sRNA antitoxin, and the mRNA-sRNA complex should be more stable than the sRNA antitoxin. Moreover, a short half-life of the protein toxin is also beneficial to the function of the toxin-antitoxin system. In addition, we study a therapeutic scenario in which a competitor mRNA is introduced to sequester the sRNA antitoxin, causing the toxic protein to be expressed. PMID:28085919

  5. Modeling sRNA-regulated Plasmid Maintenance

    CERN Document Server

    Gong, Chen Chris

    2016-01-01

    We study a theoretical model for the toxin-antitoxin (hok/sok) mechanism for plasmid maintenance in bacteria. Toxin-antitoxin systems enforce the maintenance of a plasmid through post-segregational killing of cells that have lost the plasmid. Key to their function is the tight regulation of expression of a protein toxin by an sRNA antitoxin. Here, we focus on the nonlinear nature of the regulatory circuit dynamics of the toxin-antitoxin mechanism. The mechanism relies on a transient increase in protein concentration rather than on the steady state of the genetic circuit. Through a systematic analysis of the parameter dependence of this transient increase, we confirm some known design features of this system and identify new ones: for an efficient toxin-antitoxin mechanism, the synthesis rate of the toxin's mRNA template should be lower that of the sRNA antitoxin, the mRNA template should be more stable than the sRNA antitoxin, and the mRNA-sRNA complex should be more stable than the sRNA antitoxin. Moreover, ...

  6. Rotations with Rodrigues' Vector

    Science.gov (United States)

    Pina, E.

    2011-01-01

    The rotational dynamics was studied from the point of view of Rodrigues' vector. This vector is defined here by its connection with other forms of parametrization of the rotation matrix. The rotation matrix was expressed in terms of this vector. The angular velocity was computed using the components of Rodrigues' vector as coordinates. It appears…

  7. Construction and identification of eukaryotic eukaryotic expression plasmid pcdna3.1-bace and its transient expression in cells

    Institute of Scientific and Technical Information of China (English)

    Huilin Gong; Guanjun Zhang; Weijiang Dong

    2006-01-01

    Objective: To generate eukaryotic expression vector of pcDNA3.1-BACE and obtain its transient expression in COS-7 cells and high expression in the neuroblastoma SK-N-SH cells. Methods: A 1503 bp cDNA fragment was amplified from the total RNA of human neuroblastoma by RT-PCR method and cloned into plasmid pcDNA3.1. The vector was identified by digestion with restriction enzymes BamHI and XhoI and sequenced by Sanger-dideoxy-mediated chain termination. The expression of BACE gene was detected by immunocytochemistry method. Results: The results showed that the cDNAfragment included 1503 bp total coding region. The recombinant eukaryotic cell expression vector of pcDNA3.1-BACE was constructed successfully,and the sequence of insert was identical to the published sequence. The COS-7 cells and the neuroblastoma SK-N-SH cells transfected with the pcDNA3.1-BACE plasmid expressed high level of BACE protein in cytoplasm. Conclusion: The recombinant plasmid pcDNA3.1-BACE can provide very useful tool for researching the reason of Alzheimer's disease and lays the important foundation for preventing the AD laterly.

  8. Gram negative shuttle BAC vector for heterologous expression of metagenomic libraries.

    Science.gov (United States)

    Kakirde, Kavita S; Wild, Jadwiga; Godiska, Ronald; Mead, David A; Wiggins, Andrew G; Goodman, Robert M; Szybalski, Waclaw; Liles, Mark R

    2011-04-15

    Bacterial artificial chromosome (BAC) vectors enable stable cloning of large DNA fragments from single genomes or microbial assemblages. A novel shuttle BAC vector was constructed that permits replication of BAC clones in diverse Gram-negative species. The "Gram-negative shuttle BAC" vector (pGNS-BAC) uses the F replicon for stable single-copy replication in E. coli and the broad-host-range RK2 mini-replicon for high-copy replication in diverse Gram-negative bacteria. As with other BAC vectors containing the oriV origin, this vector is capable of an arabinose-inducible increase in plasmid copy number. Resistance to both gentamicin and chloramphenicol is encoded on pGNS-BAC, permitting selection for the plasmid in diverse bacterial species. The oriT from an IncP plasmid was cloned into pGNS-BAC to enable conjugal transfer, thereby allowing both electroporation and conjugation of pGNS-BAC DNA into bacterial hosts. A soil metagenomic library was constructed in pGNS-BAC-1 (the first version of the vector, lacking gentamicin resistance and oriT), and recombinant clones were demonstrated to replicate in diverse Gram-negative hosts, including Escherichia coli, Pseudomonas spp., Salmonella enterica, Serratia marcescens, Vibrio vulnificus and Enterobacter nimipressuralis. This shuttle BAC vector can be utilized to clone genomic DNA from diverse sources, and then transfer it into diverse Gram-negative bacterial species to facilitate heterologous expression of recombinant pathways.

  9. Genes from plasmid pKM101 in Haemophilus influenzae: separation of functions of mucA and mucB

    Energy Technology Data Exchange (ETDEWEB)

    Balganesh, M.; Setlow, J.K.

    1985-11-01

    Haemophilus influenzae, normally not mutable by UV, became UV mutable with a recombinant plasmid insertion. A 7.8-kilobase-pair (kbp) fragment of the plasmid pKM101 containing the mucA and mucB genes was ligated to the shuttle vector pDM2, and a Rec- strain of H. influenzae was transformed with the ligated mixture. All of the transformants, unlike the parent Rec- strain, were resistant to UV, could carry out postreplication repair and Weigle reactivation, showed greatly increased spontaneous mutation, and contained a plasmid carrying an insert of only 1.2 rather than 7.8 kbp. This plasmid in a umuC mutant strain of Escherichia coli complemented a pKM101 derivative lacking mucA function but with an intact mucB gene, although there was no complementation with a mucA+ mucB- plasmid, suggesting that the newly constructed plasmid coded for the mucA protein; this is in accord with the restriction analysis and hybridization between the plasmid and a probe containing all of the mucA gene but only a small fraction of mucB. When one of the H. influenzae Rec- transformants lost the plasmid, the resistance to UV was retained but the high spontaneous mutation and UV mutability were not. The fact that there was hybridization between the chromosome of the cured strain and a probe containing both muc genes but none when almost no mucB was present suggested that at least part of the mucB gene had been integrated into the Rec- chromosome. Five different postreplication repair-proficient strains became UV mutable and had high spontaneous mutation rates caused by the putative mucA plasmid, indicating that these strains already possessed a chromosomal equivalent of the mucB gene.

  10. Non-viral S/MAR vectors replicate episomally in vivo when provided with a selective advantage.

    Science.gov (United States)

    Wong, S P; Argyros, O; Coutelle, C; Harbottle, R P

    2011-01-01

    The ideal gene therapy vector should enable persistent expression without the limitations of safety and reproducibility. We previously reported that a prototype plasmid vector, containing a scaffold matrix attachment region (S/MAR) domain and the luciferase reporter gene, showed transgene expression for at least 6 months following a single administration to MF1 mice. Following partial hepatectomy of the animals, however, we found no detectable vector replication and subsequent propagation in vivo. To overcome this drawback, we have now developed an in vivo liver selection strategy by which liver cells transfected with an S/MAR plasmid are provided with a survival advantage over non-transfected cells. This allows an enrichment of vectors that are capable of replicating and establishing themselves as extra-chromosomal entities in the liver. Accordingly, a novel S/MAR plasmid encoding the Bcl-2 gene was constructed; Bcl-2 expression confers resistance against apoptosis-mediated challenges by the Fas-activating antibody Jo2. Following hydrodynamic delivery to the livers of mice and frequent Jo2 administrations, we demonstrate that this Bcl-luciferase S/MAR plasmid is indeed capable of providing sustained luciferase reporter gene expression for over 3 months and that this plasmid replicates as an episomal entity in vivo. These results provide proof-of-principle that S/MAR vectors are capable of preventing transgene silencing, are resistant to integration and are able to confer mitotic stability in vivo when provided with a selective advantage.

  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. Plasmid transfer between bacteria in soil microcosms and the field

    Directory of Open Access Journals (Sweden)

    Eric Smit

    1997-01-01

    Full Text Available In ibis review factors influencing conjugal plasmid transfer between bacteria and the possible role of naturally occurring selftransmissible plasmide for the dissemination of recombinant DNA in soil will be discussed. In microcosm studies, plasmid transfer between various species of introduced bacteria has been detected. Moreover, plamid transfer to indigenous soil micoorganisms was observed. Soil is an oligotrophic environment and plasmid transfer occurred mainly under conditions which were nutritionally favourable for bacteria, such as in the plant rhizosphere and in the presence of clay minerais or added nutrients. Mobilizable plasmids, lacking the ability to transfer themselves, have been reported to be transferred in the presence of selftransmissible plasmids. A study comparing conjugal transfer in microcosme with those in the field revealed that the transfer rates found in microcosme and in the field were similar. Transfer of chromosomal DNA by plasmid RP4 could only be shown on filters and was not observed in soil. Transfer of plasmids carrying biodegradative genes appeared to be favoured in the presence of the compound that can be degraded. Evidence was found for the presence of naturally-occurring selftransmissible plasmids in bacteria in the rhizosphere which could mobilize recombinant plasmids.

  13. Isolation and screening of plasmids from the epilithon which mobilize recombinant plasmid pD10.

    OpenAIRE

    Hill, K E; A. J. Weightman; Fry, J C

    1992-01-01

    This study examined the potential of bacteria from river epilithon to mobilize a recombinant catabolic plasmid, pD10, encoding 3-chlorobenzoate degradation and kanamycin resistance. Fifty-four mobilizing plasmids were exogenously isolated by triparental matings between strains of Pseudomonas putida and epilithic bacteria from the River Taff (South Wales, United Kingdom). Frequencies for mobilization ranged from 1.7 x 10(-8) to 4.5 x 10(-3) per recipient at 20 degrees C. The sizes of the mobil...

  14. Fed-batch microbioreactor platform for scale down and analysis of a plasmid DNA production process.

    Science.gov (United States)

    Bower, Diana M; Lee, Kevin S; Ram, Rajeev J; Prather, Kristala L J

    2012-08-01

    The rising costs of bioprocess research and development emphasize the need for high-throughput, low-cost alternatives to bench-scale bioreactors for process development. In particular, there is a need for platforms that can go beyond simple batch growth of the organism of interest to include more advanced monitoring, control, and operation schemes such as fed-batch or continuous. We have developed a 1-mL microbioreactor capable of monitoring and control of dissolved oxygen, pH, and temperature. Optical density can also be measured online for continuous monitoring of cell growth. To test our microbioreactor platform, we used production of a plasmid DNA vaccine vector (pVAX1-GFP) in Escherichia coli via a fed-batch temperature-inducible process as a model system. We demonstrated that our platform can accurately predict growth, glycerol and acetate concentrations, as well as plasmid copy number and quality obtained in a bench-scale bioreactor. The predictive abilities of the micro-scale system were robust over a range of feed rates as long as key process parameters, such as dissolved oxygen, were kept constant across scales. We have highlighted plasmid DNA production as a potential application for our microbioreactor, but the device has broad utility for microbial process development in other industries as well. Copyright © 2012 Wiley Periodicals, Inc.

  15. Construction of recombinant eukaryotic expression plasmid of AMA1 gene of Neospora caninum and its expression in Vero cells%犬新孢子虫AMA1基因重组真核表达质粒的构建及其在Vero细胞中的表达

    Institute of Scientific and Technical Information of China (English)

    郭焕平; 贾立军; 薛书江; 钱年超; 张守发

    2013-01-01

    为构建犬新孢子虫AMA1基因的重组真核表达质粒,了解其在Vero细胞中的表达情况,采用RT-PCR方法扩增牛源犬新孢子虫吉林株AMA1全长基因,构建了重组真核表达质粒pVAX1-NcAMA1;利用脂质体介导转染法将该重组质粒转染至Vero细胞,应用间接免疫荧光方法(IFA)和Western-blot技术检测AMA1基因在Vero细胞中的表达情况.结果显示,扩增的牛源犬新孢子虫吉林株AMA1基因的长度为1 695 bp,与GenBank中相应基因序列(AB265823.1)的同源性为99.9%.成功构建了重组真核表达质粒pVAX1-NcAMA1,IFA检测发现AMA1基因在Vero细胞中获得了瞬时表达,表达产物经Western-blot鉴定,其分子质量约为68 ku,且具有较好的反应原性.本试验为新孢子虫病核酸疫苗的进一步研究奠定了基础.%To construct recombinant eukaryotic expression plasmid of AMA1 gene of Neospora canihum and express it in Vero cells,the full-length fragment of NcAMA1 gene from bovine N.caninum Jilin strain was amplified by RT-PCR,and then subcloned into the expression vector pVAX-1 to construct a eukaryotic expression plasmid pVAX1-AMA1.The recombinant was then transfected into the Vero cells by liposome method.The expressed product in Vero cells was analyzed by indirect immunofluorescence assay (IFA) and Western-blot.The results showed that the amplified fragment was 1 695 bp,and shared 99.9% homology with the corresponding gene sequence in GenBank (AB265823.1).The recombinant eukaryotic expression plasmid pVAX1-AMA1 was successfully constructed.IFA result showed that the AMA1 gene was transiently expressed,and Western-blotting result showed that expressed product was 68 ku in molecular weight and had a positive reaction with anti-AMA1 serum.These results may provide a foundation for further studies on the development of nucleic acid vaccines against N.caninum infections.

  16. CONSTRUCTION OF RECOMBINANT PLASMID PIRES-BAX-HGF%pIRES-Bax-HGF重组质粒的构建

    Institute of Scientific and Technical Information of China (English)

    黄涛; 常青; 徐平

    2011-01-01

    Objective To construct the plasmid vector of pIRES-Bax-HGF. Methods The Bax gene sequence was cloned from ovarian cancer samples, and HGF gene sequence from pBluescript Ⅱ SK+HGF plasmid derived from ATCC. Bax and HGF coding sequence were inserted into pIRES plasmid by step double enzyme digestion. Results Enzyme digestion and sequencing indicated that the construction of recombinant pIRES-Bax-HGF plasmid was successful. Conclusion Obtaining the coding genes of both Bax and HGF, and successfully creating the plasmid vector of plRES-Bax-HGF establish a foundation for further study of the effects of HGF and Bax on vascular endothelial cells, smooth muscle cells, and fibrocytes, which steps out an important step to treat post-CABG restenosis at gene level.%目的 构建pIRES-Bax-HGF质粒载体.方法 采用RT-PCR方法从卵巢癌标本中克隆Bax的基因序列,从ATCC来源的pBlueseriptⅡ SK+HGF质粒中克隆HGF的基因序列.分步双酶切插入到plRES载体质粒中.结果 酶切鉴定及测序表明,重组pIRES-Bax-HGF质粒构建成功.结论 获取HGF及Bax编码基因并成功构建重组pIRES-Bax-HGF质粒载体,为进一步研究HGF及Bax对血管内皮细胞、平滑肌细胞、纤维细胞等的影响奠定了基础,也向基因水平干预冠状动脉旁路移植术术后再狭窄的形成迈出了重要一步.

  17. Engineering BioBrick vectors from BioBrick parts

    Directory of Open Access Journals (Sweden)

    Knight Thomas F

    2008-04-01

    Full Text Available Abstract Background The underlying goal of synthetic biology is to make the process of engineering biological systems easier. Recent work has focused on defining and developing standard biological parts. The technical standard that has gained the most traction in the synthetic biology community is the BioBrick standard for physical composition of genetic parts. Parts that conform to the BioBrick assembly standard are BioBrick standard biological parts. To date, over 2,000 BioBrick parts have been contributed to, and are available from, the Registry of Standard Biological Parts. Results Here we extended the same advantages of BioBrick standard biological parts to the plasmid-based vectors that are used to provide and propagate BioBrick parts. We developed a process for engineering BioBrick vectors from BioBrick parts. We designed a new set of BioBrick parts that encode many useful vector functions. We combined the new parts to make a BioBrick base vector that facilitates BioBrick vector construction. We demonstrated the utility of the process by constructing seven new BioBrick vectors. We also successfully used the resulting vectors to assemble and propagate other BioBrick standard biological parts. Conclusion We extended the principles of part reuse and standardization to BioBrick vectors. As a result, myriad new BioBrick vectors can be readily produced from all existing and newly designed BioBrick parts. We invite the synthetic biology community to (1 use the process to make and share new BioBrick vectors; (2 expand the current collection of BioBrick vector parts; and (3 characterize and improve the available collection of BioBrick vector parts.

  18. Nonviral Vectors for Gene Delivery

    Science.gov (United States)

    Baoum, Abdulgader Ahmed

    2011-12-01

    The development of nonviral vectors for safe and efficient gene delivery has been gaining considerable attention recently. An ideal nonviral vector must protect the gene against degradation by nuclease in the extracellular matrix, internalize the plasma membrane, escape from the endosomal compartment, unpackage the gene at some point and have no detrimental effects. In comparison to viruses, nonviral vectors are relatively easy to synthesize, less immunogenic, low in cost, and have no limitation in the size of a gene that can be delivered. Significant progress has been made in the basic science and applications of various nonviral gene delivery vectors; however, the majority of nonviral approaches are still inefficient and often toxic. To this end, two nonviral gene delivery systems using either biodegradable poly(D,L-lactide- co-glycolide) (PLG) nanoparticles or cell penetrating peptide (CPP) complexes have been designed and studied using A549 human lung epithelial cells. PLG nanoparticles were optimized for gene delivery by varying particle surface chemistry using different coating materials that adsorb to the particle surface during formation. A variety of cationic coating materials were studied and compared to more conventional surfactants used for PLG nanoparticle fabrication. Nanoparticles (˜200 nm) efficiently encapsulated plasmids encoding for luciferase (80-90%) and slowly released the same for two weeks. After a delay, moderate levels of gene expression appeared at day 5 for certain positively charged PLG particles and gene expression was maintained for at least two weeks. In contrast, gene expression mediated by polyethyleneimine (PEI) ended at day 5. PLG particles were also significantly less cytotoxic than PEI suggesting the use of these vehicles for localized, sustained gene delivery to the pulmonary epithelium. On the other hand, a more simple method to synthesize 50-200 nm complexes capable of high transfection efficiency or high gene knockdown was

  19. Concise vector analysis

    CERN Document Server

    Eliezer, C J; Maxwell, E A; Sneddon, I N

    1963-01-01

    Concise Vector Analysis is a five-chapter introductory account of the methods and techniques of vector analysis. These methods are indispensable tools in mathematics, physics, and engineering. The book is based on lectures given by the author in the University of Ceylon.The first two chapters deal with vector algebra. These chapters particularly present the addition, representation, and resolution of vectors. The next two chapters examine the various aspects and specificities of vector calculus. The last chapter looks into some standard applications of vector algebra and calculus.This book wil

  20. Light axial vector mesons

    CERN Document Server

    Chen, Kan; Liu, Xiang; Matsuki, Takayuki

    2015-01-01

    Inspired by the abundant experimental observation of axial vector states, we study whether the observed axial vector states can be categorized into the conventional axial vector meson family. In this paper we carry out analysis based on the mass spectra and two-body Okubo-Zweig-Iizuka-allowed decays. Besides testing the possible axial vector meson assignments, we also predict abundant information for their decays and the properties of some missing axial vector mesons, which are valuable to further experimental exploration of the observed and predicted axial vector mesons.

  1. Isolation of clinical strains of Pseudomonas aeruginosa harboring different plasmids.

    Science.gov (United States)

    Ranjbar, R; Owlia, P; Saderi, H; Bameri, Z; Izadi, M; Jonaidi, N; Morovvati, S

    2007-09-01

    Aim of this study was to investigate the presence of plasmids among the strains of P. aeruginosa isolated from clinically diagnosed cases in Tehran in 2006. A total of 38 strains of P. aeruginosa were isolated. With the exception of one isolate, all P. aeruginosa strains harbored at least one plasmid band. The electrophoretic analysis of plasmid DNAs showed different number of plasmid bands among the strains tested. The DNA band of 1.4 kbp was evident in 84.2% of the strains. Approximately 71 and 21% of the isolates harbored concomitantly two and three plasmids, respectively. Isolation of strains with diverse types of plasmids suggests the different cluster of P. aeruginosa might be disseminated during the current study period.

  2. Transformation of Haemophilus influenzae by plasmid RSF0885

    Energy Technology Data Exchange (ETDEWEB)

    Notani, N.K.; Setlow, J.K.; McCarthy, D.; Clayton, N.L.

    1981-12-01

    Plasmid RSF0885, which conferred ampicillin resistance, transformed competent Haemophilus influenzae cells with low efficiency (maximun, less than 0.01%). As judged by competition experiments and uptake of radioactivity, plasmid RSF0885 deoxyribonucleic acid was taken up into competent H. influenzae cells several orders of magnitude less efficiently than H. influenzae chromosomal deoxyribonucleic acid. Plasmid RSF0885 transformed cells with even lower efficiency than could be accounted for by the low uptake. Transformation was not affected by rec-1 and rec-2 mutations in the recipient, and strains cured of the plasmid did not show increased transformation. Plasmid molecules cut once with a restriction enzyme that made blunt ends did not transform. Transformation was favored by the closed circular form of the plasmid.

