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Sample records for genome engineering synthetic

  1. Synthetic Biology to Engineer Bacteriophage Genomes.

    Science.gov (United States)

    Rita Costa, Ana; Milho, Catarina; Azeredo, Joana; Pires, Diana Priscila

    2018-01-01

    Recent advances in the synthetic biology field have enabled the development of new molecular biology techniques used to build specialized bacteriophages with new functionalities. Bacteriophages have been engineered towards a wide range of applications including pathogen control and detection, targeted drug delivery, or even assembly of new materials.In this chapter, two strategies that have been successfully used to genetically engineer bacteriophage genomes are addressed: a yeast-based platform and bacteriophage recombineering of electroporated DNA.

  2. Genome Engineering and Modification Toward Synthetic Biology for the Production of Antibiotics.

    Science.gov (United States)

    Zou, Xuan; Wang, Lianrong; Li, Zhiqiang; Luo, Jie; Wang, Yunfu; Deng, Zixin; Du, Shiming; Chen, Shi

    2018-01-01

    Antibiotic production is often governed by large gene clusters composed of genes related to antibiotic scaffold synthesis, tailoring, regulation, and resistance. With the expansion of genome sequencing, a considerable number of antibiotic gene clusters has been isolated and characterized. The emerging genome engineering techniques make it possible towards more efficient engineering of antibiotics. In addition to genomic editing, multiple synthetic biology approaches have been developed for the exploration and improvement of antibiotic natural products. Here, we review the progress in the development of these genome editing techniques used to engineer new antibiotics, focusing on three aspects of genome engineering: direct cloning of large genomic fragments, genome engineering of gene clusters, and regulation of gene cluster expression. This review will not only summarize the current uses of genomic engineering techniques for cloning and assembly of antibiotic gene clusters or for altering antibiotic synthetic pathways but will also provide perspectives on the future directions of rebuilding biological systems for the design of novel antibiotics. © 2017 Wiley Periodicals, Inc.

  3. Synthetic genome engineering forging new frontiers for wine yeast.

    Science.gov (United States)

    Pretorius, Isak S

    2017-02-01

    Over the past 15 years, the seismic shifts caused by the convergence of biomolecular, chemical, physical, mathematical, and computational sciences alongside cutting-edge developments in information technology and engineering have erupted into a new field of scientific endeavor dubbed Synthetic Biology. Recent rapid advances in high-throughput DNA sequencing and DNA synthesis techniques are enabling the design and construction of new biological parts (genes), devices (gene networks) and modules (biosynthetic pathways), and the redesign of biological systems (cells and organisms) for useful purposes. In 2014, the budding yeast Saccharomyces cerevisiae became the first eukaryotic cell to be equipped with a fully functional synthetic chromosome. This was achieved following the synthesis of the first viral (poliovirus in 2002 and bacteriophage Phi-X174 in 2003) and bacterial (Mycoplasma genitalium in 2008 and Mycoplasma mycoides in 2010) genomes, and less than two decades after revealing the full genome sequence of a laboratory (S288c in 1996) and wine (AWRI1631 in 2008) yeast strain. A large international project - the Synthetic Yeast Genome (Sc2.0) Project - is now underway to synthesize all 16 chromosomes (∼12 Mb carrying ∼6000 genes) of the sequenced S288c laboratory strain by 2018. If successful, S. cerevisiae will become the first eukaryote to cross the horizon of in silico design of complex cells through de novo synthesis, reshuffling, and editing of genomes. In the meantime, yeasts are being used as cell factories for the semi-synthetic production of high-value compounds, such as the potent antimalarial artemisinin, and food ingredients, such as resveratrol, vanillin, stevia, nootkatone, and saffron. As a continuum of previously genetically engineered industrially important yeast strains, precision genome engineering is bound to also impact the study and development of wine yeast strains supercharged with synthetic DNA. The first taste of what the future

  4. Multiplexed genome engineering and genotyping methods applications for synthetic biology and metabolic engineering.

    Science.gov (United States)

    Wang, Harris H; Church, George M

    2011-01-01

    Engineering at the scale of whole genomes requires fundamentally new molecular biology tools. Recent advances in recombineering using synthetic oligonucleotides enable the rapid generation of mutants at high efficiency and specificity and can be implemented at the genome scale. With these techniques, libraries of mutants can be generated, from which individuals with functionally useful phenotypes can be isolated. Furthermore, populations of cells can be evolved in situ by directed evolution using complex pools of oligonucleotides. Here, we discuss ways to utilize these multiplexed genome engineering methods, with special emphasis on experimental design and implementation. Copyright © 2011 Elsevier Inc. All rights reserved.

  5. Directed evolution combined with synthetic biology strategies expedite semi-rational engineering of genes and genomes.

    Science.gov (United States)

    Kang, Zhen; Zhang, Junli; Jin, Peng; Yang, Sen

    2015-01-01

    Owing to our limited understanding of the relationship between sequence and function and the interaction between intracellular pathways and regulatory systems, the rational design of enzyme-coding genes and de novo assembly of a brand-new artificial genome for a desired functionality or phenotype are difficult to achieve. As an alternative approach, directed evolution has been widely used to engineer genomes and enzyme-coding genes. In particular, significant developments toward DNA synthesis, DNA assembly (in vitro or in vivo), recombination-mediated genetic engineering, and high-throughput screening techniques in the field of synthetic biology have been matured and widely adopted, enabling rapid semi-rational genome engineering to generate variants with desired properties. In this commentary, these novel tools and their corresponding applications in the directed evolution of genomes and enzymes are discussed. Moreover, the strategies for genome engineering and rapid in vitro enzyme evolution are also proposed.

  6. Bacterial genome engineering and synthetic biology: combating pathogens.

    Science.gov (United States)

    Krishnamurthy, Malathy; Moore, Richard T; Rajamani, Sathish; Panchal, Rekha G

    2016-11-04

    The emergence and prevalence of multidrug resistant (MDR) pathogenic bacteria poses a serious threat to human and animal health globally. Nosocomial infections and common ailments such as pneumonia, wound, urinary tract, and bloodstream infections are becoming more challenging to treat due to the rapid spread of MDR pathogenic bacteria. According to recent reports by the World Health Organization (WHO) and Centers for Disease Control and Prevention (CDC), there is an unprecedented increase in the occurrence of MDR infections worldwide. The rise in these infections has generated an economic strain worldwide, prompting the WHO to endorse a global action plan to improve awareness and understanding of antimicrobial resistance. This health crisis necessitates an immediate action to target the underlying mechanisms of drug resistance in bacteria. The advent of new bacterial genome engineering and synthetic biology (SB) tools is providing promising diagnostic and treatment plans to monitor and treat widespread recalcitrant bacterial infections. Key advances in genetic engineering approaches can successfully aid in targeting and editing pathogenic bacterial genomes for understanding and mitigating drug resistance mechanisms. In this review, we discuss the application of specific genome engineering and SB methods such as recombineering, clustered regularly interspaced short palindromic repeats (CRISPR), and bacterial cell-cell signaling mechanisms for pathogen targeting. The utility of these tools in developing antibacterial strategies such as novel antibiotic production, phage therapy, diagnostics and vaccine production to name a few, are also highlighted. The prevalent use of antibiotics and the spread of MDR bacteria raise the prospect of a post-antibiotic era, which underscores the need for developing novel therapeutics to target MDR pathogens. The development of enabling SB technologies offers promising solutions to deliver safe and effective antibacterial therapies.

  7. Genome engineering using a synthetic gene circuit in Bacillus subtilis.

    Science.gov (United States)

    Jeong, Da-Eun; Park, Seung-Hwan; Pan, Jae-Gu; Kim, Eui-Joong; Choi, Soo-Keun

    2015-03-31

    Genome engineering without leaving foreign DNA behind requires an efficient counter-selectable marker system. Here, we developed a genome engineering method in Bacillus subtilis using a synthetic gene circuit as a counter-selectable marker system. The system contained two repressible promoters (B. subtilis xylA (Pxyl) and spac (Pspac)) and two repressor genes (lacI and xylR). Pxyl-lacI was integrated into the B. subtilis genome with a target gene containing a desired mutation. The xylR and Pspac-chloramphenicol resistant genes (cat) were located on a helper plasmid. In the presence of xylose, repression of XylR by xylose induced LacI expression, the LacIs repressed the Pspac promoter and the cells become chloramphenicol sensitive. Thus, to survive in the presence of chloramphenicol, the cell must delete Pxyl-lacI by recombination between the wild-type and mutated target genes. The recombination leads to mutation of the target gene. The remaining helper plasmid was removed easily under the chloramphenicol absent condition. In this study, we showed base insertion, deletion and point mutation of the B. subtilis genome without leaving any foreign DNA behind. Additionally, we successfully deleted a 2-kb gene (amyE) and a 38-kb operon (ppsABCDE). This method will be useful to construct designer Bacillus strains for various industrial applications. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  8. Teaching Synthetic Biology, Bioinformatics and Engineering to Undergraduates: The Interdisciplinary Build-a-Genome Course

    Science.gov (United States)

    Dymond, Jessica S.; Scheifele, Lisa Z.; Richardson, Sarah; Lee, Pablo; Chandrasegaran, Srinivasan; Bader, Joel S.; Boeke, Jef D.

    2009-01-01

    A major challenge in undergraduate life science curricula is the continual evaluation and development of courses that reflect the constantly shifting face of contemporary biological research. Synthetic biology offers an excellent framework within which students may participate in cutting-edge interdisciplinary research and is therefore an attractive addition to the undergraduate biology curriculum. This new discipline offers the promise of a deeper understanding of gene function, gene order, and chromosome structure through the de novo synthesis of genetic information, much as synthetic approaches informed organic chemistry. While considerable progress has been achieved in the synthesis of entire viral and prokaryotic genomes, fabrication of eukaryotic genomes requires synthesis on a scale that is orders of magnitude higher. These high-throughput but labor-intensive projects serve as an ideal way to introduce undergraduates to hands-on synthetic biology research. We are pursuing synthesis of Saccharomyces cerevisiae chromosomes in an undergraduate laboratory setting, the Build-a-Genome course, thereby exposing students to the engineering of biology on a genomewide scale while focusing on a limited region of the genome. A synthetic chromosome III sequence was designed, ordered from commercial suppliers in the form of oligonucleotides, and subsequently assembled by students into ∼750-bp fragments. Once trained in assembly of such DNA “building blocks” by PCR, the students accomplish high-yield gene synthesis, becoming not only technically proficient but also constructively critical and capable of adapting their protocols as independent researchers. Regular “lab meeting” sessions help prepare them for future roles in laboratory science. PMID:19015540

  9. Genome scale engineering techniques for metabolic engineering.

    Science.gov (United States)

    Liu, Rongming; Bassalo, Marcelo C; Zeitoun, Ramsey I; Gill, Ryan T

    2015-11-01

    Metabolic engineering has expanded from a focus on designs requiring a small number of genetic modifications to increasingly complex designs driven by advances in genome-scale engineering technologies. Metabolic engineering has been generally defined by the use of iterative cycles of rational genome modifications, strain analysis and characterization, and a synthesis step that fuels additional hypothesis generation. This cycle mirrors the Design-Build-Test-Learn cycle followed throughout various engineering fields that has recently become a defining aspect of synthetic biology. This review will attempt to summarize recent genome-scale design, build, test, and learn technologies and relate their use to a range of metabolic engineering applications. Copyright © 2015 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

  10. Modularization of genetic elements promotes synthetic metabolic engineering.

    Science.gov (United States)

    Qi, Hao; Li, Bing-Zhi; Zhang, Wen-Qian; Liu, Duo; Yuan, Ying-Jin

    2015-11-15

    In the context of emerging synthetic biology, metabolic engineering is moving to the next stage powered by new technologies. Systematical modularization of genetic elements makes it more convenient to engineer biological systems for chemical production or other desired purposes. In the past few years, progresses were made in engineering metabolic pathway using synthetic biology tools. Here, we spotlighted the topic of implementation of modularized genetic elements in metabolic engineering. First, we overviewed the principle developed for modularizing genetic elements and then discussed how the genetic modules advanced metabolic engineering studies. Next, we picked up some milestones of engineered metabolic pathway achieved in the past few years. Last, we discussed the rapid raised synthetic biology field of "building a genome" and the potential in metabolic engineering. Copyright © 2015 Elsevier Inc. All rights reserved.

  11. Efficient fdCas9 Synthetic Endonuclease with Improved Specificity for Precise Genome Engineering

    KAUST Repository

    Aouida, Mustapha

    2015-07-30

    The Cas9 endonuclease is used for genome editing applications in diverse eukaryotic species. A high frequency of off-target activity has been reported in many cell types, limiting its applications to genome engineering, especially in genomic medicine. Here, we generated a synthetic chimeric protein between the catalytic domain of the FokI endonuclease and the catalytically inactive Cas9 protein (fdCas9). A pair of guide RNAs (gRNAs) that bind to sense and antisense strands with a defined spacer sequence range can be used to form a catalytically active dimeric fdCas9 protein and generate double-strand breaks (DSBs) within the spacer sequence. Our data demonstrate an improved catalytic activity of the fdCas9 endonuclease, with a spacer range of 15–39 nucleotides, on surrogate reporters and genomic targets. Furthermore, we observed no detectable fdCas9 activity at known Cas9 off-target sites. Taken together, our data suggest that the fdCas9 endonuclease variant is a superior platform for genome editing applications in eukaryotic systems including mammalian cells.

  12. Efficient fdCas9 Synthetic Endonuclease with Improved Specificity for Precise Genome Engineering

    KAUST Repository

    Aouida, Mustapha; Eid, Ayman; Ali, Zahir; Cradick, Thomas; Lee, Ciaran; Deshmukh, Harshavardhan; Atef, Ahmed; Abu Samra, Dina Bashir Kamil; Gadhoum, Samah Zeineb; Merzaban, Jasmeen; Bao, Gang; Mahfouz, Magdy M.

    2015-01-01

    The Cas9 endonuclease is used for genome editing applications in diverse eukaryotic species. A high frequency of off-target activity has been reported in many cell types, limiting its applications to genome engineering, especially in genomic medicine. Here, we generated a synthetic chimeric protein between the catalytic domain of the FokI endonuclease and the catalytically inactive Cas9 protein (fdCas9). A pair of guide RNAs (gRNAs) that bind to sense and antisense strands with a defined spacer sequence range can be used to form a catalytically active dimeric fdCas9 protein and generate double-strand breaks (DSBs) within the spacer sequence. Our data demonstrate an improved catalytic activity of the fdCas9 endonuclease, with a spacer range of 15–39 nucleotides, on surrogate reporters and genomic targets. Furthermore, we observed no detectable fdCas9 activity at known Cas9 off-target sites. Taken together, our data suggest that the fdCas9 endonuclease variant is a superior platform for genome editing applications in eukaryotic systems including mammalian cells.

  13. Genome-wide engineering of an infectious clone of herpes simplex virus type 1 using synthetic genomics assembly methods.

    Science.gov (United States)

    Oldfield, Lauren M; Grzesik, Peter; Voorhies, Alexander A; Alperovich, Nina; MacMath, Derek; Najera, Claudia D; Chandra, Diya Sabrina; Prasad, Sanjana; Noskov, Vladimir N; Montague, Michael G; Friedman, Robert M; Desai, Prashant J; Vashee, Sanjay

    2017-10-17

    Here, we present a transformational approach to genome engineering of herpes simplex virus type 1 (HSV-1), which has a large DNA genome, using synthetic genomics tools. We believe this method will enable more rapid and complex modifications of HSV-1 and other large DNA viruses than previous technologies, facilitating many useful applications. Yeast transformation-associated recombination was used to clone 11 fragments comprising the HSV-1 strain KOS 152 kb genome. Using overlapping sequences between the adjacent pieces, we assembled the fragments into a complete virus genome in yeast, transferred it into an Escherichia coli host, and reconstituted infectious virus following transfection into mammalian cells. The virus derived from this yeast-assembled genome, KOS YA , replicated with kinetics similar to wild-type virus. We demonstrated the utility of this modular assembly technology by making numerous modifications to a single gene, making changes to two genes at the same time and, finally, generating individual and combinatorial deletions to a set of five conserved genes that encode virion structural proteins. While the ability to perform genome-wide editing through assembly methods in large DNA virus genomes raises dual-use concerns, we believe the incremental risks are outweighed by potential benefits. These include enhanced functional studies, generation of oncolytic virus vectors, development of delivery platforms of genes for vaccines or therapy, as well as more rapid development of countermeasures against potential biothreats.

  14. Freedom and Responsibility in Synthetic Genomics: The Synthetic Yeast Project

    OpenAIRE

    Sliva, Anna; Yang, Huanming; Boeke, Jef D.; Mathews, Debra J. H.

    2015-01-01

    First introduced in 2011, the Synthetic Yeast Genome (Sc2.0) Project is a large international synthetic genomics project that will culminate in the first eukaryotic cell (Saccharomyces cerevisiae) with a fully synthetic genome. With collaborators from across the globe and from a range of institutions spanning from do-it-yourself biology (DIYbio) to commercial enterprises, it is important that all scientists working on this project are cognizant of the ethical and policy issues associated with...

  15. Synthetic biology and metabolic engineering.

    Science.gov (United States)

    Stephanopoulos, Gregory

    2012-11-16

    Metabolic engineering emerged 20 years ago as the discipline occupied with the directed modification of metabolic pathways for the microbial synthesis of various products. As such, it deals with the engineering (design, construction, and optimization) of native as well as non-natural routes of product synthesis, aided in this task by the availability of synthetic DNA, the core enabling technology of synthetic biology. The two fields, however, only partially overlap in their interest in pathway engineering. While fabrication of biobricks, synthetic cells, genetic circuits, and nonlinear cell dynamics, along with pathway engineering, have occupied researchers in the field of synthetic biology, the sum total of these areas does not constitute a coherent definition of synthetic biology with a distinct intellectual foundation and well-defined areas of application. This paper reviews the origins of the two fields and advances two distinct paradigms for each of them: that of unit operations for metabolic engineering and electronic circuits for synthetic biology. In this context, metabolic engineering is about engineering cell factories for the biological manufacturing of chemical and pharmaceutical products, whereas the main focus of synthetic biology is fundamental biological research facilitated by the use of synthetic DNA and genetic circuits.

  16. Targeted genome regulation via synthetic programmable transcriptional regulators

    KAUST Repository

    Piatek, Agnieszka Anna

    2016-04-19

    Regulation of gene transcription controls cellular functions and coordinates responses to developmental, physiological and environmental cues. Precise and efficient molecular tools are needed to characterize the functions of single and multiple genes in linear and interacting pathways in a native context. Modular DNA-binding domains from zinc fingers (ZFs) and transcriptional activator-like proteins (TALE) are amenable to bioengineering to bind DNA target sequences of interest. As a result, ZF and TALE proteins were used to develop synthetic programmable transcription factors. However, these systems are limited by the requirement to re-engineer proteins for each new target sequence. The clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR associated 9 (Cas9) genome editing tool was recently repurposed for targeted transcriptional regulation by inactivation of the nuclease activity of Cas9. Due to the facile engineering, simplicity, precision and amenability to library construction, the CRISPR/Cas9 system is poised to revolutionize the functional genomics field across diverse eukaryotic species. In this review, we discuss the development of synthetic customizable transcriptional regulators and provide insights into their current and potential applications, with special emphasis on plant systems, in characterization of gene functions, elucidation of molecular mechanisms and their biotechnological applications. © 2016 Informa UK Limited, trading as Taylor & Francis Group

  17. Freedom and Responsibility in Synthetic Genomics: The Synthetic Yeast Project.

    Science.gov (United States)

    Sliva, Anna; Yang, Huanming; Boeke, Jef D; Mathews, Debra J H

    2015-08-01

    First introduced in 2011, the Synthetic Yeast Genome (Sc2.0) PROJECT is a large international synthetic genomics project that will culminate in the first eukaryotic cell (Saccharomyces cerevisiae) with a fully synthetic genome. With collaborators from across the globe and from a range of institutions spanning from do-it-yourself biology (DIYbio) to commercial enterprises, it is important that all scientists working on this project are cognizant of the ethical and policy issues associated with this field of research and operate under a common set of principles. In this commentary, we survey the current ethics and regulatory landscape of synthetic biology and present the Sc2.0 Statement of Ethics and Governance to which all members of the project adhere. This statement focuses on four aspects of the Sc2.0 PROJECT: societal benefit, intellectual property, safety, and self-governance. We propose that such project-level agreements are an important, valuable, and flexible model of self-regulation for similar global, large-scale synthetic biology projects in order to maximize the benefits and minimize potential harms. Copyright © 2015 by the Genetics Society of America.

  18. Mammalian Synthetic Biology: Engineering Biological Systems.

    Science.gov (United States)

    Black, Joshua B; Perez-Pinera, Pablo; Gersbach, Charles A

    2017-06-21

    The programming of new functions into mammalian cells has tremendous application in research and medicine. Continued improvements in the capacity to sequence and synthesize DNA have rapidly increased our understanding of mechanisms of gene function and regulation on a genome-wide scale and have expanded the set of genetic components available for programming cell biology. The invention of new research tools, including targetable DNA-binding systems such as CRISPR/Cas9 and sensor-actuator devices that can recognize and respond to diverse chemical, mechanical, and optical inputs, has enabled precise control of complex cellular behaviors at unprecedented spatial and temporal resolution. These tools have been critical for the expansion of synthetic biology techniques from prokaryotic and lower eukaryotic hosts to mammalian systems. Recent progress in the development of genome and epigenome editing tools and in the engineering of designer cells with programmable genetic circuits is expanding approaches to prevent, diagnose, and treat disease and to establish personalized theranostic strategies for next-generation medicines. This review summarizes the development of these enabling technologies and their application to transforming mammalian synthetic biology into a distinct field in research and medicine.

  19. Synthetic biology approaches in cancer immunotherapy, genetic network engineering, and genome editing.

    Science.gov (United States)

    Chakravarti, Deboki; Cho, Jang Hwan; Weinberg, Benjamin H; Wong, Nicole M; Wong, Wilson W

    2016-04-18

    Investigations into cells and their contents have provided evolving insight into the emergence of complex biological behaviors. Capitalizing on this knowledge, synthetic biology seeks to manipulate the cellular machinery towards novel purposes, extending discoveries from basic science to new applications. While these developments have demonstrated the potential of building with biological parts, the complexity of cells can pose numerous challenges. In this review, we will highlight the broad and vital role that the synthetic biology approach has played in applying fundamental biological discoveries in receptors, genetic circuits, and genome-editing systems towards translation in the fields of immunotherapy, biosensors, disease models and gene therapy. These examples are evidence of the strength of synthetic approaches, while also illustrating considerations that must be addressed when developing systems around living cells.

  20. Synthetic biology: an emerging engineering discipline.

    Science.gov (United States)

    Cheng, Allen A; Lu, Timothy K

    2012-01-01

    Over the past decade, synthetic biology has emerged as an engineering discipline for biological systems. Compared with other substrates, biology poses a unique set of engineering challenges resulting from an incomplete understanding of natural biological systems and tools for manipulating them. To address these challenges, synthetic biology is advancing from developing proof-of-concept designs to focusing on core platforms for rational and high-throughput biological engineering. These platforms span the entire biological design cycle, including DNA construction, parts libraries, computational design tools, and interfaces for manipulating and probing synthetic circuits. The development of these enabling technologies requires an engineering mindset to be applied to biology, with an emphasis on generalizable techniques in addition to application-specific designs. This review aims to discuss the progress and challenges in synthetic biology and to illustrate areas where synthetic biology may impact biomedical engineering and human health.

  1. Synthetic biology meets tissue engineering.

    Science.gov (United States)

    Davies, Jamie A; Cachat, Elise

    2016-06-15

    Classical tissue engineering is aimed mainly at producing anatomically and physiologically realistic replacements for normal human tissues. It is done either by encouraging cellular colonization of manufactured matrices or cellular recolonization of decellularized natural extracellular matrices from donor organs, or by allowing cells to self-organize into organs as they do during fetal life. For repair of normal bodies, this will be adequate but there are reasons for making unusual, non-evolved tissues (repair of unusual bodies, interface to electromechanical prostheses, incorporating living cells into life-support machines). Synthetic biology is aimed mainly at engineering cells so that they can perform custom functions: applying synthetic biological approaches to tissue engineering may be one way of engineering custom structures. In this article, we outline the 'embryological cycle' of patterning, differentiation and morphogenesis and review progress that has been made in constructing synthetic biological systems to reproduce these processes in new ways. The state-of-the-art remains a long way from making truly synthetic tissues, but there are now at least foundations for future work. © 2016 Authors; published by Portland Press Limited.

  2. A simple and effective method for construction of Escherichia coli strains proficient for genome engineering.

    Directory of Open Access Journals (Sweden)

    Young Shin Ryu

    Full Text Available Multiplex genome engineering is a standalone recombineering tool for large-scale programming and accelerated evolution of cells. However, this advanced genome engineering technique has been limited to use in selected bacterial strains. We developed a simple and effective strain-independent method for effective genome engineering in Escherichia coli. The method involves introducing a suicide plasmid carrying the λ Red recombination system into the mutS gene. The suicide plasmid can be excised from the chromosome via selection in the absence of antibiotics, thus allowing transient inactivation of the mismatch repair system during genome engineering. In addition, we developed another suicide plasmid that enables integration of large DNA fragments into the lacZ genomic locus. These features enable this system to be applied in the exploitation of the benefits of genome engineering in synthetic biology, as well as the metabolic engineering of different strains of E. coli.

  3. Biotechnology and synthetic biology approaches for metabolic engineering of bioenergy crops.

    Science.gov (United States)

    Shih, Patrick M; Liang, Yan; Loqué, Dominique

    2016-07-01

    The Green Revolution has fuelled an exponential growth in human population since the mid-20th century. Due to population growth, food and energy demands will soon surpass supply capabilities. To overcome these impending problems, significant improvements in genetic engineering will be needed to complement breeding efforts in order to accelerate the improvement of agronomical traits. The new field of plant synthetic biology has emerged in recent years and is expected to support rapid, precise, and robust engineering of plants. In this review, we present recent advances made in the field of plant synthetic biology, specifically in genome editing, transgene expression regulation, and bioenergy crop engineering, with a focus on traits related to lignocellulose, oil, and soluble sugars. Ultimately, progress and innovation in these fields may facilitate the development of beneficial traits in crop plants to meet society's bioenergy needs. © 2016 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.

  4. Incorporating comparative genomics into the design-test-learn cycle of microbial strain engineering.

    Science.gov (United States)

    Sardi, Maria; Gasch, Audrey P

    2017-08-01

    Engineering microbes with new properties is an important goal in industrial engineering, to establish biological factories for production of biofuels, commodity chemicals and pharmaceutics. But engineering microbes to produce new compounds with high yield remains a major challenge toward economically viable production. Incorporating several modern approaches, including synthetic and systems biology, metabolic modeling and regulatory rewiring, has proven to significantly advance industrial strain engineering. This review highlights how comparative genomics can also facilitate strain engineering, by identifying novel genes and pathways, regulatory mechanisms and genetic background effects for engineering. We discuss how incorporating comparative genomics into the design-test-learn cycle of strain engineering can provide novel information that complements other engineering strategies. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  5. Plant synthetic biology.

    Science.gov (United States)

    Liu, Wusheng; Stewart, C Neal

    2015-05-01

    Plant synthetic biology is an emerging field that combines engineering principles with plant biology toward the design and production of new devices. This emerging field should play an important role in future agriculture for traditional crop improvement, but also in enabling novel bioproduction in plants. In this review we discuss the design cycles of synthetic biology as well as key engineering principles, genetic parts, and computational tools that can be utilized in plant synthetic biology. Some pioneering examples are offered as a demonstration of how synthetic biology can be used to modify plants for specific purposes. These include synthetic sensors, synthetic metabolic pathways, and synthetic genomes. We also speculate about the future of synthetic biology of plants. Copyright © 2015 Elsevier Ltd. All rights reserved.

  6. Characterization of Thermal Stability of Synthetic and Semi-Synthetic Engine Oils

    Directory of Open Access Journals (Sweden)

    Anand Kumar Tripathi

    2015-03-01

    Full Text Available Engine oils undergo oxidative degradation and wears out during service. Hence it is important to characterize ageing of engine oils at different simulated conditions to evaluate the performance of existing oils and also design new formulations. This work focuses on characterizing the thermo-oxidative degradation of synthetic and semi-synthetic engine oils aged at 120, 149 and 200 °C. Apparent activation energy of decomposition of aged oils evaluated using the isoconversional Kissinger-Akahira-Sunose technique was used as a thermal stability marker. The temporal variation of stability at different ageing temperatures was corroborated with kinematic viscosity, oxidation, sulfation and nitration indices, total base number, antiwear additive content and molecular structure of the organic species present in the oils. At the lowest temperature employed, synthetic oil underwent higher rate of oxidation, while semi-synthetic oil was stable for longer time periods. At higher temperatures, the initial rate of change of average apparent activation energy of synthetic oil correlated well with a similar variation in oxidation number. A mixture of long chain linear, branched, and cyclic hydrocarbons were observed when semi-synthetic oil was degraded at higher temperatures.

  7. Programming cells by multiplex genome engineering and accelerated evolution.

    Science.gov (United States)

    Wang, Harris H; Isaacs, Farren J; Carr, Peter A; Sun, Zachary Z; Xu, George; Forest, Craig R; Church, George M

    2009-08-13

    The breadth of genomic diversity found among organisms in nature allows populations to adapt to diverse environments. However, genomic diversity is difficult to generate in the laboratory and new phenotypes do not easily arise on practical timescales. Although in vitro and directed evolution methods have created genetic variants with usefully altered phenotypes, these methods are limited to laborious and serial manipulation of single genes and are not used for parallel and continuous directed evolution of gene networks or genomes. Here, we describe multiplex automated genome engineering (MAGE) for large-scale programming and evolution of cells. MAGE simultaneously targets many locations on the chromosome for modification in a single cell or across a population of cells, thus producing combinatorial genomic diversity. Because the process is cyclical and scalable, we constructed prototype devices that automate the MAGE technology to facilitate rapid and continuous generation of a diverse set of genetic changes (mismatches, insertions, deletions). We applied MAGE to optimize the 1-deoxy-D-xylulose-5-phosphate (DXP) biosynthesis pathway in Escherichia coli to overproduce the industrially important isoprenoid lycopene. Twenty-four genetic components in the DXP pathway were modified simultaneously using a complex pool of synthetic DNA, creating over 4.3 billion combinatorial genomic variants per day. We isolated variants with more than fivefold increase in lycopene production within 3 days, a significant improvement over existing metabolic engineering techniques. Our multiplex approach embraces engineering in the context of evolution by expediting the design and evolution of organisms with new and improved properties.

  8. Basic science through engineering? Synthetic modeling and the idea of biology-inspired engineering.

    Science.gov (United States)

    Knuuttila, Tarja; Loettgers, Andrea

    2013-06-01

    Synthetic biology is often understood in terms of the pursuit for well-characterized biological parts to create synthetic wholes. Accordingly, it has typically been conceived of as an engineering dominated and application oriented field. We argue that the relationship of synthetic biology to engineering is far more nuanced than that and involves a sophisticated epistemic dimension, as shown by the recent practice of synthetic modeling. Synthetic models are engineered genetic networks that are implanted in a natural cell environment. Their construction is typically combined with experiments on model organisms as well as mathematical modeling and simulation. What is especially interesting about this combinational modeling practice is that, apart from greater integration between these different epistemic activities, it has also led to the questioning of some central assumptions and notions on which synthetic biology is based. As a result synthetic biology is in the process of becoming more "biology inspired." Copyright © 2013 Elsevier Ltd. All rights reserved.

  9. Meeting Report: Synthetic Biology Jamboree for Undergraduates

    Science.gov (United States)

    Campbell, A. Malcolm

    2005-01-01

    The field of synthetic biology (the name is derived from an analogy to synthetic chemistry) has recognized itself as a "field" only since about 2002. Synthetic biology has gotten some high-profile attention recently, but most people are not aware the field even exists. Synthetic biologists apply engineering principles to genomic circuits to…

  10. Efficient engineering of marker-free synthetic allotetraploids of Saccharomyces.

    Science.gov (United States)

    Alexander, William G; Peris, David; Pfannenstiel, Brandon T; Opulente, Dana A; Kuang, Meihua; Hittinger, Chris Todd

    2016-04-01

    Saccharomyces interspecies hybrids are critical biocatalysts in the fermented beverage industry, including in the production of lager beers, Belgian ales, ciders, and cold-fermented wines. Current methods for making synthetic interspecies hybrids are cumbersome and/or require genome modifications. We have developed a simple, robust, and efficient method for generating allotetraploid strains of prototrophic Saccharomyces without sporulation or nuclear genome manipulation. S. cerevisiae×S. eubayanus, S. cerevisiae×S. kudriavzevii, and S. cerevisiae×S. uvarum designer hybrid strains were created as synthetic lager, Belgian, and cider strains, respectively. The ploidy and hybrid nature of the strains were confirmed using flow cytometry and PCR-RFLP analysis, respectively. This method provides an efficient means for producing novel synthetic hybrids for beverage and biofuel production, as well as for constructing tetraploids to be used for basic research in evolutionary genetics and genome stability. Copyright © 2015 Elsevier Inc. All rights reserved.

  11. Synthetic Biology: Engineering Living Systems from Biophysical Principles.

    Science.gov (United States)

    Bartley, Bryan A; Kim, Kyung; Medley, J Kyle; Sauro, Herbert M

    2017-03-28

    Synthetic biology was founded as a biophysical discipline that sought explanations for the origins of life from chemical and physical first principles. Modern synthetic biology has been reinvented as an engineering discipline to design new organisms as well as to better understand fundamental biological mechanisms. However, success is still largely limited to the laboratory and transformative applications of synthetic biology are still in their infancy. Here, we review six principles of living systems and how they compare and contrast with engineered systems. We cite specific examples from the synthetic biology literature that illustrate these principles and speculate on their implications for further study. To fully realize the promise of synthetic biology, we must be aware of life's unique properties. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  12. CRISPR and the Rebirth of Synthetic Biology

    NARCIS (Netherlands)

    Heidari, Raheleh; Shaw, David Martin; Elger, Bernice Simone

    Emergence of novel genome engineering technologies such as clustered regularly interspaced short palindromic repeat (CRISPR) has refocused attention on unresolved ethical complications of synthetic biology. Biosecurity concerns, deontological issues and human right aspects of genome editing have

  13. Synthetic biology approaches to engineer T cells.

    Science.gov (United States)

    Wu, Chia-Yung; Rupp, Levi J; Roybal, Kole T; Lim, Wendell A

    2015-08-01

    There is rapidly growing interest in learning how to engineer immune cells, such as T lymphocytes, because of the potential of these engineered cells to be used for therapeutic applications such as the recognition and killing of cancer cells. At the same time, our knowhow and capability to logically engineer cellular behavior is growing rapidly with the development of synthetic biology. Here we describe how synthetic biology approaches are being used to rationally alter the behavior of T cells to optimize them for therapeutic functions. We also describe future developments that will be important in order to construct safe and precise T cell therapeutics. Copyright © 2015 Elsevier Ltd. All rights reserved.