  3. Conjugative botulinum neurotoxin-encoding plasmids in Clostridium botulinum.

    Directory of Open Access Journals (Sweden)

    Kristin M Marshall

    Full Text Available BACKGROUND: Clostridium botulinum produces seven distinct serotypes of botulinum neurotoxins (BoNTs. The genes encoding different subtype neurotoxins of serotypes A, B, F and several dual neurotoxin-producing strains have been shown to reside on plasmids, suggesting that intra- and interspecies transfer of BoNT-encoding plasmids may occur. The objective of the present study was to determine whether these C. botulinum BoNT-encoding plasmids are conjugative. METHODOLOGY/PRINCIPAL FINDINGS: C. botulinum BoNT-encoding plasmids pBotCDC-A3 (strain CDC-A3, pCLJ (strain 657Ba and pCLL (strain Eklund 17B were tagged with the erythromycin resistance marker (Erm using the ClosTron mutagenesis system by inserting a group II intron into the neurotoxin genes carried on these plasmids. Transfer of the tagged plasmids from the donor strains CDC-A3, 657Ba and Eklund 17B to tetracycline-resistant recipient C. botulinum strains was evaluated in mating experiments. Erythromycin and tetracycline resistant transconjugants were isolated from donor:recipient mating pairs tested. Transfer of the plasmids to the transconjugants was confirmed by pulsed-field gel electrophoresis (PFGE and Southern hybridizations. Transfer required cell-to-cell contact and was DNase resistant. This indicates that transfer of these plasmids occurs via a conjugation mechanism. CONCLUSIONS/SIGNIFICANCE: This is the first evidence supporting conjugal transfer of native botulinum neurotoxin-encoding plasmids in C. botulinum, and provides a probable mechanism for the lateral distribution of BoNT-encoding plasmids to other C. botulinum strains. The potential transfer of C. botulinum BoNT-encoding plasmids to other bacterial hosts in the environment or within the human intestine is of great concern for human pathogenicity and necessitates further characterization of these plasmids.

  4. Photonic plasmid stability of transformed Salmonella Typhimurium: A comparison of three unique plasmids

    Directory of Open Access Journals (Sweden)

    Lay Donald

    2009-07-01

    Full Text Available Abstract Background Acquiring a highly stable photonic plasmid in transformed Salmonella Typhimurium for use in biophotonic studies of bacterial tracking in vivo is critical to experimental paradigm development. The objective of this study was to determine stability of transformed Salmonella Typhimurium (S. typh-lux using three different plasmids and characterize their respective photonic properties. Results In presence of ampicillin (AMP, S. typh-lux with pCGLS-1, pAK1-lux and pXEN-1 plasmids exhibited 100% photon-emitting colonies over a 10-d study period. Photon emitters of S. typh-lux with pCGLS-1, pAK1-lux and pXEN-1 without AMP selection decreased over time (P 7 to 1 × 109 CFU, P 0.05; although photonic emissions across a range of bacterial concentrations were not different (1 × 104 to 1 × 106 CFU, P > 0.05. For very low density bacterial concentrations imaged in 96 well plates photonic emissions were positively correlated with bacterial concentration (P 3 to 1 × 105 CFU low to high were different in the 96-well plate format (P Conclusion These data characterize photon stability properties for S. typh-lux transformed with three different photon generating plasmids that may facilitate real-time Salmonella tracking using in vivo or in situ biophotonic paradigms.

  5. Construction of Recombinant Plasmid Harboring APP717 Mutation and Preliminary Study of APP Proteolysis

    Institute of Scientific and Technical Information of China (English)

    LI Xiaoqing; XUE Zheng; ZHANG Suming

    2007-01-01

    In order to investigate the pathogenesis of Alzheimer disease (AD) and study the enzy-matic progress of amyloid precursor protein (APP), the fluorescent eukaryotic expression plasmid of C99 was constructed containing APP717 mutation. The fragment encoding the last 99-aa of APP(which was named C99 containing APP717 mutation), together with the fragment encoding yellow fluorescence protein (which was named YFP) were amplified by PCR. The two fragments (YFP andC99) were inserted into the vector pcDNA3.0. The recombinant plasmid pcDNA3.0-YFP-C99 was accomplished and its authenticity was confirmed by enzyme digestion and sequencing. Then SH-SY5Y cells were transiently transfected with the recombinant plasmid pcDNA3.0-YFP-C99. The expression of the fusion gene was detected by laser confocalmicroscopy. Amyloid-β (Aβ) was de- tected by both microscopy and immunochemistry. The authenticity of the construct was confirmed by the endonuclease digestion and DNA sequencing. The YFP fluorescence could be seen and proved the expression of fusion gene. Aβ labeled by YFP was detected by confocalmicroscopy and confirmed by immunocytochemistry. It was found that Aβ accumulated and deposited in the intracytoplasm, mem-brane and outside of the cell. Furthermore, Aβ accumulated mainly within the cell ahead of the depo-sition in the cell space and the cell shape was rough. It was suggested that Aβ could be generatedwithin the cells. Aβ accumulated in the cell at the early stage before the deposition outside of the cells.Intracellular Aβ accumulation induced the secondary damage to the cells and caused the cell shape rough. Taken together, the recombinant plasmid, pcDNA3.0-YFP-C99 could be a useful tool to fur-ther study the cleavage mechanism of APP and to explore the pathogenesis of AD.

  6. Fluoride enhances transfection activity of carbonate apatite by increasing cytoplasmic stability of plasmid DNA

    Energy Technology Data Exchange (ETDEWEB)

    Chowdhury, E.H., E-mail: md.ezharul.hoque@med.monash.edu.my [Jeffrey Cheah School of Medicine and Health Sciences, Monash University Sunway Campus, Jalan Lagoon Selatan, Bandar Sunway, Selangor Darul Ehsan (Malaysia)

    2011-06-17

    Highlights: {yields} Cytoplasmic stability of plasmid DNA is enhanced by fluoride incorporation into carbonate apatite carrier. {yields} Fluoridated carbonate apatite promotes a robust increase in transgene expression. {yields} Controlled dissolution of fluoridated carbonate apatite in endosomal acidic environment might buffer the endosomes and prevent degradation of the released DNA. -- Abstract: Intracellular delivery of a functional gene or a nucleic acid sequence to specifically knockdown a harmful gene is a potential approach to precisely treat a critical human disease. The intensive efforts in the last few decades led to the development of a number of viral and non-viral synthetic vectors. However, an ideal delivery tool in terms of the safety and efficacy has yet to be established. Recently, we have developed pH-sensing inorganic nanocrystals of carbonate apatite for efficient and cell-targeted delivery of gene and gene-silencing RNA. Here we show that addition of very low level of fluoride to the particle-forming medium facilitates a robust increase in transgene expression following post-incubation of the particles with HeLa cells. Confocal microscopic observation and Southern blotting prove the cytoplasmic existence of plasmid DNA delivered by likely formed fluoridated carbonate apatite particles while degradation of plasmid DNA presumably by cytoplasmic nucleases was noticed following delivery with apatite particles alone. The beneficial role of fluoride in enhancing carbonate apatite-mediated gene expression might be due to the buffering potential of generated fluoridated apatite in endosomal acidic environment, thereby increasing the half-life of delivered plasmid DNA.

  7. Encapsulation of plasmid DNA in calcium phosphate nanoparticles: stem cell uptake and gene transfer efficiency

    Directory of Open Access Journals (Sweden)

    Cao X

    2011-12-01

    Full Text Available Xia Cao*, Wenwen Deng*, Yuan Wei*, Weiyan Su, Yan Yang, Yawei Wei, Jiangnan Yu, Ximing XuDepartment of Pharmaceutics, School of Pharmacy, and Center for Nano Drug/Gene Delivery and Tissue Engineering, Jiangsu University, Jingkou District, Zhenjiang, People's Republic of China*These authors contributed equally to this workBackground: The purpose of this study was to develop calcium phosphate nanocomposite particles encapsulating plasmid DNA (CP-pDNA nanoparticles as a nonviral vector for gene delivery.Methods: CP-pDNA nanoparticles employing plasmid transforming growth factor beta 1 (TGF-β1 were prepared and characterized. The transfection efficiency and cell viability of the CP-pDNA nanoparticles were evaluated in mesenchymal stem cells, which were identified by immunofluorescence staining. Cytotoxicity of plasmid TGF-β1 and calcium phosphate to mesenchymal stem cells were evaluated by MTT assay.Results: The integrity of TGF-β1 encapsulated in the CP-pDNA nanoparticles was maintained. The well dispersed CP-pDNA nanoparticles exhibited an ultralow particle size (20–50 nm and significantly lower cytotoxicity than Lipofectamine™ 2000. Immunofluorescence staining revealed that the cultured cells in this study were probably mesenchymal stem cells. The cellular uptake and transfection efficiency of the CP-pDNA nanoparticles into the mesenchymal stem cells were higher than that of needle-like calcium phosphate nanoparticles and a standard calcium phosphate transfection kit. Furthermore, live cell imaging and confocal laser microscopy vividly showed the transportation process of the CP-pDNA nanoparticles in mesenchymal stem cells. The results of a cytotoxicity assay found that both plasmid TGF-β1 and calcium phosphate were not toxic to mesenchymal stem cells.Conclusion: CP-pDNA nanoparticles can be developed into an effective alternative as a nonviral gene delivery system that is highly efficient and has low cytotoxicity.Keywords: calcium

  8. 喉癌来源的MAGE-A3基因真核表达载体及其稳定表达模型的构建和鉴定%Construction and identification of laryngocarcinoma-derived melanoma antigen-encoding-A3 gene eukaryotic expression vector and steady expression model

    Institute of Scientific and Technical Information of China (English)

    吉晓滨; 吕景礼; 陈敬贤; 刘启才; 谢景华

    2012-01-01

    目的:克隆人黑色素瘤相关抗原MAGE-A3基因,构建真核表达载体,检测、鉴定其在细胞中的表达.方法:MAGE-A3基因进行PCR扩增,凝胶回收PCR产物片段并连接至pMD18-T载体.测序后将MAGE-A3基因片段亚克隆至真核表达载体,构建成pIRES2-EGFP/MAGE-A3重组质粒.经脂质体转染至293T细胞株后,荧光定量PCR、Western-blotting法鉴定MAGE-A3基因的表达.结果:MAGE-A3基因正确克隆在plRES2-EGFP/MAGE-A3,测序结果与GenBank公布的MAGE-A3序列一致.转染293T细胞后能检测到MAGE-A3基因和蛋白的表达.结论:成功构建plRES2-EGFP/MAGE-A3真核表达质粒,并能在293T细胞中有效表达MAGE-A3蛋白,奠定了其作为DNA疫苗应用的基础.%Objective; To construct human melanoma antigen-encoding gene ( MAGE-A3 ) eukaryotic expression vector, and to identify and determine the expression in cell lines. Methods; MAGE-A3 gene was amplified by using polymerase chain reaction (PCR) and was linked to pMD18-T vector after harvesting PCR products via gel extraction. After confirmation by sequencing, the gene segments of MAGE-A3 were subcloned into eukaryotic expression vector for construction of pIRES2-EGFP/MAGE-A3, the recombinant plasmid. This was followed by transfection into the 293 T cell lines using liposomes for further determination of MAGE-A3 gene expression via fluorescent quantitative PCR and Western blotting. Results; MAGE-A3 gene was successfully cloned in the plasmid pIRES2-EGFP/MAGE-A3 , showing identical sequence of MAGE-A3 as compared with that reported in GenBank. The expression of MAGE-A3 genes and proteins were identified after transfection into the 293T cells. Conclusion;The successful construction of eukaryotic expression plasmid pIRES2-EGFP/MAGE-A3 was associated with effective expression of MAGE-A3 protein in 293T cell lines. This may offer grounds for the application of pIRES2-EGFP/MAGE-A3 as a DNA vaccine candidate in laryngocarcinoma immunotherapy.

  9. mIL-18腺病毒载体构建及在大鼠骨髓间充质干细胞中的表达%Construction of adenovirus vectors contained mIL-18 and expression in rat mesenchymal stem cells

    Institute of Scientific and Technical Information of China (English)

    许刚; 郭燕舞; 徐如祥; 姜晓丹

    2012-01-01

    目的 构建表达mIL-18基因的腺病毒载体,并转染大鼠骨髓间充质干细胞(rBMSCs),观察其在rBMSCs中的表达,为脑胶质瘤的基因治疗实验研究奠定基础.方法 mIL-18基因亚克隆于穿梭质粒pAdtrackCMV上,构建的pAdtrackCMV-mIL-18线性化后转化已含有腺病毒骨架质粒pAdEasy-1的大肠杆菌BJ5183感受态细胞,挑选同源重组质粒,线性化后HEK293细胞包装,CsCI纯化.体外分离培养大鼠骨髓间充质干细胞,流式细胞仪检测细胞免疫表型;然后转染rBMSCs,通过荧光显微镜、RT-PCR、ELISA等方法检测mIL-18表达.结果 成功构建了绿色荧光蛋白标记的腺病毒Ad-mIL-18,并在rBMSCs中高效表达.结论 pAdEasy-1是基因转染的高效系统,腺病毒Ad- mIL-18转染的rBMSCs可以作为脑肿瘤基因治疗研究的种子细胞.%Objective To construct adenovirus vectors contained mIL-18 and observation the expression in rBMSCs and explore experiment study for gene therapy of gliorna. Methods mIL-18 gene was subcloned into the shuttle plasmid pAdtrackCMV, then linearized the vector and convert competent cell BJ5183 which contain the adenovirus frame plasmid pAdEasy-1. The homologous recombination plasmid was packaged by HEK293 cells and purified by CsCL The cell im-munophenotype was detected by flow cytometry. The expression of mIL-18 was measured by fluorescence microscope. RT-PCR and ELISA. Results The adenovirus vector Ad- mIL-18 was successfully constructed, which was labeled by green fluorescent protein (GFP) and effectively expressed in rBMSCs, Conclusion pAdEasyl is an effective gene trans-fection system. rBMSCs transfected with Ad- mIL-18 can serve as the seeds cells for the gene therapy of glioma.

  10. Bacteriophages Limit the Existence Conditions for Conjugative Plasmids

    Science.gov (United States)

    Wood, A. Jamie; Dytham, Calvin; Pitchford, Jonathan W.; Truman, Julie; Spiers, Andrew; Paterson, Steve; Brockhurst, Michael A.

    2015-01-01

    ABSTRACT Bacteriophages are a major cause of bacterial mortality and impose strong selection on natural bacterial populations, yet their effects on the dynamics of conjugative plasmids have rarely been tested. We combined experimental evolution, mathematical modeling, and individual-based simulations to explain how the ecological and population genetics effects of bacteriophages upon bacteria interact to determine the dynamics of conjugative plasmids and their persistence. The ecological effects of bacteriophages on bacteria are predicted to limit the existence conditions for conjugative plasmids, preventing persistence under weak selection for plasmid accessory traits. Experiments showed that phages drove faster extinction of plasmids in environments where the plasmid conferred no benefit, but they also revealed more complex effects of phages on plasmid dynamics under these conditions, specifically, the temporary maintenance of plasmids at fixation followed by rapid loss. We hypothesized that the population genetic effects of bacteriophages, specifically, selection for phage resistance mutations, may have caused this. Further mathematical modeling and individual-based simulations supported our hypothesis, showing that conjugative plasmids may hitchhike with phage resistance mutations in the bacterial chromosome. PMID:26037122

  11. Plasmid genes required for microcin B17 production.

    Science.gov (United States)

    San Millán, J L; Kolter, R; Moreno, F

    1985-09-01

    The production of the antibiotic substance microcin B17 (Mcc) is determined by a 3.5-kilobase DNA fragment from plasmid pMccB17. Several Mcc- mutations on plasmid pMccB17 were obtained by both transposon insertion and nitrosoguanidine mutagenesis. Plasmids carrying these mutations were tested for their ability to complement Mcc- insertion or deletion mutations on pMM102 (pMM102 is a pBR322 derivative carrying the region encoding microcin B17). Results from these experiments indicate that at least four plasmid genes are required for microcin production.

  12. [Isolation of the R'his plasmids of Vibrio cholerae].

    Science.gov (United States)

    Rusina, O Iu; Tiganova, I G; Aleshkin, G I; Andreeva, I V; Skavronskaia, A G

    1987-06-01

    V. cholerae strain VT5104 capable of donor activity in conjugation has been constructed by the genetic technique based on plasmid RP4::Mucts62 integration into V. cholerae chromosome due to plasmid homology with Mucts62 inserted into the chromosome. The gene for histidine synthesis has been mobilized and transferred into the recipient cells from VT5104 donor. The conjugants obtained are able to efficiently transfer his+ gene included into the plasmid structure in conjugation with eltor recipient. Thus, the constructed strain VT5104 generates R' plasmids carrying V. cholerae chromosomal genes.

  13. Degradative Plasmid and Heavy Metal Resistance Plasmid Naturally Coexist in Phenol and Cyanide Assimilating Bacteria

    Directory of Open Access Journals (Sweden)

    Bahig E.  Deeb

    2009-01-01

    Full Text Available Problem statement: Heavy metals are known to be powerful inhibitors of xenobiotics biodegradation activities. Alleviation the inhibitory effect of these metals on the phenol biodegradation activities in presence of heavy metals resistant plasmid was investigated. Approach: Combination of genetic systems of degradation of xenobiotic compound and heavy metal resistance was one of the approaches to the creation of polyfunctional strains for bioremediation of soil after co-contamination with organic pollutants and heavy metals. Results: A bacterial strain Pseudomonas putida PhCN (pPhCN1, pPhCN2 had been obtained. This bacterium contained two plasmids, a 120 Kb catabolic plasmid that encode for breakdown of phenol (pPhCN1 and pPhCN2 plasmid (100 Kb that code for cadmium and copper resistant. Cyanide assimilation by this bacterium was encoded by chromosomal genes. The inhibitory effect of cadmium (Cd2+ or copper (Cu2+ on the degradation of phenol and cyanide by P. putida strains PhCN and PhCN1 (contained pPhCN1 were investigated. The resistant strain PhCN showed high ability to degrade phenol and cyanide in presence of Cd2+ or Cu2+ comparing with the sensitive strain PhCN1. In addition, Cd2+ or Cu2+ was also found to exert a strong inhibitory effect on the C23O dioxygenase enzyme activity in the presence of cyanide as a nitrogen source. Conclusion: The presence of heavy metal resistance plasmid alleviated the inhibitory effect of metals on the phenol and cyanide assimilation by resistant strain.

  14. VectorBase

    Data.gov (United States)

    U.S. Department of Health & Human Services — VectorBase is a Bioinformatics Resource Center for invertebrate vectors. It is one of four Bioinformatics Resource Centers funded by NIAID to provide web-based...

  15. A plasmid carrying mucA and mucB genes from pKM101 in Haemophilus influenzae and Escherichia coli

    Energy Technology Data Exchange (ETDEWEB)

    Spikes, D.; Setlow, J.K. (Brookhaven National Lab., Upton, NY (USA))

    1989-10-01

    The plasmid pMucAMucB, constructed from the Haemophilus influenzae vector pDM2, and a similar plasmid, constructed from pBR322, increased the survival after UV irradiation of Escherichia coli AB1157 with the umu-36 mutation and also caused UV-induced mutation in the E. coli strain. In H. influenzae, pMucAMucB caused a small but reproducible increase in survival after UV irradiation in wild-type cells and in a rec-1 mutant, but there was no increase in spontaneous mutation in the wild type or in the rec-1 mutant and no UV-induced mutation.

  16. Unmarked gene deletion and host-vector system for the hyperthermophilic crenarchaeon Sulfolobus islandicus

    DEFF Research Database (Denmark)

    Deng, Ling; Zhu, Haojun; Chen, Zhengjun

    2009-01-01

    , and unmarked lacS mutants were obtained by each method. A new alternative recombination mechanism, i.e., marker circularization and integration, was shown to operate in the latter method, which did not yield the designed deletion mutation. Subsequently, Sulfolobus-E. coli plasmid shuttle vectors were...

  17. Construction and shuttling of novel bifunctional vectors for Streptomyces spp. and Escherichia coli.

    OpenAIRE

    Neesen, K; Volckaert, G.

    1989-01-01

    Shuttle vectors for gene transfer between Streptomyces spp. and Escherichia coli have been constructed by fusion of an artificial multicopy E. coli replicon and DNA fragments of pIJ702. Stable transfer to Streptomyces lividans was obtained. Marked differences in transformation efficiency were observed when plasmid DNA isolated from E. coli GM119 was used instead of that from strain HB101.

  18. Construction of thiostrepton-inducible, high-copy-number expression vectors for use in Streptomyces spp.

    NARCIS (Netherlands)

    Takano, Eriko; White, Janet; Thompson, Charles J.; Bibb, Mervyn J.

    1995-01-01

    A high-copy-number plasmid expression vector (pIJ6021) was constructed that contains a thiostrepton-inducible promoter, PtipA, from Streptomyces lividans 66. The promoter and ribosome-binding site of tipA lie immediately upstream from a multiple cloning site (MCS) which begins with a NdeI site (5'-C

  19. Construction of shuttle vectors capable of conjugative transfer from Escherichia coli to nitrogen-fixing filamentous cyanobacteria

    Energy Technology Data Exchange (ETDEWEB)

    Wolk, C.P.; Vonshak, A.; Kehoe, P.; Elhai, J.