  14. Cell-free synthetic biology for in vitro prototype engineering.

    Science.gov (United States)

    Moore, Simon J; MacDonald, James T; Freemont, Paul S

    2017-06-15

    Cell-free transcription-translation is an expanding field in synthetic biology as a rapid prototyping platform for blueprinting the design of synthetic biological devices. Exemplar efforts include translation of prototype designs into medical test kits for on-site identification of viruses (Zika and Ebola), while gene circuit cascades can be tested, debugged and re-designed within rapid turnover times. Coupled with mathematical modelling, this discipline lends itself towards the precision engineering of new synthetic life. The next stages of cell-free look set to unlock new microbial hosts that remain slow to engineer and unsuited to rapid iterative design cycles. It is hoped that the development of such systems will provide new tools to aid the transition from cell-free prototype designs to functioning synthetic genetic circuits and engineered natural product pathways in living cells. © 2017 The Author(s).

  15. PGASO: A synthetic biology tool for engineering a cellulolytic yeast

    Directory of Open Access Journals (Sweden)

    Chang Jui-Jen

    2012-07-01

    Full Text Available Abstract Background To achieve an economical cellulosic ethanol production, a host that can do both cellulosic saccharification and ethanol fermentation is desirable. However, to engineer a non-cellulolytic yeast to be such a host requires synthetic biology techniques to transform multiple enzyme genes into its genome. Results A technique, named Promoter-based Gene Assembly and Simultaneous Overexpression (PGASO, that employs overlapping oligonucleotides for recombinatorial assembly of gene cassettes with individual promoters, was developed. PGASO was applied to engineer Kluyveromycesmarxianus KY3, which is a thermo- and toxin-tolerant yeast. We obtained a recombinant strain, called KR5, that is capable of simultaneously expressing exoglucanase and endoglucanase (both of Trichodermareesei, a beta-glucosidase (from a cow rumen fungus, a neomycin phosphotransferase, and a green fluorescent protein. High transformation efficiency and accuracy were achieved as ~63% of the transformants was confirmed to be correct. KR5 can utilize beta-glycan, cellobiose or CMC as the sole carbon source for growth and can directly convert cellobiose and beta-glycan to ethanol. Conclusions This study provides the first example of multi-gene assembly in a single step in a yeast species other than Saccharomyces cerevisiae. We successfully engineered a yeast host with a five-gene cassette assembly and the new host is capable of co-expressing three types of cellulase genes. Our study shows that PGASO is an efficient tool for simultaneous expression of multiple enzymes in the kefir yeast KY3 and that KY3 can serve as a host for developing synthetic biology tools.

  16. Challenges and opportunities in synthetic biology for chemical engineers.

    Science.gov (United States)

    Luo, Yunzi; Lee, Jung-Kul; Zhao, Huimin

    2013-11-15

    Synthetic biology provides numerous great opportunities for chemical engineers in the development of new processes for large-scale production of biofuels, value-added chemicals, and protein therapeutics. However, challenges across all scales abound. In particular, the modularization and standardization of the components in a biological system, so-called biological parts, remain the biggest obstacle in synthetic biology. In this perspective, we will discuss the main challenges and opportunities in the rapidly growing synthetic biology field and the important roles that chemical engineers can play in its advancement.

  17. Synthetic biology approaches: Towards sustainable exploitation of marine bioactive molecules.

    Science.gov (United States)

    Seghal Kiran, G; Ramasamy, Pasiyappazham; Sekar, Sivasankari; Ramu, Meenatchi; Hassan, Saqib; Ninawe, A S; Selvin, Joseph

    2018-06-01

    The discovery of genes responsible for the production of bioactive metabolites via metabolic pathways combined with the advances in synthetic biology tools, has allowed the establishment of numerous microbial cell factories, for instance the yeast cell factories, for the manufacture of highly useful metabolites from renewable biomass. Genome mining and metagenomics are two platforms provide base-line data for reconstruction of genomes and metabolomes which is based in the development of synthetic/semi-synthetic genomes for marine natural products discovery. Engineered biofilms are being innovated on synthetic biology platform using genetic circuits and cell signalling systems as represillators controlling biofilm formation. Recombineering is a process of homologous recombination mediated genetic engineering, includes insertion, deletion or modification of any sequence specifically. Although this discipline considered new to the scientific domain, this field has now developed as promising endeavor on the accomplishment of sustainable exploitation of marine natural products. Copyright © 2018 Elsevier B.V. All rights reserved.

  18. Synthetic biodegradable functional polymers for tissue engineering: a brief review

    OpenAIRE

    BaoLin, GUO; MA, Peter X.

    2014-01-01

    Scaffolds play a crucial role in tissue engineering. Biodegradable polymers with great processing flexibility are the predominant scaffolding materials. Synthetic biodegradable polymers with well-defined structure and without immunological concerns associated with naturally derived polymers are widely used in tissue engineering. The synthetic biodegradable polymers that are widely used in tissue engineering, including polyesters, polyanhydrides, polyphosphazenes, polyurethane, and poly (glyce...

  19. Yeast 2.0-connecting the dots in the construction of the world's first functional synthetic eukaryotic genome.

    Science.gov (United States)

    Pretorius, I S; Boeke, J D

    2018-06-01

    Historians of the future may well describe 2018 as the year that the world's first functional synthetic eukaryotic genome became a reality. Without the benefit of hindsight, it might be hard to completely grasp the long-term significance of a breakthrough moment in the history of science like this. The role of synthetic biology in the imminent birth of a budding Saccharomyces cerevisiae yeast cell carrying 16 man-made chromosomes causes the world of science to teeter on the threshold of a future-defining scientific frontier. The genome-engineering tools and technologies currently being developed to produce the ultimate yeast genome will irreversibly connect the dots between our improved understanding of the fundamentals of a complex cell containing its DNA in a specialised nucleus and the application of bioengineered eukaryotes designed for advanced biomanufacturing of beneficial products. By joining up the dots between the findings and learnings from the international Synthetic Yeast Genome project (known as the Yeast 2.0 or Sc2.0 project) and concurrent advancements in biodesign tools and smart data-intensive technologies, a future world powered by a thriving bioeconomy seems realistic. This global project demonstrates how a collaborative network of dot connectors-driven by a tinkerer's indomitable curiosity to understand how things work inside a eukaryotic cell-are using cutting-edge biodesign concepts and synthetic biology tools to advance science and to positively frame human futures (i.e. improved quality of life) in a planetary context (i.e. a sustainable environment). Explorations such as this have a rich history of resulting in unexpected discoveries and unanticipated applications for the benefit of people and planet. However, we must learn from past explorations into controversial futuristic sciences and ensure that researchers at the forefront of an emerging science such as synthetic biology remain connected to all stakeholders' concerns about the

  20. Challenges and opportunities in synthetic biology for chemical engineers

    Science.gov (United States)

    Luo, Yunzi; Lee, Jung-Kul; Zhao, Huimin

    2012-01-01

    Synthetic biology provides numerous great opportunities for chemical engineers in the development of new processes for large-scale production of biofuels, value-added chemicals, and protein therapeutics. However, challenges across all scales abound. In particular, the modularization and standardization of the components in a biological system, so-called biological parts, remain the biggest obstacle in synthetic biology. In this perspective, we will discuss the main challenges and opportunities in the rapidly growing synthetic biology field and the important roles that chemical engineers can play in its advancement. PMID:24222925

  1. Engineering synthetic TALE and CRISPR/Cas9 transcription factors for regulating gene expression.

    Science.gov (United States)

    Kabadi, Ami M; Gersbach, Charles A

    2014-09-01

    Engineered DNA-binding proteins that can be targeted to specific sites in the genome to manipulate gene expression have enabled many advances in biomedical research. This includes generating tools to study fundamental aspects of gene regulation and the development of a new class of gene therapies that alter the expression of endogenous genes. Designed transcription factors have entered clinical trials for the treatment of human diseases and others are in preclinical development. High-throughput and user-friendly platforms for designing synthetic DNA-binding proteins present innovative methods for deciphering cell biology and designing custom synthetic gene circuits. We review two platforms for designing synthetic transcription factors for manipulating gene expression: Transcription activator-like effectors (TALEs) and the RNA-guided clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system. We present an overview of each technology and a guide for designing and assembling custom TALE- and CRISPR/Cas9-based transcription factors. We also discuss characteristics of each platform that are best suited for different applications. Copyright © 2014 Elsevier Inc. All rights reserved.

  2. A Parvovirus B19 synthetic genome: sequence features and functional competence.

    Science.gov (United States)

    Manaresi, Elisabetta; Conti, Ilaria; Bua, Gloria; Bonvicini, Francesca; Gallinella, Giorgio

    2017-08-01

    Central to genetic studies for Parvovirus B19 (B19V) is the availability of genomic clones that may possess functional competence and ability to generate infectious virus. In our study, we established a new model genetic system for Parvovirus B19. A synthetic approach was followed, by design of a reference genome sequence, by generation of a corresponding artificial construct and its molecular cloning in a complete and functional form, and by setup of an efficient strategy to generate infectious virus, via transfection in UT7/EpoS1 cells and amplification in erythroid progenitor cells. The synthetic genome was able to generate virus with biological properties paralleling those of native virus, its infectious activity being dependent on the preservation of self-complementarity and sequence heterogeneity within the terminal regions. A virus of defined genome sequence, obtained from controlled cell culture conditions, can constitute a reference tool for investigation of the structural and functional characteristics of the virus. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. Methods for open innovation on a genome-design platform associating scientific, commercial, and educational communities in synthetic biology.

    Science.gov (United States)

    Toyoda, Tetsuro

    2011-01-01

    Synthetic biology requires both engineering efficiency and compliance with safety guidelines and ethics. Focusing on the rational construction of biological systems based on engineering principles, synthetic biology depends on a genome-design platform to explore the combinations of multiple biological components or BIO bricks for quickly producing innovative devices. This chapter explains the differences among various platform models and details a methodology for promoting open innovation within the scope of the statutory exemption of patent laws. The detailed platform adopts a centralized evaluation model (CEM), computer-aided design (CAD) bricks, and a freemium model. It is also important for the platform to support the legal aspects of copyrights as well as patent and safety guidelines because intellectual work including DNA sequences designed rationally by human intelligence is basically copyrightable. An informational platform with high traceability, transparency, auditability, and security is required for copyright proof, safety compliance, and incentive management for open innovation in synthetic biology. GenoCon, which we have organized and explained here, is a competition-styled, open-innovation method involving worldwide participants from scientific, commercial, and educational communities that aims to improve the designs of genomic sequences that confer a desired function on an organism. Using only a Web browser, a participating contributor proposes a design expressed with CAD bricks that generate a relevant DNA sequence, which is then experimentally and intensively evaluated by the GenoCon organizers. The CAD bricks that comprise programs and databases as a Semantic Web are developed, executed, shared, reused, and well stocked on the secure Semantic Web platform called the Scientists' Networking System or SciNetS/SciNeS, based on which a CEM research center for synthetic biology and open innovation should be established. Copyright © 2011 Elsevier Inc

  4. Proceedings of Synthetic Biology: Engineering, Evolution and Design (SEED) Conference 2015

    Energy Technology Data Exchange (ETDEWEB)

    Silver, Pamela [Harvard Univ., Cambridge, MA (United States); SEED 2015 Conference Chair; Flach, Evan [American Institute of Chemical Engineers; SEED 2015 Conference Organizer

    2016-10-27

    Synthetic Biology is an emerging discipline that seeks to accelerate the process of engineering biology. As such, the tools are broadly applicable to application areas, including chemicals and biofuels, materials, medicine and agriculture. A characteristic of the field is to look holistically at cellular design, from sensing and genetic circuitry to the manipulation of cellular processes and actuators, to controlling metabolism, to programming multicellular behaviors. Further, the types of cells that are manipulated are broad, from in vitro systems to microbes and fungi to mammalian and plant cells and living animals. Many of the projects in synthetic biology seek to move biochemical functions across organisms. The field is highly interdisciplinary with faculty and students spread across departments that focus on engineering (biological, chemical, electrical, mechanical, civil, computer science) and basic science (biology and systems biology, chemistry, physics). While there have been many one-off workshops and meeting on synthetic biology, the 2014 Synthetic Biology: Engineering, Evolution and Design (SEED) was the first of an annual conference series that serves as a reliable place to pull together the involved disciplines in order to organize and exchange advances in the science and technology in the field. Further, the SEED conferences have a strong focus on industry, with many companies represented and actively participating. A number of these companies have started major efforts in synthetic biology including large companies (e.g., Pfizer, Novartis, Dow, Dupont, BP, Total), smaller companies have recently gone public (e.g., Amyris, Gevo, Intrexon), and many start-ups (e.g., Teslagen, Refactored Materials, Pivot, Genomatica). There are a number of loosely affiliated Synthetic Biology Centers, including ones at MIT, Boston University, UCSD, UCSF, UC-Berkeley, Imperial College, Oxford, and ETH. SEED 2015 will serve as the primary meeting at which international

  5. Deriving metabolic engineering strategies from genome-scale modeling with flux ratio constraints.

    Science.gov (United States)

    Yen, Jiun Y; Nazem-Bokaee, Hadi; Freedman, Benjamin G; Athamneh, Ahmad I M; Senger, Ryan S

    2013-05-01

    Optimized production of bio-based fuels and chemicals from microbial cell factories is a central goal of systems metabolic engineering. To achieve this goal, a new computational method of using flux balance analysis with flux ratios (FBrAtio) was further developed in this research and applied to five case studies to evaluate and design metabolic engineering strategies. The approach was implemented using publicly available genome-scale metabolic flux models. Synthetic pathways were added to these models along with flux ratio constraints by FBrAtio to achieve increased (i) cellulose production from Arabidopsis thaliana; (ii) isobutanol production from Saccharomyces cerevisiae; (iii) acetone production from Synechocystis sp. PCC6803; (iv) H2 production from Escherichia coli MG1655; and (v) isopropanol, butanol, and ethanol (IBE) production from engineered Clostridium acetobutylicum. The FBrAtio approach was applied to each case to simulate a metabolic engineering strategy already implemented experimentally, and flux ratios were continually adjusted to find (i) the end-limit of increased production using the existing strategy, (ii) new potential strategies to increase production, and (iii) the impact of these metabolic engineering strategies on product yield and culture growth. The FBrAtio approach has the potential to design "fine-tuned" metabolic engineering strategies in silico that can be implemented directly with available genomic tools. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Improving magnetic properties of MgB_2 bulk superconductors by synthetic engine oil treatment

    International Nuclear Information System (INIS)

    Taylan Koparan, E.; Savaskan, B.; Yanmaz, E.

    2016-01-01

    Highlights: • The effects of synthetic engine oil treatment on magnetic properties of bulk MgB_2 superconductors has been first time investigated and reported. • Synthetic engine oil used as a product which is cheap and a rich carbon source obviously has improved the superconducting magnetic properties of MgB_2. • The critical current density of all of MgB_2 samples immersed at different standby time in engine oil in whole field range has been better than that of the pure MgB_2 sample. • The MgB_2 sample immersed at 300 min standby time in synthetic engine oil has the best performance compared to other samples. - Abstract: The present study focuses on the effects of standby time of the MgB_2 samples immersed in synthetic engine oil on the critical current density ( J_c(H)), magnetic field dependence of the pinning force density f_p(b) and T_c performances of MgB_2 bulk superconductors. Synthetic engine oil was used as a product which is cheap and a rich carbon source. Manufactured MgB_2 pellet samples were immersed at different standby time of 30 min, 120 min, 300 min and 1440 min in synthetic engine oil after the first heating process. Finally, MgB_2 samples immersed in synthetic engine oil were sintered at 1000 °C and kept for 15 min in Ar atmosphere. The critical current density of all of MgB_2 samples immersed at different standby time in engine oil in whole field range was better than that of the pure MgB_2 sample because of the number of the pinning centers. The MgB_2 sample immersed at 300 min standby time in synthetic engine oil has the best performance compared to other samples. The J_c value for the pure sample is 2.0 × 10"3 A/cm"2, whereas for the MgB_2 sample immersed at 300 min standby time in engine oil the J_c is enhanced to 4.8 × 10"3 A/cm"2 at 5 K and 3 T. The superconducting transition temperature (T_c) did not change with the increasing standby time of the samples in synthetic engine oil at all. The best diamagnetic property was

  7. Multiplex engineering of industrial yeast genomes using CRISPRm.

    Science.gov (United States)

    Ryan, Owen W; Cate, Jamie H D

    2014-01-01

    Global demand has driven the use of industrial strains of the yeast Saccharomyces cerevisiae for large-scale production of biofuels and renewable chemicals. However, the genetic basis of desired domestication traits is poorly understood because robust genetic tools do not exist for industrial hosts. We present an efficient, marker-free, high-throughput, and multiplexed genome editing platform for industrial strains of S. cerevisiae that uses plasmid-based expression of the CRISPR/Cas9 endonuclease and multiple ribozyme-protected single guide RNAs. With this multiplex CRISPR (CRISPRm) system, it is possible to integrate DNA libraries into the chromosome for evolution experiments, and to engineer multiple loci simultaneously. The CRISPRm tools should therefore find use in many higher-order synthetic biology applications to accelerate improvements in industrial microorganisms.

  8. Recent applications of synthetic biology tools for yeast metabolic engineering

    DEFF Research Database (Denmark)

    Jensen, Michael Krogh; Keasling, Jay

    2015-01-01

    to engineer microbial chemical factories has steadily decreased, improvement is still needed. Through the development of synthetic biology tools for key microbial hosts, it should be possible to further decrease the development times and improve the reliability of the resulting microorganism. Together...... with continuous decreases in price and improvements in DNA synthesis, assembly and sequencing, synthetic biology tools will rationalize time-consuming strain engineering, improve control of metabolic fluxes, and diversify screening assays for cellular metabolism. This review outlines some recently developed...... synthetic biology tools and their application to improve production of chemicals and fuels in yeast. Finally, we provide a perspective for the challenges that lie ahead....

  9. Chromatin regulation at the frontier of synthetic biology

    Science.gov (United States)

    Keung, Albert J.; Joung, J. Keith; Khalil, Ahmad S.; Collins, James J.

    2016-01-01

    As synthetic biology approaches are extended to diverse applications throughout medicine, biotechnology and basic biological research, there is an increasing need to engineer yeast, plant and mammalian cells. Eukaryotic genomes are regulated by the diverse biochemical and biophysical states of chromatin, which brings distinct challenges, as well as opportunities, over applications in bacteria. Recent synthetic approaches, including `epigenome editing', have allowed the direct and functional dissection of many aspects of physiological chromatin regulation. These studies lay the foundation for biomedical and biotechnological engineering applications that could take advantage of the unique combinatorial and spatiotemporal layers of chromatin regulation to create synthetic systems of unprecedented sophistication. PMID:25668787

  10. Mammalian Synthetic Biology: Time for Big MACs.

    Science.gov (United States)

    Martella, Andrea; Pollard, Steven M; Dai, Junbiao; Cai, Yizhi

    2016-10-21

    The enabling technologies of synthetic biology are opening up new opportunities for engineering and enhancement of mammalian cells. This will stimulate diverse applications in many life science sectors such as regenerative medicine, development of biosensing cell lines, therapeutic protein production, and generation of new synthetic genetic regulatory circuits. Harnessing the full potential of these new engineering-based approaches requires the design and assembly of large DNA constructs-potentially up to chromosome scale-and the effective delivery of these large DNA payloads to the host cell. Random integration of large transgenes, encoding therapeutic proteins or genetic circuits into host chromosomes, has several drawbacks such as risks of insertional mutagenesis, lack of control over transgene copy-number and position-specific effects; these can compromise the intended functioning of genetic circuits. The development of a system orthogonal to the endogenous genome is therefore beneficial. Mammalian artificial chromosomes (MACs) are functional, add-on chromosomal elements, which behave as normal chromosomes-being replicating and portioned to daughter cells at each cell division. They are deployed as useful gene expression vectors as they remain independent from the host genome. MACs are maintained as a single-copy and can accommodate multiple gene expression cassettes of, in theory, unlimited DNA size (MACs up to 10 megabases have been constructed). MACs therefore enabled control over ectopic gene expression and represent an excellent platform to rapidly prototype and characterize novel synthetic gene circuits without recourse to engineering the host genome. This review describes the obstacles synthetic biologists face when working with mammalian systems and how the development of improved MACs can overcome these-particularly given the spectacular advances in DNA synthesis and assembly that are fuelling this research area.

  11. Bacterial Genome Engineering and Synthetic Biology: Combating Pathogens

    Science.gov (United States)

    2016-11-04

    extremely high genome sequence similarity between non-pathogenic and pathogenic strains by targeting small sequence variations present in the...Microbiol 2011, 14(5):524-531. 46. Bikard D, Euler CW, Jiang W, Nussenzweig PM, Goldberg GW, Duportet X, Fischetti VA, Marraffini LA: Exploiting

  12. Natural - synthetic - artificial!

    DEFF Research Database (Denmark)

    Nielsen, Peter E

    2010-01-01

    The terms "natural," "synthetic" and "artificial" are discussed in relation to synthetic and artificial chromosomes and genomes, synthetic and artificial cells and artificial life.......The terms "natural," "synthetic" and "artificial" are discussed in relation to synthetic and artificial chromosomes and genomes, synthetic and artificial cells and artificial life....

  13. Improving magnetic properties of MgB{sub 2} bulk superconductors by synthetic engine oil treatment

    Energy Technology Data Exchange (ETDEWEB)

    Taylan Koparan, E., E-mail: etaylan20@gmail.com [Department of Science Education, Eregli Faculty of Education, Bulent Ecevit University, TR-67300, Zonguldak (Turkey); Savaskan, B. [Energy Systems Engineering, Faculty of Technology, Karadeniz Technical University, 61830, Of, Trabzon (Turkey); Yanmaz, E. [Department of Mechatronics, Faculty of Engineering and Architecture, İstanbul Gelişim University, İstanbul (Turkey)

    2016-08-15

    Highlights: • The effects of synthetic engine oil treatment on magnetic properties of bulk MgB{sub 2} superconductors has been first time investigated and reported. • Synthetic engine oil used as a product which is cheap and a rich carbon source obviously has improved the superconducting magnetic properties of MgB{sub 2}. • The critical current density of all of MgB{sub 2} samples immersed at different standby time in engine oil in whole field range has been better than that of the pure MgB{sub 2} sample. • The MgB{sub 2} sample immersed at 300 min standby time in synthetic engine oil has the best performance compared to other samples. - Abstract: The present study focuses on the effects of standby time of the MgB{sub 2} samples immersed in synthetic engine oil on the critical current density ( J{sub c}(H)), magnetic field dependence of the pinning force density f{sub p}(b) and T{sub c} performances of MgB{sub 2} bulk superconductors. Synthetic engine oil was used as a product which is cheap and a rich carbon source. Manufactured MgB{sub 2} pellet samples were immersed at different standby time of 30 min, 120 min, 300 min and 1440 min in synthetic engine oil after the first heating process. Finally, MgB{sub 2} samples immersed in synthetic engine oil were sintered at 1000 °C and kept for 15 min in Ar atmosphere. The critical current density of all of MgB{sub 2} samples immersed at different standby time in engine oil in whole field range was better than that of the pure MgB{sub 2} sample because of the number of the pinning centers. The MgB{sub 2} sample immersed at 300 min standby time in synthetic engine oil has the best performance compared to other samples. The J{sub c} value for the pure sample is 2.0 × 10{sup 3} A/cm{sup 2}, whereas for the MgB{sub 2} sample immersed at 300 min standby time in engine oil the J{sub c} is enhanced to 4.8 × 10{sup 3} A/cm{sup 2} at 5 K and 3 T. The superconducting transition temperature (T{sub c}) did not change

  14. Recent advances of molecular toolbox construction expand Pichia pastoris in synthetic biology applications.

    Science.gov (United States)

    Kang, Zhen; Huang, Hao; Zhang, Yunfeng; Du, Guocheng; Chen, Jian

    2017-01-01

    Pichia pastoris: (reclassified as Komagataella phaffii), a methylotrophic yeast strain has been widely used for heterologous protein production because of its unique advantages, such as readily achievable high-density fermentation, tractable genetic modifications and typical eukaryotic post-translational modifications. More recently, P. pastoris as a metabolic pathway engineering platform has also gained much attention. In this mini-review, we addressed recent advances of molecular toolboxes, including synthetic promoters, signal peptides, and genome engineering tools that established for P. pastoris. Furthermore, the applications of P. pastoris towards synthetic biology were also discussed and prospected especially in the context of genome-scale metabolic pathway analysis.

  15. Evaluation of synthetic promoters in Physcomitrella patens

    DEFF Research Database (Denmark)

    Peramuna, Anantha; Bae, Hansol; Rasmussen, Erling Koch

    2018-01-01

    Securing a molecular toolbox including diverse promoters is essential for genome engineering. However, native promoters have limitations such as the available number or the length of the promoter. In this work, three short synthetic promoters were characterized by using the yellow fluorescent...

  16. Synthetic lubrication oil influences on performance and emission characteristic of coated diesel engine fuelled by biodiesel blends

    International Nuclear Information System (INIS)

    Mohamed Musthafa, M.

    2016-01-01

    Highlights: • Synthetic lubricant provides the maximum performance benefits. • Synthetic lubricant is capable of retaining satisfactory viscosity. • Synthetic lubricant is to increase the life of the engine. • Improvement in efficiency of the coated engine with synthetic lubrication. • No significant changes in the coated engine emission with synthetic lubricants. - Abstract: In this study, the effects of using synthetic lubricating oil on the performance and exhaust emissions in a low heat rejection diesel engine running on Pongamia methyl ester blends and diesel have been investigated experimentally compared to those obtained from a conventional diesel engine with SAE 40 lubrication oil fuelled by diesel. For this purpose, direct injection diesel engine was converted to Yttria-stabilized zirconia (YSZ) coated engine. The results showed 5–9% increase in engine efficiency and 8–17% decrease in specific fuel consumption, as well as significant improvements in exhaust gas emissions (except NO_X) for all tested fuels (pure diesel, B10 and B20) used in coated engine with synthetic lubricants compared to that of the uncoated engine with SAE 40 lubricant running on diesel fuel.

  17. Genome engineering in Vibrio cholerae

    DEFF Research Database (Denmark)

    Val, Marie-Eve; Skovgaard, Ole; Ducos-Galand, Magaly

    2012-01-01

    Although bacteria with multipartite genomes are prevalent, our knowledge of the mechanisms maintaining their genome is very limited, and much remains to be learned about the structural and functional interrelationships of multiple chromosomes. Owing to its bi-chromosomal genome architecture and its....... This difficulty was surmounted using a unique and powerful strategy based on massive rearrangement of prokaryotic genomes. We developed a site-specific recombination-based engineering tool, which allows targeted, oriented, and reciprocal DNA exchanges. Using this genetic tool, we obtained a panel of V. cholerae...

  18. CRISPR and the Rebirth of Synthetic Biology.

    Science.gov (United States)

    Heidari, Raheleh; Shaw, David Martin; Elger, Bernice Simone

    2017-04-01

    Emergence of novel genome engineering technologies such as clustered regularly interspaced short palindromic repeat (CRISPR) has refocused attention on unresolved ethical complications of synthetic biology. Biosecurity concerns, deontological issues and human right aspects of genome editing have been the subject of in-depth debate; however, a lack of transparent regulatory guidelines, outdated governance codes, inefficient time-consuming clinical trial pathways and frequent misunderstanding of the scientific potential of cutting-edge technologies have created substantial obstacles to translational research in this area. While a precautionary principle should be applied at all stages of genome engineering research, the stigma of germline editing, synthesis of new life forms and unrealistic presentation of current technologies should not arrest the transition of new therapeutic, diagnostic or preventive tools from research to clinic. We provide a brief review on the present regulation of CRISPR and discuss the translational aspect of genome engineering research and patient autonomy with respect to the "right to try" potential novel non-germline gene therapies.

  19. Genome Engineering for Personalized Arthritis Therapeutics.

    Science.gov (United States)

    Adkar, Shaunak S; Brunger, Jonathan M; Willard, Vincent P; Wu, Chia-Lung; Gersbach, Charles A; Guilak, Farshid

    2017-10-01

    Arthritis represents a family of complex joint pathologies responsible for the majority of musculoskeletal conditions. Nearly all diseases within this family, including osteoarthritis, rheumatoid arthritis, and juvenile idiopathic arthritis, are chronic conditions with few or no disease-modifying therapeutics available. Advances in genome engineering technology, most recently with CRISPR-Cas9, have revolutionized our ability to interrogate and validate genetic and epigenetic elements associated with chronic diseases such as arthritis. These technologies, together with cell reprogramming methods, including the use of induced pluripotent stem cells, provide a platform for human disease modeling. We summarize new evidence from genome-wide association studies and genomics that substantiates a genetic basis for arthritis pathogenesis. We also review the potential contributions of genome engineering in the development of new arthritis therapeutics. Copyright © 2017 Elsevier Ltd. All rights reserved.

  20. Cell-free protein synthesis enabled rapid prototyping for metabolic engineering and synthetic biology

    Directory of Open Access Journals (Sweden)

    Lihong Jiang

    2018-06-01

    Full Text Available Advances in metabolic engineering and synthetic biology have facilitated the manufacturing of many valuable-added compounds and commodity chemicals using microbial cell factories in the past decade. However, due to complexity of cellular metabolism, the optimization of metabolic pathways for maximal production represents a grand challenge and an unavoidable barrier for metabolic engineering. Recently, cell-free protein synthesis system (CFPS has been emerging as an enabling alternative to address challenges in biomanufacturing. This review summarizes the recent progresses of CFPS in rapid prototyping of biosynthetic pathways and genetic circuits (biosensors to speed up design-build-test (DBT cycles of metabolic engineering and synthetic biology. Keywords: Cell-free protein synthesis, Metabolic pathway optimization, Genetic circuits, Metabolic engineering, Synthetic biology

  1. Chemical genomic guided engineering of gamma-valerolactone tolerant yeast.

    Science.gov (United States)

    Bottoms, Scott; Dickinson, Quinn; McGee, Mick; Hinchman, Li; Higbee, Alan; Hebert, Alex; Serate, Jose; Xie, Dan; Zhang, Yaoping; Coon, Joshua J; Myers, Chad L; Landick, Robert; Piotrowski, Jeff S

    2018-01-12

    Gamma valerolactone (GVL) treatment of lignocellulosic bomass is a promising technology for degradation of biomass for biofuel production; however, GVL is toxic to fermentative microbes. Using a combination of chemical genomics with the yeast (Saccharomyces cerevisiae) deletion collection to identify sensitive and resistant mutants, and chemical proteomics to monitor protein abundance in the presence of GVL, we sought to understand the mechanism toxicity and resistance to GVL with the goal of engineering a GVL-tolerant, xylose-fermenting yeast. Chemical genomic profiling of GVL predicted that this chemical affects membranes and membrane-bound processes. We show that GVL causes rapid, dose-dependent cell permeability, and is synergistic with ethanol. Chemical genomic profiling of GVL revealed that deletion of the functionally related enzymes Pad1p and Fdc1p, which act together to decarboxylate cinnamic acid and its derivatives to vinyl forms, increases yeast tolerance to GVL. Further, overexpression of Pad1p sensitizes cells to GVL toxicity. To improve GVL tolerance, we deleted PAD1 and FDC1 in a xylose-fermenting yeast strain. The modified strain exhibited increased anaerobic growth, sugar utilization, and ethanol production in synthetic hydrolysate with 1.5% GVL, and under other conditions. Chemical proteomic profiling of the engineered strain revealed that enzymes involved in ergosterol biosynthesis were more abundant in the presence of GVL compared to the background strain. The engineered GVL strain contained greater amounts of ergosterol than the background strain. We found that GVL exerts toxicity to yeast by compromising cellular membranes, and that this toxicity is synergistic with ethanol. Deletion of PAD1 and FDC1 conferred GVL resistance to a xylose-fermenting yeast strain by increasing ergosterol accumulation in aerobically grown cells. The GVL-tolerant strain fermented sugars in the presence of GVL levels that were inhibitory to the unmodified strain

  2. Precision genome engineering and agriculture: opportunities and regulatory challenges.

    Science.gov (United States)

    Voytas, Daniel F; Gao, Caixia

    2014-06-01

    Plant agriculture is poised at a technological inflection point. Recent advances in genome engineering make it possible to precisely alter DNA sequences in living cells, providing unprecedented control over a plant's genetic material. Potential future crops derived through genome engineering include those that better withstand pests, that have enhanced nutritional value, and that are able to grow on marginal lands. In many instances, crops with such traits will be created by altering only a few nucleotides among the billions that comprise plant genomes. As such, and with the appropriate regulatory structures in place, crops created through genome engineering might prove to be more acceptable to the public than plants that carry foreign DNA in their genomes. Public perception and the performance of the engineered crop varieties will determine the extent to which this powerful technology contributes towards securing the world's food supply.

  3. Engineering emergent multicellular behavior through synthetic adhesion

    Science.gov (United States)

    Glass, David; Riedel-Kruse, Ingmar

    In over a decade, synthetic biology has developed increasingly robust gene networks within single cells, but constructed very few systems that demonstrate multicellular spatio-temporal dynamics. We are filling this gap in synthetic biology's toolbox by developing an E. coli self-assembly platform based on modular cell-cell adhesion. We developed a system in which adhesive selectivity is provided by a library of outer membrane-displayed peptides with intra-library specificities, while affinity is provided by consistent expression across the entire library. We further provide a biophysical model to help understand the parameter regimes in which this tool can be used to self-assemble into cellular clusters, filaments, or meshes. The combined platform will enable future development of synthetic multicellular systems for use in consortia-based metabolic engineering, in living materials, and in controlled study of minimal multicellular systems. Stanford Bio-X Bowes Fellowship.

  4. Transcription control engineering and applications in synthetic biology

    Directory of Open Access Journals (Sweden)

    Michael D. Engstrom

    2017-09-01

    Full Text Available In synthetic biology, researchers assemble biological components in new ways to produce systems with practical applications. One of these practical applications is control of the flow of genetic information (from nucleic acid to protein, a.k.a. gene regulation. Regulation is critical for optimizing protein (and therefore activity levels and the subsequent levels of metabolites and other cellular properties. The central dogma of molecular biology posits that information flow commences with transcription, and accordingly, regulatory tools targeting transcription have received the most attention in synthetic biology. In this mini-review, we highlight many past successes and summarize the lessons learned in developing tools for controlling transcription. In particular, we focus on engineering studies where promoters and transcription terminators (cis-factors were directly engineered and/or isolated from DNA libraries. We also review several well-characterized transcription regulators (trans-factors, giving examples of how cis- and trans-acting factors have been combined to create digital and analogue switches for regulating transcription in response to various signals. Last, we provide examples of how engineered transcription control systems have been used in metabolic engineering and more complicated genetic circuits. While most of our mini-review focuses on the well-characterized bacterium Escherichia coli, we also provide several examples of the use of transcription control engineering in non-model organisms. Similar approaches have been applied outside the bacterial kingdom indicating that the lessons learned from bacterial studies may be generalized for other organisms.

  5. Transcription control engineering and applications in synthetic biology.

    Science.gov (United States)

    Engstrom, Michael D; Pfleger, Brian F

    2017-09-01

    In synthetic biology, researchers assemble biological components in new ways to produce systems with practical applications. One of these practical applications is control of the flow of genetic information (from nucleic acid to protein), a.k.a. gene regulation. Regulation is critical for optimizing protein (and therefore activity) levels and the subsequent levels of metabolites and other cellular properties. The central dogma of molecular biology posits that information flow commences with transcription, and accordingly, regulatory tools targeting transcription have received the most attention in synthetic biology. In this mini-review, we highlight many past successes and summarize the lessons learned in developing tools for controlling transcription. In particular, we focus on engineering studies where promoters and transcription terminators ( cis -factors) were directly engineered and/or isolated from DNA libraries. We also review several well-characterized transcription regulators ( trans- factors), giving examples of how cis- and trans -acting factors have been combined to create digital and analogue switches for regulating transcription in response to various signals. Last, we provide examples of how engineered transcription control systems have been used in metabolic engineering and more complicated genetic circuits. While most of our mini-review focuses on the well-characterized bacterium Escherichia coli , we also provide several examples of the use of transcription control engineering in non-model organisms. Similar approaches have been applied outside the bacterial kingdom indicating that the lessons learned from bacterial studies may be generalized for other organisms.

  6. Synthetic biology in cell-based cancer immunotherapy.

    Science.gov (United States)

    Chakravarti, Deboki; Wong, Wilson W

    2015-08-01

    The adoptive transfer of genetically engineered T cells with cancer-targeting receptors has shown tremendous promise for eradicating tumors in clinical trials. This form of cellular immunotherapy presents a unique opportunity to incorporate advanced systems and synthetic biology approaches to create cancer therapeutics with novel functions. We first review the development of synthetic receptors, switches, and circuits to control the location, duration, and strength of T cell activity against tumors. In addition, we discuss the cellular engineering and genome editing of host cells (or the chassis) to improve the efficacy of cell-based cancer therapeutics, and to reduce the time and cost of manufacturing. Copyright © 2015 Elsevier Ltd. All rights reserved.

  7. Regulatory RNA-assisted genome engineering in microorganisms.

    Science.gov (United States)

    Si, Tong; HamediRad, Mohammad; Zhao, Huimin

    2015-12-01

    Regulatory RNAs are increasingly recognized and utilized as key modulators of gene expression in diverse organisms. Thanks to their modular and programmable nature, trans-acting regulatory RNAs are especially attractive in genome-scale applications. Here we discuss the recent examples in microbial genome engineering implementing various trans-acting RNA platforms, including sRNA, RNAi, asRNA and CRISRP-Cas. In particular, we focus on how the scalable and multiplex nature of trans-acting RNAs has been used to tackle the challenges in creating genome-wide and combinatorial diversity for functional genomics and metabolic engineering applications. Advances in computational design and context-dependent regulation are also discussed for their contribution in improving fine-tuning capabilities of trans-acting RNAs. Copyright © 2015 Elsevier Ltd. All rights reserved.

  8. The path to next generation biofuels: successes and challenges in the era of synthetic biology

    Science.gov (United States)

    2010-01-01

    Volatility of oil prices along with major concerns about climate change, oil supply security and depleting reserves have sparked renewed interest in the production of fuels from renewable resources. Recent advances in synthetic biology provide new tools for metabolic engineers to direct their strategies and construct optimal biocatalysts for the sustainable production of biofuels. Metabolic engineering and synthetic biology efforts entailing the engineering of native and de novo pathways for conversion of biomass constituents to short-chain alcohols and advanced biofuels are herewith reviewed. In the foreseeable future, formal integration of functional genomics and systems biology with synthetic biology and metabolic engineering will undoubtedly support the discovery, characterization, and engineering of new metabolic routes and more efficient microbial systems for the production of biofuels. PMID:20089184

  9. Millstone: software for multiplex microbial genome analysis and engineering.

    Science.gov (United States)

    Goodman, Daniel B; Kuznetsov, Gleb; Lajoie, Marc J; Ahern, Brian W; Napolitano, Michael G; Chen, Kevin Y; Chen, Changping; Church, George M

    2017-05-25

    Inexpensive DNA sequencing and advances in genome editing have made computational analysis a major rate-limiting step in adaptive laboratory evolution and microbial genome engineering. We describe Millstone, a web-based platform that automates genotype comparison and visualization for projects with up to hundreds of genomic samples. To enable iterative genome engineering, Millstone allows users to design oligonucleotide libraries and create successive versions of reference genomes. Millstone is open source and easily deployable to a cloud platform, local cluster, or desktop, making it a scalable solution for any lab.

  10. Synthetic biology: Novel approaches for microbiology.

    Science.gov (United States)

    Padilla-Vaca, Felipe; Anaya-Velázquez, Fernando; Franco, Bernardo

    2015-06-01

    In the past twenty years, molecular genetics has created powerful tools for genetic manipulation of living organisms. Whole genome sequencing has provided necessary information to assess knowledge on gene function and protein networks. In addition, new tools permit to modify organisms to perform desired tasks. Gene function analysis is speed up by novel approaches that couple both high throughput data generation and mining. Synthetic biology is an emerging field that uses tools for generating novel gene networks, whole genome synthesis and engineering. New applications in biotechnological, pharmaceutical and biomedical research are envisioned for synthetic biology. In recent years these new strategies have opened up the possibilities to study gene and genome editing, creation of novel tools for functional studies in virus, parasites and pathogenic bacteria. There is also the possibility to re-design organisms to generate vaccine subunits or produce new pharmaceuticals to combat multi-drug resistant pathogens. In this review we provide our opinion on the applicability of synthetic biology strategies for functional studies of pathogenic organisms and some applications such as genome editing and gene network studies to further comprehend virulence factors and determinants in pathogenic organisms. We also discuss what we consider important ethical issues for this field of molecular biology, especially for potential misuse of the new technologies. Copyright© by the Spanish Society for Microbiology and Institute for Catalan Studies.

  11. Synthetic Biology and Personalized Medicine

    Science.gov (United States)

    Jain, K.K.

    2013-01-01

    Synthetic biology, application of synthetic chemistry to biology, is a broad term that covers the engineering of biological systems with structures and functions not found in nature to process information, manipulate chemicals, produce energy, maintain cell environment and enhance human health. Synthetic biology devices contribute not only to improve our understanding of disease mechanisms, but also provide novel diagnostic tools. Methods based on synthetic biology enable the design of novel strategies for the treatment of cancer, immune diseases metabolic disorders and infectious diseases as well as the production of cheap drugs. The potential of synthetic genome, using an expanded genetic code that is designed for specific drug synthesis as well as delivery and activation of the drug in vivo by a pathological signal, was already pointed out during a lecture delivered at Kuwait University in 2005. Of two approaches to synthetic biology, top-down and bottom-up, the latter is more relevant to the development of personalized medicines as it provides more flexibility in constructing a partially synthetic cell from basic building blocks for a desired task. PMID:22907209

  12. Experiential Engineering through iGEM--An Undergraduate Summer Competition in Synthetic Biology

    Science.gov (United States)

    Mitchell, Rudolph; Dori, Yehudit Judy; Kuldell, Natalie H.

    2011-01-01

    Unlike students in other engineering disciplines, undergraduates in biological engineering typically have limited opportunity to develop design competencies, and even fewer chances to implement their designed projects. The international Genetically Engineered Machines (iGEM) competition is a student Synthetic Biology competition that, in 2009,…

  13. CRISPR-Cas: Revolutionising genome engineering

    African Journals Online (AJOL)

    Within the repeating As, Cs, Gs and Ts of the human genome is the .... Medicine, South African Medical Research Council Extramural Unit for Stem Cell Research and Therapy, ... Second, should we allow embryonic/germline engineering, or.

  14. Rational synthetic pathway refactoring of natural products biosynthesis in actinobacteria.

    Science.gov (United States)

    Tan, Gao-Yi; Liu, Tiangang

    2017-01-01

    Natural products (NPs) and their derivatives are widely used as frontline treatments for many diseases. Actinobacteria spp. are used to produce most of NP antibiotics and have also been intensively investigated for NP production, derivatization, and discovery. However, due to the complicated transcriptional and metabolic regulation of NP biosynthesis in Actinobacteria, especially in the cases of genome mining and heterologous expression, it is often difficult to rationally and systematically engineer synthetic pathways to maximize biosynthetic efficiency. With the emergence of new tools and methods in metabolic engineering, the synthetic pathways of many chemicals, such as fatty acids and biofuels, in model organisms (e.g. Escherichia coli ), have been refactored to realize precise and flexible control of production. These studies also offer a promising approach for synthetic pathway refactoring in Actinobacteria. In this review, the great potential of Actinobacteria as a microbial cell factory for biosynthesis of NPs is discussed. To this end, recent progress in metabolic engineering of NP synthetic pathways in Actinobacteria are summarized and strategies and perspectives to rationally and systematically refactor synthetic pathways in Actinobacteria are highlighted. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  15. Synthetic Biology: Applications in the Food Sector.

    Science.gov (United States)

    Tyagi, Ashish; Kumar, Ashwani; Aparna, S V; Mallappa, Rashmi H; Grover, Sunita; Batish, Virender Kumar

    2016-08-17

    Synthetic biology also termed as "genomic alchemy" represents a powerful area of science that is based on the convergence of biological sciences with systems engineering. It has been fittingly described as "moving from reading the genetic code to writing it" as it focuses on building, modeling, designing and fabricating novel biological systems using customized gene components that result in artificially created genetic circuitry. The scientifically compelling idea of the technological manipulation of life has been advocated since long time. Realization of this idea has gained momentum with development of high speed automation and the falling cost of gene sequencing and synthesis following the completion of the human genome project. Synthetic biology will certainly be instrumental in shaping the development of varying areas ranging from biomedicine, biopharmaceuticals, chemical production, food and dairy quality monitoring, packaging, and storage of food and dairy products, bioremediation and bioenergy production, etc. However, potential dangers of using synthetic life forms have to be acknowledged and adoption of policies by the scientific community to ensure safe practice while making important advancements in the ever expanding field of synthetic biology is to be fully supported and implemented.

  16. Systems Biology as an Integrated Platform for Bioinformatics, Systems Synthetic Biology, and Systems Metabolic Engineering

    Science.gov (United States)

    Chen, Bor-Sen; Wu, Chia-Chou

    2013-01-01

    Systems biology aims at achieving a system-level understanding of living organisms and applying this knowledge to various fields such as synthetic biology, metabolic engineering, and medicine. System-level understanding of living organisms can be derived from insight into: (i) system structure and the mechanism of biological networks such as gene regulation, protein interactions, signaling, and metabolic pathways; (ii) system dynamics of biological networks, which provides an understanding of stability, robustness, and transduction ability through system identification, and through system analysis methods; (iii) system control methods at different levels of biological networks, which provide an understanding of systematic mechanisms to robustly control system states, minimize malfunctions, and provide potential therapeutic targets in disease treatment; (iv) systematic design methods for the modification and construction of biological networks with desired behaviors, which provide system design principles and system simulations for synthetic biology designs and systems metabolic engineering. This review describes current developments in systems biology, systems synthetic biology, and systems metabolic engineering for engineering and biology researchers. We also discuss challenges and future prospects for systems biology and the concept of systems biology as an integrated platform for bioinformatics, systems synthetic biology, and systems metabolic engineering. PMID:24709875

  17. Systems Biology as an Integrated Platform for Bioinformatics, Systems Synthetic Biology, and Systems Metabolic Engineering

    Directory of Open Access Journals (Sweden)

    Bor-Sen Chen

    2013-10-01

    Full Text Available Systems biology aims at achieving a system-level understanding of living organisms and applying this knowledge to various fields such as synthetic biology, metabolic engineering, and medicine. System-level understanding of living organisms can be derived from insight into: (i system structure and the mechanism of biological networks such as gene regulation, protein interactions, signaling, and metabolic pathways; (ii system dynamics of biological networks, which provides an understanding of stability, robustness, and transduction ability through system identification, and through system analysis methods; (iii system control methods at different levels of biological networks, which provide an understanding of systematic mechanisms to robustly control system states, minimize malfunctions, and provide potential therapeutic targets in disease treatment; (iv systematic design methods for the modification and construction of biological networks with desired behaviors, which provide system design principles and system simulations for synthetic biology designs and systems metabolic engineering. This review describes current developments in systems biology, systems synthetic biology, and systems metabolic engineering for engineering and biology researchers. We also discuss challenges and future prospects for systems biology and the concept of systems biology as an integrated platform for bioinformatics, systems synthetic biology, and systems metabolic engineering.

  18. Phage Therapy in the Era of Synthetic Biology.

    Science.gov (United States)

    Barbu, E Magda; Cady, Kyle C; Hubby, Bolyn

    2016-10-03

    For more than a century, bacteriophage (or phage) research has enabled some of the most important discoveries in biological sciences and has equipped scientists with many of the molecular biology tools that have advanced our understanding of replication, maintenance, and expression of genetic material. Phages have also been recognized and exploited as natural antimicrobial agents and nanovectors for gene therapy, but their potential as therapeutics has not been fully exploited in Western medicine because of challenges such as narrow host range, bacterial resistance, and unique pharmacokinetics. However, increasing concern related to the emergence of bacteria resistant to multiple antibiotics has heightened interest in phage therapy and the development of strategies to overcome hurdles associated with bacteriophage therapeutics. Recent progress in sequencing technologies, DNA manipulation, and synthetic biology allowed scientists to refactor the entire bacterial genome of Mycoplasma mycoides, thereby creating the first synthetic cell. These new strategies for engineering genomes may have the potential to accelerate the construction of designer phage genomes with superior therapeutic potential. Here, we discuss the use of phage as therapeutics, as well as how synthetic biology can create bacteriophage with desirable attributes. Copyright © 2016 Cold Spring Harbor Laboratory Press; all rights reserved.

  19. Review of Microfluidic Photobioreactor Technology for Metabolic Engineering and Synthetic Biology of Cyanobacteria and Microalgae

    Directory of Open Access Journals (Sweden)

    Ya-Tang Yang

    2016-10-01

    Full Text Available One goal of metabolic engineering and synthetic biology for cyanobacteria and microalgae is to engineer strains that can optimally produce biofuels and commodity chemicals. However, the current workflow is slow and labor intensive with respect to assembly of genetic parts and characterization of production yields because of the slow growth rates of these organisms. Here, we review recent progress in the microfluidic photobioreactors and identify opportunities and unmet needs in metabolic engineering and synthetic biology. Because of the unprecedented experimental resolution down to the single cell level, long-term real-time monitoring capability, and high throughput with low cost, microfluidic photobioreactor technology will be an indispensible tool to speed up the development process, advance fundamental knowledge, and realize the full potential of metabolic engineering and synthetic biology for cyanobacteria and microalgae.

  20. Genome-Wide Tuning of Protein Expression Levels to Rapidly Engineer Microbial Traits.

    Science.gov (United States)

    Freed, Emily F; Winkler, James D; Weiss, Sophie J; Garst, Andrew D; Mutalik, Vivek K; Arkin, Adam P; Knight, Rob; Gill, Ryan T

    2015-11-20

    The reliable engineering of biological systems requires quantitative mapping of predictable and context-independent expression over a broad range of protein expression levels. However, current techniques for modifying expression levels are cumbersome and are not amenable to high-throughput approaches. Here we present major improvements to current techniques through the design and construction of E. coli genome-wide libraries using synthetic DNA cassettes that can tune expression over a ∼10(4) range. The cassettes also contain molecular barcodes that are optimized for next-generation sequencing, enabling rapid and quantitative tracking of alleles that have the highest fitness advantage. We show these libraries can be used to determine which genes and expression levels confer greater fitness to E. coli under different growth conditions.

  1. Use of genome-scale microbial models for metabolic engineering

    DEFF Research Database (Denmark)

    Patil, Kiran Raosaheb; Åkesson, M.; Nielsen, Jens

    2004-01-01

    Metabolic engineering serves as an integrated approach to design new cell factories by providing rational design procedures and valuable mathematical and experimental tools. Mathematical models have an important role for phenotypic analysis, but can also be used for the design of optimal metaboli...... network structures. The major challenge for metabolic engineering in the post-genomic era is to broaden its design methodologies to incorporate genome-scale biological data. Genome-scale stoichiometric models of microorganisms represent a first step in this direction....

  2. Genome engineering in human cells.

    Science.gov (United States)

    Song, Minjung; Kim, Young-Hoon; Kim, Jin-Soo; Kim, Hyongbum

    2014-01-01

    Genome editing in human cells is of great value in research, medicine, and biotechnology. Programmable nucleases including zinc-finger nucleases, transcription activator-like effector nucleases, and RNA-guided engineered nucleases recognize a specific target sequence and make a double-strand break at that site, which can result in gene disruption, gene insertion, gene correction, or chromosomal rearrangements. The target sequence complexities of these programmable nucleases are higher than 3.2 mega base pairs, the size of the haploid human genome. Here, we briefly introduce the structure of the human genome and the characteristics of each programmable nuclease, and review their applications in human cells including pluripotent stem cells. In addition, we discuss various delivery methods for nucleases, programmable nickases, and enrichment of gene-edited human cells, all of which facilitate efficient and precise genome editing in human cells.

  3. Genomics-Based Discovery of Plant Genes for Synthetic Biology of Terpenoid Fragrances: A Case Study in Sandalwood oil Biosynthesis.

    Science.gov (United States)

    Celedon, J M; Bohlmann, J

    2016-01-01

    Terpenoid fragrances are powerful mediators of ecological interactions in nature and have a long history of traditional and modern industrial applications. Plants produce a great diversity of fragrant terpenoid metabolites, which make them a superb source of biosynthetic genes and enzymes. Advances in fragrance gene discovery have enabled new approaches in synthetic biology of high-value speciality molecules toward applications in the fragrance and flavor, food and beverage, cosmetics, and other industries. Rapid developments in transcriptome and genome sequencing of nonmodel plant species have accelerated the discovery of fragrance biosynthetic pathways. In parallel, advances in metabolic engineering of microbial and plant systems have established platforms for synthetic biology applications of some of the thousands of plant genes that underlie fragrance diversity. While many fragrance molecules (eg, simple monoterpenes) are abundant in readily renewable plant materials, some highly valuable fragrant terpenoids (eg, santalols, ambroxides) are rare in nature and interesting targets for synthetic biology. As a representative example for genomics/transcriptomics enabled gene and enzyme discovery, we describe a strategy used successfully for elucidation of a complete fragrance biosynthetic pathway in sandalwood (Santalum album) and its reconstruction in yeast (Saccharomyces cerevisiae). We address questions related to the discovery of specific genes within large gene families and recovery of rare gene transcripts that are selectively expressed in recalcitrant tissues. To substantiate the validity of the approaches, we describe the combination of methods used in the gene and enzyme discovery of a cytochrome P450 in the fragrant heartwood of tropical sandalwood, responsible for the fragrance defining, final step in the biosynthesis of (Z)-santalols. © 2016 Elsevier Inc. All rights reserved.

  4. Gene interactions in the DNA damage-response pathway identified by genome-wide RNA-interference analysis of synthetic lethality

    NARCIS (Netherlands)

    van Haaften, Gijs; Vastenhouw, Nadine L; Nollen, Ellen A A; Plasterk, Ronald H A; Tijsterman, Marcel

    2004-01-01

    Here, we describe a systematic search for synthetic gene interactions in a multicellular organism, the nematode Caenorhabditis elegans. We established a high-throughput method to determine synthetic gene interactions by genome-wide RNA interference and identified genes that are required to protect

  5. An att site-based recombination reporter system for genome engineering and synthetic DNA assembly.

    Science.gov (United States)

    Bland, Michael J; Ducos-Galand, Magaly; Val, Marie-Eve; Mazel, Didier

    2017-07-14

    Direct manipulation of the genome is a widespread technique for genetic studies and synthetic biology applications. The tyrosine and serine site-specific recombination systems of bacteriophages HK022 and ΦC31 are widely used for stable directional exchange and relocation of DNA sequences, making them valuable tools in these contexts. We have developed site-specific recombination tools that allow the direct selection of recombination events by embedding the attB site from each system within the β-lactamase resistance coding sequence (bla). The HK and ΦC31 tools were developed by placing the attB sites from each system into the signal peptide cleavage site coding sequence of bla. All possible open reading frames (ORFs) were inserted and tested for recombination efficiency and bla activity. Efficient recombination was observed for all tested ORFs (3 for HK, 6 for ΦC31) as shown through a cointegrate formation assay. The bla gene with the embedded attB site was functional for eight of the nine constructs tested. The HK/ΦC31 att-bla system offers a simple way to directly select recombination events, thus enhancing the use of site-specific recombination systems for carrying out precise, large-scale DNA manipulation, and adding useful tools to the genetics toolbox. We further show the power and flexibility of bla to be used as a reporter for recombination.

  6. Engineered Viruses as Genome Editing Devices

    Science.gov (United States)

    Chen, Xiaoyu; Gonçalves, Manuel A F V

    2016-01-01

    Genome editing based on sequence-specific designer nucleases, also known as programmable nucleases, seeks to modify in a targeted and precise manner the genetic information content of living cells. Delivering into cells designer nucleases alone or together with donor DNA templates, which serve as surrogate homologous recombination (HR) substrates, can result in gene knockouts or gene knock-ins, respectively. As engineered replication-defective viruses, viral vectors are having an increasingly important role as delivery vehicles for donor DNA templates and designer nucleases, namely, zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated Cas9 (CRISPR−Cas9) nucleases, also known as RNA-guided nucleases (RGNs). We review this dual role played by engineered viral particles on genome editing while focusing on their main scaffolds, consisting of lentiviruses, adeno-associated viruses, and adenoviruses. In addition, the coverage of the growing body of research on the repurposing of viral vectors as delivery systems for genome editing tools is complemented with information regarding their main characteristics, pros, and cons. Finally, this information is framed by a concise description of the chief principles, tools, and applications of the genome editing field as a whole. PMID:26336974

  7. CRISPR Genome Engineering for Human Pluripotent Stem Cell Research.

    Science.gov (United States)

    Chaterji, Somali; Ahn, Eun Hyun; Kim, Deok-Ho

    2017-01-01

    The emergence of targeted and efficient genome editing technologies, such as repurposed bacterial programmable nucleases (e.g., CRISPR-Cas systems), has abetted the development of cell engineering approaches. Lessons learned from the development of RNA-interference (RNA-i) therapies can spur the translation of genome editing, such as those enabling the translation of human pluripotent stem cell engineering. In this review, we discuss the opportunities and the challenges of repurposing bacterial nucleases for genome editing, while appreciating their roles, primarily at the epigenomic granularity. First, we discuss the evolution of high-precision, genome editing technologies, highlighting CRISPR-Cas9. They exist in the form of programmable nucleases, engineered with sequence-specific localizing domains, and with the ability to revolutionize human stem cell technologies through precision targeting with greater on-target activities. Next, we highlight the major challenges that need to be met prior to bench-to-bedside translation, often learning from the path-to-clinic of complementary technologies, such as RNA-i. Finally, we suggest potential bioinformatics developments and CRISPR delivery vehicles that can be deployed to circumvent some of the challenges confronting genome editing technologies en route to the clinic.

  8. Genome engineering for microbial natural product discovery.

    Science.gov (United States)

    Choi, Si-Sun; Katsuyama, Yohei; Bai, Linquan; Deng, Zixin; Ohnishi, Yasuo; Kim, Eung-Soo

    2018-03-03

    The discovery and development of microbial natural products (MNPs) have played pivotal roles in the fields of human medicine and its related biotechnology sectors over the past several decades. The post-genomic era has witnessed the development of microbial genome mining approaches to isolate previously unsuspected MNP biosynthetic gene clusters (BGCs) hidden in the genome, followed by various BGC awakening techniques to visualize compound production. Additional microbial genome engineering techniques have allowed higher MNP production titers, which could complement a traditional culture-based MNP chasing approach. Here, we describe recent developments in the MNP research paradigm, including microbial genome mining, NP BGC activation, and NP overproducing cell factory design. Copyright © 2018 Elsevier Ltd. All rights reserved.

  9. TALENs: customizable molecular DNA scissors for genome engineering of plants.

    Science.gov (United States)

    Chen, Kunling; Gao, Caixia

    2013-06-20

    Precise genome modification with engineered nucleases is a powerful tool for studying basic biology and applied biotechnology. Transcription activator-like effector nucleases (TALENs), consisting of an engineered specific (TALE) DNA binding domain and a Fok I cleavage domain, are newly developed versatile reagents for genome engineering in different organisms. Because of the simplicity of the DNA recognition code and their modular assembly, TALENs can act as customizable molecular DNA scissors inducing double-strand breaks (DSBs) at given genomic location. Thus, they provide a valuable approach to targeted genome modifications such as mutations, insertions, replacements or chromosome rearrangements. In this article, we review the development of TALENs, and summarize the principles and tools for TALEN-mediated gene targeting in plant cells, as well as current and potential strategies for use in plant research and crop improvement. Copyright © 2013. Published by Elsevier Ltd.

  10. An evaluation of multiple annealing and looping based genome amplification using a synthetic bacterial community

    KAUST Repository

    Wang, Yong; Gao, Zhaoming; Xu, Ying; Li, Guangyu; He, Lisheng; Qian, Peiyuan

    2016-01-01

    -generation-sequencing technology. Using a synthetic bacterial community, the amplification efficiency of the Multiple Annealing and Looping Based Amplification Cycles (MALBAC) kit that is originally developed to amplify the single-cell genomic DNA of mammalian organisms

  11. Metabolic Engineering for Production of Biorenewable Fuels and Chemicals: Contributions of Synthetic Biology

    Directory of Open Access Journals (Sweden)

    Laura R. Jarboe

    2010-01-01

    Full Text Available Production of fuels and chemicals through microbial fermentation of plant material is a desirable alternative to petrochemical-based production. Fermentative production of biorenewable fuels and chemicals requires the engineering of biocatalysts that can quickly and efficiently convert sugars to target products at a cost that is competitive with existing petrochemical-based processes. It is also important that biocatalysts be robust to extreme fermentation conditions, biomass-derived inhibitors, and their target products. Traditional metabolic engineering has made great advances in this area, but synthetic biology has contributed and will continue to contribute to this field, particularly with next-generation biofuels. This work reviews the use of metabolic engineering and synthetic biology in biocatalyst engineering for biorenewable fuels and chemicals production, such as ethanol, butanol, acetate, lactate, succinate, alanine, and xylitol. We also examine the existing challenges in this area and discuss strategies for improving biocatalyst tolerance to chemical inhibitors.

  12. Reverse engineering validation using a benchmark synthetic gene circuit in human cells.

    Science.gov (United States)

    Kang, Taek; White, Jacob T; Xie, Zhen; Benenson, Yaakov; Sontag, Eduardo; Bleris, Leonidas

    2013-05-17

    Multicomponent biological networks are often understood incompletely, in large part due to the lack of reliable and robust methodologies for network reverse engineering and characterization. As a consequence, developing automated and rigorously validated methodologies for unraveling the complexity of biomolecular networks in human cells remains a central challenge to life scientists and engineers. Today, when it comes to experimental and analytical requirements, there exists a great deal of diversity in reverse engineering methods, which renders the independent validation and comparison of their predictive capabilities difficult. In this work we introduce an experimental platform customized for the development and verification of reverse engineering and pathway characterization algorithms in mammalian cells. Specifically, we stably integrate a synthetic gene network in human kidney cells and use it as a benchmark for validating reverse engineering methodologies. The network, which is orthogonal to endogenous cellular signaling, contains a small set of regulatory interactions that can be used to quantify the reconstruction performance. By performing successive perturbations to each modular component of the network and comparing protein and RNA measurements, we study the conditions under which we can reliably reconstruct the causal relationships of the integrated synthetic network.

  13. Genome Partitioner: A web tool for multi-level partitioning of large-scale DNA constructs for synthetic biology applications.

    Science.gov (United States)

    Christen, Matthias; Del Medico, Luca; Christen, Heinz; Christen, Beat

    2017-01-01

    Recent advances in lower-cost DNA synthesis techniques have enabled new innovations in the field of synthetic biology. Still, efficient design and higher-order assembly of genome-scale DNA constructs remains a labor-intensive process. Given the complexity, computer assisted design tools that fragment large DNA sequences into fabricable DNA blocks are needed to pave the way towards streamlined assembly of biological systems. Here, we present the Genome Partitioner software implemented as a web-based interface that permits multi-level partitioning of genome-scale DNA designs. Without the need for specialized computing skills, biologists can submit their DNA designs to a fully automated pipeline that generates the optimal retrosynthetic route for higher-order DNA assembly. To test the algorithm, we partitioned a 783 kb Caulobacter crescentus genome design. We validated the partitioning strategy by assembling a 20 kb test segment encompassing a difficult to synthesize DNA sequence. Successful assembly from 1 kb subblocks into the 20 kb segment highlights the effectiveness of the Genome Partitioner for reducing synthesis costs and timelines for higher-order DNA assembly. The Genome Partitioner is broadly applicable to translate DNA designs into ready to order sequences that can be assembled with standardized protocols, thus offering new opportunities to harness the diversity of microbial genomes for synthetic biology applications. The Genome Partitioner web tool can be accessed at https://christenlab.ethz.ch/GenomePartitioner.

  14. Genome Partitioner: A web tool for multi-level partitioning of large-scale DNA constructs for synthetic biology applications.

    Directory of Open Access Journals (Sweden)

    Matthias Christen

    Full Text Available Recent advances in lower-cost DNA synthesis techniques have enabled new innovations in the field of synthetic biology. Still, efficient design and higher-order assembly of genome-scale DNA constructs remains a labor-intensive process. Given the complexity, computer assisted design tools that fragment large DNA sequences into fabricable DNA blocks are needed to pave the way towards streamlined assembly of biological systems. Here, we present the Genome Partitioner software implemented as a web-based interface that permits multi-level partitioning of genome-scale DNA designs. Without the need for specialized computing skills, biologists can submit their DNA designs to a fully automated pipeline that generates the optimal retrosynthetic route for higher-order DNA assembly. To test the algorithm, we partitioned a 783 kb Caulobacter crescentus genome design. We validated the partitioning strategy by assembling a 20 kb test segment encompassing a difficult to synthesize DNA sequence. Successful assembly from 1 kb subblocks into the 20 kb segment highlights the effectiveness of the Genome Partitioner for reducing synthesis costs and timelines for higher-order DNA assembly. The Genome Partitioner is broadly applicable to translate DNA designs into ready to order sequences that can be assembled with standardized protocols, thus offering new opportunities to harness the diversity of microbial genomes for synthetic biology applications. The Genome Partitioner web tool can be accessed at https://christenlab.ethz.ch/GenomePartitioner.

  15. Ortholog prediction of the Aspergillus genus applicable for synthetic biology

    DEFF Research Database (Denmark)

    Rasmussen, Jane Lind Nybo; Vesth, Tammi Camilla; Theobald, Sebastian

    of genotype-to-phenotype. To achieve this, we have developed orthologous protein prediction software that utilizes genus-wide genetic diversity. The approach is optimized for large data sets, based on BLASTp considering protein identity and alignment coverage, and clustering using single linkage of bi......The Aspergillus genus contains leading industrial microorganisms, excelling in producing bioactive compounds and enzymes. Using synthetic biology and bioinformatics, we aim to re-engineer these organisms for applications within human health, pharmaceuticals, environmental engineering, and food......-directional hits. The result is orthologous protein families describing the genomic and functional features of individual species, clades and the core/pan genome of Aspergillus; and applicable to genotype-to-phenotype analyses in other microbial genera....

  16. Plant synthetic biology for molecular engineering of signalling and development.

    Science.gov (United States)

    Nemhauser, Jennifer L; Torii, Keiko U

    2016-03-02

    Molecular genetic studies of model plants in the past few decades have identified many key genes and pathways controlling development, metabolism and environmental responses. Recent technological and informatics advances have led to unprecedented volumes of data that may uncover underlying principles of plants as biological systems. The newly emerged discipline of synthetic biology and related molecular engineering approaches is built on this strong foundation. Today, plant regulatory pathways can be reconstituted in heterologous organisms to identify and manipulate parameters influencing signalling outputs. Moreover, regulatory circuits that include receptors, ligands, signal transduction components, epigenetic machinery and molecular motors can be engineered and introduced into plants to create novel traits in a predictive manner. Here, we provide a brief history of plant synthetic biology and significant recent examples of this approach, focusing on how knowledge generated by the reference plant Arabidopsis thaliana has contributed to the rapid rise of this new discipline, and discuss potential future directions.

  17. A photoactivatable Cre-loxP recombination system for optogenetic genome engineering.

    Science.gov (United States)

    Kawano, Fuun; Okazaki, Risako; Yazawa, Masayuki; Sato, Moritoshi

    2016-12-01

    Genome engineering techniques represented by the Cre-loxP recombination system have been used extensively for biomedical research. However, powerful and useful techniques for genome engineering that have high spatiotemporal precision remain elusive. Here we develop a highly efficient photoactivatable Cre recombinase (PA-Cre) to optogenetically control genome engineering in vivo. PA-Cre is based on the reassembly of split Cre fragments by light-inducible dimerization of the Magnet system. PA-Cre enables sharp induction (up to 320-fold) of DNA recombination and is efficiently activated even by low-intensity illumination (∼0.04 W m -2 ) or short periods of pulsed illumination (∼30 s). We demonstrate that PA-Cre allows for efficient DNA recombination in an internal organ of living mice through noninvasive external illumination using a LED light source. The present PA-Cre provides a powerful tool to greatly facilitate optogenetic genome engineering in vivo.