    1984-03-01

    Wild-type cyanobacteria of the genus Anabaena are capable of oxygenic photosynthesis, differentiation of cells called heterocysts at semiregular intervals along the cyanobacterial filaments, and aerobic nitrogen fixation by the heterocysts. To foster analysis of the physiological processes characteristic of these cyanobacteria, we have constructed a family of shuttle vectors capable of replication and selection in Escherichia coli and, in unaltered form, in several strains of Anabaena. Highly efficient conjugative transfer of these vectors from E. coli to Anabaena is dependent upon the presence of broad host-range plasmid RP-4 and of helper plasmids. The shuttle vectors contain portions of plasmid pBR322 required for replication and mobilization, with sites for Anabaena restriction enzymes deleted; cyanobacterial replicon pDU1, which lacks such sites; and determinants for resistance to chloramphenicol, streptomycin, neomycin, and erythromycin. 38 references, 7 figures, 1 table.

  20. On Double Vector Bundles

    Institute of Scientific and Technical Information of China (English)

    Zhuo CHEN; Zhang Ju LIU; Yun He SHENG

    2014-01-01

    In this paper, we construct a category of short exact sequences of vector bundles and prove that it is equivalent to the category of double vector bundles. Moreover, operations on double vector bundles can be transferred to operations on the corresponding short exact sequences. In particular, we study the duality theory of double vector bundles in term of the corresponding short exact sequences. Examples including the jet bundle and the Atiyah algebroid are discussed.

  1. On Double Vector Bundles

    OpenAIRE

    Chen, Zhuo; Liu, Zhangju; Sheng, Yunhe

    2011-01-01

    In this paper, we construct a category of short exact sequences of vector bundles and prove that it is equivalent to the category of double vector bundles. Moreover, operations on double vector bundles can be transferred to operations on the corresponding short exact sequences. In particular, we study the duality theory of double vector bundles in term of the corresponding short exact sequences. Examples including the jet bundle and the Atiyah algebroid are discussed.

  2. Construction and Characterization of an in-vivo Linear Covalently Closed DNA Vector Production System

    Science.gov (United States)

    2012-01-01

    Background While safer than their viral counterparts, conventional non-viral gene delivery DNA vectors offer a limited safety profile. They often result in the delivery of unwanted prokaryotic sequences, antibiotic resistance genes, and the bacterial origins of replication to the target, which may lead to the stimulation of unwanted immunological responses due to their chimeric DNA composition. Such vectors may also impart the potential for chromosomal integration, thus potentiating oncogenesis. We sought to engineer an in vivo system for the quick and simple production of safer DNA vector alternatives that were devoid of non-transgene bacterial sequences and would lethally disrupt the host chromosome in the event of an unwanted vector integration event. Results We constructed a parent eukaryotic expression vector possessing a specialized manufactured multi-target site called “Super Sequence”, and engineered E. coli cells (R-cell) that conditionally produce phage-derived recombinase Tel (PY54), TelN (N15), or Cre (P1). Passage of the parent plasmid vector through R-cells under optimized conditions, resulted in rapid, efficient, and one step in vivo generation of mini lcc—linear covalently closed (Tel/TelN-cell), or mini ccc—circular covalently closed (Cre-cell), DNA constructs, separated from the backbone plasmid DNA. Site-specific integration of lcc plasmids into the host chromosome resulted in chromosomal disruption and 105 fold lower viability than that seen with the ccc counterpart. Conclusion We offer a high efficiency mini DNA vector production system that confers simple, rapid and scalable in vivo production of mini lcc DNA vectors that possess all the benefits of “minicircle” DNA vectors and virtually eliminate the potential for undesirable vector integration events. PMID:23216697

  3. Custodial vector model

    DEFF Research Database (Denmark)

    Becciolini, Diego; Franzosi, Diogo Buarque; Foadi, Roshan;

    2015-01-01

    We analyze the Large Hadron Collider (LHC) phenomenology of heavy vector resonances with a $SU(2)_L\\times SU(2)_R$ spectral global symmetry. This symmetry partially protects the electroweak S-parameter from large contributions of the vector resonances. The resulting custodial vector model spectrum...

  4. Custodial vector model

    DEFF Research Database (Denmark)

    Becciolini, Diego; Franzosi, Diogo Buarque; Foadi, Roshan

    2015-01-01

    We analyze the Large Hadron Collider (LHC) phenomenology of heavy vector resonances with a $SU(2)_L\\times SU(2)_R$ spectral global symmetry. This symmetry partially protects the electroweak S-parameter from large contributions of the vector resonances. The resulting custodial vector model spectrum...

  5. Effect of plasmid R391 and other IncJ plasmids on the survival of Escherichia coli after UV irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Pembroke, J.T.; Stevens, E. (University Coll., Galway (Ireland))

    1984-07-01

    The presence of the IncJ plasmids R391, R997, R705, R706, R748, and R749 was shown to sensitize Escherichia coli AB1157 and both its uvr A and lexA derivatives to UV irradiation. No alteration in post-irradiation survival was observed in a recA mutant containing these plasmids, compared with the non-plasmid-containing recA strain. Analysis of recombination frequency in Hfr crosses to recA/sup +/ cells containing plasmid R391 indicated a reduction in recombination frequency compared with that obtained in similar crosses to a non-plasmid-containing strain. This effect was not due to plasmid-encoded restriction or entry exclusion systems and therefore must be considered as a real block in recombination. When cells containing plasmid R391 were irradiated and allowed to photoreactivate, an increase in survival was observed which was comparable to that observed in the non-plasmid-containing derivative. This indicated that post-irradiation processing of UV-induced damage, or lack of such processing, by mechanisms other than photoreactivation was responsible for the UV sensitivity associated with plasmid R391.

  6. Physical structure and genetic expression of the sulfonamide-resistance plasmid pLS80 and its derivatives in Streptococcus pneumoniae and Bacillus subtilis

    Energy Technology Data Exchange (ETDEWEB)

    Lopez, P.; Espinosa, M.; Lacks, S.A.

    1984-01-01

    The 10-kb chromosomal fragment of Streptococcus pneumoniae cloned in pLS80 contains the sul-d allele of the pneumococcal gene for dihydropteroate synthase. As a single copy in the chromosome this allele confers resistance to sulfanilamide at 0.2 mg/ml; in the multicopy plasmid it confers resistance to 2.0 mg/ml. The sul-d mutation was mapped by restriction analysis to a 0.4-kb region. A spontaneous deletion beginning approx. 1.5 kb to the right of the sul-d mutation prevented gene function, possibly by removing a promoter. This region could be restored by chromosomal facilitation and be demonstrated in the plasmid by selection for sulfonamide resistance. Under selection for a vector marker, tetracycline resistance, only the deleted plasmid was detectable, apparently as a result of plasmid segregation and the advantageous growth rates of cells with smaller plasmids. When such cells were selected for sulfonamide resistance, the deleted region returned to the plasmid, presumably by equilibration between the chromosome and the plasmid pool, to give a low frequency (approx. 10/sup -3/) of cells resistant to sulfanilamide at 2.0 mg/ml. Models for the mechanisms of chromosomal facilitation and equilibration are proposed. Several derivatives of pLS80 could be transferred to Bacillus subtilis, where they conferred resistance to sulfanilamide at 2 mg/ml, thereby demonstrating cross-species expression of the pneumococcal gene. Transfer of the plasmids to B. subtilis gave rise to large deletions to the left of the sul-d marker, but these deletions did not interfere with the sul-d gene function. Restriction maps of pLS80 and its variously deleted derivatives are presented.

  7. The contribution of plasmid design and release to in vivo gene expression following delivery from cationic polymer modified scaffolds.

    Science.gov (United States)

    Avilés, Misael O; Lin, Chia-Hsuan; Zelivyanskaya, Marina; Graham, John G; Boehler, Ryan M; Messersmith, Phillip B; Shea, Lonnie D

    2010-02-01

    Tissue engineering scaffolds capable of gene delivery can provide a structure that supports tissue formation while also inducing the expression of inductive factors. Sustained release strategies are hypothesized to maintain elevated plasmid concentrations locally that can enhance gene transfer. In this report, we investigate the relationship between plasmid release kinetics and the extent and duration of transgene expression. Scaffolds were fabricated from polymer microspheres modified with cationic polymers (polyethylenimine, poly(L-lysine), poly(allylamine hydrochloride), polydiallyldimethylammonium) or polydopamine (PD), with PD enhancing incorporation and slowing release. In vivo implantation of scaffolds into the peritoneal fat pad had no significant changes in the level and duration of transgene expression between PD and unmodified scaffolds. Control studies with plasmid dried onto scaffolds, which exhibited a rapid release, and scaffolds with extended leaching to reduce initial quantities released had similar levels and duration of expression. Changing the plasmid design, from a cytomegalovirus (CMV) to an ubiquitin C (UbC) promoter substantially altered the duration of expression. These studies suggest that the initial dose released and vector design affect the extent and duration of transgene expression, which may be sustained over several weeks, potentially leading to numerous applications in cell transplantation and regenerative medicine. (c) 2009 Elsevier Ltd. All rights reserved.

  8. Construction and expression of mouse chemokine receptor-7 green fluorescent lentiviral expression vector in immature dendritic cells%趋化因子受体7基因绿色荧光慢病毒载体构建及在未成熟树突状细胞中的表达

    Institute of Scientific and Technical Information of China (English)

    宋立孝; 张璞; 李德鹏; 曾令宇; 陈翀; 潘秀英; 徐开林; 黄一虹

    2012-01-01

    DCs.METHODS: The CCR7 was amplified from the mouse thymus by RT-PCR and transfected into pCR-Blunt carrier. The CCR7 DNA fragment and IRES-GFP were cloned into lentiviral expression vector LV-Lac, to form recombinant lentivial plasmid LV-CCR7. Three plasmids of the lentivial system (recombinant lentivial plasmid LV-CCR7, package plasmid ?NRF, envelope plasmid pVSVG) were co-transfected with package lentivirus by lipofectamine. The imDCs were infected by the recombinant lentivial, The morphology of transfected imDCs was examined under a fluorescent microscope and flow cytometry was used to determine the expression of CCR7.RESULTS AND CONCLUSION: The CCR7 fragment was amplified from cDNA of the mouse thymus and subcloned to pCR-Blunt carrier, and lentivi ral expression vector LV-CCR7 was constructed successfully. After infected with 293 FT cells for 24 hours through three plasmid package system, positive expression of GFP was observed under fluorescence microscope. The titer of the lentivirus was above 108 U/L, and to obtain the recombinant lentivial with mouse CCR7 gene. The lentivial plasmid could infect imDCs effectively, a large amount of GFP and CCR7 expression was observed under fluorescence microscope and flow cytometry, respectively, and the positive rate was 50%. It indicates that GFP lentiviral vector LV-CCR7 with mouse CCR7 gene were successfully constructed and expressed in imDCs.

  9. [Effect of endonuclease G depletion on plasmid DNA uptake and levels of homologous recombination in hela cells].

    Science.gov (United States)

    Misic, V; El-Mogy, M; Geng, S; Haj-Ahmad, Y

    2016-01-01

    Endonuclease G (EndoG) is a mitochondrial apoptosis regulator that also has roles outside of programmed cell death. It has been implicated as a defence DNase involved in the degradation of exogenous DNA after transfection of mammalian cells and in homologous recombination of viral and endogenous DNA. In this study, we looked at the effect of EndoG depletion on plasmid DNA uptake and the levels of homologous recombination in HeLa cells. We show that the proposed defence role of EndoG against uptake of non-viral DNA vectors does not extend to the cervical carcinoma HeLa cells, as targeting of EndoG expression by RNA interference failed to increase intracellular plasmid DNA levels. However, reducing EndoG levels in HeLa cells resulted in a statistically significant reduction of homologous recombination between two plasmid DNA substrates. These findings suggest that non-viral DNA vectors are also substrates for EndoG in its role in homologous recombination.

  10. PROTECTION AGAINST LEPTOSPIROSIS BY IMMUNIZATION WITH PLASMID DNA ENCODING 33 kDa ENDOFLAGELLIN OF L.INTERROGANS SEROVAR LAI

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Objective. To evaluate how the efficacy of DNA inocutation affects the ability to raise protective immunity against Leptospira.Methods. A pair of oligonucleotide primers were designed to amplify the endoflagellar gene of L. interrogans sensu stricto serovar lai. An approximately 840bp fragment was generated with PCR and inserted into VR1012, a plasmid DNA expression vector, after the fragment and VR1012 were digested respectively with EcoRV and Sal I. A recombinant plasmid designated as VR1012+flaB2 was obtained. The vector, VR1012 consits of a pUC18 backbone with the cytomegalovirus(CMV) IE1 enhancer, promoter, and intron A, transcription regulatory elements and the BGH polyadenylation sequences driving the expressing of leptospiral endoflagellar gene of L. interrogans sensu stricto serovar lai. Plasmid encoding leptospiral endoflagellin gene was injected into quadriceps of NZW rabbits.Results.This resulted in the generation of specific leptospiral antibody with high ELISA titer (1:32768) in the rabbits. Immuno/protection was performed in guinea pigs without adjuvant. The group"VR1012+flaB2" showed higher survival rate(90%,9/10 animals),compared with the group "VR1012 lack flaB2" and the group "normal saline".Conclusion.The technique of DNA vaccine has potential advantages over certain other vaccine preparation technologies. However whether DNA vaccine will be useful for vaccine development remains to be tested.

  11. Chromosomal targeting of replicating plasmids in the yeast Hansenula polymorpha

    NARCIS (Netherlands)

    Faber, Klaas Nico; Swaving, Gert Jan; Faber, Folkert; Ab, Geert; Harder, Willem; Veenhuis, Marten; Haima, Pieter

    1992-01-01

    Using an optimized transformation protocol we have studied the possible interactions between transforming plasmid DNA and the Hansenula polymorpha genome. Plasmids consisting only of a pBR322 replicon, an antibiotic resistance marker for Escherichia coli and the Saccharomyces cerevisiae LEU2 gene we

  12. Genomic comparison of archaeal conjugative plasmids from Sulfolobus

    DEFF Research Database (Denmark)

    Greve, Bo Bjørn

    2004-01-01

    All of the known self-transmissable plasmids of the Archaea have been found in the genus Sulfolobus. To gain more insight into archaeal conjugative processes, four newly isolated self-transmissable plasmids, pKEF9, pHVE14, pARN3 and pARN4, were sequenced and subjected to a comparative sequence...

  13. Linear plasmid in the genome of Clavibacter michiganensis subsp. sepedonicus.

    Science.gov (United States)

    Brown, Susan E; Knudson, Dennis L; Ishimaru, Carol A

    2002-05-01

    Contour-clamped homogeneous electric field gel analysis of genomic DNA of the plant pathogen Clavibacter michiganensis subsp. sepedonicus revealed the presence of a previously unreported extrachromosomal element. This new element was demonstrated to be a linear plasmid. Of 11 strains evaluated, all contained either a 90-kb (pCSL1) or a 140-kb (pCSL2) linear plasmid.

  14. Homology of plasmids in strains of unicellular cyanobacteria

    NARCIS (Netherlands)

    Hondel, C.A.M.J.J. van den; Keegstra, W.; Borrias, W.E.; Arkel, G.A. van

    1979-01-01

    Six strains of unicellular cyanobacteria were examined for the presence of plasmids. Analysis of lysates of these strains by CsCl-ethidium bromide density centrifugation yielded a major chromosomal DNA band and a minor band containing covalently closed circular plasmid DNA, as shown by electron micr

  15. Examination of uropathogenic Escherichia coli strains conferring large plasmids

    Directory of Open Access Journals (Sweden)

    SUHARTONO

    2010-04-01

    Full Text Available Suhartono (2010 Examination of uropathogenic Escherichia coli strains conferring large plasmids. Biodiversitas 11: 59-64. Of major uropathogens, Escherichia coli has been widely known as a main pathogen of UTIs globally and has considerable medical and financial consequences. A strain of UPEC, namely E. coli ST131, confers a large plasmid encoding cephalosporinases (class C β-lactamase or AmpC that may be disseminated through horizontal transfer among bacterial populations. Therefore, it is worth examining such large plasmids by isolating, purifying, and digesting the plasmid with restriction enzymes. The examination of the large plasmids was conducted by isolating plasmid DNA visualized by agarose gel electrophoresis as well as by PFGE. The relationship of plasmids among isolates was carried out by HpaI restriction enzyme digestion. Of 36 isolates of E. coli ST 131, eight isolates possessed large plasmids, namely isolates 3, 9, 10, 12, 17, 18, 26 and 30 with the largest molecular size confirmed by agarose gel electrophoresis and PFGE was ~42kb and ~118kb respectively. Restriction enzyme analysis revealed that isolates 9, 10, 12, 17 and 18 have the common restriction patterns and those isolates might be closely related.

  16. 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 papil

  17. 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 papil

  18. Plasmid cloning vehicle for Haemophilus influenzae and Escherichia coli

    Energy Technology Data Exchange (ETDEWEB)

    McCarthy, D.; Clayton, N.L.; Setlow, J.K.

    1982-09-01

    A new plasmid cloning vehicle (pDM2) was used to introduce a library of Haemophilus influenzae chromosomal fragments into H. influenzae. Transformants of the higly recombination-defective rec-1 mutant were more likely to contain exclusively recombinant plasmids after exposure to ligated DNA mixtures than was the wild type. pDM2 could replicate in Escherichia coli K-12.

  19. 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. Th

  20. 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 blaCMY and blaNDM Current typing methods for IncA/C plasmids offer limited resolution. In this study, we present the complete sequence of a blaNDM-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 blaNDM-positive plasmids examined (15/17; 88%) fall into ST1, suggesting acquisition and subsequent expansion of this blaNDM-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.

  1. Twist基因逆转录病毒载体的构建及其诱导正常乳腺上皮细胞发生上皮间质转化%Construction of retroviral vector carrying Twist gene and its induction of epithelialmesenchymal transition in human mammary epithelial cells

    Institute of Scientific and Technical Information of China (English)

    杨佳佳; 胡萍; 周明莉; 黄杰涛; 柳满然

    2013-01-01

    Objective To construct a retroviral vector carrying Twist gene and investigate its effect on human mammary MCF10A epithelial cells.Methods Myc-Twist was digested from pcDNA3/myc-Twist and subcloned into the retroviral vector pBABE-puro to construct a recombinant plasmid (pBABE-myc-Twist).The inserted Twist gene was confirmed by restriction enzyme digestion and DNA sequencing.The plasmid pBABE-myc-Twist and the packaging plasmid pAmpho were co-transfected into HEK293T cells for packaging of retrovirus.Meanwhile,the control plasmid pBABE-puro and the packaging plasmid were co-transfected into the other HEK293T cells as a control group.Human mammary MCF10A epithelial cells were infected with the retroviruses carrying Twist gene or the controls,and selected by puromycin.The expression of Twist in the MCF10A-Twist and MCF10A-Vector cells was determined by RT-PCR and Westem blotting.The expressions of epithelial-mesenchymal transition (EMT) marker proteins induced by Twist in MCF10A cells were detected using immunofluorescence cytochemistry and Westem blotting.Cell migration and invasion abilities were analyzed by Transwell(R) assay.Results The myc-tagged Twist gene was correctly inserted into the retroviral expression vector as a recombinant plasmid (pBABE-myc-Twist) as identified by restriction analysis and DNA sequencing.The Twist gene was efficiently delivered into human mammary MCF10A epithelial cells by the retrovirus,resulting in the stable expression of Twist mRNA and myc-tagged Twist protein as shown by RT-PCR and Western blotting,respectively.The expression of the epithelial biomarker E-cadherin was downregulated whereas,the mesenchymal marker vimentin upregulated in MCF10A-Twist cells as shown by immunofluorescence cytochemistry and Western blotting.Cell migration and invasion abilities were enhanced notably in MCF10A-Twist cells as compared with MCF10A-Vector control cells (P <0.01).Conclusion Twist induces EMT of MCF10A cells and plays an important role in

  2. Deciphering conjugative plasmid permissiveness in wastewater microbiomes

    DEFF Research Database (Denmark)

    Jacquiod, Samuel Jehan Auguste; Brejnrod, Asker Daniel; Milani, Stefan Morberg

    2017-01-01

    Wastewater treatment plants (WWTPs) are designed to robustly treat polluted water. They are characterized by ceaseless flows of organic, chemical and microbial matter, followed by treatment steps before environmental release. WWTPs are hotspots of horizontal gene transfer between bacteria via...... still remains largely uncharted. Furthermore, current in vitro methods used to assess conjugation in complex microbiomes do not include in situ behaviours of recipient cells, resulting in partial understanding of transfers. We investigated the in vitro conjugation capacities of WWTP microbiomes from...... diversity of recipient bacterial phyla for the plasmid was observed, especially in WWTP outlets. We also identified permissive bacteria potentially able to cross WWTPs and engage in conjugation before and after water treatment. Bacterial activity and lifestyle seem to influence conjugation extent...

  3. 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...

  4. [Influence of spv plasmid genes group in Salmonella Enteritidis virulence for chickens. I. Occurrence of spv plasmid genes group in Salmonella Enteritidis large virulence plasmid].

    Science.gov (United States)

    Madajczak, Grzegorz; Binek, Marian

    2005-01-01

    Many Salmonella Enteritidis virulence factors are encoded by genes localized on plasmids, especially large virulence plasmid, in highly conserved fragment, they create spv plasmid gene group. The aims of realized researches were spv genes occurrence evaluation and composition analysis among Salmonella Enteritidis strains caused infection in chickens. Researches were realized on 107 isolates, where in every cases large virulence plasmid 59 kbp size were detected. Specific nucleotides sequences of spv genes (spvRABCD) were detected in 47.7% of isolates. In the rest of examined bacteria spv genes occurred variably. Most often extreme genes of spv group, like spvR and spvD were absent, what could indicate that factors encoded by them are not most important for Salmonella Enteritidis live and their expressed virulence.

  5. Local Application of BMP-2 Specific Plasmids in Fibrin Glue does not Promote Implant Fixation

    Directory of Open Access Journals (Sweden)

    Plank Christan

    2011-07-01

    Full Text Available Abstract Background BMP-2 is known to accelerate fracture healing and might also enhance osseointegration and implant fixation. Application of recombinant BMP-2 has a time-limited effect. Therefore, a gene transfer approach with a steady production of BMP-2 appears to be attractive. The aim of this study was to examine the effect of locally applied BMP-2 plasmids on the bone-implant integration in a non-weight bearing rabbit tibia model using a comparatively new non-viral copolymer-protected gene vector (COPROG. Methods Sixty rabbits were divided into 4 groups. All of them received nailing of both tibiae. The verum group had the nails inserted with the COPROG vector and BMP-2 plasmids using fibrin glue as a carrier. Controls were a group with fibrin glue only and a blank group. After 28 and 56 days, these three groups were sacrificed and one tibia was randomly chosen for biomechanical testing, while the other tibia underwent histomorphometrical examination. In a fourth group, a reporter-gene was incorporated in the fibrin glue instead of the BMP-2 formula to prove that transfection was successful. Results Implant fixation strength was significantly lower after 28 and 56 days in the verum group. Histomorphometry supported the findings after 28 days, showing less bone-implant contact. In the fourth group, successful transfection could be confirmed by detection of the reporter-gene in 20 of 22 tibiae. But, also systemic reporter-gene expression was found in heterotopic locations, showing an undesired spreading of the locally applied gene formula. Conclusion Our results underline the transfecting capability of this vector and support the idea that BMP-2 might diminish osseointegration. Further studies are necessary to specify the exact mechanisms and the systemic effects.

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

    DEFF Research Database (Denmark)

    Klümper, Uli

    larger than previously assumed. I was able to show abundant plasmid transfer from the Gram negative donor strains to a wide diversity of Gram positive soil bacteria, formerly thought to constitute distinct clusters of gene transfer. Moreover, among the observed transconjugants, I identified a core super...... environmental factors that modulate plasmid transfer in soil microbial communities. In order to attain these goals, I developed a high-throughput method that enabled me to evaluate the permissiveness of bacterial communities towards introduced plasmids. This new approach is based on the introduction...... fraction of soil the bacteria (up to 1 in 10,000) were able to take up any of these broad host range conjugal plasmids. The transconjugal pools comprised 11 bacterial phyla. This finding indicates that the realized transfer range of broad host range plasmids in environmental microbial communities is much...

  7. Main features of DNA-based immunization vectors

    Directory of Open Access Journals (Sweden)

    V. Azevedo

    1999-02-01

    Full Text Available DNA-based immunization has initiated a new era of vaccine research. One of the main goals of gene vaccine development is the control of the levels of expression in vivo for efficient immunization. Modifying the vector to modulate expression or immunogenicity is of critical importance for the improvement of DNA vaccines. The most frequently used vectors for genetic immunization are plasmids. In this article, we review some of the main elements relevant to their design such as strong promoter/enhancer region, introns, genes encoding antigens of interest from the pathogen (how to choose and modify them, polyadenylation termination sequence, origin of replication for plasmid production in Escherichia coli, antibiotic resistance gene as selectable marker, convenient cloning sites, and the presence of immunostimulatory sequences (ISS that can be added to the plasmid to enhance adjuvanticity and to activate the immune system. In this review, the specific modifications that can increase overall expression as well as the potential of DNA-based vaccination are also discussed.

  8. Complex nature of enterococcal pheromone-responsive plasmids.

    Science.gov (United States)

    Wardal, Ewa; Sadowy, Ewa; Hryniewicz, Waleria

    2010-01-01

    Pheromone-responsive plasmids constitute a unique group of approximately 20 plasmids identified, as yet, only among enterococcal species. Several of their representatives, e.g. pAD1, pCF10, pPD1 and pAM373 have been extensively studied. These plasmids possess a sophisticated conjugation mechanism based on response to sex pheromones--small peptides produced by plasmid-free recipient cells. Detailed analysis of regulation and function of the pheromone response process revealed its great complexity and dual role--in plasmid conjugation and modulation of enterococcal virulence. Among other functional modules identified in pheromone plasmids, the stabilization/partition systems play a crucial role in stable maintenance of the plasmid molecule in host bacteria. Among them, the par locus of pAD1 is one of the exceptional RNA addiction systems. Pheromone-responsive plasmids contribute also to enterococcal phenotype being an important vehicle of antibiotic resistance in this genus. Both types of acquired vancomycin resistance determinants, vanA and vanB, as well many other resistant phenotypes, were found to be located on these plasmids. They also encode two basic agents of enterococcal virulence, i.e. aggregation substance (AS) and cytolysin. AS participates in mating-pair formation during conjugation but can also facilitate the adherence ofenterococci to human tissues during infection. The second protein, cytolysin, displays hemolytic activity and helps to invade eukaryotic cells. There are still many aspects of the nature of pheromone plasmids that remain unclear and more detailed studies are needed to understand their uniqueness and complexity.

  9. Antibiotic resistance of vibrio cholerae: special considerations of R-plasmids.

    Science.gov (United States)

    Kuwahara, S

    1978-09-01

    Studies on the transmission of R plasmid by conjugation between enterobacteria and vibrio or related bacteria were reviewed. The majority of the reports confirmed successful transmission from enterobacteria to Vibrio cholerae and related species, although the transmission frequencies were extremely low and the transmitted R plasmid was very unstable except for thermosensitive kanamycin plasmid and usual R plasmid coexisting with P plasmid. Strains of V. cholerae and Aeromonas liquefaciens as well as A. salmonicida bearing R plasmid were detected in nature. R plasmid was relatively unstable in V. cholerae strains with which transmission of R plasmid to enterobacteria was confirmed. At present, only 3 R plasmids have been obtained from naturally occurring strains of V. cholerae. Although the 2 European plasmids belong to the C incompatibility group with 98 megadalton closed covalent circular DNA molecule, one plasmid belongs to the J group with more than 25 megadalton molecular weight, and no CCC of satelite DNA was detected in bacteria harboring this plasmid.

  10. Transfection of bone marrow mesenchymal stem cells using green fluorescence protein labeled hVEGF165 recombinant plasmid mediated by liposome

    Institute of Scientific and Technical Information of China (English)

    Tao Wang; Tian-An Liao; Shao-Bo Zhong

    2013-01-01

    Objective:To study the role of bone marrow mesenchymal stem cells (BMSCs) in construction of vascularized engineered tissue. Methods: hVEGF165 was amplified via RT-PCR before recombinant with pShuttle-green fluorescence protein;green fluorescent protein (GFP)-CMV. Then the recombinant shuttle plasmid was transfected into BMSCs with LipofectamineTM 2000 for packaging and amplifying. hVEGF165 mRNA expression in BMSCs cells was tested. Results:The sequence of hVEGF165 in pShuttle-GFP-hVEGF165 plasmid was confirmed by double-enzyme cleavage method and sequencing. hVEGF165 was highly expressed in BMSCs. Conclusions:The GFP/hVEGF165 recombinant plasmid vector was constructed successfully and expressed effectively in host cells, which may be helpful for discussing the possibility of the application of VEGF165-BMSCs in tissue engineering and ischemic disease cure.

  11. Studies on Saccharomyces cerevisiae under carbon-limiting growth transformed with plasmid pCYG4 that carries the gene for NADP-GDH.

    Science.gov (United States)

    Lima Filho, J L; Ledingham, W M

    1990-02-01

    The gene (GDH1) coding for the NADP-linked glutamate dehydrogenase system (NADP-GDH) has been cloned from Saccharomyces cerevisiae strain. Cells being transformed by the NADP-GDH gene on a 2 micron bared vector (pCYG4) plasmid confering 11-fold higher level on expressed GDH activity over the wild-type cells. The behavior of these cells was investigated under chemostatic growth with a carbon rate-limiting nutrient. Specific growth rates of cells carrying plasmid pCYG4 were found to be slightly slower than wild type cells. Furthermore, the NADP-GDH activity increases proportionally with the dilution rate. In addition, oscillations in the NADP-GDH activity, especially at a dilution rate up to 0.15/h, are probably consequential on the appearance of a changing mixed population (cells with and without plasmids).

  12. Microcalorimetric Studies on Gene Promoter Function of Cloned DNA Fragements from Halobacterium halobium J7 Plasmid pHH205 in Escherichia coli TG1

    Institute of Scientific and Technical Information of China (English)

    LEI,Ke-Lin; HOU,Han-Na; LIU,Yi; YE,Xue-Cheng; SHEN,Ping

    2007-01-01

    Halobacterium halobium is a typical kind of extremely halophilic bacterium. Combined with the antibiotic resistance assay, the microcalorimetric method was used to study the promoter function of the cloned DNA fragments from Halobacterium halobium J7 plasmid pHH205 in Escherichia coli TG1. The promoter probe vector, plasmid pKK232-8, was used to form the recombinants. The DNA fragment, which is the promoter for the chloramphenicol acetyl transferase (CAT) gene in plasmid pKK232-8, is about 800 bp, and the chloramphenicol resistance level presented by IC50 is about 200 μg·mL-1, which suggests a high promoter activity. The conclusions show that there probably exist double-function or trinary-function gene promoters in Halobacterium halobium, and Archaea may contain rich genetic resources.

  13. Bacterial Mitosis: ParM of Plasmid R1 Moves Plasmid DNA by an Actin-like Insertional Polymerization Mechanism

    DEFF Research Database (Denmark)

    Møller-Jensen, Jakob; Borch, Jonas; Dam, Mette

    2003-01-01

    Bacterial DNA segregation takes place in an active and ordered fashion. In the case of Escherichia coli plasmid R1, the partitioning system (par) separates paired plasmid copies and moves them to opposite cell poles. Here we address the mechanism by which the three components of the R1 par system...

  14. Construction, production, and purification of recombinant adenovirus vectors.

    Science.gov (United States)

    Miravet, Susana; Ontiveros, Maria; Piedra, Jose; Penalva, Cristina; Monfar, Mercè; Chillón, Miguel

    2014-01-01

    Recombinant adenoviruses provide a versatile system for gene expression studies and therapeutic applications. In this chapter, a standard procedure for their generation and small-scale production is described. Homologous recombination in E. coli between shuttle plasmids and full-length adenovirus backbones (E1-deleted) is used for the generation of recombinant adenoviral vectors genomes. The adenovirus genomes are then analyzed to confirm their identity and integrity, and further linearized and transfected to generate a recombinant adenoviral vector in permissive human cells. These vectors are then purified by two sequential CsCl gradient centrifugations and subjected to a chromatography step in order to eliminate the CsCl and exchange buffers. Finally, the viral stock is characterized through the quantification of its viral particle content and its infectivity.

  15. Promising plasmid DNA vector based on APTES-modified silica nanoparticles

    OpenAIRE

    Cheang TY; Tang B; Xu AW; Chang GQ; Hu ZJ; He WL; Xing ZH; Xu JB; Wang M; Wang SM

    2012-01-01

    Tuck-yun Cheang1,*, Bing Tang1,*, An-wu Xu2, Guang-qi Chang1, Zuo-jun Hu1, Wei-ling He1, Zhou-hao Xing2, Jian-bo Xu1, Mian Wang1, Shen-ming Wang11Department of Vascular Surgery, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China; 2Division of Nanomaterials and Chemistry, Hefei National Laboratory for Physical Sciences at Microscale, University of Science and Technology of China, Hefei, China  *Both authors contributed equally to this workAbstract: Nanoparticles...

  16. 大鼠Kir6.2基因真核表达载体的构建及其在HEK293细胞中的表达%Construction of a eukaryotic expression vector containing the Kir6.2 gene and its expression in HEK293 cells

    Institute of Scientific and Technical Information of China (English)

    任晓燕; 谭淑慧; 马国诏; 杜怡峰

    2011-01-01

    Objective To clone the Kir6.2 gene, construct its eukaryotic expression vector, and obtain positive HEK293 cell clones stably expressing the Kir6.2 gene.Methods Total RNA was extracted from the SD rat brain.Full-length cDNA encoding the Kir6.2 gene was obtained by RT-PCR and inserted into the T1-simple cloning vector.After the sequencing was confirmed , the gene was subcloned into pcDNA3 to construct the recombinant eukaryotic expression vector pcDNA3.0/Kir6.2.The recombinant plasmid was transfected into HEK293 cells by lipofectamin and positive cell clones were screened with G418.Expression of the Kir6.2 gene in the transfected cells was confirmed by RT-PCR and Western blot.Results PCR and sequencing showed that the target gene was cloned into the recombinant vector.Overexpression of the Kir6.2 gene in the transfected HEK293 cells was identified by RT-PCR and Western blot.Conclusion The eukaryotic expression plasmid containing the Kir6.2 gene was successfully constructed.Positive HEK293 cell clones stably expressing the Kir6.2 gene were obtained.%目的 克隆Kit6.2基因,构建真核表达载体pcDNA3.0/Kir6.2,将重组质粒转染HEK293细胞获得高表达Kir6.2基因的细胞克隆.方法 从SD大鼠脑组织中提取总RNA,采用RT-PCR方法获得Kit6.2基因的全长cDNA,插入TI-simple克隆载体中进行序列测定,测序正确后将其亚克隆至表达载体pcDNA3.0,利用脂质体将重组质粒转染HEK293细胞,经G418筛选获得抗性细胞克隆,采用RT-PCR和Western blot方法,鉴定Kir6.2基因在HEK293细胞中的过表达.结果 经PCR和DNA序列测定证实,目的 基因已插入重组质粒,RT-PCR和West-em blot证明,经G418筛选得到的转基因HEK293细胞克隆中存在Kir6.2基因的表达.结论 成功构建Kir6.2基因的真核表达载体,获得稳定表达Kil6.2基因的HEK293 细胞克隆.

  17. Paired cloning vectors for complementation of mutations in the cyanobacterium Anabaena sp. strain PCC 7120

    Energy Technology Data Exchange (ETDEWEB)

    Wolk, C. Peter Wolk [Michigan State University, East Lansing; Fan, Qing [Northwestern University, Evanston; Zhou, Ruanbao [Anhui Normal University, People' s Republic of China; Huang, Guocun [University of Texas Southwestern Medical; Lechno-Yossef, Sigal [Michigan State University, East Lansing; Kuritz, Tanya [ORNL; Wojciuch, Elizabeth [Michigan State University, East Lansing

    2007-01-01

    The clones generated in a sequencing project represent a resource for subsequent analysis of the organism whose genome has been sequenced. We describe an interrelated group of cloning vectors that either integrate into the genome or replicate, and that enhance the utility, for developmental and other studies, of the clones used to determine the genomic sequence of the cyanobacterium, Anabaena sp. strain PCC 7120. One integrating vector is a mobilizable BAC vector that was used both to generate bridging clones and to complement transposon mutations. Upon addition of a cassette that permits mobilization and selection, pUC-based sequencing clones can also integrate into the genome and thereupon complement transposon mutations. The replicating vectors are based on cyanobacterial plasmid pDU1, whose sequence we report, and on broad-host-range plasmid RSF1010. The RSF1010- and pDU1-based vectors provide the opportunity to express different genes from either cell-type-specific or -generalist promoters, simultaneously from different plasmids in the same cyanobacterial cells. We show that pDU1 ORF4 and its upstream region play an essential role in the replication and copy number of pDU1, and that ORFs alr2887 and alr3546 (hetF{sub A}) of Anabaena sp. are required specifically for fixation of dinitrogen under oxic conditions.

  18. Construction of recombinant adenovirus vector containing AFP and generation of adenovirus-mediated AFP gene modified dendritic cells vaccine%含人AFP基因重组腺病毒载体的构建及其转染树突状细胞瘤苗的制备

    Institute of Scientific and Technical Information of China (English)

    杨静悦; 曹大勇; 刘文超; 斯小明

    2009-01-01

    Objective:To construct recombinant adenovirus vectors containing human AFP genes,and infect dendritic cell. Methods: Full length AFP cDNAs were subcloned into pIND vector,followed by being cloned into shuttle2 vector.The AFP gene fragments resulted from the shuttle2-AFP digested with PI-Sce and I-Ceu were linked to the linear adeno-X virus DNA.After packaged with HEK293 cells,the adenovirus expression vector was obtained.The plasmid pAdeno-AFP was identified by endonuclease and PCR.After dendritic cells were infected pAdeno-AFP,the surface molecules of pAdeno-AFP/DC were analysed by flow cytometry.AFP levels in culture supernatant of pAdeno-AFP/DC were measured by ELISA. Results: AFP gene in the inserted DNA of adeno-AFP was confirmed by PCR,and predictive fragments proved by restriction enzyme digestion analysis were exhibited.All the above results indicated that human AFP gene had been connected with pAdeno-X vectors correctly.The recombinant adenovirus vector of human AFP gene packaged in HEK293 cells,it will be used to introduce the target gene into dendritic cell.pAdeno-AFP/DC were able to upregulate CD1a,CD11c,CD80,CD86 and HLA-DR.And pAdeno-AFP/DC could secrete high level of AFP in vitro. Conclusion: The recombinant adenovirus vector of human AFP gene have been constructed successfully.The established AFP -DC vaccine may be a tool of the hepatocellular carcinoma immunotherapy,and it will be the foundation of future clinical use of DC vaccine.%目的:构建含人AFP基因的腺病毒载体,体外转染树突状细胞,制备树突状细胞肝癌瘤苗.方法: 将AFP基因亚克隆到pIND 载体和Shuttle2载体中,构建穿梭载体Shuttle2-AFP.用PI-Sce Ⅰ和I-CeuⅠ双酶切后将所获AFP基因片段再与线性化的腺病毒载体pAdeno-X连接,构成pAdeno-AFP重组腺病毒载体.其后,用重组腺病毒载体转染HEK293细胞,包装腺病毒表达载体.通过酶切、PCR对腺病毒载体进行鉴定.包装好的重组病毒载体pAdeno-AFP体外

  19. Construction of new cloning, lacZ reporter and scarless-markerless suicide vectors for genetic studies in Aggregatibacter actinomycetemcomitans.

    Science.gov (United States)

    Juárez-Rodríguez, María Dolores; Torres-Escobar, Ascención; Demuth, Donald R

    2013-05-01

    To elucidate the putative function of a gene, effective tools are required for genetic characterization that facilitate its inactivation, deletion or modification on the bacterial chromosome. In the present study, the nucleotide sequence of the Escherichia coli/Aggregatibacter actinomycetemcomitans shuttle vector pYGK was determined, allowing us to redesign and construct a new shuttle cloning vector, pJT4, and promoterless lacZ transcriptional/translational fusion plasmids, pJT3 and pJT5. Plasmids pJT4 and pJT5 contain the origin of replication necessary to maintain shuttle vector replication. In addition, a new suicide vector, pJT1, was constructed for the generation of scarless and markerless deletion mutations of genes in the oral pathogen A. actinomycetemcomitans. Plasmid pJT1 is a pUC-based suicide vector that is counter-selectable for sucrose sensitivity. This vector does not leave antibiotic markers or scars on the chromosome after gene deletion and thus provides the option to combine several mutations in the same genetic background. The effectiveness of pJT1 was demonstrated by the construction of A. actinomycetemcomitans isogenic qseB single deletion (ΔqseB) mutant and lsrRK double deletion mutants (ΔlsrRK). These new vectors may offer alternatives for genetic studies in A. actinomycetemcomitans and other members of the HACEK (Haemophilus spp., A. actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, and Kingella kingae) group of Gram-negative bacteria.

  20. Vector Soliton Fiber Lasers

    CERN Document Server

    Zhang, Han

    2011-01-01

    Solitons, as stable localized wave packets that can propagate long distance in dispersive media without changing their shapes, are ubiquitous in nonlinear physical systems. Since the first experimental realization of optical bright solitons in the anomalous dispersion single mode fibers (SMF) by Mollenauer et al. in 1980 and optical dark solitons in the normal dispersion SMFs by P. Emplit et al. in 1987, optical solitons in SMFs had been extensively investigated. In reality a SMF always supports two orthogonal polarization modes. Taking fiber birefringence into account, it was later theoretically predicted that various types of vector solitons, including the bright-bright vector solitons, dark-dark vector solitons and dark-bright vector solitons, could be formed in SMFs. However, except the bright-bright type of vector solitons, other types of vector solitons are so far lack of clear experimental evidence. Optical solitons have been observed not only in the SMFs but also in mode locked fiber lasers. It has be...