  18. Synthetic biology to access and expand nature’s chemical diversity

    Science.gov (United States)

    Smanski, Michael J.; Zhou, Hui; Claesen, Jan; Shen, Ben; Fischbach, Michael; Voigt, Christopher A.

    2016-01-01

    Bacterial genomes encode the biosynthetic potential to produce hundreds of thousands of complex molecules with diverse applications, from medicine to agriculture and materials. Economically accessing the potential encoded within sequenced genomes promises to reinvigorate waning drug discovery pipelines and provide novel routes to intricate chemicals. This is a tremendous undertaking, as the pathways often comprise dozens of genes spanning as much as 100+ kiliobases of DNA, are controlled by complex regulatory networks, and the most interesting molecules are made by non-model organisms. Advances in synthetic biology address these issues, including DNA construction technologies, genetic parts for precision expression control, synthetic regulatory circuits, computer aided design, and multiplexed genome engineering. Collectively, these technologies are moving towards an era when chemicals can be accessed en mass based on sequence information alone. This will enable the harnessing of metagenomic data and massive strain banks for high-throughput molecular discovery and, ultimately, the ability to forward design pathways to complex chemicals not found in nature. PMID:26876034

  19. CRISPR Mediated Genome Engineering and its Application in Industry.

    Science.gov (United States)

    Kaboli, Saeed; Babazada, Hasan

    2018-01-01

    The CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 (CRISPR-associated nuclease 9) method has been dramatically changing the field of genome engineering. It is a rapid, highly efficient and versatile tool for precise modification of genome that uses a guide RNA (gRNA) to target Cas9 to a specific sequence. This novel RNA-guided genome-editing technique has become a revolutionary tool in biomedical science and has many innovative applications in different fields. In this review, we briefly introduce the Cas9-mediated genome-editing tool, summarize the recent advances in CRISPR/Cas9 technology to engineer the genomes of a wide variety of organisms, and discuss their applications to treatment of fungal and viral disease. We also discuss advantageous of CRISPR/Cas9 technology to drug design, creation of animal model, and to food, agricultural and energy sciences. Adoption of the CRISPR/Cas9 technology in biomedical and biotechnological researches would create innovative applications of it not only for breeding of strains exhibiting desired traits for specific industrial and medical applications, but also for investigation of genome function.

  20. Chemical and Biophysical Modulation of Cas9 for Tunable Genome Engineering.

    Science.gov (United States)

    Nuñez, James K; Harrington, Lucas B; Doudna, Jennifer A

    2016-03-18

    The application of the CRISPR-Cas9 system for genome engineering has revolutionized the ability to interrogate genomes of mammalian cells. Programming the Cas9 endonuclease to induce DNA breaks at specified sites is achieved by simply modifying the sequence of its cognate guide RNA. Although Cas9-mediated genome editing has been shown to be highly specific, cleavage events at off-target sites have also been reported. Minimizing, and eventually abolishing, unwanted off-target cleavage remains a major goal of the CRISPR-Cas9 technology before its implementation for therapeutic use. Recent efforts have turned to chemical biology and biophysical approaches to engineer inducible genome editing systems for controlling Cas9 activity at the transcriptional and protein levels. Here, we review recent advancements to modulate Cas9-mediated genome editing by engineering split-Cas9 constructs, inteins, small molecules, protein-based dimerizing domains, and light-inducible systems.

  1. Programming biological operating systems: genome design, assembly and activation.

    Science.gov (United States)

    Gibson, Daniel G

    2014-05-01

    The DNA technologies developed over the past 20 years for reading and writing the genetic code converged when the first synthetic cell was created 4 years ago. An outcome of this work has been an extraordinary set of tools for synthesizing, assembling, engineering and transplanting whole bacterial genomes. Technical progress, options and applications for bacterial genome design, assembly and activation are discussed.

  2. TMTI Task 1.6 Genetic Engineering Methods and Detection

    Energy Technology Data Exchange (ETDEWEB)

    Slezak, T; Lenhoff, R; Allen, J; Borucki, M; Vitalis, E; Gardner, S

    2009-12-04

    A large number of GE techniques can be adapted from other microorganisms to biothreat bacteria and viruses. Detection of GE in a microorganism increases in difficulty as the size of the genetic change decreases. In addition to the size of the engineered change, the consensus genomic sequence of the microorganism can impact the difficulty of detecting an engineered change in genomes that are highly variable from strain to strain. This problem will require comprehensive databases of whole genome sequences for more genetically variable biothreat bacteria and viruses. Preliminary work with microarrays for detecting synthetic elements or virulence genes and analytic bioinformatic approaches for whole genome sequence comparison to detect genetic engineering show promise for attacking this difficult problem but a large amount of future work remains.

  3. Development of synthetic selfish elements based on modular nucleases in Drosophila melanogaster

    OpenAIRE

    Simoni, A; Siniscalchi, C; Chan, Y-S; Huen, DS; Russell, S; Windbichler, N; Crisanti, A

    2014-01-01

    Selfish genes are DNA elements that increase their rate of genetic transmission at the expense of other genes in the genome and can therefore quickly spread within a population. It has been suggested that selfish elements could be exploited to modify the genome of entire populations for medical and ecological applications. Here we report that transcription activator-like effector nuclease (TALEN) and zinc finger nuclease (ZFN) can be engineered into site-specific synthetic selfish elements (S...

  4. The fusion of biology, computer science, and engineering: towards efficient and successful synthetic biology.

    Science.gov (United States)

    Linshiz, Gregory; Goldberg, Alex; Konry, Tania; Hillson, Nathan J

    2012-01-01

    Synthetic biology is a nascent field that emerged in earnest only around the turn of the millennium. It aims to engineer new biological systems and impart new biological functionality, often through genetic modifications. The design and construction of new biological systems is a complex, multistep process, requiring multidisciplinary collaborative efforts from "fusion" scientists who have formal training in computer science or engineering, as well as hands-on biological expertise. The public has high expectations for synthetic biology and eagerly anticipates the development of solutions to the major challenges facing humanity. This article discusses laboratory practices and the conduct of research in synthetic biology. It argues that the fusion science approach, which integrates biology with computer science and engineering best practices, including standardization, process optimization, computer-aided design and laboratory automation, miniaturization, and systematic management, will increase the predictability and reproducibility of experiments and lead to breakthroughs in the construction of new biological systems. The article also discusses several successful fusion projects, including the development of software tools for DNA construction design automation, recursive DNA construction, and the development of integrated microfluidics systems.

  5. Targeted Porcine Genome Engineering with TALENs

    DEFF Research Database (Denmark)

    Luo, Yonglun; Lin, Lin; Golas, Mariola Monika

    2015-01-01

    Genetically modified pigs are becoming an invaluable animal model for agricultural, pharmaceutical, and biomedical applications. Unlike traditional transgenesis, which is accomplished by randomly inserting an exogenous transgene cassette into the natural chromosomal context, targeted genome editing...... confers precisely editing (e.g., mutations or indels) or insertion of a functional transgenic cassette to user-designed loci. Techniques for targeted genome engineering are growing dramatically and include, e.g., zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs......), and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems. These systems provide enormous potential applications. In this chapter, we review the use of TALENs for targeted genome editing with focus on their application in pigs. In addition, a brief protocol...

  6. Synthetic Biology and Metabolic Engineering Approaches and Its Impact on Non-Conventional Yeast and Biofuel Production

    Energy Technology Data Exchange (ETDEWEB)

    Madhavan, Aravind [Biotechnology Division, National Institute for Interdisciplinary Science and Technology, Council of Scientific and Industrial Research, Trivandrum (India); Rajiv Gandhi Centre for Biotechnology, Trivandrum (India); Jose, Anju Alphonsa; Binod, Parameswaran; Sindhu, Raveendran, E-mail: sindhurgcb@gmail.com; Sukumaran, Rajeev K. [Biotechnology Division, National Institute for Interdisciplinary Science and Technology, Council of Scientific and Industrial Research, Trivandrum (India); Pandey, Ashok [Biotechnology Division, National Institute for Interdisciplinary Science and Technology, Council of Scientific and Industrial Research, Trivandrum (India); Center for Innovative and Applied Bioprocessing, Mohali, Punjab (India); Castro, Galliano Eulogio [Dpt. Ingeniería Química, Ambiental y de los Materiales Edificio, Universidad de Jaén, Jaén (Spain)

    2017-04-25

    The increasing fossil fuel scarcity has led to an urgent need to develop alternative fuels. Currently microorganisms have been extensively used for the production of first-generation biofuels from lignocellulosic biomass. Yeast is the efficient producer of bioethanol among all existing biofuels option. Tools of synthetic biology have revolutionized the field of microbial cell factories especially in the case of ethanol and fatty acid production. Most of the synthetic biology tools have been developed for the industrial workhorse Saccharomyces cerevisiae. The non-conventional yeast systems have several beneficial traits like ethanol tolerance, thermotolerance, inhibitor tolerance, genetic diversity, etc., and synthetic biology have the power to expand these traits. Currently, synthetic biology is slowly widening to the non-conventional yeasts like Hansenula polymorpha, Kluyveromyces lactis, Pichia pastoris, and Yarrowia lipolytica. Herein, we review the basic synthetic biology tools that can apply to non-conventional yeasts. Furthermore, we discuss the recent advances employed to develop efficient biofuel-producing non-conventional yeast strains by metabolic engineering and synthetic biology with recent examples. Looking forward, future synthetic engineering tools’ development and application should focus on unexplored non-conventional yeast species.

  7. Synthetic Biology and Metabolic Engineering Approaches and Its Impact on Non-Conventional Yeast and Biofuel Production

    Directory of Open Access Journals (Sweden)

    Raveendran Sindhu

    2017-04-01

    Full Text Available The increasing fossil fuel scarcity has led to an urgent need to develop alternative fuels. Currently microorganisms have been extensively used for the production of first-generation biofuels from lignocellulosic biomass. Yeast is the efficient producer of bioethanol among all existing biofuels option. Tools of synthetic biology have revolutionized the field of microbial cell factories especially in the case of ethanol and fatty acid production. Most of the synthetic biology tools have been developed for the industrial workhorse Saccharomyces cerevisiae. The non-conventional yeast systems have several beneficial traits like ethanol tolerance, thermotolerance, inhibitor tolerance, genetic diversity, etc., and synthetic biology have the power to expand these traits. Currently, synthetic biology is slowly widening to the non-conventional yeasts like Hansenula polymorpha, Kluyveromyces lactis, Pichia pastoris, and Yarrowia lipolytica. Herein, we review the basic synthetic biology tools that can apply to non-conventional yeasts. Furthermore, we discuss the recent advances employed to develop efficient biofuel-producing non-conventional yeast strains by metabolic engineering and synthetic biology with recent examples. Looking forward, future synthetic engineering tools’ development and application should focus on unexplored non-conventional yeast species.

  8. Synthetic Biology and Metabolic Engineering Approaches and Its Impact on Non-Conventional Yeast and Biofuel Production

    International Nuclear Information System (INIS)

    Madhavan, Aravind; Jose, Anju Alphonsa; Binod, Parameswaran; Sindhu, Raveendran; Sukumaran, Rajeev K.; Pandey, Ashok; Castro, Galliano Eulogio

    2017-01-01

    The increasing fossil fuel scarcity has led to an urgent need to develop alternative fuels. Currently microorganisms have been extensively used for the production of first-generation biofuels from lignocellulosic biomass. Yeast is the efficient producer of bioethanol among all existing biofuels option. Tools of synthetic biology have revolutionized the field of microbial cell factories especially in the case of ethanol and fatty acid production. Most of the synthetic biology tools have been developed for the industrial workhorse Saccharomyces cerevisiae. The non-conventional yeast systems have several beneficial traits like ethanol tolerance, thermotolerance, inhibitor tolerance, genetic diversity, etc., and synthetic biology have the power to expand these traits. Currently, synthetic biology is slowly widening to the non-conventional yeasts like Hansenula polymorpha, Kluyveromyces lactis, Pichia pastoris, and Yarrowia lipolytica. Herein, we review the basic synthetic biology tools that can apply to non-conventional yeasts. Furthermore, we discuss the recent advances employed to develop efficient biofuel-producing non-conventional yeast strains by metabolic engineering and synthetic biology with recent examples. Looking forward, future synthetic engineering tools’ development and application should focus on unexplored non-conventional yeast species.

  9. Synthetic biology: engineering molecular computers

    CERN Multimedia

    CERN. Geneva

    2018-01-01

    Complicated systems cannot survive the rigors of a chaotic environment, without balancing mechanisms that sense, decide upon and counteract the exerted disturbances. Especially so with living organisms, forced by competition to incredible complexities, escalating also their self-controlling plight. Therefore, they compute. Can we harness biological mechanisms to create artificial computing systems? Biology offers several levels of design abstraction: molecular machines, cells, organisms... ranging from the more easily-defined to the more inherently complex. At the bottom of this stack we find the nucleic acids, RNA and DNA, with their digital structure and relatively precise interactions. They are central enablers of designing artificial biological systems, in the confluence of engineering and biology, that we call Synthetic biology. In the first part, let us follow their trail towards an overview of building computing machines with molecules -- and in the second part, take the case study of iGEM Greece 201...

  10. Genome Engineering with TALE and CRISPR Systems in Neuroscience.

    Science.gov (United States)

    Lee, Han B; Sundberg, Brynn N; Sigafoos, Ashley N; Clark, Karl J

    2016-01-01

    Recent advancement in genome engineering technology is changing the landscape of biological research and providing neuroscientists with an opportunity to develop new methodologies to ask critical research questions. This advancement is highlighted by the increased use of programmable DNA-binding agents (PDBAs) such as transcription activator-like effector (TALE) and RNA-guided clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated (Cas) systems. These PDBAs fused or co-expressed with various effector domains allow precise modification of genomic sequences and gene expression levels. These technologies mirror and extend beyond classic gene targeting methods contributing to the development of novel tools for basic and clinical neuroscience. In this Review, we discuss the recent development in genome engineering and potential applications of this technology in the field of neuroscience.

  11. Efficient Genome Editing in Induced Pluripotent Stem Cells with Engineered Nucleases In Vitro.

    Science.gov (United States)

    Termglinchan, Vittavat; Seeger, Timon; Chen, Caressa; Wu, Joseph C; Karakikes, Ioannis

    2017-01-01

    Precision genome engineering is rapidly advancing the application of the induced pluripotent stem cells (iPSCs) technology for in vitro disease modeling of cardiovascular diseases. Targeted genome editing using engineered nucleases is a powerful tool that allows for reverse genetics, genome engineering, and targeted transgene integration experiments to be performed in a precise and predictable manner. However, nuclease-mediated homologous recombination is an inefficient process. Herein, we describe the development of an optimized method combining site-specific nucleases and the piggyBac transposon system for "seamless" genome editing in pluripotent stem cells with high efficiency and fidelity in vitro.

  12. Targeted Porcine Genome Engineering with TALENs

    DEFF Research Database (Denmark)

    Luo, Yonglun; Lin, Lin; Golas, Mariola Monika

    2015-01-01

    confers precisely editing (e.g., mutations or indels) or insertion of a functional transgenic cassette to user-designed loci. Techniques for targeted genome engineering are growing dramatically and include, e.g., zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs......, including construction of sequence-specific TALENs, delivery of TALENs into primary porcine fibroblasts, and detection of TALEN-mediated cleavage, is described. This chapter is useful for scientists who are inexperienced with TALEN engineering of porcine cells as well as of other large animals....

  13. Engineering genetic circuit interactions within and between synthetic minimal cells

    Science.gov (United States)

    Adamala, Katarzyna P.; Martin-Alarcon, Daniel A.; Guthrie-Honea, Katriona R.; Boyden, Edward S.

    2017-05-01

    Genetic circuits and reaction cascades are of great importance for synthetic biology, biochemistry and bioengineering. An open question is how to maximize the modularity of their design to enable the integration of different reaction networks and to optimize their scalability and flexibility. One option is encapsulation within liposomes, which enables chemical reactions to proceed in well-isolated environments. Here we adapt liposome encapsulation to enable the modular, controlled compartmentalization of genetic circuits and cascades. We demonstrate that it is possible to engineer genetic circuit-containing synthetic minimal cells (synells) to contain multiple-part genetic cascades, and that these cascades can be controlled by external signals as well as inter-liposomal communication without crosstalk. We also show that liposomes that contain different cascades can be fused in a controlled way so that the products of incompatible reactions can be brought together. Synells thus enable a more modular creation of synthetic biology cascades, an essential step towards their ultimate programmability.

  14. ‘Can Simple Biological Systems be Built from Standardized Interchangeable Parts?’:Negotiating Biology and Engineering in a Synthetic Biology Competition

    OpenAIRE

    Frow, Emma; Calvert, Jane

    2013-01-01

    Synthetic biology represents a recent attempt to bring engineering principles and practices to working with biology. In practice, the nature of the relationship between engineering and biology in synthetic biology is a subject of ongoing debate. The disciplines of biology and engineering are typically seen to involve differentways of knowing and doing, and to embody different assumptions and objectives. Tensions between these approaches are playing out as the field of synthetic biology is bei...

  15. Applications of cell-free protein synthesis in synthetic biology: Interfacing bio-machinery with synthetic environments.

    Science.gov (United States)

    Lee, Kyung-Ho; Kim, Dong-Myung

    2013-11-01

    Synthetic biology is built on the synthesis, engineering, and assembly of biological parts. Proteins are the first components considered for the construction of systems with designed biological functions because proteins carry out most of the biological functions and chemical reactions inside cells. Protein synthesis is considered to comprise the most basic levels of the hierarchical structure of synthetic biology. Cell-free protein synthesis has emerged as a powerful technology that can potentially transform the concept of bioprocesses. With the ability to harness the synthetic power of biology without many of the constraints of cell-based systems, cell-free protein synthesis enables the rapid creation of protein molecules from diverse sources of genetic information. Cell-free protein synthesis is virtually free from the intrinsic constraints of cell-based methods and offers greater flexibility in system design and manipulability of biological synthetic machinery. Among its potential applications, cell-free protein synthesis can be combined with various man-made devices for rapid functional analysis of genomic sequences. This review covers recent efforts to integrate cell-free protein synthesis with various reaction devices and analytical platforms. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. A Perfect Match Genomic Landscape Provides a Unified Framework for the Precise Detection of Variation in Natural and Synthetic Haploid Genomes.

    Science.gov (United States)

    Palacios-Flores, Kim; García-Sotelo, Jair; Castillo, Alejandra; Uribe, Carina; Aguilar, Luis; Morales, Lucía; Gómez-Romero, Laura; Reyes, José; Garciarubio, Alejandro; Boege, Margareta; Dávila, Guillermo

    2018-04-01

    We present a conceptually simple, sensitive, precise, and essentially nonstatistical solution for the analysis of genome variation in haploid organisms. The generation of a Perfect Match Genomic Landscape (PMGL), which computes intergenome identity with single nucleotide resolution, reveals signatures of variation wherever a query genome differs from a reference genome. Such signatures encode the precise location of different types of variants, including single nucleotide variants, deletions, insertions, and amplifications, effectively introducing the concept of a general signature of variation. The precise nature of variants is then resolved through the generation of targeted alignments between specific sets of sequence reads and known regions of the reference genome. Thus, the perfect match logic decouples the identification of the location of variants from the characterization of their nature, providing a unified framework for the detection of genome variation. We assessed the performance of the PMGL strategy via simulation experiments. We determined the variation profiles of natural genomes and of a synthetic chromosome, both in the context of haploid yeast strains. Our approach uncovered variants that have previously escaped detection. Moreover, our strategy is ideally suited for further refining high-quality reference genomes. The source codes for the automated PMGL pipeline have been deposited in a public repository. Copyright © 2018 by the Genetics Society of America.

  17. Life after the synthetic cell

    DEFF Research Database (Denmark)

    Rasmussen, Steen

    2010-01-01

    Nature asked eight synthetic-biology experts about the implications for science and society of the “synthetic cell” made by the J. Craig Venter Institute (JCVI). The institute's team assembled, modified and implanted a synthesized genome into a DNA-free bacterial shell to make a self-replicating ......Nature asked eight synthetic-biology experts about the implications for science and society of the “synthetic cell” made by the J. Craig Venter Institute (JCVI). The institute's team assembled, modified and implanted a synthesized genome into a DNA-free bacterial shell to make a self...

  18. Synthetic Biology: Engineering, Evolution and Design (SEED) Conference 2014

    Energy Technology Data Exchange (ETDEWEB)

    Voigt, Christopher [Massachusetts Institute of Technology

    2014-07-01

    SEED2014 focused on advances in the science and technology emerging from the field of synthetic biology. We broadly define this as technologies that accelerate the process of genetic engineering. It highlighted new tool development, as well as the application of these tools to diverse problems in biotechnology, including therapeutics, industrial chemicals and fuels, natural products, and agriculture. Systems spanned from in vitro experiments and viruses, through diverse bacteria, to eukaryotes (yeast, mammalian cells, plants).

  19. Genome Engineering with TALE and CRISPR Systems in Neuroscience

    Science.gov (United States)

    Lee, Han B.; Sundberg, Brynn N.; Sigafoos, Ashley N.; Clark, Karl J.

    2016-01-01

    Recent advancement in genome engineering technology is changing the landscape of biological research and providing neuroscientists with an opportunity to develop new methodologies to ask critical research questions. This advancement is highlighted by the increased use of programmable DNA-binding agents (PDBAs) such as transcription activator-like effector (TALE) and RNA-guided clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated (Cas) systems. These PDBAs fused or co-expressed with various effector domains allow precise modification of genomic sequences and gene expression levels. These technologies mirror and extend beyond classic gene targeting methods contributing to the development of novel tools for basic and clinical neuroscience. In this Review, we discuss the recent development in genome engineering and potential applications of this technology in the field of neuroscience. PMID:27092173

  20. Building Better Scientists through Cross-Disciplinary Collaboration in Synthetic Biology: A Report from the Genome Consortium for Active Teaching Workshop 2010

    Science.gov (United States)

    Wolyniak, Michael J.; Alvarez, Consuelo J.; Chandrasekaran, Vidya; Grana, Theresa M.; Holgado, Andrea; Jones, Christopher J.; Morris, Robert W.; Pereira, Anil L.; Stamm, Joyce; Washington, Talitha M.; Yang, Yixin

    2010-01-01

    Synthetic biology is the application of engineering and mathematical principles to develop novel biological devices and circuits. What separates synthetic biology from traditional molecular biology is the development of standardized interchangeable DNA "parts," just as advances in engineering in the nineteenth century brought about standardized…

  1. Synthetic biology, inspired by synthetic chemistry.

    Science.gov (United States)

    Malinova, V; Nallani, M; Meier, W P; Sinner, E K

    2012-07-16

    The topic synthetic biology appears still as an 'empty basket to be filled'. However, there is already plenty of claims and visions, as well as convincing research strategies about the theme of synthetic biology. First of all, synthetic biology seems to be about the engineering of biology - about bottom-up and top-down approaches, compromising complexity versus stability of artificial architectures, relevant in biology. Synthetic biology accounts for heterogeneous approaches towards minimal and even artificial life, the engineering of biochemical pathways on the organismic level, the modelling of molecular processes and finally, the combination of synthetic with nature-derived materials and architectural concepts, such as a cellular membrane. Still, synthetic biology is a discipline, which embraces interdisciplinary attempts in order to have a profound, scientific base to enable the re-design of nature and to compose architectures and processes with man-made matter. We like to give an overview about the developments in the field of synthetic biology, regarding polymer-based analogs of cellular membranes and what questions can be answered by applying synthetic polymer science towards the smallest unit in life, namely a cell. Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  2. Synthetic biology and its alternatives. Descartes, Kant and the idea of engineering biological machines.

    Science.gov (United States)

    Kogge, Werner; Richter, Michael

    2013-06-01

    The engineering-based approach of synthetic biology is characterized by an assumption that 'engineering by design' enables the construction of 'living machines'. These 'machines', as biological machines, are expected to display certain properties of life, such as adapting to changing environments and acting in a situated way. This paper proposes that a tension exists between the expectations placed on biological artefacts and the notion of producing such systems by means of engineering; this tension makes it seem implausible that biological systems, especially those with properties characteristic of living beings, can in fact be produced using the specific methods of engineering. We do not claim that engineering techniques have nothing to contribute to the biotechnological construction of biological artefacts. However, drawing on Descartes's and Kant's thinking on the relationship between the organism and the machine, we show that it is considerably more plausible to assume that distinctively biological artefacts emerge within a paradigm different from the paradigm of the Cartesian machine that underlies the engineering approach. We close by calling for increased attention to be paid to approaches within molecular biology and chemistry that rest on conceptions different from those of synthetic biology's engineering paradigm. Copyright © 2013 Elsevier Ltd. All rights reserved.

  3. Influence of Pichia pastoris cellular material on polymerase chain reaction performance as a synthetic biology standard for genome monitoring.

    Science.gov (United States)

    Templar, Alexander; Woodhouse, Stefan; Keshavarz-Moore, Eli; Nesbeth, Darren N

    2016-08-01

    Advances in synthetic genomics are now well underway in yeasts due to the low cost of synthetic DNA. These new capabilities also bring greater need for quantitating the presence, loss and rearrangement of loci within synthetic yeast genomes. Methods for achieving this will ideally; i) be robust to industrial settings, ii) adhere to a global standard and iii) be sufficiently rapid to enable at-line monitoring during cell growth. The methylotrophic yeast Pichia pastoris (P. pastoris) is increasingly used for industrial production of biotherapeutic proteins so we sought to answer the following questions for this particular yeast species. Is time-consuming DNA purification necessary to obtain accurate end-point polymerase chain reaction (e-pPCR) and quantitative PCR (qPCR) data? Can the novel linear regression of efficiency qPCR method (LRE qPCR), which has properties desirable in a synthetic biology standard, match the accuracy of conventional qPCR? Does cell cultivation scale influence PCR performance? To answer these questions we performed e-pPCR and qPCR in the presence and absence of cellular material disrupted by a mild 30s sonication procedure. The e-pPCR limit of detection (LOD) for a genomic target locus was 50pg (4.91×10(3) copies) of purified genomic DNA (gDNA) but the presence of cellular material reduced this sensitivity sixfold to 300pg gDNA (2.95×10(4) copies). LRE qPCR matched the accuracy of a conventional standard curve qPCR method. The presence of material from bioreactor cultivation of up to OD600=80 did not significantly compromise the accuracy of LRE qPCR. We conclude that a simple and rapid cell disruption step is sufficient to render P. pastoris samples of up to OD600=80 amenable to analysis using LRE qPCR which we propose as a synthetic biology standard. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  4. Engineering the Caenorhabditis elegans genome with CRISPR/Cas9

    NARCIS (Netherlands)

    Waaijers, Selma; Boxem, Mike

    2014-01-01

    The development in early 2013 of CRISPR/Cas9-based genome engineering promises to dramatically advance our ability to alter the genomes of model systems at will. A single, easily produced targeting RNA guides the Cas9 endonuclease to a specific DNA sequence where it creates a double strand break.

  5. CRISPR/Cas9-coupled recombineering for metabolic engineering of Corynebacterium glutamicum.

    Science.gov (United States)

    Cho, Jae Sung; Choi, Kyeong Rok; Prabowo, Cindy Pricilia Surya; Shin, Jae Ho; Yang, Dongsoo; Jang, Jaedong; Lee, Sang Yup

    2017-07-01

    Genome engineering of Corynebacterium glutamicum, an important industrial microorganism for amino acids production, currently relies on random mutagenesis and inefficient double crossover events. Here we report a rapid genome engineering strategy to scarlessly knock out one or more genes in C. glutamicum in sequential and iterative manner. Recombinase RecT is used to incorporate synthetic single-stranded oligodeoxyribonucleotides into the genome and CRISPR/Cas9 to counter-select negative mutants. We completed the system by engineering the respective plasmids harboring CRISPR/Cas9 and RecT for efficient curing such that multiple gene targets can be done iteratively and final strains will be free of plasmids. To demonstrate the system, seven different mutants were constructed within two weeks to study the combinatorial deletion effects of three different genes on the production of γ-aminobutyric acid, an industrially relevant chemical of much interest. This genome engineering strategy will expedite metabolic engineering of C. glutamicum. Copyright © 2017 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

  6. Synthetic Biology of Polyhydroxyalkanoates (PHA).

    Science.gov (United States)

    Meng, De-Chuan; Chen, Guo-Qiang

    Microbial polyhydroxyalkanoates (PHA) are a family of biodegradable and biocompatible polyesters which have been extensively studied using synthetic biology and metabolic engineering methods for improving production and for widening its diversity. Synthetic biology has allowed PHA to become composition controllable random copolymers, homopolymers, and block copolymers. Recent developments showed that it is possible to establish a microbial platform for producing not only random copolymers with controllable monomers and their ratios but also structurally defined homopolymers and block copolymers. This was achieved by engineering the genome of Pseudomonas putida or Pseudomonas entomophiles to weaken the β-oxidation and in situ fatty acid synthesis pathways, so that a fatty acid fed to the bacteria maintains its original chain length and structures when incorporated into the PHA chains. The engineered bacterium allows functional groups in a fatty acid to be introduced into PHA, forming functional PHA, which, upon grafting, generates endless PHA variety. Recombinant Escherichia coli also succeeded in producing efficiently poly(3-hydroxypropionate) or P3HP, the strongest member of PHA. Synthesis pathways of P3HP and its copolymer P3HB3HP of 3-hydroxybutyrate and 3-hydroxypropionate were assembled respectively to allow their synthesis from glucose. CRISPRi was also successfully used to manipulate simultaneously multiple genes and control metabolic flux in E. coli to obtain a series of copolymer P3HB4HB of 3-hydroxybutyrate (3HB) and 4-hydroxybutyrate (4HB). The bacterial shapes were successfully engineered for enhanced PHA accumulation.

  7. Synthetic alienation of microbial organisms by using genetic code engineering: Why and how?

    Science.gov (United States)

    Kubyshkin, Vladimir; Budisa, Nediljko

    2017-08-01

    The main goal of synthetic biology (SB) is the creation of biodiversity applicable for biotechnological needs, while xenobiology (XB) aims to expand the framework of natural chemistries with the non-natural building blocks in living cells to accomplish artificial biodiversity. Protein and proteome engineering, which overcome limitation of the canonical amino acid repertoire of 20 (+2) prescribed by the genetic code by using non-canonic amino acids (ncAAs), is one of the main focuses of XB research. Ideally, estranging the genetic code from its current form via systematic introduction of ncAAs should enable the development of bio-containment mechanisms in synthetic cells potentially endowing them with a "genetic firewall" i.e. orthogonality which prevents genetic information transfer to natural systems. Despite rapid progress over the past two decades, it is not yet possible to completely alienate an organism that would use and maintain different genetic code associations permanently. In order to engineer robust bio-contained life forms, the chemical logic behind the amino acid repertoire establishment should be considered. Starting from recent proposal of Hartman and Smith about the genetic code establishment in the RNA world, here the authors mapped possible biotechnological invasion points for engineering of bio-contained synthetic cells equipped with non-canonical functionalities. Copyright © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Synthetic biology for microbial heavy metal biosensors.

    Science.gov (United States)

    Kim, Hyun Ju; Jeong, Haeyoung; Lee, Sang Jun

    2018-02-01

    Using recombinant DNA technology, various whole-cell biosensors have been developed for detection of environmental pollutants, including heavy metal ions. Whole-cell biosensors have several advantages: easy and inexpensive cultivation, multiple assays, and no requirement of any special techniques for analysis. In the era of synthetic biology, cutting-edge DNA sequencing and gene synthesis technologies have accelerated the development of cell-based biosensors. Here, we summarize current technological advances in whole-cell heavy metal biosensors, including the synthetic biological components (bioparts), sensing and reporter modules, genetic circuits, and chassis cells. We discuss several opportunities for improvement of synthetic cell-based biosensors. First, new functional modules must be discovered in genome databases, and this knowledge must be used to upgrade specific bioparts through molecular engineering. Second, modules must be assembled into functional biosystems in chassis cells. Third, heterogeneity of individual cells in the microbial population must be eliminated. In the perspectives, the development of whole-cell biosensors is also discussed in the aspects of cultivation methods and synthetic cells.

  9. Mapping the Emergence of Synthetic Biology.

    Directory of Open Access Journals (Sweden)

    Benjamin Raimbault

    Full Text Available In this paper, we apply an original scientometric analyses to a corpus comprising synthetic biology (SynBio publications in Thomson Reuters Web of Science to characterize the emergence of this new scientific field. Three results were drawn from this empirical investigation. First, despite the exponential growth of publications, the study of population level statistics (newcomers proportion, collaboration network structure shows that SynBio has entered a stabilization process since 2010. Second, the mapping of textual and citational networks shows that SynBio is characterized by high heterogeneity and four different approaches: the central approach, where biobrick engineering is the most widespread; genome engineering; protocell creation; and metabolic engineering. We suggest that synthetic biology acts as an umbrella term allowing for the mobilization of resources, and also serves to relate scientific content and promises of applications. Third, we observed a strong intertwinement between epistemic and socio-economic dynamics. Measuring scientific production and impact and using structural analysis data, we identified a core set of mostly American scientists. Biographical analysis shows that these central and influential scientists act as "boundary spanners," meaning that their importance to the field lies not only in their academic contributions, but also in their capacity to interact with other social spaces that are outside the academic sphere.

  10. Synthetic biology and the technicity of biofuels.

    Science.gov (United States)

    Mackenzie, Adrian

    2013-06-01

    The principal existing real-world application of synthetic biology is biofuels. Several 'next generation biofuel' companies-Synthetic Genomics, Amyris and Joule Unlimited Technologies-claim to be using synthetic biology to make biofuels. The irony of this is that highly advanced science and engineering serves the very mundane and familiar realm of transport. Despite their rather prosaic nature, biofuels could offer an interesting way to highlight the novelty of synthetic biology from several angles at once. Drawing on the French philosopher of technology and biology Gilbert Simondon, we can understand biofuels as technical objects whose genesis involves processes of concretisation that negotiate between heterogeneous geographical, biological, technical, scientific and commercial realities. Simondon's notion of technicity, the degree of concretisation of a technical object, usefully conceptualises this relationality. Viewed in terms of technicity, we might understand better how technical entities, elements, and ensembles are coming into being in the name of synthetic biology. The broader argument here is that when we seek to identify the newness of disciplines, their newness might be less epistemic and more logistic. Crown Copyright © 2013. Published by Elsevier Ltd. All rights reserved.