  1. Vehicle Based Vector Sensor

    Science.gov (United States)

    2015-09-28

    300001 1 of 16 VEHICLE-BASED VECTOR SENSOR STATEMENT OF GOVERNMENT INTEREST [0001] The invention described herein may be manufactured and...unmanned underwater vehicle that can function as an acoustic vector sensor . (2) Description of the Prior Art [0004] It is known that a propagating...mechanics. An acoustic vector sensor measures the particle motion via an accelerometer and combines Attorney Docket No. 300001 2 of 16 the

  2. Implicit Real Vector Automata

    Directory of Open Access Journals (Sweden)

    Jean-François Degbomont

    2010-10-01

    Full Text Available This paper addresses the symbolic representation of non-convex real polyhedra, i.e., sets of real vectors satisfying arbitrary Boolean combinations of linear constraints. We develop an original data structure for representing such sets, based on an implicit and concise encoding of a known structure, the Real Vector Automaton. The resulting formalism provides a canonical representation of polyhedra, is closed under Boolean operators, and admits an efficient decision procedure for testing the membership of a vector.

  3. siRNA干扰人BMP9重组腺病毒载体的构建及其促进乳腺癌SK-BR-3细胞增殖%Construction of recombinant adenovirus vector interfering the expression of human BMP9 and promoting the proliferation of breast cancer SK-BR-3 cells

    Institute of Scientific and Technical Information of China (English)

    刘月红; 王科; 孙笑笑; 万绍恒; 王维; 陈莹莹; 张彦

    2013-01-01

    目的 筛选特异性干扰人BMP9基因的siRNA序列并制备重组腺病毒AdsiBMP9,探讨RNAi人BMP9基因后对乳腺癌SK-BR-3细胞增殖能力的影响.方法 设计制备3对干扰人BMP9的双链DNA序列,亚克隆至Pses-Hus质粒中获得Pses-Hus-siBMP9质粒,脂质体转染乳腺上皮细胞HBL-100筛选有效干扰质粒,构建重组腺病毒并感染SK-BR-3细胞,RT-PCR,Western blot检测BMP9表达,MTT检测细胞增殖能力.结果 成功构建并筛选出针对人BMP9基因的有效干扰质粒并包装成腺病毒,病毒滴度为1×1010IU/mL,感染SK-BR-3细胞显示BMP9在转录水平和翻译水平表达量显著低于对照组和空白组(P<0.05),第5天AdsiBMP9组细胞的增殖率显著高于AdsiNC组(P<0.05).结论 成功构建特异性沉默人BMP9基因的siRNA腺病毒载体,可有效抑制SK-BR-3细胞中BMP9基因的表达从而促进该细胞增殖.%Objective To screen specific small interfering RNA(siRNA) target human BMP9 gene and to prepare recombinant adenovirus vector AdsiBMP9 for investigation of its effects on the proliferation of breast cancer SK-BR-3 cells. Methods Three pairs of double-stranded DNA fragments for silencing human BMP9 were designed and synthesized, then subcloned into the shuttle plasmid Pses-Hus. The recombinant plasmids Pses-Hus-siBMP9 were transfected into the breast epithelial cells HBL-100 by lipofectamine transfection reagent, screened the effective interfering plasmid, constructed AdsiBMP9 and infected SK-BR-3 cells. The expression level of BMP9 mRNA and protein were detected by RT-PCR and western blot. The proliferation of SK-BR-3 cells were observed with MTT assay. Results The recombinant plasmid Pses-Hus-siBMP9 and recombinant adenovirus AdsiBMP9 were successfully constructed and its titer was 1 × 1010 IU/mL. Compared to the negative and non-infected controls, the expres- sion of BMP9 gene was significantly inhibited after the SK-BR-3 cells were infected by AdsiBMP9. SK-BR-3 cells infected with

  4. Advances in the research of adjuvants for plasmid DNA vaccines%DNA疫苗佐剂的研究进展

    Institute of Scientific and Technical Information of China (English)

    蒋丽明; 叶琳

    2009-01-01

    DNA疫苗是一种很有希望的免疫方法,经多途径接种质粒DNA能引起有效的免疫应答,重复给予不会产生抗载体免疫.然而,质粒DNA疫苗在小型实验动物中诱导的免疫应答远强于在人类和其他非人灵长类动物中.已设计多种佐剂通过直接刺激免疫系统或增强DNA表达来提高疫苗的免疫原性,这些佐剂包括免疫协同刺激分子、细胞因子、补体分子、脂质体、核酸、聚合物、纳米粒和微粒类佐剂.此文对DNA疫苗佐剂的研究进展作一综述.%Plasmid DNA vaccine is a promising modality for immunization. Immunization with plasmid DNA by various routes can trigger effective iimnune responses. The immunogens can be administered repeatedly without inducing anti-vector immunity. However, the immune responses induced by plasmid DNA vaccines are much stronger in small laboratory animal models than in non-human primates and humans. A number of adjuvants, including immune co-stimulatory molecules, cytokines, complement molecules, liposomes, nucleic acids, polymers, micro-and nano-particles, have been designed to improve the immunogenicity of DNA vaccines by directly stimulating the immune system or by enhancing plasmid DNA expression. This review introduces the progress in development of these adjuvants for plasmid DNA vaccines.

  5. Vectors and their applications

    CERN Document Server

    Pettofrezzo, Anthony J

    2005-01-01

    Geared toward undergraduate students, this text illustrates the use of vectors as a mathematical tool in plane synthetic geometry, plane and spherical trigonometry, and analytic geometry of two- and three-dimensional space. Its rigorous development includes a complete treatment of the algebra of vectors in the first two chapters.Among the text's outstanding features are numbered definitions and theorems in the development of vector algebra, which appear in italics for easy reference. Most of the theorems include proofs, and coordinate position vectors receive an in-depth treatment. Key concept

  6. Complex Polynomial Vector Fields

    DEFF Research Database (Denmark)

    Dias, Kealey

    vector fields. Since the class of complex polynomial vector fields in the plane is natural to consider, it is remarkable that its study has only begun very recently. There are numerous fundamental questions that are still open, both in the general classification of these vector fields, the decomposition...... or meromorphic (allowing poles as singularities) functions. There already exists a well-developed theory for iterative holomorphic dynamical systems, and successful relations found between iteration theory and flows of vector fields have been one of the main motivations for the recent interest in holomorphic...

  7. In vitro replication of cyanobacterial plasmids from Synechocystis PCC 6803.

    Science.gov (United States)

    Yang, X; Daniell, H; McFadden, B

    1994-09-01

    Little knowledge of DNA replication in cyanobacteria is available. In this study, we report the development and characterization of an in vitro system for studies of replication of the endogenous plasmids from the unicellular cyanobacterium Synechocystis 6803. This system (fraction III) was isolated at high salt concentrations and partially purified on a heparin-agarose column. DNA polymerases in Synechocystis 6803 appeared to be associated with membranes and could be released by the addition of ammonium sulfate to 20% saturation. DNA synthesis in fraction III was dependent on the addition of cyanobacterial plasmids isolated from the same strain. The in vitro replication products consist mostly of the supercoiled form of the plasmids. Unlike replication of many Escherichia coli plasmids, replication of cyanobacterial plasmids did not require added ATP, was not inhibited by omission of the ribonucleotides, and was insensitive to the RNA polymerase inhibitor rifampicin and the gyrase inhibitor novobiocin, but was inhibited by ethidium bromide. These data suggest that RNA may not be involved in the initiation of replication of cyanobacterial plasmids from Synechocystis 6803. In addition, intermediates of replication have been detected by two-dimensional gel electrophoresis. Density labeling experiments also indicate that cyanobacterial plasmid synthesis in vitro occurs by a semiconservative replication.

  8. Identification and characterization of enhancer-blocking insulators to reduce retroviral vector genotoxicity.

    Directory of Open Access Journals (Sweden)

    Amy C Groth

    Full Text Available The chromatin insulator cHS4 can reduce silencing chromosomal position effects and genotoxicity associated with integrating viral vectors. However, the fully active version of this element can also reduce vector titers and is only partially effective. In order to identify alternatives to cHS4, we developed a functional lentiviral vector-based reporter screen for enhancer-blocking insulators. Using this system, we screened candidate sequences that were initially identified by chromatin profiling for binding by CTCF and for DNase hypersensitivity. All 12 analyzed candidates blocked enhancer-promoter activity. The enhancer-blocking activity of the top two candidates was confirmed in two complementary plasmid-based assays. Studies in a gammaretroviral reporter vector indicated these two candidates have little to no effect on vector titers, and do not diminish vector expression in primary mouse bone marrow cultures. Subsequent assessment in a mouse in vivo tumor formation model demonstrated that both candidates reduced the rate of gammaretroviral vector-mediated genotoxicity as effectively as the cHS4 insulator. In summary, we have developed a novel lentiviral vector-based method of screening candidate elements for insulator activity, and have used this method to identify two new insulator elements capable of improving the safety of retroviral vectors without diminishing vector titers or expression. These findings expand the limited arsenal of insulators functionally validated to reduce the rate of retroviral vector-mediated genotoxicity.

  9. Construction of a novel kind of expression plasmid by homologous recombination in Saccharomyces cerevisiae

    Institute of Scientific and Technical Information of China (English)

    CHEN; Xiangling

    2005-01-01

    [1]Brunelli, J. P., Pall, M. L., A series of yeast vectors for expression of cDNAs and other DNA sequences, Yeast, 1993, 9: 1299―1308.[2]Sikorski, R. S., Hieter, P., A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae, Genetics, 1989, 122: 19―27.[3]Bonneaud, N., Ozier-Kalogerogoulos, O., Li, G. et al., A family of low and high copy replicative, integrative and single-stranded S. cerevisiae /E. coli shuttle vector, Yeast, 1991, 7: 609―615.[4]Huo, K. K., Yu, L. L., Chen, X. J., Li, Y. Y., A stable vector for high-level expression and secretion of human interferon alpha A in yeast, Science in China, Ser. B, 1993, 36(5): 557―567.[5]Zhou, Z. X., Yuan, H. Y., He, W. et al., Expression of the modified HBsAg gene SA-28 directed by a constitutive promoter, Journal of Fudan university (Natural Science), 2000, 39(3): 264―268.[6]Paques, F., Haber, J. E., Multiple pathways of recombination induces by double-strand breaks in Saccharomyces cerevisiae, Microbiology and Molecular Biology Reviews, 1999, 63(2): 349―404.[7]Martin, K., Damage-induced recombination in the yeast Saccharomyces cerevisiae, Mutation Research, 2000, 451: 91―105.[8]Alira, S., Tomoko, O., Homologous recombination and the roles of double-strand breaks, TIBS, 1995, 20: 387―391.[9]Patrick, S., Kelly, M. T., Stephen, V. K., Recombination factor of Saccharomyces cerevisiae, Mutation Research, 2000, 451: 257―275.[10]Manivasakam, P., Weber, S. C., McElver, J., Schiestl, R. H., Micro-homology mediated PCR targeting in Saccharomyces cerevisiae, Nucleic Acids Res., 1995, 23(14): 2799―2800.[11]Baudin, A., Lacroute, F., Cullin, C., A simple and efficient method for direct gene deletion in Saccharomyces cerevisiae, Nucleic Acids Res., 1993, 21(14): 3329―3330.[12]Hua, S. B., Qiu, M., Chan, E., Zhu, L., Luo, Y., Minimum length of sequence homology required for in vivo cloning by homolo-gous recombination in yeast, Plasmid, 1997, 38

  10. Plasmid-determined resistance to fosfomycin in Serratia marcescens.

    Science.gov (United States)

    Mendoza, C; Garcia, J M; Llaneza, J; Mendez, F J; Hardisson, C; Ortiz, J M

    1980-08-01

    Multiple-antibiotic-resistant strains of Serratia marcescens isolated from hospitalized patients were examined for their ability to transfer antibiotic resistance to Escherichia coli by conjugation. Two different patterns of linked transferable resistance were found among the transconjugants. The first comprised resistance to carbenicillin, streptomycin, and fosfomycin; the second, and more common, pattern included resistance to carbenicillin, streptomycin, kanamycin, gentamicin, tetracycline, chloramphenicol, sulfonamide, and fosfomycin. The two types of transconjugant strains carried a single plasmid of either 57 or 97 megadaltons in size. Both of these plasmids are present in parental S. marcescens strains resistant to fosfomycin. The 57-megadalton plasmid was transformed into E. coli.

  11. [Epidemiologic study of 2 S. typhimurium outbreaks using plasmid fingerprints].

    Science.gov (United States)

    Baumgartner, A; Breer, C; Schopfer, K

    1989-04-05

    An outbreak of salmonellosis in an old people's home is reported. The infectious agent, S. typhi-murium, was isolated not only from several inmates but also from sick cows of the farm belonging to the home, in animal feed, from employees of the local butcher's shop, and finally in sludge from the local sewage plant. Plasmid analysis provided evidence of a common origin for the isolated S. typhi-murium strains. The incriminated strains harboured, together with two low-molecular-weight plasmids, a plasmid of approximately 50 Mdal, which was also demonstrated in some other S. typhi-murium strains isolated from clinical cases in the area around St. Gallen.

  12. Effect of Plasmid Incompatibility on DNA Transfer to Streptococcus cremoris

    OpenAIRE

    Van Der Lelie, Daniel; Vossen, Jos M.B.M. van der; Venema, Gerard

    1988-01-01

    Several Streptococcus cremoris strains were used in protoplast transformation and interspecific protoplast fusion experiments with Streptococcus lactis and Bacillus subtilis, with pGKV110, pGKV21, and ΔpAMβ1 as the marker plasmids. ΔpAMβ1 is a 15.9-kilobase nonconjugative, deletion derivative of pAMβ1, which is considerably larger than the pGKV plasmids (approximately 4.5 kilobases). In general, ΔpAMβ1 was transferred more efficiently than the pGKV plasmids. Using electroporation, we were abl...

  13. Separation of plasmid DNA topoisomers by multimodal chromatography.

    Science.gov (United States)

    Silva-Santos, A Rita; Alves, Cláudia P A; Prazeres, Duarte Miguel F; Azevedo, Ana M

    2016-06-15

    The ability to analyze the distribution of topoisomers in a plasmid DNA sample is important when evaluating the quality of preparations intended for gene therapy and DNA vaccination or when performing biochemical studies on the action of topoisomerases and gyrases. Here, we describe the separation of supercoiled (sc) and open circular (oc) topoisomers by multimodal chromatography. A medium modified with the ligand N-benzyl-N-methyl ethanolamine and an elution scheme with increasing NaCl concentration are used to accomplish the baseline separation of sc and oc plasmid. The utility of the method is demonstrated by quantitating topoisomers in a purified plasmid sample. Copyright © 2016 Elsevier Inc. All rights reserved.

  14. Heterologous viral expression systems in fosmid vectors increase the functional analysis potential of metagenomic libraries.

    Science.gov (United States)

    Terrón-González, L; Medina, C; Limón-Mortés, M C; Santero, E

    2013-01-01

    The extraordinary potential of metagenomic functional analyses to identify activities of interest present in uncultured microorganisms has been limited by reduced gene expression in surrogate hosts. We have developed vectors and specialized E. coli strains as improved metagenomic DNA heterologous expression systems, taking advantage of viral components that prevent transcription termination at metagenomic terminators. One of the systems uses the phage T7 RNA-polymerase to drive metagenomic gene expression, while the other approach uses the lambda phage transcription anti-termination protein N to limit transcription termination. A metagenomic library was constructed and functionally screened to identify genes conferring carbenicillin resistance to E. coli. The use of these enhanced expression systems resulted in a 6-fold increase in the frequency of carbenicillin resistant clones. Subcloning and sequence analysis showed that, besides β-lactamases, efflux pumps are not only able contribute to carbenicillin resistance but may in fact be sufficient by themselves to convey carbenicillin resistance.

  15. Development of a site-directed integration plasmid for heterologous gene expression in Mycoplasma gallisepticum.

    Directory of Open Access Journals (Sweden)

    Isolde Nieszner

    Full Text Available Deciphering the molecular basis of the interactions between the parasite Mycoplasma gallisepticum and its avian hosts suffers from the lack of genetic tools available for the pathogen. In the absence of well established methods for targeted disruption of relevant M. gallisepticum genes, we started to develop suicide vectors and equipped them with a short fragment of M. gallisepticum origin or replication (oriC MG. We failed to create a disruption vector, although by adding a further short fragment of the M. gallisepticum tufB upstream region we created a "Trojan horse" plasmid. This is fully integrated into the genomic DNA of M. gallisepticum, always at the same site, oriC MG, and is able to carry and express any gene of interest in the genetic background of M. gallisepticum. Successful expression of a heterologous gene was shown with the lacZ gene of E. coli. When used for gene complementation or expression of hybrid genes in M. gallisepticum, a site-specific combined integration/expression vector constitutes an improvement on randomly integrating transposons, which might have unexpected effects on the expression of chromosomal genes.

  16. Cloning of Bacillus subtilis leucina A, B and C genes with Escherichia coli plasmids and expression of the leuC gene in E. coli.

    Science.gov (United States)

    Nagahari, K; Sakaguchi, K

    1978-01-17

    The leucine genes of Bacillus subtilis have been cloned directly from the chromosomal DNA into Escherichia coli leuB cells by selection for the Leu+ phenotype using RSF2124 as a vector plasmid. The hybrid plasmid designated RSF2124-B.leu contained a 4.2 megadalton fragment derived from B. subtilis DNA, including the leu genes. The fragment had one site susceptible to EcoRI* and another site susceptible to BamNI endonuclease. Among the three fragments produced by EcoRI* and BamNI endonucleases, the 1.2 megadalton fragment had the ability to transform B. subtilis leuA, leuB and leuC auxotrophs to leu+. However, B. subtilis ilvB and ilvc auxotrophs were not rescued even by the whole 4.2 megadalton fragment present in the hybrid plasmid. beta-Isopropylmalate dehydrogenase (leuB gene product) activity found in E. coli cells containing the hybrid plasmid was about 60% of that in E. coli wild type cells, despite the high copy number (7.8) of the plasmid per chromosome observed.

  17. Thermostable DNA ligase-mediated PCR production of circular plasmid (PPCP) and its application in directed evolution via in situ error-prone PCR.

    Science.gov (United States)

    Le, Yilin; Chen, Huayou; Zagursky, Robert; Wu, J H David; Shao, Weilan

    2013-08-01

    Polymerase chain reaction (PCR) is a powerful method to produce linear DNA fragments. Here we describe the Tma thermostable DNA ligase-mediated PCR production of circular plasmid (PPCP) and its application in directed evolution via in situ error-prone PCR. In this thermostable DNA ligase-mediated whole-plasmid amplification method, the resultant DNA nick between the 5' end of the PCR primer and the extended newly synthesized DNA 3' end of each PCR cycle is ligated by Tma DNA ligase, resulting in circular plasmid DNA product that can be directly transformed. The template plasmid DNA is eliminated by 'selection marker swapping' upon transformation. When performed under an error-prone condition with Taq DNA polymerase, PPCP allows one-step construction of mutagenesis libraries based on in situ error-prone PCR so that random mutations are introduced into the target gene without altering the expression vector plasmid. A significant difference between PPCP and previously published methods is that PPCP allows exponential amplification of circular DNA. We used this method to create random mutagenesis libraries of a xylanase gene and two cellulase genes. Screening of these libraries resulted in mutant proteins with desired properties, demonstrating the usefulness of in situ error-prone PPCP for creating random mutagenesis libraries for directed evolution.

  18. Estimating the number of plasmids taken up by a eukaryotic cell during transfection and evidence that antisense RNA abolishes gene expression in Physarum polycephalum.

    Science.gov (United States)

    Materna, Stefan C; Marwan, Wolfgang

    2005-02-01

    We have estimated the statistical distribution of the number of plasmids taken up by individual Jurkat lymphoma cells during electroporation in the presence of two plasmids, one encoding for yellow (EYFP) the other for cyan (ECFP) fluorescent protein. The plasmid concentration at which most of the cells take up only one plasmid or several molecules was determined by statistical analysis. We found that cells behaved slightly heterogeneous in plasmid uptake and describe how the homogeneity of a cell population can be quantified by Poisson statistics in order to identify experimental conditions that yield homogeneously transfection-competent cell populations. The experimental procedure worked out with Jurkat cells was applied to assay the effectiveness of antisense RNA in knocking down gene expression in Physarum polycephalum. Double transfection of flagellates with vectors encoding EYFP and antisense-EYFP revealed for the first time that gene expression can be suppressed by co-expression of antisense RNA in Physarum. Quantitative analysis revealed that one copy of antisense expressing gene per EYFP gene was sufficient to completely suppress formation of the EYFP protein in Physarum.

  19. Genetic modification of bone-marrow mesenchymal stem cells and hematopoietic cells with human coagulation factor IX-expressing plasmids.

    Science.gov (United States)

    Sam, Mohammad Reza; Azadbakhsh, Azadeh Sadat; Farokhi, Farrah; Rezazadeh, Kobra; Sam, Sohrab; Zomorodipour, Alireza; Haddad-Mashadrizeh, Aliakbar; Delirezh, Nowruz; Mokarizadeh, Aram

    2016-05-01

    Ex-vivo gene therapy of hemophilias requires suitable bioreactors for secretion of hFIX into the circulation and stem cells hold great potentials in this regard. Viral vectors are widely manipulated and used to transfer hFIX gene into stem cells. However, little attention has been paid to the manipulation of hFIX transgene itself. Concurrently, the efficacy of such a therapeutic approach depends on determination of which vectors give maximal transgene expression. With this in mind, TF-1 (primary hematopoietic lineage) and rat-bone marrow mesenchymal stem cells (BMSCs) were transfected with five hFIX-expressing plasmids containing different combinations of two human β-globin (hBG) introns inside the hFIX-cDNA and Kozak element and hFIX expression was evaluated by different methods. In BMSCs and TF-1 cells, the highest hFIX level was obtained from the intron-less and hBG intron-I,II containing plasmids respectively. The highest hFIX activity was obtained from the cells that carrying the hBG intron-I,II containing plasmids. BMSCs were able to produce higher hFIX by 1.4 to 4.7-fold increase with activity by 2.4 to 4.4-fold increase compared to TF-1 cells transfected with the same constructs. BMSCs and TF-1 cells could be effectively bioengineered without the use of viral vectors and hFIX minigene containing hBG introns could represent a particular interest in stem cell-based gene therapy of hemophilias.

  20. Bacterial mitosis: ParM of plasmid R1 moves plasmid DNA by an actin-like insertional polymerization mechanism.

    Science.gov (United States)

    Møller-Jensen, Jakob; Borch, Jonas; Dam, Mette; Jensen, Rasmus B; Roepstorff, Peter; Gerdes, Kenn

    2003-12-01

    Bacterial DNA segregation takes place in an active and ordered fashion. In the case of Escherichia coli plasmid R1, the partitioning system (par) separates paired plasmid copies and moves them to opposite cell poles. Here we address the mechanism by which the three components of the R1 par system act together to generate the force required for plasmid movement during segregation. ParR protein binds cooperatively to the centromeric parC DNA region, thereby forming a complex that interacts with the filament-forming actin-like ParM protein in an ATP-dependent manner, suggesting that plasmid movement is powered by insertional polymerization of ParM. Consistently, we find that segregating plasmids are positioned at the ends of extending ParM filaments. Thus, the process of R1 plasmid segregation in E. coli appears to be mechanistically analogous to the actin-based motility operating in eukaryotic cells. In addition, we find evidence suggesting that plasmid pairing is required for ParM polymerization.

  1. Influenza Plasmid DNA Vaccines: Progress and Prospects.

    Science.gov (United States)

    Bicho, Diana; Queiroz, João António; Tomaz, Cândida Teixeira

    2015-01-01

    Current influenza vaccines have long been used to fight flu infectious; however, recent advances highlight the importance of produce new alternatives. Even though traditional influenza vaccines are safe and usually effective, they need to be uploaded every year to anticipate circulating flu viruses. This limitation together with the use of embryonated chicken eggs as the substrate for vaccine production, is time-consuming and could involve potential biohazards in growth of new virus strains. Plasmid DNA produced by prokaryote microorganisms and encoding foreign proteins had emerged as a promising therapeutic tool. This technology allows the expression of a gene of interest by eukaryotic cells in order to induce protective immune responses against the pathogen of interest. In this review, we discuss the strategies to choose the best DNA vaccine to be applied in the treatment and prevention of influenza. Specifically, we give an update of influenza DNA vaccines developments, all involved techniques, their main characteristics, applicability and technical features to obtain the best option against influenza infections.

  2. Advancing vector biology research

    NARCIS (Netherlands)

    Kohl, Alain; Pondeville, Emilie; Schnettler, Esther; Crisanti, Andrea; Supparo, Clelia; Christophides, George K.; Kersey, Paul J.; Maslen, Gareth L.; Takken, Willem; Koenraadt, Constantianus J.M.; Oliva, Clelia F.; Busquets, Núria; Abad, F.X.; Failloux, Anna Bella; Levashina, Elena A.; Wilson, Anthony J.; Veronesi, Eva; Pichard, Maëlle; Arnaud Marsh, Sarah; Simard, Frédéric; Vernick, Kenneth D.

    2016-01-01

    Vector-borne pathogens impact public health, animal production, and animal welfare. Research on arthropod vectors such as mosquitoes, ticks, sandflies, and midges which transmit pathogens to humans and economically important animals is crucial for development of new control measures that target t

  3. The Use of Viral Vectors in Gene Transfer Therapy

    Directory of Open Access Journals (Sweden)

    A. Dziaková

    2016-05-01

    Full Text Available Gene therapy is strategy based on using genes as pharmaceuticals. Gene therapy is a treatment that involves altering the genes inside body's cells to stop disease. Genes contain DNA- the code controlling body form and function. Genes that do not work properly can cause disease. Gene therapy replaces a faulty gene or adds a new gene in an attempt to cure disease or improve the ability of the body to fight disease. Gene therapy holds promise for treating a wide range of diseases, including cancer, cystic fibrosis, heart disease, diabetes, hemophilia and AIDS. Various types of genetic material are used in gene therapy; double-stranded DNA (dsDNA, single-stranded DNA (ssDNA, plasmid DNA and antisense oligodeoxynucleotides (ASON. The success of gene therapy depends on assuring the entrance of the therapeutic gene to targeted cells without any form of biodegradation. Commonly used vectors in gene therapy are: adenoviruses (400 clinical studies; 23.8%, retroviruses (344 clinical studies; 20.5%, unenveloped/plasmid DNA (304 clinical studies, 17.7%, adeno-associated viruses (75 clinical studies; 4.5% and others. In this paper, we have reviewed the major gene delivery vectors and recent improvements made in their design meant to overcome the issues that commonly arise with the use of gene therapy vectors.

  4. Data set for describing the elaboration of a compatible Gateway-based co-expression vector set and supporting its validation

    OpenAIRE

    Loubna Salim; Claire Feger; Didier Busso

    2016-01-01

    This article contains Supplementary Data including methods and figures that relate to the article entitled “Construction of a compatible Gateway-based co-expression vector set for expressing multiprotein complexes in E. coli” (L. Salim, C. Feger, D. Busso, 2016) [1] that describes the elaboration and the validation of a set of versatile compatible plasmids for co-expression studies in Escherichia coli. Here, we describe experimental procedures for plasmid construction and recombinant prote...

  5. Complex Polynomial Vector Fields

    DEFF Research Database (Denmark)

    Dias, Kealey

    or meromorphic (allowing poles as singularities) functions. There already exists a well-developed theory for iterative holomorphic dynamical systems, and successful relations found between iteration theory and flows of vector fields have been one of the main motivations for the recent interest in holomorphic...... vector fields. Since the class of complex polynomial vector fields in the plane is natural to consider, it is remarkable that its study has only begun very recently. There are numerous fundamental questions that are still open, both in the general classification of these vector fields, the decomposition...... of parameter spaces into structurally stable domains, and a description of the bifurcations. For this reason, the talk will focus on these questions for complex polynomial vector fields....

  6. Complex Polynomial Vector Fields

    DEFF Research Database (Denmark)

    The two branches of dynamical systems, continuous and discrete, correspond to the study of differential equations (vector fields) and iteration of mappings respectively. In holomorphic dynamics, the systems studied are restricted to those described by holomorphic (complex analytic) functions...... vector fields. Since the class of complex polynomial vector fields in the plane is natural to consider, it is remarkable that its study has only begun very recently. There are numerous fundamental questions that are still open, both in the general classification of these vector fields, the decomposition...... of parameter spaces into structurally stable domains, and a description of the bifurcations. For this reason, the talk will focus on these questions for complex polynomial vector fields....

  7. Complex Polynomial Vector Fields

    DEFF Research Database (Denmark)

    Dias, Kealey

    The two branches of dynamical systems, continuous and discrete, correspond to the study of differential equations (vector fields) and iteration of mappings respectively. In holomorphic dynamics, the systems studied are restricted to those described by holomorphic (complex analytic) functions...... or meromorphic (allowing poles as singularities) functions. There already exists a well-developed theory for iterative holomorphic dynamical systems, and successful relations found between iteration theory and flows of vector fields have been one of the main motivations for the recent interest in holomorphic...... vector fields. Since the class of complex polynomial vector fields in the plane is natural to consider, it is remarkable that its study has only begun very recently. There are numerous fundamental questions that are still open, both in the general classification of these vector fields, the decomposition...

  8. A set of vectors for introduction of antibiotic resistance genes by in vitro Cre-mediated recombination

    OpenAIRE

    Vassetzky Yegor S; Dmitriev Petr V

    2008-01-01

    Abstract Background Introduction of new antibiotic resistance genes in the plasmids of interest is a frequent task in molecular cloning practice. Classical approaches involving digestion with restriction endonucleases and ligation are time-consuming. Findings We have created a set of insertion vectors (pINS) carrying genes that provide resistance to various antibiotics (puromycin, blasticidin and G418) and containing a loxP site. Each vector (pINS-Puro, pINS-Blast or pINS-Neo) contains either...

  9. Does Nonlinear Modeling Play a Role in Plasmid Bioprocess Monitoring using Fourier Transform Infrared Spectra?

    Science.gov (United States)

    Lopes, Marta B; Calado, Cecília R C; Figueiredo, Mário A T; Bioucas-Dias, José M

    2016-11-16

    The monitoring of biopharmaceutical products using Fourier transform infrared (FT-IR) spectroscopy relies on calibration techniques involving the acquisition of spectra of bioprocess samples along the process. The most commonly used method for that purpose is partial least squares (PLS) regression, under the assumption that a linear model is valid. Despite being successful in the presence of small nonlinearities, linear methods may fail in the presence of strong nonlinearities. This paper studies the potential usefulness of nonlinear regression methods for predicting, from in situ near-infrared (NIR) and mid-infrared (MIR) spectra acquired in high-throughput mode, biomass and plasmid concentrations in Escherichia coli DH5-α cultures producing the plasmid model pVAX-LacZ. The linear methods PLS and ridge regression (RR) are compared with their kernel (nonlinear) versions, kPLS and kRR, as well as with the (also nonlinear) relevance vector machine (RVM) and Gaussian process regression (GPR). For the systems studied, RR provided better predictive performances compared to the remaining methods. Moreover, the results point to further investigation based on larger data sets whenever differences in predictive accuracy between a linear method and its kernelized version could not be found. The use of nonlinear methods, however, shall be judged regarding the additional computational cost required to tune their additional parameters, especially when the less computationally demanding linear methods herein studied are able to successfully monitor the variables under study.

  10. Regulation of deleted in liver cancer-1 gene domains on the proliferation of human colon cancer HT29 cell

    Institute of Scientific and Technical Information of China (English)

    吴平平

    2013-01-01

    Objective To study the role of deleted in liver cancer-1(DLC-1) gene main domains on the regulation of hu-man colon cancer HT29 cell proliferation. Methods Subcloning recombinant plasmid vectors with Rho GTPase activating protein(RhoGAP),sterile alpha motif(SAM)

  11. 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.

  12. Plasmid Conjugation in E. coli and Drug Resistance

    African Journals Online (AJOL)

    Prof. Ogunji

    respiratory infections etc) or prescribing the 'newest' antibiotics in the market when older “brands” may ..... influence an increase in mortality rate; high economic burden and longer hospital ... Conjugating plasmids into bacteria; Tri Parental.

  13. Construction and Identification of Plasmid pTA-TUB2

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    An about 1.40 Kb target gene fragment was yielded by PCR amplification with the plasmid pRB 129,which was identified by restriction enzyme digestion that the PCR product was TU B2 gene.The gene was digested by the restriction enzyme and was linked with pTA plasmid to construct pTA-TU B2 plasmid.The plasmid was transformed into Chaetomium spp.by PEG method and the transformation rate was 27/(2×105) and it is nine times higher than that of pRB 129.The transformants can grow on the PDA containing 1 000 μg*mL-1 carbendazim,which is 1 000 times higher than the original Chaetomium spp.The resistance was stable after 10 times transfer on non-selective medium.

  14. Generation of Partially Reprogrammed Cells and Fully Reprogrammed iPS Cells by Plasmid Transfection.

    Science.gov (United States)

    Kim, Jong Soo; Choi, Hyun Woo; Hong, Yean Ju; Do, Jeong Tae

    2016-01-01

    Induced pluripotent stem (iPS) cells can be directly generated from somatic cells by overexpression of defined transcription factors. iPS cells can perpetually self-renew and differentiate into all cell types of an organism. iPS cells were first generated through infection with retroviruses that contain reprogramming factors. However, development of an exogene-free iPS cell generation method is crucial for future therapeutic applications, because integrated exogenes result in the formation of tumors in chimeras and regain pluripotency after differentiation in vitro. Here, we describe a method to generate iPS cells by transfection of plasmid vectors and to convert partially reprogrammed cells into fully reprogrammed iPS cells by switching from mouse ESC culture conditions to KOSR-based media with bFGF. We also describe basic methods used to characterize fully reprogrammed iPS cells.

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

    DEFF Research Database (Denmark)

    Musovic, Sanin

    at spredningskapacitet af en konjugerbare plasmid, der koder for kviksølv resistens via merA genet, finder sted under substrat begrænsede forhold til syntetisk bakterielt samfund. Plasmid overførsel var meget forhøjet ved kontinuert udsættelse af mikrokosms for en høj koncentration af kviksølv. De forskellige vækstrater...

  16. The Native Plasmid pML21 Plays a Role in Stress Tolerance in Enterococcus faecalis ML21, as Analyzed by Plasmid Curing Using Plasmid Incompatibility.

    Science.gov (United States)

    Zuo, Fang-Lei; Chen, Li-Li; Zeng, Zhu; Feng, Xiu-Juan; Yu, Rui; Lu, Xiao-Ming; Ma, Hui-Qin; Chen, Shang-Wu

    2016-02-01

    To investigate the role of the native plasmid pML21 in Enterococcus faecalis ML21's response to abiotic stresses, the plasmid pML21 was cured based on the principle of plasmid incompatibility and segregational instability, generating E. faecalis mutant strain ML0. The mutant and the wild strains were exposed to abiotic stresses: bile salts, low pH, H2O2, ethanol, heat, and NaCl, and their survival rate was measured. We found that curing of pML21 lead to reduced tolerance to stress in E. faecalis ML0, especially oxidative and osmotic stress. Complementation analysis suggested that the genes from pML21 played different role in stress tolerance. The result indicated that pML21 plays a role in E. faecalis ML21's response to abiotic stresses.

  17. Low-copy episomal vector pFY20 and high-saturation coverage genomic libraries for the fission yeast Schizosaccharomyces pombe.

    Science.gov (United States)

    Wahls, Wayne P; Davidson, Mari K

    2008-09-01

    In fission yeast, as in many organisms, episomally replicating plasmid DNA molecules can be used for a wide variety of applications. However, replicating plasmids described previously are each propagated at a high copy number per cell. Plasmid fission yeast twenty (pFY20) contains the ura4(+) gene for positive and negative selection, an origin of replication (ars1) and a stability element (stb). Although this plasmid does not have a centromere, it is propagated with a copy number of about two plasmids per haploid genome equivalent and it is transmitted with relatively high fidelity in mitosis and meiosis. This low-copy vector is useful for screens and mutational studies where overexpression (e.g. from high copy plasmids) is undesirable. We therefore constructed multiple partial-digest, size-fractionated, fission yeast genomic DNA libraries in pFY20 and in the cloning vector pBluescript KS(+). These libraries have sufficient complexity (average of 2100 genome equivalents each) for saturation screening by complementation, plasmid shuffle or hybridization.

  18. Optimization of vesicular stomatitis virus-G pseudotyped feline immunodeficiency virus vector for minimized cytotoxicity with efficient gene transfer.

    Science.gov (United States)

    Song, Jae Jin; Lee, Boyoung; Chang, Jin Woo; Kim, Joo-Hang; Kwon, Yunhee Kim; Lee, Heuiran

    2003-05-01

    FIV-based lentiviral vector has shown a unique opportunity as an efficient gene delivery vehicle, especially to nondividing human cells. Here, we genetically reconstructed the FIV-based vector by serially deleting residual virus genes of gag and vif, leading to minimized cytotoxicity together with efficient virus production and gene transfer. The modified FIV- based vector was generated by transiently transfecting 293T cells with three plasmids of the gene transfer vector with minimal gag region, the packaging plasmid without vif and the VSV-G-expressing plasmid. The vector was routinely generated as many as 1 x 10(7) transducing particles per ml and easily concentrated by simple centrifugation. The cytotoxic effect significantly decreased in sensitive cells to FIV infection even at high multiplicity of infection (MOI), such as 500. Moreover, the transduction efficiency was consistently retained after cell cycle was arrested in a variety of human cells. Taken together, our results suggest that the modified VSV-G pseudotyped FIV-based vector efficiently transduce dividing and nondividing human cells with minimal cytotoxicity.

  19. A New Shuttle Plasmid That Stably Replicates in Clostridium acetobutylicum.

    Science.gov (United States)

    Lee, Sang-Hyun; Kwon, Min-A; Choi, Sunwha; Kim, Sooah; Kim, Jungyeon; Shin, Yong-An; Kim, Kyoung Heon

    2015-10-01

    We have developed a new shuttle plasmid, designated as pLK1-MCS that can replicate in both Clostridium acetobutylicum and Escherichia coli, by combining the pUB110 and pUC19 plasmids. Plasmid pLK1-MCS replicated more stably than previously reported plasmids containing either the pIM13 or the pAMβ1 replicon in the absence of antibiotic selective pressure. The transfer frequency of pLK1-MCS into C. acetobutylicum was similar to the transfer frequency of other shuttle plasmids. We complemented C. acetobutylicum ML1 (that does not produce solvents such as acetone, butanol, and ethanol owing to loss of the megaplasmid pSOL1 harboring the adhE1-ctfAB-adc operon) by introducing pLK1-MCS carrying the adhE1-ctfAB-adc operon into C. acetobutylicum ML1. The transformed cells were able to resume anaerobic solvent production, indicating that the new shuttle plasmid has the potential for practical use in microbial biotechnology.

  20. Plasmid copy number noise in monoclonal populations of bacteria

    Science.gov (United States)

    Wong Ng, Jérôme; Chatenay, Didier; Robert, Jérôme; Poirier, Michael Guy

    2010-01-01

    Plasmids are extra chromosomal DNA that can confer to their hosts’ supplementary characteristics such as antibiotic resistance. Plasmids code for their copy number through their own replication frequency. Even though the biochemical networks underlying the plasmid copy number (PCN) regulation processes have been studied and modeled, no measurement of the heterogeneity in PCN within a whole population has been done. We have developed a fluorescent-based measurement system, which enables determination of the mean and noise in PCN within a monoclonal population of bacteria. Two different fluorescent protein reporters were inserted: one on the chromosome and the other on the plasmid. The fluorescence of these bacteria was measured with a microfluidic flow cytometry device. We show that our measurements are consistent with known plasmid characteristics. We find that the partitioning system lowers the PCN mean and standard deviation. Finally, bacterial populations were allowed to grow without selective pressure. In this case, we were able to determine the plasmid loss rate and growth inhibition effect.

  1. Construction and Use of Flow Cytometry Optimized Plasmid-Sensor Strains

    DEFF Research Database (Denmark)

    Bahl, Martin Iain; Oregaard, Gunnar; Sørensen, Søren Johannes;

    2009-01-01

    stability of the plasmid is high. The method presented here relies on a phenotypic (green fluorescence protein) marker, which is switched on if the host bacteria loses the residing plasmid. The incorporation of flow cytometry for single-cell detection and discrimination between plasmid-free and plasmid...

  2. Stable transformation of the gram-positive phytopathogenic bacterium Clavibacter michiganensis subsp. sepedonicus with several cloning vectors.

    Science.gov (United States)

    Laine, M J; Nakhei, H; Dreier, J; Lehtilä, K; Meletzus, D; Eichenlaub, R; Metzler, M C

    1996-05-01

    In this paper we describe transformation of Clavibacter michiganensis subsp. sepedonicus, the potato ring rot bacterium, with plasmid vectors. Three of the plasmids used, pDM100, pDM302, and pDM306, contain the origin of replication from pCM1, a native plasmid of C. michiganensis subsp. michiganensis. We constructed two new cloning vectors, pHN205 and pHN216, by using the origin of replication of pCM2, another native plasmid of C. michiganensis subsp. michiganensis. Plasmids pDM302, pHN205, and pHN216 were stably maintained without antibiotic selection in various strains of C. michiganensis subsp. sepedonicus. We observed that for a single plasmid, different strains of C. michiganensis subsp. sepedonicus showed significantly different transformation efficiencies. We also found unexplained strain-to-strain differences in stability with various plasmid constructions containing different arrangements of antibiotic resistance genes and origins of replication. We examined the effect of a number of factors on transformation efficiency. The best transformation efficiencies were obtained when C. michiganensis subsp. sepedonicus cells were grown on DM agar plates, harvested during the early exponential growth phase, and used fresh (without freezing) for electroporation. The maximal transformation efficiency obtained was 4.6 x 10(4) CFU/microgram of pHN216 plasmid DNA. To demonstrate the utility of this transformation system, we cloned a beta-1,4-endoglucanase-encoding gene from C. michiganensis subsp. sepedonicus into pHN216. When this construction, pHN216:C8, was electroporated into competent cells of a cellulase-deficient mutant, it restored cellulase production to almost wild-type levels.