  11. Insights into structural variations and genome rearrangements in prokaryotic genomes.

    Science.gov (United States)

    Periwal, Vinita; Scaria, Vinod

    2015-01-01

    Structural variations (SVs) are genomic rearrangements that affect fairly large fragments of DNA. Most of the SVs such as inversions, deletions and translocations have been largely studied in context of genetic diseases in eukaryotes. However, recent studies demonstrate that genome rearrangements can also have profound impact on prokaryotic genomes, leading to altered cell phenotype. In contrast to single-nucleotide variations, SVs provide a much deeper insight into organization of bacterial genomes at a much better resolution. SVs can confer change in gene copy number, creation of new genes, altered gene expression and many other functional consequences. High-throughput technologies have now made it possible to explore SVs at a much refined resolution in bacterial genomes. Through this review, we aim to highlight the importance of the less explored field of SVs in prokaryotic genomes and their impact. We also discuss its potential applicability in the emerging fields of synthetic biology and genome engineering where targeted SVs could serve to create sophisticated and accurate genome editing. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  12. Genome Writing: Current Progress and Related Applications

    Directory of Open Access Journals (Sweden)

    Yueqiang Wang

    2018-02-01

    Full Text Available The ultimate goal of synthetic biology is to build customized cells or organisms to meet specific industrial or medical needs. The most important part of the customized cell is a synthetic genome. Advanced genomic writing technologies are required to build such an artificial genome. Recently, the partially-completed synthetic yeast genome project represents a milestone in this field. In this mini review, we briefly introduce the techniques for de novo genome synthesis and genome editing. Furthermore, we summarize recent research progresses and highlight several applications in the synthetic genome field. Finally, we discuss current challenges and future prospects. Keywords: Synthetic biology, Genome writing, Genome editing, Bioethics, Biosafety

  13. Origins of Programmable Nucleases for Genome Engineering.

    Science.gov (United States)

    Chandrasegaran, Srinivasan; Carroll, Dana

    2016-02-27

    Genome engineering with programmable nucleases depends on cellular responses to a targeted double-strand break (DSB). The first truly targetable reagents were the zinc finger nucleases (ZFNs) showing that arbitrary DNA sequences could be addressed for cleavage by protein engineering, ushering in the breakthrough in genome manipulation. ZFNs resulted from basic research on zinc finger proteins and the FokI restriction enzyme (which revealed a bipartite structure with a separable DNA-binding domain and a non-specific cleavage domain). Studies on the mechanism of cleavage by 3-finger ZFNs established that the preferred substrates were paired binding sites, which doubled the size of the target sequence recognition from 9 to 18bp, long enough to specify a unique genomic locus in plant and mammalian cells. Soon afterwards, a ZFN-induced DSB was shown to stimulate homologous recombination in cells. Transcription activator-like effector nucleases (TALENs) that are based on bacterial TALEs fused to the FokI cleavage domain expanded this capability. The fact that ZFNs and TALENs have been used for genome modification of more than 40 different organisms and cell types attests to the success of protein engineering. The most recent technology platform for delivering a targeted DSB to cellular genomes is that of the RNA-guided nucleases, which are based on the naturally occurring Type II prokaryotic CRISPR-Cas9 system. Unlike ZFNs and TALENs that use protein motifs for DNA sequence recognition, CRISPR-Cas9 depends on RNA-DNA recognition. The advantages of the CRISPR-Cas9 system-the ease of RNA design for new targets and the dependence on a single, constant Cas9 protein-have led to its wide adoption by research laboratories around the world. These technology platforms have equipped scientists with an unprecedented ability to modify cells and organisms almost at will, with wide-ranging implications across biology and medicine. However, these nucleases have also been shown to cut

  14. Efficient CRISPR/Cas9-Based Genome Engineering in Human Pluripotent Stem Cells.

    Science.gov (United States)

    Kime, Cody; Mandegar, Mohammad A; Srivastava, Deepak; Yamanaka, Shinya; Conklin, Bruce R; Rand, Tim A

    2016-01-01

    Human pluripotent stem cells (hPS cells) are rapidly emerging as a powerful tool for biomedical discovery. The advent of human induced pluripotent stem cells (hiPS cells) with human embryonic stem (hES)-cell-like properties has led to hPS cells with disease-specific genetic backgrounds for in vitro disease modeling and drug discovery as well as mechanistic and developmental studies. To fully realize this potential, it will be necessary to modify the genome of hPS cells with precision and flexibility. Pioneering experiments utilizing site-specific double-strand break (DSB)-mediated genome engineering tools, including zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), have paved the way to genome engineering in previously recalcitrant systems such as hPS cells. However, these methods are technically cumbersome and require significant expertise, which has limited adoption. A major recent advance involving the clustered regularly interspaced short palindromic repeats (CRISPR) endonuclease has dramatically simplified the effort required for genome engineering and will likely be adopted widely as the most rapid and flexible system for genome editing in hPS cells. In this unit, we describe commonly practiced methods for CRISPR endonuclease genomic editing of hPS cells into cell lines containing genomes altered by insertion/deletion (indel) mutagenesis or insertion of recombinant genomic DNA. Copyright © 2016 John Wiley & Sons, Inc.

  15. Enzymatic engineering of the porcine genome with transposons and recombinases

    Directory of Open Access Journals (Sweden)

    Carlson Daniel F

    2007-07-01

    Full Text Available Abstract Background Swine is an important agricultural commodity and biomedical model. Manipulation of the pig genome provides opportunity to improve production efficiency, enhance disease resistance, and add value to swine products. Genetic engineering can also expand the utility of pigs for modeling human disease, developing clinical treatment methodologies, or donating tissues for xenotransplantation. Realizing the full potential of pig genetic engineering requires translation of the complete repertoire of genetic tools currently employed in smaller model organisms to practical use in pigs. Results Application of transposon and recombinase technologies for manipulation of the swine genome requires characterization of their activity in pig cells. We tested four transposon systems- Sleeping Beauty, Tol2, piggyBac, and Passport in cultured porcine cells. Transposons increased the efficiency of DNA integration up to 28-fold above background and provided for precise delivery of 1 to 15 transgenes per cell. Both Cre and Flp recombinase were functional in pig cells as measured by their ability to remove a positive-negative selection cassette from 16 independent clones and over 20 independent genomic locations. We also demonstrated a Cre-dependent genetic switch capable of eliminating an intervening positive-negative selection cassette and activating GFP expression from episomal and genome-resident transposons. Conclusion We have demonstrated for the first time that transposons and recombinases are capable of mobilizing DNA into and out of the porcine genome in a precise and efficient manner. This study provides the basis for developing transposon and recombinase based tools for genetic engineering of the swine genome.

  16. Synthetic biology and biosecurity: challenging the "myths".

    OpenAIRE

    Jefferson, C; Lentzos, F; Marris, C

    2014-01-01

    15.10.14 KB. Ok to add published version to spiral, OA paper under cc by Synthetic biology, a field that aims to make biology easier to engineer, is routinely described as leading to an increase in the dual-use threat, i.e., the potential for the same scientific research to be used for peaceful purposes or misused for warfare or terrorism. Fears have been expressed that the de-skilling of biology, combined with online access to the genomic DNA sequences of pathogenic organisms an...

  17. Synthetic Biology: Life, Jim, but Not As We Know It

    Science.gov (United States)

    Hallinan, Jennifer

    Frankenstein, Mary Shelley's classic tale of horror, warns of the perils of hubris: of the terrible fate that awaits when Man plays God and attempts to create life. Molecular biologists are clearly not listening. Not content with merely inserting the occasional gene into the genome of an existing organism. they are developing a whole new field, Synthetic Biology, which aims to engineer from first principles organisms with desirable, controllable qualities.

  18. Eight new genomes and synthetic controls increase the accessibility of rapid melt-MAMA SNP typing of Coxiella burnetii.

    Directory of Open Access Journals (Sweden)

    Edvin Karlsson

    Full Text Available The case rate of Q fever in Europe has increased dramatically in recent years, mainly because of an epidemic in the Netherlands in 2009. Consequently, there is a need for more extensive genetic characterization of the disease agent Coxiella burnetii in order to better understand the epidemiology and spread of this disease. Genome reference data are essential for this purpose, but only thirteen genome sequences are currently available. Current methods for typing C. burnetii are criticized for having problems in comparing results across laboratories, require the use of genomic control DNA, and/or rely on markers in highly variable regions. We developed in this work a method for single nucleotide polymorphism (SNP typing of C. burnetii isolates and tissue samples based on new assays targeting ten phylogenetically stable synonymous canonical SNPs (canSNPs. These canSNPs represent previously known phylogenetic branches and were here identified from sequence comparisons of twenty-one C. burnetii genomes, eight of which were sequenced in this work. Importantly, synthetic control templates were developed, to make the method useful to laboratories lacking genomic control DNA. An analysis of twenty-one C. burnetii genomes confirmed that the species exhibits high sequence identity. Most of its SNPs (7,493/7,559 shared by >1 genome follow a clonal inheritance pattern and are therefore stable phylogenetic typing markers. The assays were validated using twenty-six genetically diverse C. burnetii isolates and three tissue samples from small ruminants infected during the epidemic in the Netherlands. Each sample was assigned to a clade. Synthetic controls (vector and PCR amplified gave identical results compared to the corresponding genomic controls and are viable alternatives to genomic DNA. The results from the described method indicate that it could be useful for cheap and rapid disease source tracking at non-specialized laboratories, which requires accurate

  19. Investigation of engine performance and emissions of a diesel engine with a blend of marine gas oil and synthetic diesel fuel.

    Science.gov (United States)

    Nabi, Md Nurun; Hustad, Johan Einar

    2012-01-01

    This paper investigates diesel engine performance and exhaust emissions with marine gas oil (MGO) and a blend of MGO and synthetic diesel fuel. Ten per cent by volume of Fischer-Tropsch (FT), a synthetic diesel fuel, was added to MGO to investigate its influence on the diesel engine performance and emissions. The blended fuel was termed as FT10 fuel, while the neat (100 vol%) MGO was termed as MGO fuel. The experiments were conducted with a fourstroke, six-cylinder, turbocharged, direct injection, Scania DC 1102 diesel engine. It is interesting to note that all emissions including smoke (filter smoke number), total particulate matter (TPM), carbon monoxide (CO), total unburned hydrocarbon (THC), oxides of nitrogen (NOx) and engine noise were reduced with FT10 fuel compared with the MGO fuel. Diesel fine particle number and mass emissions were measured with an electrical low pressure impactor. Like other exhaust emissions, significant reductions in fine particles and mass emissions were observed with the FT10 fuel. The reduction was due to absence of sulphur and aromatic compounds in the FT fuel. In-cylinder gas pressure and engine thermal efficiency were identical for both FT10 and MGO fuels.

  20. Accuracy of Genomic Selection in a Rice Synthetic Population Developed for Recurrent Selection Breeding.

    Science.gov (United States)

    Grenier, Cécile; Cao, Tuong-Vi; Ospina, Yolima; Quintero, Constanza; Châtel, Marc Henri; Tohme, Joe; Courtois, Brigitte; Ahmadi, Nourollah

    2015-01-01

    Genomic selection (GS) is a promising strategy for enhancing genetic gain. We investigated the accuracy of genomic estimated breeding values (GEBV) in four inter-related synthetic populations that underwent several cycles of recurrent selection in an upland rice-breeding program. A total of 343 S2:4 lines extracted from those populations were phenotyped for flowering time, plant height, grain yield and panicle weight, and genotyped with an average density of one marker per 44.8 kb. The relative effect of the linkage disequilibrium (LD) and minor allele frequency (MAF) thresholds for selecting markers, the relative size of the training population (TP) and of the validation population (VP), the selected trait and the genomic prediction models (frequentist and Bayesian) on the accuracy of GEBVs was investigated in 540 cross validation experiments with 100 replicates. The effect of kinship between the training and validation populations was tested in an additional set of 840 cross validation experiments with a single genomic prediction model. LD was high (average r2 = 0.59 at 25 kb) and decreased slowly, distribution of allele frequencies at individual loci was markedly skewed toward unbalanced frequencies (MAF average value 15.2% and median 9.6%), and differentiation between the four synthetic populations was low (FST ≤0.06). The accuracy of GEBV across all cross validation experiments ranged from 0.12 to 0.54 with an average of 0.30. Significant differences in accuracy were observed among the different levels of each factor investigated. Phenotypic traits had the biggest effect, and the size of the incidence matrix had the smallest. Significant first degree interaction was observed for GEBV accuracy between traits and all the other factors studied, and between prediction models and LD, MAF and composition of the TP. The potential of GS to accelerate genetic gain and breeding options to increase the accuracy of predictions are discussed.

  1. GIGGLE: a search engine for large-scale integrated genome analysis.

    Science.gov (United States)

    Layer, Ryan M; Pedersen, Brent S; DiSera, Tonya; Marth, Gabor T; Gertz, Jason; Quinlan, Aaron R

    2018-02-01

    GIGGLE is a genomics search engine that identifies and ranks the significance of genomic loci shared between query features and thousands of genome interval files. GIGGLE (https://github.com/ryanlayer/giggle) scales to billions of intervals and is over three orders of magnitude faster than existing methods. Its speed extends the accessibility and utility of resources such as ENCODE, Roadmap Epigenomics, and GTEx by facilitating data integration and hypothesis generation.

  2. Bacteriophage-based synthetic biology for the study of infectious diseases

    Science.gov (United States)

    Lu, Timothy K.

    2014-01-01

    Since their discovery, bacteriophages have contributed enormously to our understanding of molecular biology as model systems. Furthermore, bacteriophages have provided many tools that have advanced the fields of genetic engineering and synthetic biology. Here, we discuss bacteriophage-based technologies and their application to the study of infectious diseases. New strategies for engineering genomes have the potential to accelerate the design of novel phages as therapies, diagnostics, and tools. Though almost a century has elapsed since their discovery, bacteriophages continue to have a major impact on modern biological sciences, especially with the growth of multidrug-resistant bacteria and interest in the microbiome. PMID:24997401

  3. European governance challenges in 21st century bio-engineering

    NARCIS (Netherlands)

    Est, van R.; Stemerding, D.; Est, van R.; Stemerding, D.

    2012-01-01

    This Final Report discussed four case studies (whole genome sequencing, neuromodulation, biocybernetic adaptation and standardization in synthetic biology) focusing on particular developments in the four fields of 21st-century bio-engineering that were explored more extensively in the earlier

  4. Using Synthetic Biology to Engineer Living Cells That Interface with Programmable Materials.

    Science.gov (United States)

    Heyde, Keith C; Scott, Felicia Y; Paek, Sung-Ho; Zhang, Ruihua; Ruder, Warren C

    2017-03-09

    We have developed an abiotic-biotic interface that allows engineered cells to control the material properties of a functionalized surface. This system is made by creating two modules: a synthetically engineered strain of E. coli cells and a functionalized material interface. Within this paper, we detail a protocol for genetically engineering selected behaviors within a strain of E. coli using molecular cloning strategies. Once developed, this strain produces elevated levels of biotin when exposed to a chemical inducer. Additionally, we detail protocols for creating two different functionalized surfaces, each of which is able to respond to cell-synthesized biotin. Taken together, we present a methodology for creating a linked, abiotic-biotic system that allows engineered cells to control material composition and assembly on nonliving substrates.

  5. Microbial production of natural and non-natural flavonoids: Pathway engineering, directed evolution and systems/synthetic biology.

    Science.gov (United States)

    Pandey, Ramesh Prasad; Parajuli, Prakash; Koffas, Mattheos A G; Sohng, Jae Kyung

    2016-01-01

    In this review, we address recent advances made in pathway engineering, directed evolution, and systems/synthetic biology approaches employed in the production and modification of flavonoids from microbial cells. The review is divided into two major parts. In the first, various metabolic engineering and system/synthetic biology approaches used for production of flavonoids and derivatives are discussed broadly. All the manipulations/engineering accomplished on the microorganisms since 2000 are described in detail along with the biosynthetic pathway enzymes, their sources, structures of the compounds, and yield of each product. In the second part of the review, post-modifications of flavonoids by four major reactions, namely glycosylations, methylations, hydroxylations and prenylations using recombinant strains are described. Copyright © 2016 Elsevier Inc. All rights reserved.

  6. Programming Morphogenesis through Systems and Synthetic Biology.

    Science.gov (United States)

    Velazquez, Jeremy J; Su, Emily; Cahan, Patrick; Ebrahimkhani, Mo R

    2018-04-01

    Mammalian tissue development is an intricate, spatiotemporal process of self-organization that emerges from gene regulatory networks of differentiating stem cells. A major goal in stem cell biology is to gain a sufficient understanding of gene regulatory networks and cell-cell interactions to enable the reliable and robust engineering of morphogenesis. Here, we review advances in synthetic biology, single cell genomics, and multiscale modeling, which, when synthesized, provide a framework to achieve the ambitious goal of programming morphogenesis in complex tissues and organoids. Copyright © 2017 Elsevier Ltd. All rights reserved.

  7. GIGGLE: a search engine for large-scale integrated genome analysis

    Science.gov (United States)

    Layer, Ryan M; Pedersen, Brent S; DiSera, Tonya; Marth, Gabor T; Gertz, Jason; Quinlan, Aaron R

    2018-01-01

    GIGGLE is a genomics search engine that identifies and ranks the significance of genomic loci shared between query features and thousands of genome interval files. GIGGLE (https://github.com/ryanlayer/giggle) scales to billions of intervals and is over three orders of magnitude faster than existing methods. Its speed extends the accessibility and utility of resources such as ENCODE, Roadmap Epigenomics, and GTEx by facilitating data integration and hypothesis generation. PMID:29309061

  8. Suitability of Different Natural and Synthetic Biomaterials for Dental Pulp Tissue Engineering.

    Science.gov (United States)

    Galler, Kerstin M; Brandl, Ferdinand P; Kirchhof, Susanne; Widbiller, Matthias; Eidt, Andreas; Buchalla, Wolfgang; Göpferich, Achim; Schmalz, Gottfried

    2018-02-01

    Dental pulp tissue engineering is possible after insertion of pulpal stem cells combined with a scaffold into empty root canals. Commonly used biomaterials are collagen or poly(lactic) acid, which are either difficult to modify or to insert into such a narrow space. New hydrogel scaffolds with bioactive, specifically tailored functions could optimize the conditions for this approach. Different synthetic and natural hydrogels were tested for their suitability to engineer dental pulp. Two functionalized modifications of polyethylene glycol were developed in this study and compared to a self-assembling peptide, as well as to collagen and fibrin. Cell viability of dental pulp stem cells in test materials was assessed over two weeks. Cells in selected test materials laden with dentin-derived growth factors were inserted into human tooth roots and implanted subcutaneously into immunocompromised mice. In vitro cell culture exhibited distinct differences between scaffold types, where viability was significantly higher in natural compared to synthetic materials. In vivo experiments showed considerable differences regarding scaffold degradation, soft tissue formation, vascularization, and odontoblast-like cell differentiation. Fibrin appeared most suitable to enable generation of a pulp-like tissue and differentiation of cells into odontoblasts at the cell-dentin interface. In conclusion, natural materials, especially fibrin, proved to be superior compared to synthetic scaffolds regarding cell viability and dental pulp-like tissue formation.

  9. Rapid Prototyping of Microbial Cell Factories via Genome-scale Engineering

    Science.gov (United States)

    Si, Tong; Xiao, Han; Zhao, Huimin

    2014-01-01

    Advances in reading, writing and editing genetic materials have greatly expanded our ability to reprogram biological systems at the resolution of a single nucleotide and on the scale of a whole genome. Such capacity has greatly accelerated the cycles of design, build and test to engineer microbes for efficient synthesis of fuels, chemicals and drugs. In this review, we summarize the emerging technologies that have been applied, or are potentially useful for genome-scale engineering in microbial systems. We will focus on the development of high-throughput methodologies, which may accelerate the prototyping of microbial cell factories. PMID:25450192

  10. Biofuel production in Escherichia coli. The role of metabolic engineering and synthetic biology

    Energy Technology Data Exchange (ETDEWEB)

    Clomburg, James M. [Rice Univ., Houston, TX (United States). Dept. of Chemical and Biomolecular Engineering; Gonzalez, Ramon [Rice Univ., Houston, TX (United States). Dept. of Chemical and Biomolecular Engineering; Rice Univ., Houston, TX (United States). Dept. of Bioengineering

    2010-03-15

    The microbial production of biofuels is a promising avenue for the development of viable processes for the generation of fuels from sustainable resources. In order to become cost and energy effective, these processes must utilize organisms that can be optimized to efficiently produce candidate fuels from a variety of feedstocks. Escherichia coli has become a promising host organism for the microbial production of biofuels in part due to the ease at which this organism can be manipulated. Advancements in metabolic engineering and synthetic biology have led to the ability to efficiently engineer E. coli as a biocatalyst for the production of a wide variety of potential biofuels from several biomass constituents. This review focuses on recent efforts devoted to engineering E. coli for the production of biofuels, with emphasis on the key aspects of both the utilization of a variety of substrates as well as the synthesis of several promising biofuels. Strategies for the efficient utilization of carbohydrates, carbohydrate mixtures, and noncarbohydrate carbon sources will be discussed along with engineering efforts for the exploitation of both fermentative and nonfermentative pathways for the production of candidate biofuels such as alcohols and higher carbon biofuels derived from fatty acid and isoprenoid pathways. Continued advancements in metabolic engineering and synthetic biology will help improve not only the titers, yields, and productivities of biofuels discussed herein, but also increase the potential range of compounds that can be produced. (orig.)

  11. Efficient engineering of a bacteriophage genome using the type I-E CRISPR-Cas system.

    Science.gov (United States)

    Kiro, Ruth; Shitrit, Dror; Qimron, Udi

    2014-01-01

    The clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) system has recently been used to engineer genomes of various organisms, but surprisingly, not those of bacteriophages (phages). Here we present a method to genetically engineer the Escherichia coli phage T7 using the type I-E CRISPR-Cas system. T7 phage genome is edited by homologous recombination with a DNA sequence flanked by sequences homologous to the desired location. Non-edited genomes are targeted by the CRISPR-Cas system, thus enabling isolation of the desired recombinant phages. This method broadens CRISPR Cas-based editing to phages and uses a CRISPR-Cas type other than type II. The method may be adjusted to genetically engineer any bacteriophage genome.

  12. Lentiviral Delivery of Proteins for Genome Engineering.

    Science.gov (United States)

    Cai, Yujia; Mikkelsen, Jacob Giehm

    2016-01-01

    Viruses have evolved to traverse cellular barriers and travel to the nucleus by mechanisms that involve active transport through the cytoplasm and viral quirks to resist cellular restriction factors and innate immune responses. Virus-derived vector systems exploit the capacity of viruses to ferry genetic information into cells, and now - more than three decades after the discovery of HIV - lentiviral vectors based on HIV-1 have become instrumental in biomedical research and gene therapies that require genomic insertion of transgenes. By now, the efficacy of lentiviral gene delivery to stem cells, cells of the immune system including T cells, hepatic cells, and many other therapeutically relevant cell types is well established. Along with nucleic acids, HIV-1 virions carry the enzymatic tools that are essential for early steps of infection. Such capacity to package enzymes, even proteins of nonviral origin, has unveiled new ways of exploiting cellular intrusion of HIV-1. Based on early findings demonstrating the packaging of heterologous proteins into virus particles as part of the Gag and GagPol polypeptides, we have established lentiviral protein transduction for delivery of DNA transposases and designer nucleases. This strategy for delivering genome-engineering proteins facilitates high enzymatic activity within a short time frame and may potentially improve the safety of genome editing. Exploiting the full potential of lentiviral vectors, incorporation of foreign protein can be combined with the delivery of DNA transposons or a donor sequence for homology-directed repair in so-called 'all-in-one' lentiviral vectors. Here, we briefly describe intracellular restrictions that may affect lentiviral gene and protein delivery and review the current status of lentiviral particles as carriers of tool kits for genome engineering.

  13. GeNemo: a search engine for web-based functional genomic data.

    Science.gov (United States)

    Zhang, Yongqing; Cao, Xiaoyi; Zhong, Sheng

    2016-07-08

    A set of new data types emerged from functional genomic assays, including ChIP-seq, DNase-seq, FAIRE-seq and others. The results are typically stored as genome-wide intensities (WIG/bigWig files) or functional genomic regions (peak/BED files). These data types present new challenges to big data science. Here, we present GeNemo, a web-based search engine for functional genomic data. GeNemo searches user-input data against online functional genomic datasets, including the entire collection of ENCODE and mouse ENCODE datasets. Unlike text-based search engines, GeNemo's searches are based on pattern matching of functional genomic regions. This distinguishes GeNemo from text or DNA sequence searches. The user can input any complete or partial functional genomic dataset, for example, a binding intensity file (bigWig) or a peak file. GeNemo reports any genomic regions, ranging from hundred bases to hundred thousand bases, from any of the online ENCODE datasets that share similar functional (binding, modification, accessibility) patterns. This is enabled by a Markov Chain Monte Carlo-based maximization process, executed on up to 24 parallel computing threads. By clicking on a search result, the user can visually compare her/his data with the found datasets and navigate the identified genomic regions. GeNemo is available at www.genemo.org. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  14. Rapid prototyping of microbial cell factories via genome-scale engineering.

    Science.gov (United States)

    Si, Tong; Xiao, Han; Zhao, Huimin

    2015-11-15

    Advances in reading, writing and editing genetic materials have greatly expanded our ability to reprogram biological systems at the resolution of a single nucleotide and on the scale of a whole genome. Such capacity has greatly accelerated the cycles of design, build and test to engineer microbes for efficient synthesis of fuels, chemicals and drugs. In this review, we summarize the emerging technologies that have been applied, or are potentially useful for genome-scale engineering in microbial systems. We will focus on the development of high-throughput methodologies, which may accelerate the prototyping of microbial cell factories. Copyright © 2014 Elsevier Inc. All rights reserved.

  15. Transcription Activator-Like Effectors (TALEs) Hybrid Nucleases for Genome Engineering Application

    KAUST Repository

    Wibowo, Anjar

    2011-06-06

    Gene targeting is a powerful genome engineering tool that can be used for a variety of biotechnological applications. Genomic double-strand DNA breaks generated by engineered site-specific nucleases can stimulate gene targeting. Hybrid nucleases are composed of DNA binding module and DNA cleavage module. Zinc Finger Nucleases were used to generate double-strand DNA breaks but it suffers from failures and lack of reproducibility. The transcription activator–like effectors (TALEs) from plant pathogenic Xanthomonas contain a unique type of DNA-binding domain that bind specific DNA targets. The purpose of this study is to generate novel sequence specific nucleases by fusing a de novo engineered Hax3 TALE-based DNA binding domain to a FokI cleavage domain. Our data show that the de novo engineered TALE nuclease can bind to its target sequence and create double-strand DNA breaks in vitro. We also show that the de novo engineered TALE nuclease is capable of generating double-strand DNA breaks in its target sequence in vivo, when transiently expressed in Nicotiana benthamiana leaves. In conclusion, our data demonstrate that TALE-based hybrid nucleases can be tailored to bind a user-selected DNA sequence and generate site-specific genomic double-strand DNA breaks. TALE-based hybrid nucleases hold much promise as powerful molecular tools for gene targeting applications.

  16. Next-generation genome-scale models for metabolic engineering

    DEFF Research Database (Denmark)

    King, Zachary A.; Lloyd, Colton J.; Feist, Adam M.

    2015-01-01

    Constraint-based reconstruction and analysis (COBRA) methods have become widely used tools for metabolic engineering in both academic and industrial laboratories. By employing a genome-scale in silico representation of the metabolic network of a host organism, COBRA methods can be used to predict...... examples of applying COBRA methods to strain optimization are presented and discussed. Then, an outlook is provided on the next generation of COBRA models and the new types of predictions they will enable for systems metabolic engineering....

  17. Genome Editing in Escherichia coli with Cas9 and synthetic CRISPRs

    Energy Technology Data Exchange (ETDEWEB)

    Peng, Ze; Richardson, Sarah; Robinson, David; Deutsch, Samuel; Cheng, Jan-Fang

    2014-03-14

    Recently, the Cas9-CRISPR system has proven to be a useful tool for genome editing in eukaryotes, which repair the double stranded breaks made by Cas9 with non-homologous end joining or homologous recombination. Escherichia coli lacks non-homologous end joining and has a very low homologous recombination rate, effectively rendering targeted Cas9 activity lethal. We have developed a heat curable, serializable, plasmid based system for selectionless Cas9 editing in arbitrary E. coli strains that uses synthetic CRISPRs for targeting and -red to effect repairs of double stranded breaks. We have demonstrated insertions, substitutions, and multi-target deletions with our system, which we have tested in several strains.

  18. Towards a synthetic chloroplast.

    Directory of Open Access Journals (Sweden)

    Christina M Agapakis

    2011-04-01

    Full Text Available The evolution of eukaryotic cells is widely agreed to have proceeded through a series of endosymbiotic events between larger cells and proteobacteria or cyanobacteria, leading to the formation of mitochondria or chloroplasts, respectively. Engineered endosymbiotic relationships between different species of cells are a valuable tool for synthetic biology, where engineered pathways based on two species could take advantage of the unique abilities of each mutualistic partner.We explored the possibility of using the photosynthetic bacterium Synechococcus elongatus PCC 7942 as a platform for studying evolutionary dynamics and for designing two-species synthetic biological systems. We observed that the cyanobacteria were relatively harmless to eukaryotic host cells compared to Escherichia coli when injected into the embryos of zebrafish, Danio rerio, or taken up by mammalian macrophages. In addition, when engineered with invasin from Yersinia pestis and listeriolysin O from Listeria monocytogenes, S. elongatus was able to invade cultured mammalian cells and divide inside macrophages.Our results show that it is possible to engineer photosynthetic bacteria to invade the cytoplasm of mammalian cells for further engineering and applications in synthetic biology. Engineered invasive but non-pathogenic or immunogenic photosynthetic bacteria have great potential as synthetic biological devices.

  19. Engineered CRISPR/Cas9 system for multiplex genome engineering of polyploid industrial yeast strains.

    Science.gov (United States)

    Lian, Jiazhang; Bao, Zehua; Hu, Sumeng; Zhao, Huimin

    2018-06-01

    The CRISPR/Cas9 system has been widely used for multiplex genome engineering of Saccharomyces cerevisiae. However, its application in manipulating industrial yeast strains is less successful, probably due to the genome complexity and low copy numbers of gRNA expression plasmids. Here we developed an efficient CRISPR/Cas9 system for industrial yeast strain engineering by using our previously engineered plasmids with increased copy numbers. Four genes in both a diploid strain (Ethanol Red, 8 alleles in total) and a triploid strain (ATCC 4124, 12 alleles in total) were knocked out in a single step with 100% efficiency. This system was used to construct xylose-fermenting, lactate-producing industrial yeast strains, in which ALD6, PHO13, LEU2, and URA3 were disrupted in a single step followed by the introduction of a xylose utilization pathway and a lactate biosynthetic pathway on auxotrophic marker plasmids. The optimized CRISPR/Cas9 system provides a powerful tool for the development of industrial yeast based microbial cell factories. © 2018 Wiley Periodicals, Inc.

  20. Accuracy of Genomic Selection in a Rice Synthetic Population Developed for Recurrent Selection Breeding.

    Directory of Open Access Journals (Sweden)

    Cécile Grenier

    Full Text Available Genomic selection (GS is a promising strategy for enhancing genetic gain. We investigated the accuracy of genomic estimated breeding values (GEBV in four inter-related synthetic populations that underwent several cycles of recurrent selection in an upland rice-breeding program. A total of 343 S2:4 lines extracted from those populations were phenotyped for flowering time, plant height, grain yield and panicle weight, and genotyped with an average density of one marker per 44.8 kb. The relative effect of the linkage disequilibrium (LD and minor allele frequency (MAF thresholds for selecting markers, the relative size of the training population (TP and of the validation population (VP, the selected trait and the genomic prediction models (frequentist and Bayesian on the accuracy of GEBVs was investigated in 540 cross validation experiments with 100 replicates. The effect of kinship between the training and validation populations was tested in an additional set of 840 cross validation experiments with a single genomic prediction model. LD was high (average r2 = 0.59 at 25 kb and decreased slowly, distribution of allele frequencies at individual loci was markedly skewed toward unbalanced frequencies (MAF average value 15.2% and median 9.6%, and differentiation between the four synthetic populations was low (FST ≤0.06. The accuracy of GEBV across all cross validation experiments ranged from 0.12 to 0.54 with an average of 0.30. Significant differences in accuracy were observed among the different levels of each factor investigated. Phenotypic traits had the biggest effect, and the size of the incidence matrix had the smallest. Significant first degree interaction was observed for GEBV accuracy between traits and all the other factors studied, and between prediction models and LD, MAF and composition of the TP. The potential of GS to accelerate genetic gain and breeding options to increase the accuracy of predictions are discussed.

  1. Designing synthetic biology.

    Science.gov (United States)

    Agapakis, Christina M

    2014-03-21

    Synthetic biology is frequently defined as the application of engineering design principles to biology. Such principles are intended to streamline the practice of biological engineering, to shorten the time required to design, build, and test synthetic gene networks. This streamlining of iterative design cycles can facilitate the future construction of biological systems for a range of applications in the production of fuels, foods, materials, and medicines. The promise of these potential applications as well as the emphasis on design has prompted critical reflection on synthetic biology from design theorists and practicing designers from many fields, who can bring valuable perspectives to the discipline. While interdisciplinary connections between biologists and engineers have built synthetic biology via the science and the technology of biology, interdisciplinary collaboration with artists, designers, and social theorists can provide insight on the connections between technology and society. Such collaborations can open up new avenues and new principles for research and design, as well as shed new light on the challenging context-dependence-both biological and social-that face living technologies at many scales. This review is inspired by the session titled "Design and Synthetic Biology: Connecting People and Technology" at Synthetic Biology 6.0 and covers a range of literature on design practice in synthetic biology and beyond. Critical engagement with how design is used to shape the discipline opens up new possibilities for how we might design the future of synthetic biology.

  2. Genome engineering via TALENs and CRISPR/Cas9 systems: challenges and perspectives

    KAUST Repository

    Mahfouz, Magdy M.