  3. Physical Characterization of Gemini Surfactant-Based Synthetic Vectors for the Delivery of Linear Covalently Closed (LCC DNA Ministrings.

    Directory of Open Access Journals (Sweden)

    Chi Hong Sum

    Full Text Available In combination with novel linear covalently closed (LCC DNA minivectors, referred to as DNA ministrings, a gemini surfactant-based synthetic vector for gene delivery has been shown to exhibit enhanced delivery and bioavailability while offering a heightened safety profile. Due to topological differences from conventional circular covalently closed (CCC plasmid DNA vectors, the linear topology of LCC DNA ministrings may present differences with regards to DNA interaction and the physicochemical properties influencing DNA-surfactant interactions in the formulation of lipoplexed particles. In this study, N,N-bis(dimethylhexadecyl-α,ω-propanediammonium(16-3-16gemini-based synthetic vectors, incorporating either CCC plasmid or LCC DNA ministrings, were characterized and compared with respect to particle size, zeta potential, DNA encapsulation, DNase sensitivity, and in vitro transgene delivery efficacy. Through comparative analysis, differences between CCC plasmid DNA and LCC DNA ministrings led to variations in the physical properties of the resulting lipoplexes after complexation with 16-3-16 gemini surfactants. Despite the size disparities between the plasmid DNA vectors (CCC and DNA ministrings (LCC, differences in DNA topology resulted in the generation of lipoplexes of comparable particle sizes. The capacity for ministring (LCC derived lipoplexes to undergo complete counterion release during lipoplex formation contributed to improved DNA encapsulation, protection from DNase degradation, and in vitro transgene delivery.

  4. Allelopathy of plasmid-bearing and plasmid-free organisms competing for two complementary resources in a chemostat.

    Science.gov (United States)

    Bhattacharyya, Joydeb; Smith, Hal L; Pal, Samares

    2012-01-01

    We consider a model of competition between plasmid-bearing and plasmid-free organisms for two complementary nutrients in a chemostat. We assume that the plasmid-bearing organism produces an allelopathic agent at the cost of its reproductive abilities which is lethal to plasmid-free organism. Our analysis leads to different thresholds in terms of the model parameters acting as conditions under which the organisms associated with the system cannot thrive even in the absence of competition. Local stability of the system is obtained in the absence of one or both the organisms. Also, global stability of the system is obtained in the presence of both the organisms. Computer simulations have been carried out to illustrate various analytical results.

  5. ON VECTOR NETWORK EQUILIBRIUM PROBLEMS

    Institute of Scientific and Technical Information of China (English)

    Guangya CHEN

    2005-01-01

    In this paper we define a concept of weak equilibrium for vector network equilibrium problems.We obtain sufficient conditions of weak equilibrium points and establish relation with vector network equilibrium problems and vector variational inequalities.

  6. 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

    of these plasmids within pathogenic hosts are poorly understood. Here we study plasmid-host adaptations following transfer of a 73 kb conjugative multidrug resistance plasmid to naïve clinical isolates of Klebsiella pneumoniae and Escherichia coli We use experimental evolution, mathematical modelling and population...... 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...... of plasmid adaptation. While insertion sequences are well known to supply plasmids with adaptive traits, our findings suggest that they also play an important role in plasmid evolution by maintaining the plasticity necessary to alleviate plasmid-host constrains. Further, the observed evolutionary strategy...

  7. Use of Integrase-Minus Lentiviral Vector for Transient Expression

    Directory of Open Access Journals (Sweden)

    Hossein Azadeh

    2012-01-01

    Full Text Available Objective: Lentivirus-derived vectors are among the most promising viral vectors for gene therapy which is currently available, but their use in clinical practice is limited due to associated risk of insertional mutagenesis. Gene targeting is an ideal method for gene therapy, but it has low efficiency in comparison to viral vector methods. In this study, we are going to design and construct an integrase-minus lentiviral vector. This vector is suitable for transient expression of gene and gene targeting with viral vector.Materials and Methods: In this experimental study, three missense mutations were induced in the catalytic domain of Integrase gene in the pLP1 plasmid and resulted D64V, D116A and E152G changes in the amino acid sequence through site directed mutagenesis. The pLenti6.2-GW/EmGFP transfer vector, associated with native and mutated packaging mix, was transfected into 293T cell line. In order to titer the lentivirus stock, the viruses were harvested. Finally, the viruses transduced into COS-7 cell line to assess green fluorescent protein (GFP gene expression by a fluorescence microscopy.Results: Recombinant and wild lentiviruses titer was about 5~8×106 transducing units/ml in COS-7 cell line. The number of GFP-positive cells transduced with native viruses was decreased slightly during two weeks after viral transduction. In contrast, in the case of integrase-minus viruses, a dramatic decrease in the number of GFP positive cells was observed.Conclusion: This study was conducted to overcome the integration of lentiviral genome into a host genome. Nonintegrating lentiviral vectors can be used for transient gene expression and gene targeting if a Target gene cassette is placed in the lentivirus gene structure. This combination method decreases disadvantages of both processes, such as random integration of lentiviruses and low efficiency of gene targeting.

  8. Multiple Pathways of Plasmid DNA Transfer in Helicobacter pylori

    Science.gov (United States)

    Rohrer, Stefanie; Holsten, Lea; Weiss, Evelyn; Benghezal, Mohammed; Fischer, Wolfgang; Haas, Rainer

    2012-01-01

    Many Helicobacter pylori (Hp) strains carry cryptic plasmids of different size and gene content, the function of which is not well understood. A subgroup of these plasmids (e.g. pHel4, pHel12), contain a mobilisation region, but no cognate type IV secretion system (T4SS) for conjugative transfer. Instead, certain H. pylori strains (e.g. strain P12 carrying plasmid pHel12) can harbour up to four T4SSs in their genome (cag-T4SS, comB, tfs3, tfs4). Here, we show that such indigenous plasmids can be efficiently transferred between H. pylori strains, even in the presence of extracellular DNaseI eliminating natural transformation. Knockout of a plasmid-encoded mobA relaxase gene significantly reduced plasmid DNA transfer in the presence of DNaseI, suggesting a DNA conjugation or mobilisation process. To identify the T4SS involved in this conjugative DNA transfer, each individual T4SS was consecutively deleted from the bacterial chromosome. Using a marker-free counterselectable gene deletion procedure (rpsL counterselection method), a P12 mutant strain was finally obtained with no single T4SS (P12ΔT4SS). Mating experiments using these mutants identified the comB T4SS in the recipient strain as the major mediator of plasmid DNA transfer between H. pylori strains, both in a DNaseI-sensitive (natural transformation) as well as a DNaseI-resistant manner (conjugative transfer). However, transfer of a pHel12::cat plasmid from a P12ΔT4SS donor strain into a P12ΔT4SS recipient strain provided evidence for the existence of a third, T4SS-independent mechanism of DNA transfer. This novel type of plasmid DNA transfer, designated as alternate DNaseI-Resistant (ADR) mechanism, is observed at a rather low frequency under in vitro conditions. Taken together, our study describes for the first time the existence of three distinct pathways of plasmid DNA transfer between H. pylori underscoring the importance of horizontal gene transfer for this species. PMID:23029142

  9. Construction of shuttle, expression vector of human tumor necrosis factor alpha (hTNF-α) gene and its expression in a cyanobacterium, Anabaena sp. PCC 7120

    Institute of Scientific and Technical Information of China (English)

    刘凤龙; 施定基; 商之狄; 邵宁; 徐旭东; 钟泽璞; 张宏斌; 吴锦银; 王捷; 江悦华; 赵树进; 林晨; 张雪艳; 吴旻; 彭国宏; 张海霞; 曾呈奎

    1999-01-01

    The construction of the shuttle, expression vector of human tumor necrosis factor alpha (hTNF-a) gene and its expression in a cyanobacterium Anabaena sp. PCC 7120 was reported. The 700-bp hTNF cDNA fragments have been recovered from plasmid pRL-rhTNF, then inserted downstream of the promoter PpsbA in the plasmid pRL439. The resultant intermediary plasmid pRL-TC has further been combined with the shuttle vector pDC-8 to get the shuttle, expression vector pDC-TNF. The expression of the rhTNF gene in Escherichia coli has been analyzed by SDS-PAGE and thin-layer scanning, and the results show that the expressed TNF protein with these two vectors is 16.9 percent (pRL-TC) and 15.0 percent (pDC-TNF) of the total proteins in the cells, respectively, while the expression level of TNF gene in plasmid pRL-rhTNF is only 11.8 percent. Combined with the participation of the conjugal and helper plasmids, pDC-TNF has been introduced into Anabaena sp PCC 7120 by triparental conjugative transfer, and the stable transgenic

  10. Horizontal Transfer of Plasmid-Mediated Cephalosporin Resistance Genes in the Intestine of Houseflies (Musca domestica).

    Science.gov (United States)

    Fukuda, Akira; Usui, Masaru; Okubo, Torahiko; Tamura, Yutaka

    2016-06-01

    Houseflies are a mechanical vector for various types of bacteria, including antimicrobial-resistant bacteria (ARB). If the intestine of houseflies is a suitable site for the transfer of antimicrobial resistance genes (ARGs), houseflies could also serve as a biological vector for ARB. To clarify whether cephalosporin resistance genes are transferred efficiently in the housefly intestine, we compared with conjugation experiments in vivo (in the intestine) and in vitro by using Escherichia coli with eight combinations of four donor and two recipient strains harboring plasmid-mediated cephalosporin resistance genes and chromosomal-encoded rifampicin resistance genes, respectively. In the in vivo conjugation experiment, houseflies ingested donor strains for 6 hr and then recipient strains for 3 hr, and 24 hr later, the houseflies were surface sterilized and analyzed. In vitro conjugation experiments were conducted using the broth-mating method. In 3/8 combinations, the in vitro transfer frequency (Transconjugants/Donor) was ≥1.3 × 10(-4); the in vivo transfer rates of cephalosporin resistance genes ranged from 2.0 × 10(-4) to 5.7 × 10(-5). Moreover, cephalosporin resistance genes were transferred to other species of enteric bacteria of houseflies such as Achromobacter sp. and Pseudomonas fluorescens. These results suggest that houseflies are not only a mechanical vector for ARB but also a biological vector for the occurrence of new ARB through the horizontal transfer of ARGs in their intestine.

  11. 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.

  12. Optimization of supercoiled HPV-16 E6/E7 plasmid DNA purification with arginine monolith using design of experiments.

    Science.gov (United States)

    Almeida, A M; Queiroz, J A; Sousa, F; Sousa, A

    2015-01-26

    The progress of DNA vaccines is dependent on the development of suitable chromatographic procedures to successfully purify genetic vectors, such as plasmid DNA. Human Papillomavirus is associated with the development of tumours due to the oncogenic power of E6 and E7 proteins, produced by this virus. The supercoiled HPV-16 E6/E7 plasmid-based vaccine was recently purified with the arginine monolith, with 100% of purity, but only 39% of recovery was achieved. Therefore, the present study describes the application of experimental design tools, a newly explored methodology in preparative chromatography, in order to improve the supercoiled plasmid DNA recovery with the arginine monolith, maintaining the high purity degree. In addition, the importance and influence of pH in the pDNA retention to the arginine ligand was also demonstrated. The Composite Central Face design was validated and the recovery of the target molecule was successfully improved from 39% to 83.5%, with an outstanding increase of more than double, while maintaining 100% of purity.

  13. Gene therapy of experimental autoimmune thyroiditis mice by in vivo administration of plasmid DNA coding for human interleukin-10

    Institute of Scientific and Technical Information of China (English)

    ZHANGZhen-Lin; LINBo; YULu-Yang; SHENShui-Xian; ZHULi-Hua; WANGWui-Ping; GUOLi-He

    2003-01-01

    AIM: To investigate the effect of interleukin-10 (IL-10) gene on experimental autoimmune thyroiditis mice.METHODS: Mice were immunized to induce autoimmune thyroiditis with porcine thyroglobulin (pTg), and thyroids of mice were injected with IL-10 DNA. On d 28 after immunization with pTg, mRNA expression of IL-10 inthyroid glands was detected and thyroid specimens were histopathological studied. RESULTS: The mRNA expression of IL-10 was detected in thyroid glands on d 7 and 14 after injection of IL-10 plasmid DNA or on COS-7 cells48 h after IL-10 plasmid DNA transfection. In addition, hlL-10 levels in culture media significantly increased 48 hand 72 h after IL-10 plasmid DNA transfection. Infiltration index of lymphocytes (1.1±0.4) in thyroids ofIL-10-treated mice was significantly lower than that of pcDNA3-null-treated mice (2.2±0.5) (P<0.01). Comparedwith pcDNA3-null control mice, IL-10-treated mice had lower levels of serum IFN-γ(P<0.01). CONCLUSION:The direct injection of DNA expression vectors encoding IL-10 into thyroid significantly inhibited development oflymphocytic infiltration of thyroid of autoimmune th,yroiditis mice, and alleviated the progression of this disease.

  14. Self-Assembled Functional Nanostructure of Plasmid DNA with Ionic Liquid [Bmim][PF₆]: Enhanced Efficiency in Bacterial Gene Transformation.

    Science.gov (United States)

    Soni, Sarvesh K; Sarkar, Sampa; Mirzadeh, Nedaossadat; Selvakannan, P R; Bhargava, Suresh K

    2015-04-28

    The electrostatic interaction between the negatively charged phosphate groups of plasmid DNA and the cationic part of hydrophobic ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate ([Bmim][PF6]), initiates spontaneous self-assembly to form the functional nanostructures made up of DNA and ionic liquid (IL). These functional nanostructures were demonstrated as promising synthetic nonviral vectors for the efficient bacterial pGFP gene transformation in cells. In particular, the functional nanostructures that were made up of 1 μL of IL ([Bmim][PF6]) and 1 μg of plasmid DNA can increase the transformation efficiency by 300-400% in microbial systems, without showing any toxicity for E. coli DH5α cells. (31)P nuclear magnetic resonance (NMR), Fourier transform infrared (FTIR) spectroscopy, and X-ray photoelectron (XPS) spectroscopic analysis revealed that the electrostatic interaction between negatively charged phosphate oxygen and cationic Bmim(+) tends to initiate the self-assembly process. Thermogravimetric analysis of the DNA-IL functional nanostructures showed that these nanostructures consist of ∼16 wt % ionic liquid, which is considered to provide the stability to the plasmid DNA that eventually enhanced the transformation efficiency.

  15. Construction and use of a versatile set of broad-host-range cloning and expression vectors based on the RK2 replicon.

    Science.gov (United States)

    Blatny, J M; Brautaset, T; Winther-Larsen, H C; Haugan, K; Valla, S

    1997-01-01

    The plasmid vectors described in this report are derived from the broad-host-range RK2 replicon and can be maintained in many gram-negative bacterial species. The complete nucleotide sequences of all of the cloning and expression vectors are known. Important characteristics of the cloning vectors are as follows: a size range of 4.8 to 7.1 kb, unique cloning sites, different antibiotic resistance markers for selection of plasmid-containing cells, oriT-mediated conjugative plasmid transfer, plasmid stabilization functions, and a means for a simple method for modification of plasmid copy number. Expression vectors were constructed by insertion of the inducible Pu or Pm promoter together with its regulatory gene xylR or xylS, respectively, from the TOL plasmid of Pseudomonas putida. One of these vectors was used in an analysis of the correlation between phosphoglucomutase activity and amylose accumulation in Escherichia coli. The experiments showed that amylose synthesis was only marginally affected by the level of basal expression from the Pm promoter of the Acetobacter xylinum phosphoglucomutase gene (celB). In contrast, amylose accumulation was strongly reduced when transcription from Pm was induced. CelB was also expressed with a very high induction ratio in Xanthomonas campestris. These experiments showed that the A. xylinum celB gene could not complement the role of the bifunctional X. campestris phosphoglucomutase-phosphomannomutase gene in xanthan biosynthesis. We believe that the vectors described here are useful for cloning experiments, gene expression, and physiological studies with a wide range of bacteria and presumably also for analysis of gene transfer in the environment. PMID:9023917

  16. Construction of a cytomegalovirus-based amplicon: a vector with a unique transfer capacity.

    Science.gov (United States)

    Borst, Eva Maria; Messerle, Martin

    2003-07-01

    Cytomegalovirus (CMV) has a number of interesting properties that qualifies it as a vector for gene transfer. Especially appealing is the ability of the CMV genome to persist in hematopoietic progenitor cells and the packaging capacity of the viral capsid that accommodates a DNA genome of 230 kbp. In order to exploit the packaging capacity of the CMV capsid we investigated whether the principles of an amplicon vector can be applied to CMV. Amplicons are herpesviral vectors, which contain only the cis-active sequences required for replication and packaging of the vector genome. For construction of a CMV amplicon the sequences comprising the lytic origin of replication (orilyt) and the cleavage packaging recognition sites (pac) of human CMV were cloned onto a plasmid. A gene encoding the green fluorescent protein was used as a model transgene. The amplicon plasmid replicated in the presence of a CMV helper virus and was packaged into CMV particles, with replication and packaging being dependent on the presence of the orilyt and pac sequences. The packaged amplicon could be transferred to recipient cells and reisolated from the transduced cells. Analysis of the DNA isolated from CMV capsids revealed that the CMV amplicon was packaged as a concatemer with a size of approximately 210 kbp. The CMV amplicon vector has the potential to transfer therapeutic genes with a size of more than 200 kbp and thus provides a unique transfer capacity among viral vectors.

  17. Altered Murine Tissue Colonization by Borrelia burgdorferi following Targeted Deletion of Linear Plasmid 17-Carried Genes

    OpenAIRE

    Casselli, Timothy; Tourand, Yvonne; Bankhead, Troy

    2012-01-01

    The causative agent of Lyme disease, Borrelia burgdorferi, possesses a segmented genome comprised of a single linear chromosome and upwards of 23 linear and circular plasmids. Much of what is known about plasmid-borne genes comes from studying laboratory clones that have spontaneously lost one or more plasmids during in vitro passage. Some plasmids, including the linear plasmid lp17, are never or rarely reported to be lost during routine culture; therefore, little is known about the requireme...

  18. Light composite vector bosons

    CERN Document Server

    Kosower, David A.

    1993-01-01

    In gauge theories with slowly-running coupling constants, it may be possible for four-fermion operators to be nearly marginal. Such operators can possess asymptotically weak couplings, and can plausibly give rise to light composite vector mesons.

  19. GAP Land Cover - Vector

    Data.gov (United States)

    Minnesota Department of Natural Resources — This vector dataset is a detailed (1-acre minimum), hierarchically organized vegetation cover map produced by computer classification of combined two-season pairs of...

  20. Tagged Vector Contour (TVC)

    Data.gov (United States)

    Kansas Data Access and Support Center — The Kansas Tagged Vector Contour (TVC) dataset consists of digitized contours from the 7.5 minute topographic quadrangle maps. Coverage for the state is incomplete....

  1. Construction of gene targeting vectors from lambda KOS genomic libraries.

    Science.gov (United States)

    Wattler, S; Kelly, M; Nehls, M

    1999-06-01

    We describe a highly redundant murine genomic library in a new lambda phage, lambda knockout shuttle (lambda KOS) that facilitates the very rapid construction of replacement-type gene targeting vectors. The library consists of 94 individually amplified subpools, each containing an average of 40,000 independent genomic clones. The subpools are arrayed into a 96-well format that allows a PCR-based efficient recovery of independent genomic clones. The lambda KOS vector backbone permits the CRE-mediated conversion into high-copy number pKOS plasmids, wherein the genomic inserts are automatically flanked by negative-selection cassettes. The lambda KOS vector system exploits the yeast homologous recombination machinery to simplify the construction of replacement-type gene targeting vectors independent of restriction sites within the genomic insert. We outline procedures that allow the generation of simple and more sophisticated conditional gene targeting vectors within 3-4 weeks, beginning with the screening of the lambda KOS genomic library.