    2014-09-24

    The ability to precisely modify genome sequence and regulate gene expression patterns in a site-specific manner holds much promise in plant biotechnology. Genome-engineering technologies that enable such highly specific and efficient modification are advancing with unprecedented pace. Transcription activator-like effectors (TALEs) provide customizable DNA-binding modules designed to bind to any sequence of interest. Thus, TALEs have been used as a DNA targeting module fused to functional domains for a variety of targeted genomic and epigenomic modifications. TALE nucleases (TALENs) have been used with much success across eukaryotic species to edit genomes. Recently, clustered regularly interspaced palindromic repeats (CRISPRs) that are used as guide RNAs for Cas9 nuclease-specific digestion has been introduced as a highly efficient DNA-targeting platform for genome editing and regulation. Here, we review the discovery, development and limitations of TALENs and CRIPSR/Cas9 systems as genome-engineering platforms in plants. We discuss the current questions, potential improvements and the development of the next-generation genome-editing platforms with an emphasis on producing designer plants to address the needs of agriculture and basic plant biology.

  3. Genome engineering via TALENs and CRISPR/Cas9 systems: challenges and perspectives

    KAUST Repository

    Mahfouz, Magdy M.; Piatek, Agnieszka Anna; Stewart, Charles Neal

    2014-01-01

    The ability to precisely modify genome sequence and regulate gene expression patterns in a site-specific manner holds much promise in plant biotechnology. Genome-engineering technologies that enable such highly specific and efficient modification are advancing with unprecedented pace. Transcription activator-like effectors (TALEs) provide customizable DNA-binding modules designed to bind to any sequence of interest. Thus, TALEs have been used as a DNA targeting module fused to functional domains for a variety of targeted genomic and epigenomic modifications. TALE nucleases (TALENs) have been used with much success across eukaryotic species to edit genomes. Recently, clustered regularly interspaced palindromic repeats (CRISPRs) that are used as guide RNAs for Cas9 nuclease-specific digestion has been introduced as a highly efficient DNA-targeting platform for genome editing and regulation. Here, we review the discovery, development and limitations of TALENs and CRIPSR/Cas9 systems as genome-engineering platforms in plants. We discuss the current questions, potential improvements and the development of the next-generation genome-editing platforms with an emphasis on producing designer plants to address the needs of agriculture and basic plant biology.

  4. Engineering Therapeutic T Cells: From Synthetic Biology to Clinical Trials.

    Science.gov (United States)

    Esensten, Jonathan H; Bluestone, Jeffrey A; Lim, Wendell A

    2017-01-24

    Engineered T cells are currently in clinical trials to treat patients with cancer, solid organ transplants, and autoimmune diseases. However, the field is still in its infancy. The design, and manufacturing, of T cell therapies is not standardized and is performed mostly in academic settings by competing groups. Reliable methods to define dose and pharmacokinetics of T cell therapies need to be developed. As of mid-2016, there are no US Food and Drug Administration (FDA)-approved T cell therapeutics on the market, and FDA regulations are only slowly adapting to the new technologies. Further development of engineered T cell therapies requires advances in immunology, synthetic biology, manufacturing processes, and government regulation. In this review, we outline some of these challenges and discuss the contributions that pathologists can make to this emerging field.

  5. Delivering the Goods for Genome Engineering and Editing.

    Science.gov (United States)

    Skipper, Kristian Alsbjerg; Mikkelsen, Jacob Giehm

    2015-08-01

    A basic understanding of genome evolution and the life and impact of microorganisms, like viruses and bacteria, has been fundamental in the quest for efficient genetic therapies. The expanding tool box for genetic engineering now contains transposases, recombinases, and nucleases, all created from naturally occurring genome-modifying proteins. Whereas conventional gene therapies have sought to establish sustained expression of therapeutic genes, genomic tools are needed only in a short time window and should be delivered to cells ideally in a balanced "hit-and-run" fashion. Current state-of-the-art delivery strategies are based on intracellular production of protein from transfected plasmid DNA or in vitro-transcribed RNA, or from transduced viral templates. Here, we discuss advantages and challenges of intracellular production strategies and describe emerging approaches based on the direct delivery of protein either by transfer of recombinant protein or by lentiviral protein transduction. With focus on adapting viruses for protein delivery, we describe the concept of "all-in-one" lentiviral particles engineered to codeliver effector proteins and donor sequences for DNA transposition or homologous recombination. With optimized delivery methods-based on transferring DNA, RNA, or protein-it is no longer far-fetched that researchers in the field will indeed deliver the goods for somatic gene therapies.

  6. Viral Cre-LoxP tools aid genome engineering in mammalian cells.

    Science.gov (United States)

    Sengupta, Ranjita; Mendenhall, Amy; Sarkar, Nandita; Mukherjee, Chandreyee; Afshari, Amirali; Huang, Joseph; Lu, Biao

    2017-01-01

    Targeted nucleases have transformed genome editing technology, providing more efficient methods to make targeted changes in mammalian genome. In parallel, there is an increasing demand of Cre-LoxP technology for complex genome manipulation such as large deletion, addition, gene fusion and conditional removal of gene sequences at the target site. However, an efficient and easy-to-use Cre-recombinase delivery system remains lacking. We designed and constructed two sets of expression vectors for Cre-recombinase using two highly efficient viral systems, the integrative lentivirus and non-integrative adeno associated virus. We demonstrate the effectiveness of those methods in Cre-delivery into stably-engineered HEK293 cells harboring LoxP-floxed red fluorescent protein (RFP) and puromycin (Puro) resistant reporters. The delivered Cre recombinase effectively excised the floxed RFP-Puro either directly or conditionally, therefore validating the function of these molecular tools. Given the convenient options of two selections markers, these viral-based systems offer a robust and easy-to-use tool for advanced genome editing, expanding complicated genome engineering to a variety of cell types and conditions. We have developed and functionally validated two viral-based Cre-recombinase delivery systems for efficient genome manipulation in various mammalian cells. The ease of gene delivery with the built-in reporters and inducible element enables live cell monitoring, drug selection and temporal knockout, broadening applications of genome editing.

  7. Combined protein construct and synthetic gene engineering for heterologous protein expression and crystallization using Gene Composer

    Directory of Open Access Journals (Sweden)

    Walchli John

    2009-04-01

    Full Text Available Abstract Background With the goal of improving yield and success rates of heterologous protein production for structural studies we have developed the database and algorithm software package Gene Composer. This freely available electronic tool facilitates the information-rich design of protein constructs and their engineered synthetic gene sequences, as detailed in the accompanying manuscript. Results In this report, we compare heterologous protein expression levels from native sequences to that of codon engineered synthetic gene constructs designed by Gene Composer. A test set of proteins including a human kinase (P38α, viral polymerase (HCV NS5B, and bacterial structural protein (FtsZ were expressed in both E. coli and a cell-free wheat germ translation system. We also compare the protein expression levels in E. coli for a set of 11 different proteins with greatly varied G:C content and codon bias. Conclusion The results consistently demonstrate that protein yields from codon engineered Gene Composer designs are as good as or better than those achieved from the synonymous native genes. Moreover, structure guided N- and C-terminal deletion constructs designed with the aid of Gene Composer can lead to greater success in gene to structure work as exemplified by the X-ray crystallographic structure determination of FtsZ from Bacillus subtilis. These results validate the Gene Composer algorithms, and suggest that using a combination of synthetic gene and protein construct engineering tools can improve the economics of gene to structure research.

  8. Synthetic constructs in/for the environment: managing the interplay between natural and engineered Biology.

    Science.gov (United States)

    Schmidt, Markus; de Lorenzo, Víctor

    2012-07-16

    The plausible release of deeply engineered or even entirely synthetic/artificial microorganisms raises the issue of their intentional (e.g. bioremediation) or accidental interaction with the Environment. Containment systems designed in the 1980s-1990s for limiting the spread of genetically engineered bacteria and their recombinant traits are still applicable to contemporary Synthetic Biology constructs. Yet, the ease of DNA synthesis and the uncertainty on how non-natural properties and strains could interplay with the existing biological word poses yet again the challenge of designing safe and efficacious firewalls to curtail possible interactions. Such barriers may include xeno-nucleic acids (XNAs) instead of DNA as information-bearing molecules, rewriting the genetic code to make it non-understandable by the existing gene expression machineries, and/or making growth dependent on xenobiotic chemicals. Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  9. Engineering propionibacteria as versatile cell factories for the production of industrially important chemicals: advances, challenges, and prospects.

    Science.gov (United States)

    Guan, Ningzi; Zhuge, Xin; Li, Jianghua; Shin, Hyun-Dong; Wu, Jing; Shi, Zhongping; Liu, Long

    2015-01-01

    Propionibacteria are actinobacteria consisting of two principal groups: cutaneous and dairy. Cutaneous propionibacteria are considered primary pathogens to humans, whereas dairy propionibacteria are widely used in the food and pharmaceutical industries. Increasing attention has been focused on improving the performance of dairy propionibacteria for the production of industrially important chemicals, and significant advances have been made through strain engineering and process optimization in the production of flavor compounds, nutraceuticals, and antimicrobial compounds. In addition, genome sequencing of several propionibacteria species has been completed, deepening understanding of the metabolic and physiological features of these organisms. However, the metabolic engineering of propionibacteria still faces several challenges owing to the lack of efficient genome manipulation tools and the existence of various types of strong restriction-modification systems. The emergence of systems and synthetic biology provides new opportunities to overcome these bottlenecks. In this review, we first introduce the major species of propionibacteria and their properties and provide an overview of their functions and applications. We then discuss advances in the genome sequencing and metabolic engineering of these bacteria. Finally, we discuss systems and synthetic biology approaches for engineering propionibacteria as efficient and robust cell factories for the production of industrially important chemicals.

  10. Synthetic addiction extends the productive life time of engineered Escherichia coli populations

    DEFF Research Database (Denmark)

    Rugbjerg, Peter; Sarup-Lytzen, Kira; Nagy, Mariann

    2018-01-01

    range of genetic variants that disrupt the biosynthetic capacity of the engineered organism. Synthetic product addiction that couples high-yield production of a desired metabolite to expression of nonconditionally essential genes could offer a solution to this problem by selectively favoring cells...... with biosynthetic capacity in the population without constraining the medium. We constructed such synthetic product addiction by controlling the expression of two nonconditionally essential genes with a mevalonic acid biosensor. The product-addicted production organism retained high-yield mevalonic acid production...... through 95 generations of cultivation, corresponding to the number of cell generations required for >200-m3 industrial-scale production, at which time the nonaddicted strain completely abolished production. Using deep DNA sequencing, we find that the product-addicted populations do not accumulate genetic...

  11. Genome engineering and parthenocloning in the silkworm, Bombyx ...

    Indian Academy of Sciences (India)

    Genetic engineering of the silkworm, Bombyx mori, opens door to the production of new kinds of silk and to the use of silkworms as proteosynthetic bioreactors. The insertion of foreign genes into silkworm genome and the control of their expression by diverse promoters have become possible but are not yet efficient enough ...

  12. Synthetic biology and regulatory networks: where metabolic systems biology meets control engineering.

    Science.gov (United States)

    He, Fei; Murabito, Ettore; Westerhoff, Hans V

    2016-04-01

    Metabolic pathways can be engineered to maximize the synthesis of various products of interest. With the advent of computational systems biology, this endeavour is usually carried out through in silico theoretical studies with the aim to guide and complement further in vitro and in vivo experimental efforts. Clearly, what counts is the result in vivo, not only in terms of maximal productivity but also robustness against environmental perturbations. Engineering an organism towards an increased production flux, however, often compromises that robustness. In this contribution, we review and investigate how various analytical approaches used in metabolic engineering and synthetic biology are related to concepts developed by systems and control engineering. While trade-offs between production optimality and cellular robustness have already been studied diagnostically and statically, the dynamics also matter. Integration of the dynamic design aspects of control engineering with the more diagnostic aspects of metabolic, hierarchical control and regulation analysis is leading to the new, conceptual and operational framework required for the design of robust and productive dynamic pathways. © 2016 The Author(s).

  13. Molecular Imaging in Synthetic Biology, and Synthetic Biology in Molecular Imaging.

    Science.gov (United States)

    Gilad, Assaf A; Shapiro, Mikhail G

    2017-06-01

    Biomedical synthetic biology is an emerging field in which cells are engineered at the genetic level to carry out novel functions with relevance to biomedical and industrial applications. This approach promises new treatments, imaging tools, and diagnostics for diseases ranging from gastrointestinal inflammatory syndromes to cancer, diabetes, and neurodegeneration. As these cellular technologies undergo pre-clinical and clinical development, it is becoming essential to monitor their location and function in vivo, necessitating appropriate molecular imaging strategies, and therefore, we have created an interest group within the World Molecular Imaging Society focusing on synthetic biology and reporter gene technologies. Here, we highlight recent advances in biomedical synthetic biology, including bacterial therapy, immunotherapy, and regenerative medicine. We then discuss emerging molecular imaging approaches to facilitate in vivo applications, focusing on reporter genes for noninvasive modalities such as magnetic resonance, ultrasound, photoacoustic imaging, bioluminescence, and radionuclear imaging. Because reporter genes can be incorporated directly into engineered genetic circuits, they are particularly well suited to imaging synthetic biological constructs, and developing them provides opportunities for creative molecular and genetic engineering.

  14. Programming languages for synthetic biology.

    Science.gov (United States)

    Umesh, P; Naveen, F; Rao, Chanchala Uma Maheswara; Nair, Achuthsankar S

    2010-12-01

    In the backdrop of accelerated efforts for creating synthetic organisms, the nature and scope of an ideal programming language for scripting synthetic organism in-silico has been receiving increasing attention. A few programming languages for synthetic biology capable of defining, constructing, networking, editing and delivering genome scale models of cellular processes have been recently attempted. All these represent important points in a spectrum of possibilities. This paper introduces Kera, a state of the art programming language for synthetic biology which is arguably ahead of similar languages or tools such as GEC, Antimony and GenoCAD. Kera is a full-fledged object oriented programming language which is tempered by biopart rule library named Samhita which captures the knowledge regarding the interaction of genome components and catalytic molecules. Prominent feature of the language are demonstrated through a toy example and the road map for the future development of Kera is also presented.

  15. Design, Optimization and Application of Small Molecule Biosensor in Metabolic Engineering.

    Science.gov (United States)

    Liu, Yang; Liu, Ye; Wang, Meng

    2017-01-01

    The development of synthetic biology and metabolic engineering has painted a great future for the bio-based economy, including fuels, chemicals, and drugs produced from renewable feedstocks. With the rapid advance of genome-scale modeling, pathway assembling and genome engineering/editing, our ability to design and generate microbial cell factories with various phenotype becomes almost limitless. However, our lack of ability to measure and exert precise control over metabolite concentration related phenotypes becomes a bottleneck in metabolic engineering. Genetically encoded small molecule biosensors, which provide the means to couple metabolite concentration to measurable or actionable outputs, are highly promising solutions to the bottleneck. Here we review recent advances in the design, optimization and application of small molecule biosensor in metabolic engineering, with particular focus on optimization strategies for transcription factor (TF) based biosensors.

  16. Site-Specific Genome Engineering in Human Pluripotent Stem Cells.

    Science.gov (United States)

    Merkert, Sylvia; Martin, Ulrich

    2016-06-24

    The possibility to generate patient-specific induced pluripotent stem cells (iPSCs) offers an unprecedented potential of applications in clinical therapy and medical research. Human iPSCs and their differentiated derivatives are tools for diseases modelling, drug discovery, safety pharmacology, and toxicology. Moreover, they allow for the engineering of bioartificial tissue and are promising candidates for cellular therapies. For many of these applications, the ability to genetically modify pluripotent stem cells (PSCs) is indispensable, but efficient site-specific and safe technologies for genetic engineering of PSCs were developed only recently. By now, customized engineered nucleases provide excellent tools for targeted genome editing, opening new perspectives for biomedical research and cellular therapies.

  17. High-Efficiency Genome Editing of Streptomyces Species by an Engineered CRISPR/Cas System.

    Science.gov (United States)

    Wang, Y; Cobb, R E; Zhao, H

    2016-01-01

    Next-generation sequencing technologies have rapidly expanded the genomic information of numerous organisms and revealed a rich reservoir of natural product gene clusters from microbial genomes, especially from Streptomyces, the largest genus of known actinobacteria at present. However, genetic engineering of these bacteria is often time consuming and labor intensive, if even possible. In this chapter, we describe the design and construction of pCRISPomyces, an engineered Type II CRISPR/Cas system, for targeted multiplex gene deletions in Streptomyces lividans, Streptomyces albus, and Streptomyces viridochromogenes with editing efficiency ranging from 70% to 100%. We demonstrate pCRISPomyces as a powerful tool for genome editing in Streptomyces. © 2016 Elsevier Inc. All rights reserved.

  18. A comprehensive overview of computational resources to aid in precision genome editing with engineered nucleases.

    Science.gov (United States)

    Periwal, Vinita

    2017-07-01

    Genome editing with engineered nucleases (zinc finger nucleases, TAL effector nucleases s and Clustered regularly inter-spaced short palindromic repeats/CRISPR-associated) has recently been shown to have great promise in a variety of therapeutic and biotechnological applications. However, their exploitation in genetic analysis and clinical settings largely depends on their specificity for the intended genomic target. Large and complex genomes often contain highly homologous/repetitive sequences, which limits the specificity of genome editing tools and could result in off-target activity. Over the past few years, various computational approaches have been developed to assist the design process and predict/reduce the off-target activity of these nucleases. These tools could be efficiently used to guide the design of constructs for engineered nucleases and evaluate results after genome editing. This review provides a comprehensive overview of various databases, tools, web servers and resources for genome editing and compares their features and functionalities. Additionally, it also describes tools that have been developed to analyse post-genome editing results. The article also discusses important design parameters that could be considered while designing these nucleases. This review is intended to be a quick reference guide for experimentalists as well as computational biologists working in the field of genome editing with engineered nucleases. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  19. Models for synthetic biology.

    Science.gov (United States)

    Kaznessis, Yiannis N

    2007-11-06

    Synthetic biological engineering is emerging from biology as a distinct discipline based on quantification. The technologies propelling synthetic biology are not new, nor is the concept of designing novel biological molecules. What is new is the emphasis on system behavior. The objective is the design and construction of new biological devices and systems to deliver useful applications. Numerous synthetic gene circuits have been created in the past decade, including bistable switches, oscillators, and logic gates, and possible applications abound, including biofuels, detectors for biochemical and chemical weapons, disease diagnosis, and gene therapies. More than fifty years after the discovery of the molecular structure of DNA, molecular biology is mature enough for real quantification that is useful for biological engineering applications, similar to the revolution in modeling in chemistry in the 1950s. With the excitement that synthetic biology is generating, the engineering and biological science communities appear remarkably willing to cross disciplinary boundaries toward a common goal.

  20. Metabolic engineering of microorganisms for biofuels production: from bugs to synthetic biology to fuels

    Energy Technology Data Exchange (ETDEWEB)

    Kuk Lee, Sung; Chou, Howard; Ham, Timothy S.; Soon Lee, Taek; Keasling, Jay D.

    2009-12-02

    The ability to generate microorganisms that can produce biofuels similar to petroleum-based transportation fuels would allow the use of existing engines and infrastructure and would save an enormous amount of capital required for replacing the current infrastructure to accommodate biofuels that have properties significantly different from petroleum-based fuels. Several groups have demonstrated the feasibility of manipulating microbes to produce molecules similar to petroleum-derived products, albeit at relatively low productivity (e.g. maximum butanol production is around 20 g/L). For cost-effective production of biofuels, the fuel-producing hosts and pathways must be engineered and optimized. Advances in metabolic engineering and synthetic biology will provide new tools for metabolic engineers to better understand how to rewire the cell in order to create the desired phenotypes for the production of economically viable biofuels.

  1. Metabolic Engineering for Probiotics and their Genome-Wide Expression Profiling.

    Science.gov (United States)

    Yadav, Ruby; Singh, Puneet K; Shukla, Pratyoosh

    2018-01-01

    Probiotic supplements in food industry have attracted a lot of attention and shown a remarkable growth in this field. Metabolic engineering (ME) approaches enable understanding their mechanism of action and increases possibility of designing probiotic strains with desired functions. Probiotic microorganisms generally referred as industrially important lactic acid bacteria (LAB) which are involved in fermenting dairy products, food, beverages and produces lactic acid as final product. A number of illustrations of metabolic engineering approaches in industrial probiotic bacteria have been described in this review including transcriptomic studies of Lactobacillus reuteri and improvement in exopolysaccharide (EPS) biosynthesis yield in Lactobacillus casei LC2W. This review summaries various metabolic engineering approaches for exploring metabolic pathways. These approaches enable evaluation of cellular metabolic state and effective editing of microbial genome or introduction of novel enzymes to redirect the carbon fluxes. In addition, various system biology tools such as in silico design commonly used for improving strain performance is also discussed. Finally, we discuss the integration of metabolic engineering and genome profiling which offers a new way to explore metabolic interactions, fluxomics and probiogenomics using probiotic bacteria like Bifidobacterium spp and Lactobacillus spp. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  2. Gene Composer in a structural genomics environment

    International Nuclear Information System (INIS)

    Lorimer, Don; Raymond, Amy; Mixon, Mark; Burgin, Alex; Staker, Bart; Stewart, Lance

    2011-01-01

    For structural biology applications, protein-construct engineering is guided by comparative sequence analysis and structural information, which allow the researcher to better define domain boundaries for terminal deletions and nonconserved regions for surface mutants. A database software application called Gene Composer has been developed to facilitate construct design. The structural genomics effort at the Seattle Structural Genomics Center for Infectious Disease (SSGCID) requires the manipulation of large numbers of amino-acid sequences and the underlying DNA sequences which are to be cloned into expression vectors. To improve efficiency in high-throughput protein structure determination, a database software package, Gene Composer, has been developed which facilitates the information-rich design of protein constructs and their underlying gene sequences. With its modular workflow design and numerous graphical user interfaces, Gene Composer enables researchers to perform all common bioinformatics steps used in modern structure-guided protein engineering and synthetic gene engineering. An example of the structure determination of H1N1 RNA-dependent RNA polymerase PB2 subunit is given

  3. Accuracy of Genomic Prediction in Synthetic Populations Depending on the Number of Parents, Relatedness, and Ancestral Linkage Disequilibrium.

    Science.gov (United States)

    Schopp, Pascal; Müller, Dominik; Technow, Frank; Melchinger, Albrecht E

    2017-01-01

    Synthetics play an important role in quantitative genetic research and plant breeding, but few studies have investigated the application of genomic prediction (GP) to these populations. Synthetics are generated by intermating a small number of parents ([Formula: see text] and thereby possess unique genetic properties, which make them especially suited for systematic investigations of factors contributing to the accuracy of GP. We generated synthetics in silico from [Formula: see text]2 to 32 maize (Zea mays L.) lines taken from an ancestral population with either short- or long-range linkage disequilibrium (LD). In eight scenarios differing in relatedness of the training and prediction sets and in the types of data used to calculate the relationship matrix (QTL, SNPs, tag markers, and pedigree), we investigated the prediction accuracy (PA) of Genomic best linear unbiased prediction (GBLUP) and analyzed contributions from pedigree relationships captured by SNP markers, as well as from cosegregation and ancestral LD between QTL and SNPs. The effects of training set size [Formula: see text] and marker density were also studied. Sampling few parents ([Formula: see text]) generates substantial sample LD that carries over into synthetics through cosegregation of alleles at linked loci. For fixed [Formula: see text], [Formula: see text] influences PA most strongly. If the training and prediction set are related, using [Formula: see text] parents yields high PA regardless of ancestral LD because SNPs capture pedigree relationships and Mendelian sampling through cosegregation. As [Formula: see text] increases, ancestral LD contributes more information, while other factors contribute less due to lower frequencies of closely related individuals. For unrelated prediction sets, only ancestral LD contributes information and accuracies were poor and highly variable for [Formula: see text] due to large sample LD. For large [Formula: see text], achieving moderate accuracy requires

  4. Toward design-based engineering of industrial microbes.

    Science.gov (United States)

    Tyo, Keith E J; Kocharin, Kanokarn; Nielsen, Jens

    2010-06-01

    Engineering industrial microbes has been hampered by incomplete knowledge of cell biology. Thus an iterative engineering cycle of modeling, implementation, and analysis has been used to increase knowledge of the underlying biology while achieving engineering goals. Recent advances in Systems Biology technologies have drastically improved the amount of information that can be collected in each iteration. As well, Synthetic Biology tools are melding modeling and molecular implementation. These advances promise to move microbial engineering from the iterative approach to a design-oriented paradigm, similar to electrical circuits and architectural design. Genome-scale metabolic models, new tools for controlling expression, and integrated -omics analysis are described as key contributors in moving the field toward Design-based Engineering. Copyright 2010 Elsevier Ltd. All rights reserved.

  5. The contribution of bacterial genome engineering to sustainable development.

    Science.gov (United States)

    Reuß, Daniel R; Commichau, Fabian M; Stülke, Jörg

    2017-09-01

    The United Nations' Sustainable Development Goals define important challenges for the prosperous development of mankind. To reach several of these goals, among them the production of value-added compounds, improved economic and ecologic balance of production processes, prevention of climate change and protection of ecosystems, the use of engineered bacteria can make valuable contributions. We discuss the strategies for genome engineering and how they can be applied to meet the United Nations' goals for sustainable development. © 2017 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

  6. The Future of Metabolic Engineering and Synthetic Biology: Towards a Systematic Practice

    Science.gov (United States)

    Yadav, Vikramaditya G.; De Mey, Marjan; Lim, Chin Giaw; Ajikumar, Parayil Kumaran; Stephanopoulos, Gregory

    2012-01-01

    Industrial biotechnology promises to revolutionize conventional chemical manufacturing in the years ahead, largely owing to the excellent progress in our ability to re-engineer cellular metabolism. However, most successes of metabolic engineering have been confined to over-producing natively synthesized metabolites in E. coli and S. cerevisiae. A major reason for this development has been the descent of metabolic engineering, particularly secondary metabolic engineering, to a collection of demonstrations rather than a systematic practice with generalizable tools. Synthetic biology, a more recent development, faces similar criticisms. Herein, we attempt to lay down a framework around which bioreaction engineering can systematize itself just like chemical reaction engineering. Central to this undertaking is a new approach to engineering secondary metabolism known as ‘multivariate modular metabolic engineering’ (MMME), whose novelty lies in its assessment and elimination of regulatory and pathway bottlenecks by re-defining the metabolic network as a collection of distinct modules. After introducing the core principles of MMME, we shall then present a number of recent developments in secondary metabolic engineering that could potentially serve as its facilitators. It is hoped that the ever-declining costs of de novo gene synthesis; the improved use of bioinformatic tools to mine, sort and analyze biological data; and the increasing sensitivity and sophistication of investigational tools will make the maturation of microbial metabolic engineering an autocatalytic process. Encouraged by these advances, research groups across the world would take up the challenge of secondary metabolite production in simple hosts with renewed vigor, thereby adding to the range of products synthesized using metabolic engineering. PMID:22629571

  7. Genome-wide analytical approaches for reverse metabolic engineering of industrially relevant phenotypes in yeast

    Science.gov (United States)

    Oud, Bart; Maris, Antonius J A; Daran, Jean-Marc; Pronk, Jack T

    2012-01-01

    Successful reverse engineering of mutants that have been obtained by nontargeted strain improvement has long presented a major challenge in yeast biotechnology. This paper reviews the use of genome-wide approaches for analysis of Saccharomyces cerevisiae strains originating from evolutionary engineering or random mutagenesis. On the basis of an evaluation of the strengths and weaknesses of different methods, we conclude that for the initial identification of relevant genetic changes, whole genome sequencing is superior to other analytical techniques, such as transcriptome, metabolome, proteome, or array-based genome analysis. Key advantages of this technique over gene expression analysis include the independency of genome sequences on experimental context and the possibility to directly and precisely reproduce the identified changes in naive strains. The predictive value of genome-wide analysis of strains with industrially relevant characteristics can be further improved by classical genetics or simultaneous analysis of strains derived from parallel, independent strain improvement lineages. PMID:22152095

  8. Genome-wide analytical approaches for reverse metabolic engineering of industrially relevant phenotypes in yeast.

    Science.gov (United States)

    Oud, Bart; van Maris, Antonius J A; Daran, Jean-Marc; Pronk, Jack T

    2012-03-01

    Successful reverse engineering of mutants that have been obtained by nontargeted strain improvement has long presented a major challenge in yeast biotechnology. This paper reviews the use of genome-wide approaches for analysis of Saccharomyces cerevisiae strains originating from evolutionary engineering or random mutagenesis. On the basis of an evaluation of the strengths and weaknesses of different methods, we conclude that for the initial identification of relevant genetic changes, whole genome sequencing is superior to other analytical techniques, such as transcriptome, metabolome, proteome, or array-based genome analysis. Key advantages of this technique over gene expression analysis include the independency of genome sequences on experimental context and the possibility to directly and precisely reproduce the identified changes in naive strains. The predictive value of genome-wide analysis of strains with industrially relevant characteristics can be further improved by classical genetics or simultaneous analysis of strains derived from parallel, independent strain improvement lineages. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  9. Synthetic biology stretching the realms of possibility in wine yeast research.

    Science.gov (United States)

    Jagtap, Umesh B; Jadhav, Jyoti P; Bapat, Vishwas A; Pretorius, Isak S

    2017-07-03

    It took several millennia to fully understand the scientific intricacies of the process through which grape juice is turned into wine. This yeast-driven fermentation process is still being perfected and advanced today. Motivated by ever-changing consumer preferences and the belief that the 'best' wine is yet to be made, numerous approaches are being pursued to improve the process of yeast fermentation and the quality of wine. Central to recent enhancements in winemaking processes and wine quality is the development of Saccharomyces cerevisiae yeast strains with improved robustness, fermentation efficiencies and sensory properties. The emerging science of Synthetic Biology - including genome engineering and DNA editing technologies - is taking yeast strain development into a totally new realm of possibility. The first example of how future wine strain development might be impacted by these new 'history-making' Synthetic Biology technologies, is the de novo production of the raspberry ketone aroma compound, 4-[4-hydroxyphenyl]butan-2-one, in a wine yeast containing a synthetic DNA cassette. This article explores how this breakthrough and the imminent outcome of the international Yeast 2.0 (or Sc2.0) project, aimed at the synthesis of the entire genome of a laboratory strain of S. cerevisiae, might accelerate the design of improved wine yeasts. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. An evaluation of multiple annealing and looping based genome amplification using a synthetic bacterial community

    KAUST Repository

    Wang, Yong

    2016-02-23

    The low biomass in environmental samples is a major challenge for microbial metagenomic studies. The amplification of a genomic DNA was frequently applied to meeting the minimum requirement of the DNA for a high-throughput next-generation-sequencing technology. Using a synthetic bacterial community, the amplification efficiency of the Multiple Annealing and Looping Based Amplification Cycles (MALBAC) kit that is originally developed to amplify the single-cell genomic DNA of mammalian organisms is examined. The DNA template of 10 pg in each reaction of the MALBAC amplification may generate enough DNA for Illumina sequencing. Using 10 pg and 100 pg templates for each reaction set, the MALBAC kit shows a stable and homogeneous amplification as indicated by the highly consistent coverage of the reads from the two amplified samples on the contigs assembled by the original unamplified sample. Although GenomePlex whole genome amplification kit allows one to generate enough DNA using 100 pg of template in each reaction, the minority of the mixed bacterial species is not linearly amplified. For both of the kits, the GC-rich regions of the genomic DNA are not efficiently amplified as suggested by the low coverage of the contigs with the high GC content. The high efficiency of the MALBAC kit is supported for the amplification of environmental microbial DNA samples, and the concerns on its application are also raised to bacterial species with the high GC content.

  11. CRISPR-cas System as a Genome Engineering Platform: Applications in Biomedicine and Biotechnology.

    Science.gov (United States)

    Hashemi, Atieh

    2018-01-01

    Genome editing mediated by Clustered Regularly Interspaced Palindromic Repeats (CRISPR) and its associated proteins (Cas) has recently been considered to be used as efficient, rapid and site-specific tool in the modification of endogenous genes in biomedically important cell types and whole organisms. It has become a predictable and precise method of choice for genome engineering by specifying a 20-nt targeting sequence within its guide RNA. Firstly, this review aims to describe the biology of CRISPR system. Next, the applications of CRISPR-Cas9 in various ways, such as efficient generation of a wide variety of biomedically important cellular models as well as those of animals, modifying epigenomes, conducting genome-wide screens, gene therapy, labelling specific genomic loci in living cells, metabolic engineering of yeast and bacteria and endogenous gene expression regulation by an altered version of this system were reviewed. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  12. GENOME-BASED MODELING AND DESIGN OF METABOLIC INTERACTIONS IN MICROBIAL COMMUNITIES

    Directory of Open Access Journals (Sweden)

    Radhakrishnan Mahadevan

    2012-10-01

    With the advent of genome sequencing, omics technologies, bioinformatics and genome-scale modeling, researchers now have unprecedented capabilities to analyze and engineer the metabolism of microbial communities. The goal of this review is to summarize recent applications of genome-scale metabolic modeling to microbial communities. A brief introduction to lumped community models is used to motivate the need for genome-level descriptions of individual species and their metabolic interactions. The review of genome-scale models begins with static modeling approaches, which are appropriate for communities where the extracellular environment can be assumed to be time invariant or slowly varying. Dynamic extensions of the static modeling approach are described, and then applications of genome-scale models for design of synthetic microbial communities are reviewed. The review concludes with a summary of metagenomic tools for analyzing community metabolism and an outlook for future research.

  13. CRISPR-Cas9 for the genome engineering of cyanobacteria and succinate production.

    Science.gov (United States)

    Li, Hung; Shen, Claire R; Huang, Chun-Hung; Sung, Li-Yu; Wu, Meng-Ying; Hu, Yu-Chen

    2016-11-01

    Cyanobacteria hold promise as a cell factory for producing biofuels and bio-derived chemicals, but genome engineering of cyanobacteria such as Synechococcus elongatus PCC 7942 poses challenges because of their oligoploidy nature and long-term instability of the introduced gene. CRISPR-Cas9 is a newly developed RNA-guided genome editing system, yet its application for cyanobacteria engineering has yet to be reported. Here we demonstrated that CRISPR-Cas9 system can effectively trigger programmable double strand break (DSB) at the chromosome of PCC 7942 and provoke cell death. With the co-transformation of template plasmid harboring the gene cassette and flanking homology arms, CRISPR-Cas9-mediated DSB enabled precise gene integration, ameliorated the homologous recombination efficiency and allowed the use of lower amount of template DNA and shorter homology arms. The CRISPR-Cas9-induced cell death imposed selective pressure and enhanced the chance of concomitant integration of gene cassettes into all chromosomes of PCC 7942, hence accelerating the process of obtaining homogeneous and stable recombinant strains. We further explored the feasibility of engineering cyanobacteria by CRISPR-Cas9-assisted simultaneous glgc knock-out and gltA/ppc knock-in, which improved the succinate titer to 435.0±35.0μg/L, an ≈11-fold increase when compared with that of the wild-type cells. These data altogether justify the use of CRISPR-Cas9 for genome engineering and manipulation of metabolic pathways in cyanobacteria. Copyright © 2016 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

  14. Under pressure: evolutionary engineering of yeast strains for improved performance in fuels and chemicals production.

    Science.gov (United States)

    Mans, Robert; Daran, Jean-Marc G; Pronk, Jack T

    2018-04-01

    Evolutionary engineering, which uses laboratory evolution to select for industrially relevant traits, is a popular strategy in the development of high-performing yeast strains for industrial production of fuels and chemicals. By integrating whole-genome sequencing, bioinformatics, classical genetics and genome-editing techniques, evolutionary engineering has also become a powerful approach for identification and reverse engineering of molecular mechanisms that underlie industrially relevant traits. New techniques enable acceleration of in vivo mutation rates, both across yeast genomes and at specific loci. Recent studies indicate that phenotypic trade-offs, which are often observed after evolution under constant conditions, can be mitigated by using dynamic cultivation regimes. Advances in research on synthetic regulatory circuits offer exciting possibilities to extend the applicability of evolutionary engineering to products of yeasts whose synthesis requires a net input of cellular energy. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  15. Synthetic biology: programming cells for biomedical applications.

    Science.gov (United States)

    Hörner, Maximilian; Reischmann, Nadine; Weber, Wilfried

    2012-01-01

    The emerging field of synthetic biology is a novel biological discipline at the interface between traditional biology, chemistry, and engineering sciences. Synthetic biology aims at the rational design of complex synthetic biological devices and systems with desired properties by combining compatible, modular biological parts in a systematic manner. While the first engineered systems were mainly proof-of-principle studies to demonstrate the power of the modular engineering approach of synthetic biology, subsequent systems focus on applications in the health, environmental, and energy sectors. This review describes recent approaches for biomedical applications that were developed along the synthetic biology design hierarchy, at the level of individual parts, of devices, and of complex multicellular systems. It describes how synthetic biological parts can be used for the synthesis of drug-delivery tools, how synthetic biological devices can facilitate the discovery of novel drugs, and how multicellular synthetic ecosystems can give insight into population dynamics of parasites and hosts. These examples demonstrate how this new discipline could contribute to novel solutions in the biopharmaceutical industry.

  16. Creating biological nanomaterials using synthetic biology

    International Nuclear Information System (INIS)

    Rice, MaryJoe K; Ruder, Warren C

    2014-01-01

    Synthetic biology is a new discipline that combines science and engineering approaches to precisely control biological networks. These signaling networks are especially important in fields such as biomedicine and biochemical engineering. Additionally, biological networks can also be critical to the production of naturally occurring biological nanomaterials, and as a result, synthetic biology holds tremendous potential in creating new materials. This review introduces the field of synthetic biology, discusses how biological systems naturally produce materials, and then presents examples and strategies for incorporating synthetic biology approaches in the development of new materials. In particular, strategies for using synthetic biology to produce both organic and inorganic nanomaterials are discussed. Ultimately, synthetic biology holds the potential to dramatically impact biological materials science with significant potential applications in medical systems. (review)

  17. Creating biological nanomaterials using synthetic biology.

    Science.gov (United States)

    Rice, MaryJoe K; Ruder, Warren C

    2014-02-01

    Synthetic biology is a new discipline that combines science and engineering approaches to precisely control biological networks. These signaling networks are especially important in fields such as biomedicine and biochemical engineering. Additionally, biological networks can also be critical to the production of naturally occurring biological nanomaterials, and as a result, synthetic biology holds tremendous potential in creating new materials. This review introduces the field of synthetic biology, discusses how biological systems naturally produce materials, and then presents examples and strategies for incorporating synthetic biology approaches in the development of new materials. In particular, strategies for using synthetic biology to produce both organic and inorganic nanomaterials are discussed. Ultimately, synthetic biology holds the potential to dramatically impact biological materials science with significant potential applications in medical systems.

  18. The future of metabolic engineering and synthetic biology: towards a systematic practice.

    Science.gov (United States)

    Yadav, Vikramaditya G; De Mey, Marjan; Lim, Chin Giaw; Ajikumar, Parayil Kumaran; Stephanopoulos, Gregory

    2012-05-01

    Industrial biotechnology promises to revolutionize conventional chemical manufacturing in the years ahead, largely owing to the excellent progress in our ability to re-engineer cellular metabolism. However, most successes of metabolic engineering have been confined to over-producing natively synthesized metabolites in E. coli and S. cerevisiae. A major reason for this development has been the descent of metabolic engineering, particularly secondary metabolic engineering, to a collection of demonstrations rather than a systematic practice with generalizable tools. Synthetic biology, a more recent development, faces similar criticisms. Herein, we attempt to lay down a framework around which bioreaction engineering can systematize itself just like chemical reaction engineering. Central to this undertaking is a new approach to engineering secondary metabolism known as 'multivariate modular metabolic engineering' (MMME), whose novelty lies in its assessment and elimination of regulatory and pathway bottlenecks by re-defining the metabolic network as a collection of distinct modules. After introducing the core principles of MMME, we shall then present a number of recent developments in secondary metabolic engineering that could potentially serve as its facilitators. It is hoped that the ever-declining costs of de novo gene synthesis; the improved use of bioinformatic tools to mine, sort and analyze biological data; and the increasing sensitivity and sophistication of investigational tools will make the maturation of microbial metabolic engineering an autocatalytic process. Encouraged by these advances, research groups across the world would take up the challenge of secondary metabolite production in simple hosts with renewed vigor, thereby adding to the range of products synthesized using metabolic engineering. Copyright © 2011 Elsevier Inc. All rights reserved.

  19. Synthetic Biology of Cyanobacteria: Unique Challenges and Opportunities

    Directory of Open Access Journals (Sweden)

    Bertram M Berla

    2013-08-01

    Full Text Available Photosynthetic organisms, and especially cyanobacteria, hold great promise as sources of renewably-produced fuels, bulk and specialty chemicals, and nutritional products. Synthetic biology tools can help unlock cyanobacteria’s potential for these functions, but unfortunately tool development for these organisms has lagged behind that for S. cerevisiae and E. coli. While these organisms may in many cases be more difficult to work with as ‘chassis’ strains for synthetic biology than certain heterotrophs, the unique advantages of autotrophs in biotechnology applications as well as the scientific importance of improved understanding of photosynthesis warrant the development of these systems into something akin to a ‘green E. coli’. In this review, we highlight unique challenges and opportunities for development of synthetic biology approaches in cyanobacteria. We review classical and recently developed methods for constructing targeted mutants in various cyanobacterial strains, and offer perspective on what genetic tools might most greatly expand the ability to engineer new functions in such strains. Similarly, we review what genetic parts are most needed for the development of cyanobacterial synthetic biology. Finally, we highlight recent methods to construct genome-scale models of cyanobacterial metabolism and to use those models to measure properties of autotrophic metabolism. Throughout this paper, we discuss some of the unique challenges of a diurnal, autotrophic lifestyle along with how the development of synthetic biology and biotechnology in cyanobacteria must fit within those constraints.

  20. Impact of synthetic biology and metabolic engineering on industrial production of fine chemicals

    DEFF Research Database (Denmark)

    Jullesson, David; David, Florian; Pfleger, Brian

    2015-01-01

    Industrial bio-processes for fine chemical production are increasingly relying on cell factories developed through metabolic engineering and synthetic biology. The use of high throughput techniques and automation for the design of cell factories, and especially platform strains, has played...... chemicals that have reached the market, key metabolic engineering tools that have allowed this to happen and some of the companies that are currently utilizing these technologies for developing industrial production processes....... an important role in the transition from laboratory research to industrial production. Model organisms such as Saccharomyces cerevisiae and Escherichia coli remain widely used host strains for industrial production due to their robust and desirable traits. This review describes some of the bio-based fine...

  1. Merlin: Computer-Aided Oligonucleotide Design for Large Scale Genome Engineering with MAGE.

    Science.gov (United States)

    Quintin, Michael; Ma, Natalie J; Ahmed, Samir; Bhatia, Swapnil; Lewis, Aaron; Isaacs, Farren J; Densmore, Douglas

    2016-06-17

    Genome engineering technologies now enable precise manipulation of organism genotype, but can be limited in scalability by their design requirements. Here we describe Merlin ( http://merlincad.org ), an open-source web-based tool to assist biologists in designing experiments using multiplex automated genome engineering (MAGE). Merlin provides methods to generate pools of single-stranded DNA oligonucleotides (oligos) for MAGE experiments by performing free energy calculation and BLAST scoring on a sliding window spanning the targeted site. These oligos are designed not only to improve recombination efficiency, but also to minimize off-target interactions. The application further assists experiment planning by reporting predicted allelic replacement rates after multiple MAGE cycles, and enables rapid result validation by generating primer sequences for multiplexed allele-specific colony PCR. Here we describe the Merlin oligo and primer design procedures and validate their functionality compared to OptMAGE by eliminating seven AvrII restriction sites from the Escherichia coli genome.

  2. Is synthetic biology mechanical biology?

    Science.gov (United States)

    Holm, Sune

    2015-12-01

    A widespread and influential characterization of synthetic biology emphasizes that synthetic biology is the application of engineering principles to living systems. Furthermore, there is a strong tendency to express the engineering approach to organisms in terms of what seems to be an ontological claim: organisms are machines. In the paper I investigate the ontological and heuristic significance of the machine analogy in synthetic biology. I argue that the use of the machine analogy and the aim of producing rationally designed organisms does not necessarily imply a commitment to mechanical biology. The ideal of applying engineering principles to biology is best understood as expressing recognition of the machine-unlikeness of natural organisms and the limits of human cognition. The paper suggests an interpretation of the identification of organisms with machines in synthetic biology according to which it expresses a strategy for representing, understanding, and constructing living systems that are more machine-like than natural organisms.

  3. Engineering of synthetic, stress-responsive yeast promoters

    DEFF Research Database (Denmark)

    Rajkumar, Arun Stephen; Liu, Guodong; Bergenholm, David

    2016-01-01

    Advances in synthetic biology and our understanding of the rules of promoter architecture have led to the development of diverse synthetic constitutive and inducible promoters in eukaryotes and prokaryotes. However, the design of promoters inducibleby specific endogenous or environmental conditions...

  4. Synthetic Genetic Targeting of Genome Instability in Cancer

    International Nuclear Information System (INIS)

    Sajesh, Babu V.; Guppy, Brent J.; McManus, Kirk J.

    2013-01-01

    Cancer is a leading cause of death throughout the World. A limitation of many current chemotherapeutic approaches is that their cytotoxic effects are not restricted to cancer cells, and adverse side effects can occur within normal tissues. Consequently, novel strategies are urgently needed to better target cancer cells. As we approach the era of personalized medicine, targeting the specific molecular defect(s) within a given patient’s tumor will become a more effective treatment strategy than traditional approaches that often target a given cancer type or sub-type. Synthetic genetic interactions are now being examined for their therapeutic potential and are designed to target the specific genetic and epigenetic phenomena associated with tumor formation, and thus are predicted to be highly selective. In general, two complementary approaches have been employed, including synthetic lethality and synthetic dosage lethality, to target aberrant expression and/or function associated with tumor suppressor genes and oncogenes, respectively. Here we discuss the concepts of synthetic lethality and synthetic dosage lethality, and explain three general experimental approaches designed to identify novel genetic interactors. We present examples and discuss the merits and caveats of each approach. Finally, we provide insight into the subsequent pre-clinical work required to validate novel candidate drug targets

  5. Blueprints for green biotech: development and application of standards for plant synthetic biology.

    Science.gov (United States)

    Patron, Nicola J

    2016-06-15

    Synthetic biology aims to apply engineering principles to the design and modification of biological systems and to the construction of biological parts and devices. The ability to programme cells by providing new instructions written in DNA is a foundational technology of the field. Large-scale de novo DNA synthesis has accelerated synthetic biology by offering custom-made molecules at ever decreasing costs. However, for large fragments and for experiments in which libraries of DNA sequences are assembled in different combinations, assembly in the laboratory is still desirable. Biological assembly standards allow DNA parts, even those from multiple laboratories and experiments, to be assembled together using the same reagents and protocols. The adoption of such standards for plant synthetic biology has been cohesive for the plant science community, facilitating the application of genome editing technologies to plant systems and streamlining progress in large-scale, multi-laboratory bioengineering projects. © 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.

  6. Impact of synthetic biology and metabolic engineering on industrial production of fine chemicals.

    Science.gov (United States)

    Jullesson, David; David, Florian; Pfleger, Brian; Nielsen, Jens

    2015-11-15

    Industrial bio-processes for fine chemical production are increasingly relying on cell factories developed through metabolic engineering and synthetic biology. The use of high throughput techniques and automation for the design of cell factories, and especially platform strains, has played an important role in the transition from laboratory research to industrial production. Model organisms such as Saccharomyces cerevisiae and Escherichia coli remain widely used host strains for industrial production due to their robust and desirable traits. This review describes some of the bio-based fine chemicals that have reached the market, key metabolic engineering tools that have allowed this to happen and some of the companies that are currently utilizing these technologies for developing industrial production processes. Copyright © 2015 Elsevier Inc. All rights reserved.

  7. CRISPR-Cas: From the Bacterial Adaptive Immune System to a Versatile Tool for Genome Engineering.

    Science.gov (United States)

    Kirchner, Marion; Schneider, Sabine

    2015-11-09

    The field of biology has been revolutionized by the recent advancement of an adaptive bacterial immune system as a universal genome engineering tool. Bacteria and archaea use repetitive genomic elements termed clustered regularly interspaced short palindromic repeats (CRISPR) in combination with an RNA-guided nuclease (CRISPR-associated nuclease: Cas) to target and destroy invading DNA. By choosing the appropriate sequence of the guide RNA, this two-component system can be used to efficiently modify, target, and edit genomic loci of interest in plants, insects, fungi, mammalian cells, and whole organisms. This has opened up new frontiers in genome engineering, including the potential to treat or cure human genetic disorders. Now the potential risks as well as the ethical, social, and legal implications of this powerful new technique move into the limelight. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. From engineering to editing the rat genome.

    Science.gov (United States)

    Meek, Stephen; Mashimo, Tomoji; Burdon, Tom

    2017-08-01

    Since its domestication over 100 years ago, the laboratory rat has been the preferred experimental animal in many areas of biomedical research (Lindsey and Baker The laboratory rat. Academic, New York, pp 1-52, 2006). Its physiology, size, genetics, reproductive cycle, cognitive and behavioural characteristics have made it a particularly useful animal model for studying many human disorders and diseases. Indeed, through selective breeding programmes numerous strains have been derived that are now the mainstay of research on hypertension, obesity and neurobiology (Okamoto and Aoki Jpn Circ J 27:282-293, 1963; Zucker and Zucker J Hered 52(6):275-278, 1961). Despite this wealth of genetic and phenotypic diversity, the ability to manipulate and interrogate the genetic basis of existing phenotypes in rat strains and the methodology to generate new rat models has lagged significantly behind the advances made with its close cousin, the laboratory mouse. However, recent technical developments in stem cell biology and genetic engineering have again brought the rat to the forefront of biomedical studies and enabled researchers to exploit the increasingly accessible wealth of genome sequence information. In this review, we will describe how a breakthrough in understanding the molecular basis of self-renewal of the pluripotent founder cells of the mammalian embryo, embryonic stem (ES) cells, enabled the derivation of rat ES cells and their application in transgenesis. We will also describe the remarkable progress that has been made in the development of gene editing enzymes that enable the generation of transgenic rats directly through targeted genetic modifications in the genomes of zygotes. The simplicity, efficiency and cost-effectiveness of the CRISPR/Cas gene editing system, in particular, mean that the ability to engineer the rat genome is no longer a limiting factor. The selection of suitable targets and gene modifications will now become a priority: a challenge where

  9. High molecular weight DNA assembly in vivo for synthetic biology applications.

    Science.gov (United States)

    Juhas, Mario; Ajioka, James W

    2017-05-01

    DNA assembly is the key technology of the emerging interdisciplinary field of synthetic biology. While the assembly of smaller DNA fragments is usually performed in vitro, high molecular weight DNA molecules are assembled in vivo via homologous recombination in the host cell. Escherichia coli, Bacillus subtilis and Saccharomyces cerevisiae are the main hosts used for DNA assembly in vivo. Progress in DNA assembly over the last few years has paved the way for the construction of whole genomes. This review provides an update on recent synthetic biology advances with particular emphasis on high molecular weight DNA assembly in vivo in E. coli, B. subtilis and S. cerevisiae. Special attention is paid to the assembly of whole genomes, such as those of the first synthetic cell, synthetic yeast and minimal genomes.

  10. Synthetic Biology Approaches to Engineer Probiotics and Members of the Human Microbiota for Biomedical Applications.

    Science.gov (United States)

    Bober, Josef R; Beisel, Chase L; Nair, Nikhil U

    2018-03-12

    An increasing number of studies have strongly correlated the composition of the human microbiota with many human health conditions and, in several cases, have shown that manipulating the microbiota directly affects health. These insights have generated significant interest in engineering indigenous microbiota community members and nonresident probiotic bacteria as biotic diagnostics and therapeutics that can probe and improve human health. In this review, we discuss recent advances in synthetic biology to engineer commensal and probiotic lactic acid bacteria, bifidobacteria, and Bacteroides for these purposes, and we provide our perspective on the future potential of these technologies. 277 Expected final online publication date for the Annual Review of Biomedical Engineering Volume 20 is June 4, 2018. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

  11. Synthetic Biology for Specialty Chemicals.

    Science.gov (United States)

    Markham, Kelly A; Alper, Hal S

    2015-01-01

    In this review, we address recent advances in the field of synthetic biology and describe how those tools have been applied to produce a wide variety of chemicals in microorganisms. Here we classify the expansion of the synthetic biology toolbox into three different categories based on their primary function in strain engineering-for design, for construction, and for optimization. Next, focusing on recent years, we look at how chemicals have been produced using these new synthetic biology tools. Advances in producing fuels are briefly described, followed by a more thorough treatment of commodity chemicals, specialty chemicals, pharmaceuticals, and nutraceuticals. Throughout this review, an emphasis is placed on how synthetic biology tools are applied to strain engineering. Finally, we discuss organism and host strain diversity and provide a future outlook in the field.

  12. Molecular Cloning Designer Simulator (MCDS: All-in-one molecular cloning and genetic engineering design, simulation and management software for complex synthetic biology and metabolic engineering projects

    Directory of Open Access Journals (Sweden)

    Zhenyu Shi

    2016-12-01

    Full Text Available Molecular Cloning Designer Simulator (MCDS is a powerful new all-in-one cloning and genetic engineering design, simulation and management software platform developed for complex synthetic biology and metabolic engineering projects. In addition to standard functions, it has a number of features that are either unique, or are not found in combination in any one software package: (1 it has a novel interactive flow-chart user interface for complex multi-step processes, allowing an integrated overview of the whole project; (2 it can perform a user-defined workflow of cloning steps in a single execution of the software; (3 it can handle multiple types of genetic recombineering, a technique that is rapidly replacing classical cloning for many applications; (4 it includes experimental information to conveniently guide wet lab work; and (5 it can store results and comments to allow the tracking and management of the whole project in one platform. MCDS is freely available from https://mcds.codeplex.com. Keywords: BioCAD, Genetic engineering software, Molecular cloning software, Synthetic biology, Workflow simulation and management

  13. Genome engineering and parthenocloning in the silkworm, Bombyx mori

    Czech Academy of Sciences Publication Activity Database

    Zabelina, V.; Klymenko, V.; Tamura, T.; Doroshenko, K.; Liang, H.; Sezutsu, H.; Sehnal, František

    2015-01-01

    Roč. 40, č. 3 (2015), s. 645-655 ISSN 0250-5991 R&D Projects: GA MŠk(CZ) EE2.3.30.0032; GA ČR GAP502/10/2382 Institutional support: RVO:60077344 Keywords : genomic cloning * genetic engineering * insect biotechnology Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.419, year: 2015 http://www. ias .ac.in/jbiosci/sep2015/645.pdf

  14. KnowEnG: a knowledge engine for genomics.

    Science.gov (United States)

    Sinha, Saurabh; Song, Jun; Weinshilboum, Richard; Jongeneel, Victor; Han, Jiawei

    2015-11-01

    We describe here the vision, motivations, and research plans of the National Institutes of Health Center for Excellence in Big Data Computing at the University of Illinois, Urbana-Champaign. The Center is organized around the construction of "Knowledge Engine for Genomics" (KnowEnG), an E-science framework for genomics where biomedical scientists will have access to powerful methods of data mining, network mining, and machine learning to extract knowledge out of genomics data. The scientist will come to KnowEnG with their own data sets in the form of spreadsheets and ask KnowEnG to analyze those data sets in the light of a massive knowledge base of community data sets called the "Knowledge Network" that will be at the heart of the system. The Center is undertaking discovery projects aimed at testing the utility of KnowEnG for transforming big data to knowledge. These projects span a broad range of biological enquiry, from pharmacogenomics (in collaboration with Mayo Clinic) to transcriptomics of human behavior. © The Author 2015. Published by Oxford University Press on behalf of the American Medical Informatics Association. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  15. Engineering the Chloroplast Genome of Oleaginous Marine Microalga Nannochloropsis oceanica

    Directory of Open Access Journals (Sweden)

    Qinhua Gan

    2018-04-01

    Full Text Available Plastid engineering offers an important tool to fill the gap between the technical and the enormous potential of microalgal photosynthetic cell factory. However, to date, few reports on plastid engineering in industrial microalgae have been documented. This is largely due to the small cell sizes and complex cell-wall structures which make these species intractable to current plastid transformation methods (i.e., biolistic transformation and polyethylene glycol-mediated transformation. Here, employing the industrial oleaginous microalga Nannochloropsis oceanica as a model, an electroporation-mediated chloroplast transformation approach was established. Fluorescent microscopy and laser confocal scanning microscopy confirmed the expression of the green fluorescence protein, driven by the endogenous plastid promoter and terminator. Zeocin-resistance selection led to an acquisition of homoplasmic strains of which a stable and site-specific recombination within the chloroplast genome was revealed by sequencing and DNA gel blotting. This demonstration of electroporation-mediated chloroplast transformation opens many doors for plastid genome editing in industrial microalgae, particularly species of which the chloroplasts are recalcitrant to chemical and microparticle bombardment transformation.

  16. Genetic Engineering In BioButanol Production And Tolerance

    Directory of Open Access Journals (Sweden)

    Ashok Rao

    Full Text Available ABSTRACT The growing need to address current energy and environmental problems has sparked an interest in developing improved biological methods to produce liquid fuels from renewable sources. Higher-chain alcohols possess chemical properties that are more similar to gasoline. Ethanol and butanol are two products which are used as biofuel. Butanol production was more concerned than ethanol because of its high octane number. Unfortunately, these alcohols are not produced efficiently in natural microorganisms, and thus economical production in industrial volumes remains a challenge. The synthetic biology, however, offers additional tools to engineer synthetic pathways in user-friendly hosts to help increase titers and productivity of bio-butanol. Knock out and over-expression of genes is the major approaches towards genetic manipulation and metabolic engineering of microbes. Yet there are TargeTron Technology, Antisense RNA and CRISPR technology has a vital role in genome manipulation of C.acetobutylicum. This review concentrates on the recent developments for efficient production of butanol and butanol tolerance by various genetically engineered microbes.

  17. Opportunities in plant synthetic biology.

    Science.gov (United States)

    Cook, Charis; Martin, Lisa; Bastow, Ruth

    2014-05-01

    Synthetic biology is an emerging field uniting scientists from all disciplines with the aim of designing or re-designing biological processes. Initially, synthetic biology breakthroughs came from microbiology, chemistry, physics, computer science, materials science, mathematics, and engineering disciplines. A transition to multicellular systems is the next logical step for synthetic biologists and plants will provide an ideal platform for this new phase of research. This meeting report highlights some of the exciting plant synthetic biology projects, and tools and resources, presented and discussed at the 2013 GARNet workshop on plant synthetic biology.

  18. Synthetic biology and metabolic engineering for marine carotenoids: new opportunities and future prospects.

    Science.gov (United States)

    Wang, Chonglong; Kim, Jung-Hun; Kim, Seon-Won

    2014-09-17

    Carotenoids are a class of diverse pigments with important biological roles such as light capture and antioxidative activities. Many novel carotenoids have been isolated from marine organisms to date and have shown various utilizations as nutraceuticals and pharmaceuticals. In this review, we summarize the pathways and enzymes of carotenoid synthesis and discuss various modifications of marine carotenoids. The advances in metabolic engineering and synthetic biology for carotenoid production are also reviewed, in hopes that this review will promote the exploration of marine carotenoid for their utilizations.

  19. Design and development of modular DNA assembly tools for Multigene Engineering and Synthetic Biology in Plants

    OpenAIRE

    Sarrión Perdigones, Manuel Alejandro

    2014-01-01

    The post-genomics era has put at the disposal of modern plant breeders an endless list of genetic building blocks for the design of new biotechnological crops. After a first wave of single-gene transgenic with controversial public acceptance, genomic information and technology is paving the way for increasingly complex designs based in multiple gene engineering. Those designs aiming at the production of inexpensive health-promoting compounds are most likely to be welcomed by consumers. In thi...

  20. Novel bio-synthetic hybrid materials and coculture systems for musculoskeletal tissue engineering

    Science.gov (United States)

    Lee, Hyeseung Janice

    Tissue Engineering is a truly exciting field of this age, trying to regenerate and repair impaired tissues. Unlike the old artificial implants, tissue engineering aims at making a long-term functional biological replacement. One strategy for such tissue engineering requires the following three components: cells, scaffolds, and soluble factors. Cells are cultured in a three-dimensional (3D) scaffold with medium containing various soluble factors. Once a tissue is developed in vitro, then it is implanted in vivo. The overall goal of this thesis was to develop novel bio-synthetic hybrid scaffolds and coculture system for musculoskeletal tissue engineering. The most abundant cartilage extracellular matrix (ECM) components are collagen and glycosaminoglycan (GAG), which are the natural scaffold for chondrocytes. As two different peptides, collagen mimetic peptide (CMP) and hyaluronic acid binding peptide (HABPep) were previously shown to bind to collagen and hyaluronic acid (HA) of GAG, respectively, it was hypothesized that immobilizing CMP and HABP on 3D scaffold would results in an interaction between ECM components and synthetic scaffolds via peptide-ECM bindings. CMP or HABPep-conjugated photopolymerizable poly(ethylene oxide) diacrylate (PEODA) hydrogels were synthesized and shown to retain encapsulated collagen or HA, respectively. This result supported that conjugated CMP and HABPep can interact with collagen and HA, respectively, and can serve as biological linkers in 3D synthetic hydrogels. When chondrocytes or mesenchymal stem cells (MSCs) were seeded, cells in CMP-conjugated scaffolds produced significantly more amount of type II collagen and GAG, compared to those in control scaffolds. Moreover, MSCs cultured in CMP-conjugated scaffolds exhibited lower level of hypertrophic markers, cbfa-1 and type X collagen. These results demonstrated that enhanced interaction between collagen and scaffold via CMP improves chondrogenesis of chondrocytes and MSCs and

  1. UVC-mutagenesis in acetogens: resistance to methanol, ethanol, acetone, or n-butanol in recombinants with tailored genomes as the step in engineering of commercial biocatalysts for continuous CO₂/H₂ blend fermentations.

    Science.gov (United States)

    Kiriukhin, Michael; Tyurin, Michael; Gak, Eugene

    2014-05-01

    Time- and cost-efficient six-step UVC-mutagenesis was developed and validated to generate acetogen mutants with preliminary reduced genomes to prevent product inhibition in the to-be-engineered commercial biocatalysts. Genome reduction was performed via elimination of pta, ack, spo0A, spo0J and some pro-phage genes. UVC-mutants such as Clostridium sp. MT1784RG, Clostridium sp. MT653RG, Clostridium sp. MT896RG, and Clostridium sp. MT1962RG (all 4 share 97 % DNA homology with Clostridium ljungdahlii ATCC 55383) were selected based on resistance to methanol (3 M), ethanol (3.6 M), acetone (2.5 M), or n-butanol (0.688 M), respectively. As a part of the biocatalyst engineering algorithm, genome reduction step was associated with integration of attTn7 recognition sequence to the chromosomes of each of the above strains to prepare the defined integration sites for future integration of multi-copy synthetic operons encoding biosynthesis of methanol, ethanol, acetone or n-butanol. Reduced genome mutants had cell duplication times decreased compared to the same for the respective parental strains. All groups of mutants had decreased share of palmitic (C16:0) and increased share of oleic (C18:1) acids along with detection of isopropylstearate (C20) compared to the parental strains. Mutants resistant to acetone and n-butanol also had monounsaturated fatty acid (C20:1) not found in parental strains. Cyclopropane fatty acid (C21) was identified only in n-butanol resistant mutants.

  2. Living GenoChemetics by hyphenating synthetic biology and synthetic chemistry in vivo.

    Science.gov (United States)

    Sharma, Sunil V; Tong, Xiaoxue; Pubill-Ulldemolins, Cristina; Cartmell, Christopher; Bogosyan, Emma J A; Rackham, Emma J; Marelli, Enrico; Hamed, Refaat B; Goss, Rebecca J M

    2017-08-09

    Marrying synthetic biology with synthetic chemistry provides a powerful approach toward natural product diversification, combining the best of both worlds: expediency and synthetic capability of biogenic pathways and chemical diversity enabled by organic synthesis. Biosynthetic pathway engineering can be employed to insert a chemically orthogonal tag into a complex natural scaffold affording the possibility of site-selective modification without employing protecting group strategies. Here we show that, by installing a sufficiently reactive handle (e.g., a C-Br bond) and developing compatible mild aqueous chemistries, synchronous biosynthesis of the tagged metabolite and its subsequent chemical modification in living culture can be achieved. This approach can potentially enable many new applications: for example, assay of directed evolution of enzymes catalyzing halo-metabolite biosynthesis in living cells or generating and following the fate of tagged metabolites and biomolecules in living systems. We report synthetic biological access to new-to-nature bromo-metabolites and the concomitant biorthogonal cross-coupling of halo-metabolites in living cultures.Coupling synthetic biology and chemical reactions in cells is a challenging task. The authors engineer bacteria capable of generating bromo-metabolites, develop a mild Suzuki-Miyaura cross-coupling reaction compatible with cell growth and carry out the cross-coupling chemistry in live cell cultures.

  3. Synthetic strong ground motions for engineering design utilizing empirical Green`s functions

    Energy Technology Data Exchange (ETDEWEB)

    Hutchings, L.J.; Jarpe, S.P.; Kasameyer, P.W.; Foxall, W.

    1996-04-11

    We present a methodology for developing realistic synthetic strong ground motions for specific sites from specific earthquakes. We analyzed the possible ground motion resulting from a M = 7.25 earthquake that ruptures 82 km of the Hayward fault for a site 1.4 km from the fault in the eastern San Francisco Bay area. We developed a suite of 100 rupture scenarios for the Hayward fault earthquake and computed the corresponding strong ground motion time histories. We synthesized strong ground motion with physics-based solutions of earthquake rupture and applied physical bounds on rupture parameters. By having a suite of rupture scenarios of hazardous earthquakes for a fixed magnitude and identifying the hazard to the site from the statistical distribution of engineering parameters, we introduce a probabilistic component into the deterministic hazard calculation. Engineering parameters of synthesized ground motions agree with those recorded from the 1995 Kobe, Japan and the 1992 Landers, California earthquakes at similar distances and site geologies.

  4. Engineering Strategies to Decode and Enhance the Genomes of Coral Symbionts

    KAUST Repository

    Levin, Rachel A.; Voolstra, Christian R.; Agrawal, Shobhit; Steinberg, Peter D.; Suggett, David J.; van Oppen, Madeleine J. H.

    2017-01-01

    Elevated sea surface temperatures from a severe and prolonged El Niño event (2014–2016) fueled by climate change have resulted in mass coral bleaching (loss of dinoflagellate photosymbionts, Symbiodinium spp., from coral tissues) and subsequent coral mortality, devastating reefs worldwide. Genetic variation within and between Symbiodinium species strongly influences the bleaching tolerance of corals, thus recent papers have called for genetic engineering of Symbiodinium to elucidate the genetic basis of bleaching-relevant Symbiodinium traits. However, while Symbiodinium has been intensively studied for over 50 years, genetic transformation of Symbiodinium has seen little success likely due to the large evolutionary divergence between Symbiodinium and other model eukaryotes rendering standard transformation systems incompatible. Here, we integrate the growing wealth of Symbiodinium next-generation sequencing data to design tailored genetic engineering strategies. Specifically, we develop a testable expression construct model that incorporates endogenous Symbiodinium promoters, terminators, and genes of interest, as well as an internal ribosomal entry site from a Symbiodinium virus. Furthermore, we assess the potential for CRISPR/Cas9 genome editing through new analyses of the three currently available Symbiodinium genomes. Finally, we discuss how genetic engineering could be applied to enhance the stress tolerance of Symbiodinium, and in turn, coral reefs.

  5. Engineering Strategies to Decode and Enhance the Genomes of Coral Symbionts

    KAUST Repository

    Levin, Rachel A.

    2017-06-30

    Elevated sea surface temperatures from a severe and prolonged El Niño event (2014–2016) fueled by climate change have resulted in mass coral bleaching (loss of dinoflagellate photosymbionts, Symbiodinium spp., from coral tissues) and subsequent coral mortality, devastating reefs worldwide. Genetic variation within and between Symbiodinium species strongly influences the bleaching tolerance of corals, thus recent papers have called for genetic engineering of Symbiodinium to elucidate the genetic basis of bleaching-relevant Symbiodinium traits. However, while Symbiodinium has been intensively studied for over 50 years, genetic transformation of Symbiodinium has seen little success likely due to the large evolutionary divergence between Symbiodinium and other model eukaryotes rendering standard transformation systems incompatible. Here, we integrate the growing wealth of Symbiodinium next-generation sequencing data to design tailored genetic engineering strategies. Specifically, we develop a testable expression construct model that incorporates endogenous Symbiodinium promoters, terminators, and genes of interest, as well as an internal ribosomal entry site from a Symbiodinium virus. Furthermore, we assess the potential for CRISPR/Cas9 genome editing through new analyses of the three currently available Symbiodinium genomes. Finally, we discuss how genetic engineering could be applied to enhance the stress tolerance of Symbiodinium, and in turn, coral reefs.