  2. RIP3基因重组质粒构建及其表达对MCF7细胞死亡方式的影响%Construction of Recombinant Plasmid of Human RIP3 Gene and Its Effects on the Death of Breast Cancer MCF7 Cells

    Institute of Scientific and Technical Information of China (English)

    路灿; 徐惠君; 贾勇圣; 佟仲生

    2014-01-01

    目的:建立稳定过表达RIP3基因的乳腺癌细胞株,并证实融合蛋白在细胞内的表达、定位及对MCF7细胞死亡方式的影响。方法逆转录聚合酶链反应(RT-PCR)检测4种乳腺癌细胞及正常乳腺上皮细胞中RIP3 mRNA的表达。以正常乳腺上皮细胞MCF10A cDNA为模板,PCR扩增RIP3基因cDNA全长,将合成的RIP3编码区序列,克隆入mCherry载体的N末端,构建重组质粒mCherry-RIP3,对重组质粒进行酶切鉴定及DNA测序。钙法转染293T细胞,收集病毒感染MCF7细胞,杀稻瘟菌素(4 mg/L)维持筛选,构建稳定表达细胞株。Western blot、荧光显微镜等检测目的基因表达效率及蛋白定位。显微镜下观察肿瘤坏死因子(TNF)-α及Caspase抑制剂Z-VAD-FMK处理下mCherry-RIP3-MCF7细胞的死亡形态及比例。结果 RIP3 mRNA在乳腺癌细胞中普遍低表达。RIP3基因成功克隆入载体。过表达mCherry-RIP3基因的MCF7细胞可见红色荧光蛋白表达,定位于胞质。目的基因RIP3在转染细胞中为过表达。mCherry-RIP3转染后增强MCF7细胞对TNF-α联合Z-VAD-FMK诱导的细胞坏死的敏感性。结论成功构建RIP3基因过表达重组质粒,获得外源性RIP3稳定过表达的乳腺癌MCF7细胞株,mCherry-RIP3定位于胞质,并在TNF-α介导的程序性坏死中起作用。%Objective To construct the recombinant RIP3 over-expressed plasmids and transfect them in breast cancer MCF7 cells, and identify the expression and localization of fusion protein, as well as its effect on the death way of MCF7 cells. Methods The expression levels of RIP3 mRNA in four breast cancer cell lines and normal mammary epithelial cells were detected by reverse transcription polymerase chain reaction (RT-PCR). The RIP 3 coding sequence was amplified by polymerase chain reaction and subcloned into mCherry vector to construct recombinant plasmids. The plasmids were transfected into MCF7 cells by lentivirus after DNA

  3. Vector Lattice Vortex Solitons

    Institute of Scientific and Technical Information of China (English)

    WANG Jian-Dong; YE Fang-Wei; DONG Liang-Wei; LI Yong-Ping

    2005-01-01

    @@ Two-dimensional vector vortex solitons in harmonic optical lattices are investigated. The stability properties of such solitons are closely connected to the lattice depth Vo. For small Vo, vector vortex solitons with the total zero-angular momentum are more stable than those with the total nonzero-angular momentum, while for large Vo, this case is inversed. If Vo is large enough, both the types of such solitons are stable.

  4. Support vector machines applications

    CERN Document Server

    Guo, Guodong

    2014-01-01

    Support vector machines (SVM) have both a solid mathematical background and good performance in practical applications. This book focuses on the recent advances and applications of the SVM in different areas, such as image processing, medical practice, computer vision, pattern recognition, machine learning, applied statistics, business intelligence, and artificial intelligence. The aim of this book is to create a comprehensive source on support vector machine applications, especially some recent advances.

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

    Directory of Open Access Journals (Sweden)

    Joseph J Gillespie

    Full Text Available BACKGROUND: 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. METHODOLOGY/PRINCIPAL FINDINGS: 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. CONCLUSION/SIGNIFICANCE: 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

  6. The Vector Curvaton

    CERN Document Server

    Navarro, Andres A

    2013-01-01

    We analyze a massive vector field with a non-canonical kinetic term in the action, minimally coupled to gravity, where the mass and kinetic function of the vector field vary as functions of time during inflation. The vector field is introduced following the same idea of a scalar curvaton, which must not affect the inflationary dynamics since its energy density during inflation is negligible compared to the total energy density in the Universe. Using this hypothesis, the vector curvaton will be solely responsible for generating the primordial curvature perturbation \\zeta. We have found that the spectra of the vector field perturbations are scale-invariant in superhorizon scales due to the suitable choice of the time dependence of the kinetic function and the effective mass during inflation. The preferred direction, generated by the vector field, makes the spectrum of \\zeta depend on the wavevector, i.e. there exists statistical anisotropy in \\zeta. This is discussed principally in the case where the mass of th...

  7. Scale-up and manufacturing of clinical-grade self-inactivating γ-retroviral vectors by transient transfection.

    Science.gov (United States)

    van der Loo, J C M; Swaney, W P; Grassman, E; Terwilliger, A; Higashimoto, T; Schambach, A; Baum, C; Thrasher, A J; Williams, D A; Nordling, D L; Reeves, L; Malik, P

    2012-03-01

    The need for γ-retroviral (gRV) vectors with a self-inactivating (SIN) design for clinical application has prompted a shift in methodology of vector manufacturing from the traditional use of stable producer lines to transient transfection-based techniques. Herein, we set out to define and optimize a scalable manufacturing process for the production of gRV vectors using transfection in a closed-system bioreactor in compliance with current good manufacturing practices (cGMP). The process was based on transient transfection of 293T cells on Fibra-Cel disks in the Wave Bioreactor. Cells were harvested from tissue culture flasks and transferred to the bioreactor containing Fibra-Cel in the presence of vector plasmid, packaging plasmids and calcium-phosphate in Dulbecco's modified Eagle's medium and 10% fetal bovine serum. Virus supernatant was harvested at 10-14 h intervals. Using optimized procedures, a total of five ecotropic cGMP-grade gRV vectors were produced (9 liters each) with titers up to 3.6 × 10(7) infectious units per milliliter on 3T3 cells. One GMP preparation of vector-like particles was also produced. These results describe an optimized process for the generation of SIN viral vectors by transfection using a disposable platform that allows for the generation of clinical-grade viral vectors without the need for cleaning validation in a cost-effective manner.

  8. Generation of virus-free induced pluripotent stem cell clones on a synthetic matrix via a single cell subcloning in the naive state.

    Directory of Open Access Journals (Sweden)

    Naoki Nishishita

    Full Text Available CD34+ cord blood cells can be reprogrammed effectively on dishes coated with a synthetic RGD motif polymer (PronectinF® using a temperature sensitive Sendai virus vector (SeV TS7 carrying reprogramming factors OCT3/4, SOX2, KLF4 and c-MYC. Dish-shaped human ES cell-like colonies emerged in serum-free primate ES cell medium (supplemented with bFGF in 20% O2 culture conditions. The copy numbers of SeV TS7 vectors in the cytoplasm were drastically reduced by a temperature shift at 38°C for three days. Then, single cells from colonies were seeded on PronectinF®-coated 96-well plates and cultured under naïve culture conditions (N2B27-based medium supplemented with LIF, forskolin, a MAPK inhibitor, and a GSK inhibitor in 5% O2 for cloning purpose. Dome-shaped mouse ES cell-like colonies from single cells emerged on PronectinF®-coated dishes. These cells were collected and cultured again in primate ES cell medium supplemented with bFGF in 20% O2 and maintained on PronectinF®-coated dishes. Cells were assessed for reprogramming, including the absence of residual SeV and their potential for three germ layer differentiation. Generation of virus-free induced pluripotent stem cell (iPSC clones from single cells under feeder-free conditions will solve some of the safety concerns related to use of xeno- or allogeneic-material in culture, and contribute to the characterization and the standardization of iPS cells intended for use in a clinical setting.

  9. 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

  10. Vector Design for Improved DNA Vaccine Efficacy, Safety and Production

    Directory of Open Access Journals (Sweden)

    James A. Williams

    2013-06-01

    Full Text Available DNA vaccination is a disruptive technology that offers the promise of a new rapidly deployed vaccination platform to treat human and animal disease with gene-based materials. Innovations such as electroporation, needle free jet delivery and lipid-based carriers increase transgene expression and immunogenicity through more effective gene delivery. This review summarizes complementary vector design innovations that, when combined with leading delivery platforms, further enhance DNA vaccine performance. These next generation vectors also address potential safety issues such as antibiotic selection, and increase plasmid manufacturing quality and yield in exemplary fermentation production processes. Application of optimized constructs in combination with improved delivery platforms tangibly improves the prospect of successful application of DNA vaccination as prophylactic vaccines for diverse human infectious disease targets or as therapeutic vaccines for cancer and allergy.

  11. Change in the plasmid copy number in acetic acid bacteria in response to growth phase and acetic acid concentration.

    Science.gov (United States)

    Akasaka, Naoki; Astuti, Wiwik; Ishii, Yuri; Hidese, Ryota; Sakoda, Hisao; Fujiwara, Shinsuke

    2015-06-01

    Plasmids pGE1 (2.5 kb), pGE2 (7.2 kb), and pGE3 (5.5 kb) were isolated from Gluconacetobacter europaeus KGMA0119, and sequence analyses revealed they harbored 3, 8, and 4 genes, respectively. Plasmid copy numbers (PCNs) were determined by real-time quantitative PCR at different stages of bacterial growth. When KGMA0119 was cultured in medium containing 0.4% ethanol and 0.5% acetic acid, PCN of pGE1 increased from 7 copies/genome in the logarithmic phase to a maximum of 12 copies/genome at the beginning of the stationary phase, before decreasing to 4 copies/genome in the late stationary phase. PCNs for pGE2 and pGE3 were maintained at 1-3 copies/genome during all phases of growth. Under a higher concentration of ethanol (3.2%) the PCN for pGE1 was slightly lower in all the growth stages, and those of pGE2 and pGE3 were unchanged. In the presence of 1.0% acetic acid, PCNs were higher for pGE1 (10 copies/genome) and pGE3 (6 copies/genome) during the logarithmic phase. Numbers for pGE2 did not change, indicating that pGE1 and pGE3 increase their PCNs in response to acetic acid. Plasmids pBE2 and pBE3 were constructed by ligating linearized pGE2 and pGE3 into pBR322. Both plasmids were replicable in Escherichia coli, Acetobacter pasteurianus and G. europaeus, highlighting their suitability as vectors for acetic acid bacteria.

  12. Plasmid mediated antibiotic resistance in isolated bacteria from burned patients.

    Science.gov (United States)

    Beige, Fahimeh; Baseri Salehi, Majid; Bahador, Nima; Mobasherzadeh, Sina

    2015-01-01

    Nowadays, the treatment of burned patients is difficult because of the high frequency of infection with antibiotic resistance bacteria. This study was conducted to evaluate the level of antibiotic resistance in Gram-negative bacteria and its relation with the existence of plasmid. The samples were collected from two hundred twenty hospitalized burned patients in Isfahan burn hospital during a three-month period (March 2012 to June 2012). The samples were isolated and the Gram-negative bacteria were identified using phenotypic method and API 20E System. Antibiotic susceptibility and plasmid profile were determined by standard Agar disc diffusion and plasmid spin column extraction methods. Totally 117 Gram-negative bacteria were isolated, the most common were Pseudomonas aerugionsa (37.6%), P. fluorescens (25.6%), Acinetobacter baumanii (20/5%) and Klebsiella pneumoniae (7.6%), respectively. The isolates showed high frequency of antibiotic resistance against ceftazidime and co-amoxiclave (100%) and low frequency of antibiotic resistance against amikacin with (70%).The results indicated that 60% of the isolates harboured plasmid. On the other hand, the patients infected with A. baumanii and P. aeruginosa were cured (with 60% frequency) whereas, those infected with P. fluorescens were not cured. Hence, probably antibiotic resistance markers of A. baumanii and P. aeruginosa are plasmid mediated; however, P. fluorescens is chromosomally mediated. Based on our findings, P. aerugionsa is a major causative agent of wound infections and amikacin could be considered as a more effective antibiotic for treatment of the burned patients.

  13. Transcription-replication collision increases recombination efficiency between plasmids.

    Science.gov (United States)

    Jialiang, Li; Feng, Chen; Zhen, Xu; Jibing, Chen; Xiang, Lv; Lingling, Zhang; Depei, Liu

    2013-11-01

    It has been proposed that the stalling of the replication forks can induce homologous recombination in several organisms, and that arrested replication forks may offer nuclease targets, thereby providing a substrate for proteins involved in double-strand repair. In this article, we constructed a plasmid with the potential for transcription-replication collision (TRC), in which DNA replication and RNA transcription occur on the same DNA template simultaneously. Theoretically, transcription will impede DNA replication and increase homologous recombination. To validate this hypothesis, another plasmid was constructed that contained a homologous sequence with the exception of some mutated sites. Co-transfection of these two plasmids into 293T cells resulted in increased recombination frequency. The ratio of these two plasmids also affected the recombination frequency. Moreover, we found high expression levels of RAD51, which indicated that the increase in the recombination rate was probably via the homologous recombination pathway. These results indicate that mutant genes in plasmids can be repaired by TRC-induced recombination.

  14. Functional amyloids as inhibitors of plasmid DNA replication

    Science.gov (United States)

    Molina-García, Laura; Gasset-Rosa, Fátima; Moreno-del Álamo, María; Fernández-Tresguerres, M. Elena; Moreno-Díaz de la Espina, Susana; Lurz, Rudi; Giraldo, Rafael

    2016-01-01

    DNA replication is tightly regulated to constrain the genetic material within strict spatiotemporal boundaries and copy numbers. Bacterial plasmids are autonomously replicating DNA molecules of much clinical, environmental and biotechnological interest. A mechanism used by plasmids to prevent over-replication is ‘handcuffing’, i.e. inactivating the replication origins in two DNA molecules by holding them together through a bridge built by a plasmid-encoded initiator protein (Rep). Besides being involved in handcuffing, the WH1 domain in the RepA protein assembles as amyloid fibres upon binding to DNA in vitro. The amyloid state in proteins is linked to specific human diseases, but determines selectable and epigenetically transmissible phenotypes in microorganisms. Here we have explored the connection between handcuffing and amyloidogenesis of full-length RepA. Using a monoclonal antibody specific for an amyloidogenic conformation of RepA-WH1, we have found that the handcuffed RepA assemblies, either reconstructed in vitro or in plasmids clustering at the bacterial nucleoid, are amyloidogenic. The replication-inhibitory RepA handcuff assembly is, to our knowledge, the first protein amyloid directly dealing with DNA. Built on an amyloid scaffold, bacterial plasmid handcuffs can bring a novel molecular solution to the universal problem of keeping control on DNA replication initiation. PMID:27147472

  15. Dcm methylation is detrimental to plasmid transformation in Clostridium thermocellum

    Energy Technology Data Exchange (ETDEWEB)

    Guss, Adam M [ORNL; Olson, Daniel G. [Thayer School of Engineering at Dartmouth; Caiazza, Nicky [Mascoma Corporation; Lynd, Lee R [Thayer School of Engineering at Dartmouth

    2012-01-01

    BACKGROUND: Industrial production of biofuels and other products by cellulolytic microorganisms is of interest but hindered by the nascent state of genetic tools. Although a genetic system for Clostridium thermocellum DSM1313 has recently been developed, available methods achieve relatively low efficiency and similar plasmids can transform C. thermocellum at dramatically different efficiencies. RESULTS: We report an increase in transformation efficiency of C. thermocellum for a variety of plasmids by using DNA that has been methylated by Escherichia coli Dam but not Dcm methylases. When isolated from a dam+ dcm+ E. coli strain, pAMG206 transforms C. thermocellum 100-fold better than the similar plasmid pAMG205, which contains an additional Dcm methylation site in the pyrF gene. Upon removal of Dcm methylation, transformation with pAMG206 showed a four- to seven-fold increase in efficiency; however, transformation efficiency of pAMG205 increased 500-fold. Removal of the Dcm methylation site from the pAM205 pyrF gene via silent mutation resulted in increased transformation efficiencies equivalent to that of pAMG206. Upon proper methylation, transformation efficiency of plasmids bearing the pMK3 and pB6A origins of replication increased ca. three orders of magnitude. CONCLUSION: E. coli Dcm methylation decreases transformation efficiency in C. thermocellum DSM1313. The use of properly methylated plasmid DNA should facilitate genetic manipulation of this industrially relevant bacterium.

  16. Dcm methylation is detrimental to plasmid transformation in Clostridium thermocellum

    Directory of Open Access Journals (Sweden)

    Guss Adam M

    2012-05-01

    Full Text Available Abstract Background Industrial production of biofuels and other products by cellulolytic microorganisms is of interest but hindered by the nascent state of genetic tools. Although a genetic system for Clostridium thermocellum DSM1313 has recently been developed, available methods achieve relatively low efficiency and similar plasmids can transform C. thermocellum at dramatically different efficiencies. Results We report an increase in transformation efficiency of C. thermocellum for a variety of plasmids by using DNA that has been methylated by Escherichia coli Dam but not Dcm methylases. When isolated from a dam+dcm+E. coli strain, pAMG206 transforms C. thermocellum 100-fold better than the similar plasmid pAMG205, which contains an additional Dcm methylation site in the pyrF gene. Upon removal of Dcm methylation, transformation with pAMG206 showed a four- to seven-fold increase in efficiency; however, transformation efficiency of pAMG205 increased 500-fold. Removal of the Dcm methylation site from the pAMG205 pyrF gene via silent mutation resulted in increased transformation efficiencies equivalent to that of pAMG206. Upon proper methylation, transformation efficiency of plasmids bearing the pMK3 and pB6A origins of replication increased ca. three orders of magnitude. Conclusions E. coli Dcm methylation decreases transformation efficiency in C. thermocellum DSM1313. The use of properly methylated plasmid DNA should facilitate genetic manipulation of this industrially relevant bacterium.

  17. Production of Fibronectin Binding Protein A at the surface of Lactococcus lactis increases plasmid transfer in vitro and in vivo.

    Directory of Open Access Journals (Sweden)

    Daniela Pontes

    Full Text Available Lactococci are noninvasive lactic acid bacteria frequently used as protein delivery vectors and, more recently, as DNA delivery vehicles. We previously showed that Lactococcus lactis (LL expressing the Fibronectin-Binding Protein A of Staphylococcus aureus (LL-FnBPA+ showed higher internalization rates in vitro in Caco-2 cells than the native (wt lactococci and were able to deliver a eukaryotic Green Fluorescent Protein (GFP expression plasmid in 1% of human Caco-2 cells. Here, using the bovine beta-lactoglobulin (BLG, one of the major cow's milk allergen, and GFP we characterized the potential of LL-FnBPA+ as an in vivo DNA vaccine delivery vehicle. We first showed that the invasive strain LL-FnBPA+ carrying the plasmid pValac:BLG (LL-FnBPA+ BLG was more invasive than LL-BLG and showed the same invasivity as LL-FnBPA+. Then we demonstrated that the Caco-2 cells, co-incubated with LL-FnBPA+ BLG produced up to 30 times more BLG than the Caco-2 cells co-incubated with the non invasive LL-BLG. Using two different gene reporters, BLG and GFP, and two different methods of detection, EIA and fluorescence microscopy, we showed in vivo that: i in order to be effective, LL-FnBPA+ required a pre-coating with Fetal Calf Serum before oral administration; ii plasmid transfer occurred in enterocytes without regard to the strains used (invasive or not; iii the use of LL-FnBPA+ increased the number of mice producing BLG, but not the level of BLG produced. We thus confirmed the good potential of invasive recombinant lactic acid bacteria as DNA delivery vector in vivo.

  18. Construction of Eukaryotic Expression Plasmid of bFGF Gene in Rats and Its Expression in Tenocytes

    Institute of Scientific and Technical Information of China (English)

    FENG Yong; ZHENG Dong; YANG Shuhua; LI Jing

    2007-01-01

    The bFGF plays an important role in embryonic development of tendons and ligaments and in the healing of injuried tendons and ligaments. The eukaryotic expression plasmid of rat basic fibroblast growth factor (bFGF) gene was constructed in order to further investigate the bFGF function in molecular regulatory mechanism in the repair of tendons and ligaments and to provide the foundation for the clinical application. The cDNA fragments of bFGF were cloned from the skin of rats by RT-PCR, and recombinated to the pMD18-T vector. The cDNA encoding bFGF was cloned from the pMD18-T vector by RT-PCR, digested with restriction enzyme EcoR Ⅰ, Pst Ⅰ and bound to eukaryotic expression plasmid pIRES2-EGFP to construct eukaryotic expression plasmid pIRES2-EGFP-bFGF. The pIRES2-EGFP-bFGF was transfected into the tenocytes by lipid-mediated ransfection technique. MTT test was used to detect the biological activity of bFGF in supematants after the transfection. The expression of type Ⅰ and Ⅲ collagen genes was detected by using RT-PCR. It was verified that the pIRES2-EGFP-bFGF was successfully constructed, and its transfection into tenocytes could significantly enhance the biological activity of bFGF, and increase the expression of type Ⅰ and Ⅲ collagen mRNA, suggesting that pIRES2-EGFP-mediated bFGF gene therapy was beneficial to the repair of tendons and ligaments.

  19. Vector cross product in n-dimensional vector space

    OpenAIRE

    Tian, Xiu-Lao; Yang, Chao; Hu, Yang; Tian, Chao

    2013-01-01

    The definition of vector cross product (VCP) introduced by Eckmann only exists in thethree- and the seven- dimensional vector space. In this paper, according to the orthogonal completeness, magnitude of basis vector cross product and all kinds of combinations of basis vector $\\hat{e}_i$, the generalized definition of VCP in the odd n-dimensional vector space is given by introducing a cross term $X_{AB}$. In addition, the definition is validated by reducing the generalization definition to the...

  20. Extended Mixed Vector Equilibrium Problems

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    Mijanur Rahaman

    2014-01-01

    Full Text Available We study extended mixed vector equilibrium problems, namely, extended weak mixed vector equilibrium problem and extended strong mixed vector equilibrium problem in Hausdorff topological vector spaces. Using generalized KKM-Fan theorem (Ben-El-Mechaiekh et al.; 2005, some existence results for both problems are proved in noncompact domain.