  6. Engineering Strategies to Decode and Enhance the Genomes of Coral Symbionts

    Directory of Open Access Journals (Sweden)

    Rachel A. Levin

    2017-06-01

    Full Text Available Elevated sea surface temperatures from a severe and prolonged El Niño event (2014–2016 fueled by climate change have resulted in mass coral bleaching (loss of dinoflagellate photosymbionts, Symbiodinium spp., from coral tissues and subsequent coral mortality, devastating reefs worldwide. Genetic variation within and between Symbiodinium species strongly influences the bleaching tolerance of corals, thus recent papers have called for genetic engineering of Symbiodinium to elucidate the genetic basis of bleaching-relevant Symbiodinium traits. However, while Symbiodinium has been intensively studied for over 50 years, genetic transformation of Symbiodinium has seen little success likely due to the large evolutionary divergence between Symbiodinium and other model eukaryotes rendering standard transformation systems incompatible. Here, we integrate the growing wealth of Symbiodinium next-generation sequencing data to design tailored genetic engineering strategies. Specifically, we develop a testable expression construct model that incorporates endogenous Symbiodinium promoters, terminators, and genes of interest, as well as an internal ribosomal entry site from a Symbiodinium virus. Furthermore, we assess the potential for CRISPR/Cas9 genome editing through new analyses of the three currently available Symbiodinium genomes. Finally, we discuss how genetic engineering could be applied to enhance the stress tolerance of Symbiodinium, and in turn, coral reefs.

  7. Engineering Strategies to Decode and Enhance the Genomes of Coral Symbionts.

    Science.gov (United States)

    Levin, Rachel A; Voolstra, Christian R; Agrawal, Shobhit; Steinberg, Peter D; Suggett, David J; van Oppen, Madeleine J H

    2017-01-01

    Elevated sea surface temperatures from a severe and prolonged El Niño event (2014-2016) fueled by climate change have resulted in mass coral bleaching (loss of dinoflagellate photosymbionts, Symbiodinium spp., from coral tissues) and subsequent coral mortality, devastating reefs worldwide. Genetic variation within and between Symbiodinium species strongly influences the bleaching tolerance of corals, thus recent papers have called for genetic engineering of Symbiodinium to elucidate the genetic basis of bleaching-relevant Symbiodinium traits. However, while Symbiodinium has been intensively studied for over 50 years, genetic transformation of Symbiodinium has seen little success likely due to the large evolutionary divergence between Symbiodinium and other model eukaryotes rendering standard transformation systems incompatible. Here, we integrate the growing wealth of Symbiodinium next-generation sequencing data to design tailored genetic engineering strategies. Specifically, we develop a testable expression construct model that incorporates endogenous Symbiodinium promoters, terminators, and genes of interest, as well as an internal ribosomal entry site from a Symbiodinium virus. Furthermore, we assess the potential for CRISPR/Cas9 genome editing through new analyses of the three currently available Symbiodinium genomes. Finally, we discuss how genetic engineering could be applied to enhance the stress tolerance of Symbiodinium , and in turn, coral reefs.

  8. Role of Synthetic and Dimensional Synthetic Organic Chemistry in Block Copolymer Micelle Nanosensor Engineering

    DEFF Research Database (Denmark)

    Ek, Pramod Kumar

    This thesis investigated the role of amphiphilic triblock copolymer micelle nanomaterials in nanosensors, with emphasis on the synthesis of micelle particle sensors. The thesis is focused on the role of synthetic and dimensional synthetic organic chemistry in amphiphilic triblock core-shellcorona...

  9. Optimizing Restriction Site Placement for Synthetic Genomes

    Science.gov (United States)

    Montes, Pablo; Memelli, Heraldo; Ward, Charles; Kim, Joondong; Mitchell, Joseph S. B.; Skiena, Steven

    Restriction enzymes are the workhorses of molecular biology. We introduce a new problem that arises in the course of our project to design virus variants to serve as potential vaccines: we wish to modify virus-length genomes to introduce large numbers of unique restriction enzyme recognition sites while preserving wild-type function by substitution of synonymous codons. We show that the resulting problem is NP-Complete, give an exponential-time algorithm, and propose effective heuristics, which we show give excellent results for five sample viral genomes. Our resulting modified genomes have several times more unique restriction sites and reduce the maximum gap between adjacent sites by three to nine-fold.

  10. Recoding aminoacyl-tRNA synthetases for synthetic biology by rational protein-RNA engineering.

    Science.gov (United States)

    Hadd, Andrew; Perona, John J

    2014-12-19

    We have taken a rational approach to redesigning the amino acid binding and aminoacyl-tRNA pairing specificities of bacterial glutaminyl-tRNA synthetase. The four-stage engineering incorporates generalizable design principles and improves the pairing efficiency of noncognate glutamate with tRNA(Gln) by over 10(5)-fold compared to the wild-type enzyme. Better optimized designs of the protein-RNA complex include substantial reengineering of the globular core region of the tRNA, demonstrating a role for specific tRNA nucleotides in specifying the identity of the genetically encoded amino acid. Principles emerging from this engineering effort open new prospects for combining rational and genetic selection approaches to design novel aminoacyl-tRNA synthetases that ligate noncanonical amino acids onto tRNAs. This will facilitate reconstruction of the cellular translation apparatus for applications in synthetic biology.

  11. Synthetic Biology and Metabolic Engineering for Marine Carotenoids: New Opportunities and Future Prospects

    Directory of Open Access Journals (Sweden)

    Chonglong Wang

    2014-09-01

    Full Text Available Carotenoids are a class of diverse pigments with important biological roles such as light capture and antioxidative activities. Many novel carotenoids have been isolated from marine organisms to date and have shown various utilizations as nutraceuticals and pharmaceuticals. In this review, we summarize the pathways and enzymes of carotenoid synthesis and discuss various modifications of marine carotenoids. The advances in metabolic engineering and synthetic biology for carotenoid production are also reviewed, in hopes that this review will promote the exploration of marine carotenoid for their utilizations.

  12. Synthetic Biology and Metabolic Engineering for Marine Carotenoids: New Opportunities and Future Prospects

    Science.gov (United States)

    Wang, Chonglong; Kim, Jung-Hun; Kim, Seon-Won

    2014-01-01

    Carotenoids are a class of diverse pigments with important biological roles such as light capture and antioxidative activities. Many novel carotenoids have been isolated from marine organisms to date and have shown various utilizations as nutraceuticals and pharmaceuticals. In this review, we summarize the pathways and enzymes of carotenoid synthesis and discuss various modifications of marine carotenoids. The advances in metabolic engineering and synthetic biology for carotenoid production are also reviewed, in hopes that this review will promote the exploration of marine carotenoid for their utilizations. PMID:25233369

  13. Synthetic biology and biosecurity: challenging the "myths".

    Science.gov (United States)

    Jefferson, Catherine; Lentzos, Filippa; Marris, Claire

    2014-01-01

    Synthetic biology, a field that aims to "make biology easier to engineer," is routinely described as leading to an increase in the "dual-use" threat, i.e., the potential for the same scientific research to be "used" for peaceful purposes or "misused" for warfare or terrorism. Fears have been expressed that the "de-skilling" of biology, combined with online access to the genomic DNA sequences of pathogenic organisms and the reduction in price for DNA synthesis, will make biology increasingly accessible to people operating outside well-equipped professional research laboratories, including people with malevolent intentions. The emergence of do-it-yourself (DIY) biology communities and of the student iGEM competition has come to epitomize this supposed trend toward greater ease of access and the associated potential threat from rogue actors. In this article, we identify five "myths" that permeate discussions about synthetic biology and biosecurity, and argue that they embody misleading assumptions about both synthetic biology and bioterrorism. We demonstrate how these myths are challenged by more realistic understandings of the scientific research currently being conducted in both professional and DIY laboratories, and by an analysis of historical cases of bioterrorism. We show that the importance of tacit knowledge is commonly overlooked in the dominant narrative: the focus is on access to biological materials and digital information, rather than on human practices and institutional dimensions. As a result, public discourse on synthetic biology and biosecurity tends to portray speculative scenarios about the future as realities in the present or the near future, when this is not warranted. We suggest that these "myths" play an important role in defining synthetic biology as a "promissory" field of research and as an "emerging technology" in need of governance.

  14. CRISPR/Cas9-mediated genome engineering of CHO cell factories: application and perspectives

    DEFF Research Database (Denmark)

    Lee, Jae Seong; Grav, Lise Marie; Lewis, Nathan E.

    2015-01-01

    repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system enables rapid,easy and efficient engineering of mammalian genomes. It has a wide range of applications frommodification of individual genes to genome-wide screening or regulation of genes. Facile genomeediting using CRISPR/Cas9 empowers...... researchers in the CHO community to elucidate the mechanisticbasis behind high level production of proteins and product quality attributes of interest. Inthis review, we describe the basis of CRISPR/Cas9-mediated genome editing and its applicationfor development of next generation CHO cell factories while...... highlighting both future perspectivesand challenges. As one of the main drivers for the CHO systems biology era, genome engineeringwith CRISPR/Cas9 will pave the way for rational design of CHO cell factories....

  15. Synthetic biological networks

    International Nuclear Information System (INIS)

    Archer, Eric; Süel, Gürol M

    2013-01-01

    Despite their obvious relationship and overlap, the field of physics is blessed with many insightful laws, while such laws are sadly absent in biology. Here we aim to discuss how the rise of a more recent field known as synthetic biology may allow us to more directly test hypotheses regarding the possible design principles of natural biological networks and systems. In particular, this review focuses on synthetic gene regulatory networks engineered to perform specific functions or exhibit particular dynamic behaviors. Advances in synthetic biology may set the stage to uncover the relationship of potential biological principles to those developed in physics. (review article)

  16. Development of synthetic selfish elements based on modular nucleases in Drosophila melanogaster.

    Science.gov (United States)

    Simoni, Alekos; Siniscalchi, Carla; Chan, Yuk-Sang; Huen, David S; Russell, Steven; Windbichler, Nikolai; Crisanti, Andrea

    2014-06-01

    Selfish genes are DNA elements that increase their rate of genetic transmission at the expense of other genes in the genome and can therefore quickly spread within a population. It has been suggested that selfish elements could be exploited to modify the genome of entire populations for medical and ecological applications. Here we report that transcription activator-like effector nuclease (TALEN) and zinc finger nuclease (ZFN) can be engineered into site-specific synthetic selfish elements (SSEs) and demonstrate their transmission of up to 70% in the Drosophila germline. We show here that SSEs can spread via DNA break-induced homologous recombination, a process known as 'homing' similar to that observed for homing endonuclease genes (HEGs), despite their fundamentally different modes of DNA binding and cleavage. We observed that TALEN and ZFN have a reduced capability of secondary homing compared to HEG as their repetitive structure had a negative effect on their genetic stability. The modular architecture of ZFNs and TALENs allows for the rapid design of novel SSEs against specific genomic sequences making them potentially suitable for the genetic engineering of wild-type populations of animals and plants, in applications such as gene replacement or population suppression of pest species. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  17. Systems metabolic engineering in an industrial setting.

    Science.gov (United States)

    Sagt, Cees M J

    2013-03-01

    Systems metabolic engineering is based on systems biology, synthetic biology, and evolutionary engineering and is now also applied in industry. Industrial use of systems metabolic engineering focuses on strain and process optimization. Since ambitious yields, titers, productivities, and low costs are key in an industrial setting, the use of effective and robust methods in systems metabolic engineering is becoming very important. Major improvements in the field of proteomics and metabolomics have been crucial in the development of genome-wide approaches in strain and process development. This is accompanied by a rapid increase in DNA sequencing and synthesis capacity. These developments enable the use of systems metabolic engineering in an industrial setting. Industrial systems metabolic engineering can be defined as the combined use of genome-wide genomics, transcriptomics, proteomics, and metabolomics to modify strains or processes. This approach has become very common since the technology for generating large data sets of all levels of the cellular processes has developed quite fast into robust, reliable, and affordable methods. The main challenge and scope of this mini review is how to translate these large data sets in relevant biological leads which can be tested for strain or process improvements. Experimental setup, heterogeneity of the culture, and sample pretreatment are important issues which are easily underrated. In addition, the process of structuring, filtering, and visualization of data is important, but also, the availability of a genetic toolbox and equipment for medium/high-throughput fermentation is a key success factor. For an efficient bioprocess, all the different components in this process have to work together. Therefore, mutual tuning of these components is an important strategy.

  18. Targeted Genome Editing Using DNA-Free RNA-Guided Cas9 Ribonucleoprotein for CHO Cell Engineering.

    Science.gov (United States)

    Shin, Jongoh; Lee, Namil; Cho, Suhyung; Cho, Byung-Kwan

    2018-01-01

    Recent advances in the CRISPR/Cas9 system have dramatically facilitated genome engineering in various cell systems. Among the protocols, the direct delivery of the Cas9-sgRNA ribonucleoprotein (RNP) complex into cells is an efficient approach to increase genome editing efficiency. This method uses purified Cas9 protein and in vitro transcribed sgRNA to edit the target gene without vector DNA. We have applied the RNP complex to CHO cell engineering to obtain desirable phenotypes and to reduce unintended insertional mutagenesis and off-target effects. Here, we describe our routine methods for RNP complex-mediated gene deletion including the protocols to prepare the purified Cas9 protein and the in vitro transcribed sgRNA. Subsequently, we also describe a protocol to confirm the edited genomic positions using the T7E1 enzymatic assay and next-generation sequencing.

  19. The Genome-Based Metabolic Systems Engineering to Boost Levan Production in a Halophilic Bacterial Model.

    Science.gov (United States)

    Aydin, Busra; Ozer, Tugba; Oner, Ebru Toksoy; Arga, Kazim Yalcin

    2018-03-01

    Metabolic systems engineering is being used to redirect microbial metabolism for the overproduction of chemicals of interest with the aim of transforming microbial hosts into cellular factories. In this study, a genome-based metabolic systems engineering approach was designed and performed to improve biopolymer biosynthesis capability of a moderately halophilic bacterium Halomonas smyrnensis AAD6 T producing levan, which is a fructose homopolymer with many potential uses in various industries and medicine. For this purpose, the genome-scale metabolic model for AAD6 T was used to characterize the metabolic resource allocation, specifically to design metabolic engineering strategies for engineered bacteria with enhanced levan production capability. Simulations were performed in silico to determine optimal gene knockout strategies to develop new strains with enhanced levan production capability. The majority of the gene knockout strategies emphasized the vital role of the fructose uptake mechanism, and pointed out the fructose-specific phosphotransferase system (PTS fru ) as the most promising target for further metabolic engineering studies. Therefore, the PTS fru of AAD6 T was restructured with insertional mutagenesis and triparental mating techniques to construct a novel, engineered H. smyrnensis strain, BMA14. Fermentation experiments were carried out to demonstrate the high efficiency of the mutant strain BMA14 in terms of final levan concentration, sucrose consumption rate, and sucrose conversion efficiency, when compared to the AAD6 T . The genome-based metabolic systems engineering approach presented in this study might be considered an efficient framework to redirect microbial metabolism for the overproduction of chemicals of interest, and the novel strain BMA14 might be considered a potential microbial cell factory for further studies aimed to design levan production processes with lower production costs.

  20. Discovery of Nigri/nox and Panto/pox site-specific recombinase systems facilitates advanced genome engineering.

    Science.gov (United States)

    Karimova, Madina; Splith, Victoria; Karpinski, Janet; Pisabarro, M Teresa; Buchholz, Frank

    2016-07-22

    Precise genome engineering is instrumental for biomedical research and holds great promise for future therapeutic applications. Site-specific recombinases (SSRs) are valuable tools for genome engineering due to their exceptional ability to mediate precise excision, integration and inversion of genomic DNA in living systems. The ever-increasing complexity of genome manipulations and the desire to understand the DNA-binding specificity of these enzymes are driving efforts to identify novel SSR systems with unique properties. Here, we describe two novel tyrosine site-specific recombination systems designated Nigri/nox and Panto/pox. Nigri originates from Vibrio nigripulchritudo (plasmid VIBNI_pA) and recombines its target site nox with high efficiency and high target-site selectivity, without recombining target sites of the well established SSRs Cre, Dre, Vika and VCre. Panto, derived from Pantoea sp. aB, is less specific and in addition to its native target site, pox also recombines the target site for Dre recombinase, called rox. This relaxed specificity allowed the identification of residues that are involved in target site selectivity, thereby advancing our understanding of how SSRs recognize their respective DNA targets.

  1. Current status of synthetic epikeratoplasty.

    Science.gov (United States)

    Thompson, K P; Hanna, K; Waring, G O; Gipson, I; Liu, Y; Gailitis, R P; Johnson-Wint, B; Green, K

    1991-01-01

    Many of the deficiencies with human tissue epikeratoplasty might be improved by the use of a suitable synthetic lenticule. Potential biomaterials for epikeratoplasty include collagen (types I, III, or IV), collagen-hydrogel copolymers, bioactive synthetics, and coated hydrogels. The biomaterial must be engineered to achieve strict specifications of optical clarity, support of epithelial migration and adhesion, permeability to solutes, and stability to corneal proteases. Attaching synthetic lenticules to the cornea without cutting Bowman's layer by adhesives, laser welding, or direct adhesion may also improve the efficacy of synthetic epikeratoplasty.

  2. Metabolite damage and repair in metabolic engineering design

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Jiayi; Jeffryes, James G.; Henry, Christopher S.; Bruner, Steven D.; Hanson, Andrew D.

    2017-11-01

    The necessarily sharp focus of metabolic engineering and metabolic synthetic biology on pathways and their fluxes has tended to divert attention from the damaging enzymatic and chemical side-reactions that pathway metabolites can undergo. Although historically overlooked and underappreciated, such metabolite damage reactions are now known to occur throughout metabolism and to generate (formerly enigmatic) peaks detected in metabolomics datasets. It is also now known that metabolite damage is often countered by dedicated repair enzymes that undo or prevent it. Metabolite damage and repair are highly relevant to engineered pathway design: metabolite damage reactions can reduce flux rates and product yields, and repair enzymes can provide robust, host-independent solutions. Herein, after introducing the core principles of metabolite damage and repair, we use case histories to document how damage and repair processes affect efficient operation of engineered pathways - particularly those that are heterologous, non-natural, or cell-free. We then review how metabolite damage reactions can be predicted, how repair reactions can be prospected, and how metabolite damage and repair can be built into genome-scale metabolic models. Lastly, we propose a versatile 'plug and play' set of well-characterized metabolite repair enzymes to solve metabolite damage problems known or likely to occur in metabolic engineering and synthetic biology projects.

  3. Genome Sequencing of Streptomyces atratus SCSIOZH16 and Activation Production of Nocardamine via Metabolic Engineering

    Directory of Open Access Journals (Sweden)

    Yan Li

    2018-06-01

    Full Text Available The Actinomycetes are metabolically flexible microorganisms capable of producing a wide range of interesting compounds, including but by no means limited to, siderophores which have high affinity for ferric iron. In this study, we report the complete genome sequence of marine-derived Streptomyces atratus ZH16 and the activation of an embedded siderophore gene cluster via the application of metabolic engineering methods. The S. atratus ZH16 genome reveals that this strain has the potential to produce 26 categories of natural products (NPs barring the ilamycins. Our activation studies revealed S. atratus SCSIO ZH16 to be a promising source of the production of nocardamine-type (desferrioxamine compounds which are important in treating acute iron intoxication and performing ecological remediation. We conclude that metabolic engineering provides a highly effective strategy by which to discover drug-like compounds and new NPs in the genomic era.

  4. Synthetic Defects for Vibrothermography

    Science.gov (United States)

    Renshaw, Jeremy; Holland, Stephen D.; Thompson, R. Bruce; Eisenmann, David J.

    2010-02-01

    Synthetic defects are an important tool used for characterizing the performance of nondestructive evaluation techniques. Viscous material-filled synthetic defects were developed for use in vibrothermography (also known as sonic IR) as a tool to improve inspection accuracy and reliability. This paper describes how the heat-generation response of these VMF synthetic defects is similar to the response of real defects. It also shows how VMF defects can be applied to improve inspection accuracy for complex industrial parts and presents a study of their application in an aircraft engine stator vane.

  5. MODEST: a web-based design tool for oligonucleotide-mediated genome engineering and recombineering

    DEFF Research Database (Denmark)

    Bonde, Mads; Klausen, Michael Schantz; Anderson, Mads Valdemar

    2014-01-01

    Recombineering and multiplex automated genome engineering (MAGE) offer the possibility to rapidly modify multiple genomic or plasmid sites at high efficiencies. This enables efficient creation of genetic variants including both single mutants with specifically targeted modifications as well......, which confers the corresponding genetic change, is performed manually. To address these challenges, we have developed the MAGE Oligo Design Tool (MODEST). This web-based tool allows designing of MAGE oligos for (i) tuning translation rates by modifying the ribosomal binding site, (ii) generating...

  6. Metabolite damage and repair in metabolic engineering design.

    Science.gov (United States)

    Sun, Jiayi; Jeffryes, James G; Henry, Christopher S; Bruner, Steven D; Hanson, Andrew D

    2017-11-01

    The necessarily sharp focus of metabolic engineering and metabolic synthetic biology on pathways and their fluxes has tended to divert attention from the damaging enzymatic and chemical side-reactions that pathway metabolites can undergo. Although historically overlooked and underappreciated, such metabolite damage reactions are now known to occur throughout metabolism and to generate (formerly enigmatic) peaks detected in metabolomics datasets. It is also now known that metabolite damage is often countered by dedicated repair enzymes that undo or prevent it. Metabolite damage and repair are highly relevant to engineered pathway design: metabolite damage reactions can reduce flux rates and product yields, and repair enzymes can provide robust, host-independent solutions. Herein, after introducing the core principles of metabolite damage and repair, we use case histories to document how damage and repair processes affect efficient operation of engineered pathways - particularly those that are heterologous, non-natural, or cell-free. We then review how metabolite damage reactions can be predicted, how repair reactions can be prospected, and how metabolite damage and repair can be built into genome-scale metabolic models. Lastly, we propose a versatile 'plug and play' set of well-characterized metabolite repair enzymes to solve metabolite damage problems known or likely to occur in metabolic engineering and synthetic biology projects. Copyright © 2017 International Metabolic Engineering Society. All rights reserved.

  7. Systems and synthetic biology approaches to alter plant cell walls and reduce biomass recalcitrance.

    Science.gov (United States)

    Kalluri, Udaya C; Yin, Hengfu; Yang, Xiaohan; Davison, Brian H

    2014-12-01

    Fine-tuning plant cell wall properties to render plant biomass more amenable to biofuel conversion is a colossal challenge. A deep knowledge of the biosynthesis and regulation of plant cell wall and a high-precision genome engineering toolset are the two essential pillars of efforts to alter plant cell walls and reduce biomass recalcitrance. The past decade has seen a meteoric rise in use of transcriptomics and high-resolution imaging methods resulting in fresh insights into composition, structure, formation and deconstruction of plant cell walls. Subsequent gene manipulation approaches, however, commonly include ubiquitous mis-expression of a single candidate gene in a host that carries an intact copy of the native gene. The challenges posed by pleiotropic and unintended changes resulting from such an approach are moving the field towards synthetic biology approaches. Synthetic biology builds on a systems biology knowledge base and leverages high-precision tools for high-throughput assembly of multigene constructs and pathways, precision genome editing and site-specific gene stacking, silencing and/or removal. Here, we summarize the recent breakthroughs in biosynthesis and remodelling of major secondary cell wall components, assess the impediments in obtaining a systems-level understanding and explore the potential opportunities in leveraging synthetic biology approaches to reduce biomass recalcitrance. Published 2014. This article is a U.S. Government work and is in the public domain in the USA. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

  8. Persistency of Prediction Accuracy and Genetic Gain in Synthetic Populations Under Recurrent Genomic Selection

    Directory of Open Access Journals (Sweden)

    Dominik Müller

    2017-03-01

    Full Text Available Recurrent selection (RS has been used in plant breeding to successively improve synthetic and other multiparental populations. Synthetics are generated from a limited number of parents ( Np , but little is known about how Np affects genomic selection (GS in RS, especially the persistency of prediction accuracy (rg , g ^ and genetic gain. Synthetics were simulated by intermating Np= 2–32 parent lines from an ancestral population with short- or long-range linkage disequilibrium (LDA and subjected to multiple cycles of GS. We determined rg , g ^ and genetic gain across 30 cycles for different training set (TS sizes, marker densities, and generations of recombination before model training. Contributions to rg , g ^ and genetic gain from pedigree relationships, as well as from cosegregation and LDA between QTL and markers, were analyzed via four scenarios differing in (i the relatedness between TS and selection candidates and (ii whether selection was based on markers or pedigree records. Persistency of rg , g ^ was high for small Np , where predominantly cosegregation contributed to rg , g ^ , but also for large Np , where LDA replaced cosegregation as the dominant information source. Together with increasing genetic variance, this compensation resulted in relatively constant long- and short-term genetic gain for increasing Np > 4, given long-range LDA in the ancestral population. Although our scenarios suggest that information from pedigree relationships contributed to rg , g ^ for only very few generations in GS, we expect a longer contribution than in pedigree BLUP, because capturing Mendelian sampling by markers reduces selective pressure on pedigree relationships. Larger TS size (NTS and higher marker density improved persistency of rg , g ^ and hence genetic gain, but additional recombinations could not increase genetic gain.

  9. Synthetic biology for manufacturing chemicals: constraints drive the use of non-conventional microbial platforms.

    Science.gov (United States)

    Czajka, Jeffrey; Wang, Qinhong; Wang, Yechun; Tang, Yinjie J

    2017-10-01

    Genetically modified microbes have had much industrial success producing protein-based products (such as antibodies and enzymes). However, engineering microbial workhorses for biomanufacturing of commodity compounds remains challenging. First, microbes cannot afford burdens with both overexpression of multiple enzymes and metabolite drainage for product synthesis. Second, synthetic circuits and introduced heterologous pathways are not yet as "robust and reliable" as native pathways due to hosts' innate regulations, especially under suboptimal fermentation conditions. Third, engineered enzymes may lack channeling capabilities for cascade-like transport of metabolites to overcome diffusion barriers or to avoid intermediate toxicity in the cytoplasmic environment. Fourth, moving engineered hosts from laboratory to industry is unreliable because genetic mutations and non-genetic cell-to-cell variations impair the large-scale fermentation outcomes. Therefore, synthetic biology strains often have unsatisfactory industrial performance (titer/yield/productivity). To overcome these problems, many different species are being explored for their metabolic strengths that can be leveraged to synthesize specific compounds. Here, we provide examples of non-conventional and genetically amenable species for industrial manufacturing, including the following: Corynebacterium glutamicum for its TCA cycle-derived biosynthesis, Yarrowia lipolytica for its biosynthesis of fatty acids and carotenoids, cyanobacteria for photosynthetic production from its sugar phosphate pathways, and Rhodococcus for its ability to biotransform recalcitrant feedstock. Finally, we discuss emerging technologies (e.g., genome-to-phenome mapping, single cell methods, and knowledge engineering) that may facilitate the development of novel cell factories.

  10. Synthetic Biology and Biosecurity: Challenging the “Myths”

    Science.gov (United States)

    Jefferson, Catherine; Lentzos, Filippa; Marris, Claire

    2014-01-01

    Synthetic biology, a field that aims to “make biology easier to engineer,” is routinely described as leading to an increase in the “dual-use” threat, i.e., the potential for the same scientific research to be “used” for peaceful purposes or “misused” for warfare or terrorism. Fears have been expressed that the “de-skilling” of biology, combined with online access to the genomic DNA sequences of pathogenic organisms and the reduction in price for DNA synthesis, will make biology increasingly accessible to people operating outside well-equipped professional research laboratories, including people with malevolent intentions. The emergence of do-it-yourself (DIY) biology communities and of the student iGEM competition has come to epitomize this supposed trend toward greater ease of access and the associated potential threat from rogue actors. In this article, we identify five “myths” that permeate discussions about synthetic biology and biosecurity, and argue that they embody misleading assumptions about both synthetic biology and bioterrorism. We demonstrate how these myths are challenged by more realistic understandings of the scientific research currently being conducted in both professional and DIY laboratories, and by an analysis of historical cases of bioterrorism. We show that the importance of tacit knowledge is commonly overlooked in the dominant narrative: the focus is on access to biological materials and digital information, rather than on human practices and institutional dimensions. As a result, public discourse on synthetic biology and biosecurity tends to portray speculative scenarios about the future as realities in the present or the near future, when this is not warranted. We suggest that these “myths” play an important role in defining synthetic biology as a “promissory” field of research and as an “emerging technology” in need of governance. PMID:25191649

  11. Design and Structure-Function Characterization of 3D Printed Synthetic Porous Biomaterials for Tissue Engineering.

    Science.gov (United States)

    Kelly, Cambre N; Miller, Andrew T; Hollister, Scott J; Guldberg, Robert E; Gall, Ken

    2018-04-01

    3D printing is now adopted for use in a variety of industries and functions. In biomedical engineering, 3D printing has prevailed over more traditional manufacturing methods in tissue engineering due to its high degree of control over both macro- and microarchitecture of porous tissue scaffolds. However, with the improved flexibility in design come new challenges in characterizing the structure-function relationships between various architectures and both mechanical and biological properties in an assortment of clinical applications. Presently, the field of tissue engineering lacks a comprehensive body of literature that is capable of drawing meaningful relationships between the designed structure and resulting function of 3D printed porous biomaterial scaffolds. This work first discusses the role of design on 3D printed porous scaffold function and then reviews characterization of these structure-function relationships for 3D printed synthetic metallic, polymeric, and ceramic biomaterials. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Genome engineering of stem cell organoids for disease modeling.

    Science.gov (United States)

    Sun, Yingmin; Ding, Qiurong

    2017-05-01

    Precision medicine emerges as a new approach that takes into account individual variability. Successful realization of precision medicine requires disease models that are able to incorporate personalized disease information and recapitulate disease development processes at the molecular, cellular and organ levels. With recent development in stem cell field, a variety of tissue organoids can be derived from patient specific pluripotent stem cells and adult stem cells. In combination with the state-of-the-art genome editing tools, organoids can be further engineered to mimic disease-relevant genetic and epigenetic status of a patient. This has therefore enabled a rapid expansion of sophisticated in vitro disease models, offering a unique system for fundamental and biomedical research as well as the development of personalized medicine. Here we summarize some of the latest advances and future perspectives in engineering stem cell organoids for human disease modeling.

  13. Engineering a palette of eukaryotic chromoproteins for bacterial synthetic biology.

    Science.gov (United States)

    Liljeruhm, Josefine; Funk, Saskia K; Tietscher, Sandra; Edlund, Anders D; Jamal, Sabri; Wistrand-Yuen, Pikkei; Dyrhage, Karl; Gynnå, Arvid; Ivermark, Katarina; Lövgren, Jessica; Törnblom, Viktor; Virtanen, Anders; Lundin, Erik R; Wistrand-Yuen, Erik; Forster, Anthony C

    2018-01-01

    Coral reefs are colored by eukaryotic chromoproteins (CPs) that are homologous to green fluorescent protein. CPs differ from fluorescent proteins (FPs) by intensely absorbing visible light to give strong colors in ambient light. This endows CPs with certain advantages over FPs, such as instrument-free detection uncomplicated by ultra-violet light damage or background fluorescence, efficient Förster resonance energy transfer (FRET) quenching, and photoacoustic imaging. Thus, CPs have found utility as genetic markers and in teaching, and are attractive for potential cell biosensor applications in the field. Most near-term applications of CPs require expression in a different domain of life: bacteria. However, it is unclear which of the eukaryotic CP genes might be suitable and how best to assay them. Here, taking advantage of codon optimization programs in 12 cases, we engineered 14 CP sequences (meffRed, eforRed, asPink, spisPink, scOrange, fwYellow, amilGFP, amajLime, cjBlue, meffBlue, aeBlue, amilCP, tsPurple and gfasPurple) into a palette of Escherichia coli BioBrick plasmids. BioBricks comply with synthetic biology's most widely used, simplified, cloning standard. Differences in color intensities, maturation times and fitness costs of expression were compared under the same conditions, and visible readout of gene expression was quantitated. A surprisingly large variation in cellular fitness costs was found, resulting in loss of color in some overnight liquid cultures of certain high-copy-plasmid-borne CPs, and cautioning the use of multiple CPs as markers in competition assays. We solved these two problems by integrating pairs of these genes into the chromosome and by engineering versions of the same CP with very different colors. Availability of 14 engineered CP genes compared in E. coli , together with chromosomal mutants suitable for competition assays, should simplify and expand CP study and applications. There was no single plasmid-borne CP that combined

  14. Biosynthesis of therapeutic natural products using synthetic biology.

    Science.gov (United States)

    Awan, Ali R; Shaw, William M; Ellis, Tom

    2016-10-01

    Natural products are a group of bioactive structurally diverse chemicals produced by microorganisms and plants. These molecules and their derivatives have contributed to over a third of the therapeutic drugs produced in the last century. However, over the last few decades traditional drug discovery pipelines from natural products have become far less productive and far more expensive. One recent development with promise to combat this trend is the application of synthetic biology to therapeutic natural product biosynthesis. Synthetic biology is a young discipline with roots in systems biology, genetic engineering, and metabolic engineering. In this review, we discuss the use of synthetic biology to engineer improved yields of existing therapeutic natural products. We further describe the use of synthetic biology to combine and express natural product biosynthetic genes in unprecedented ways, and how this holds promise for opening up completely new avenues for drug discovery and production. Copyright © 2016 Elsevier B.V. All rights reserved.

  15. Precision-engineering the Pseudomonas aeruginosa genome with two-step allelic exchange

    DEFF Research Database (Denmark)

    Hmelo, Laura R; Borlee, Bradley R; Almblad, Henrik

    2015-01-01

    Allelic exchange is an efficient method of bacterial genome engineering. This protocol describes the use of this technique to make gene knockouts and knock-ins, as well as single-nucleotide insertions, deletions and substitutions, in Pseudomonas aeruginosa. Unlike other approaches to allelic exch...

  16. Metagenomics as a tool to obtain full genomes of process-critical bacteria in engineered systems

    DEFF Research Database (Denmark)

    Albertsen, Mads; Hugenholtz, Philip; Tyson, Gene W.

    of the community. The assembled genomes include many of the process-critical bacteria involved in wastewater treatment, such as Competibacter, Tetrasphaera and TM7. The approach is not limited to different extraction methods, but can be applied to any treatment that results in different relative abundance......Bacteria play a pivotal role in engineered systems such as wastewater treatment plants. Obtaining genomes of the bacteria provides the genetic potential of the system and also allows studies of in situ functions through transcriptomics and proteomics. Hence, it enables correlations of operational......, the sequencing of bulk genomic DNA from environmental samples, has the potential to provide genomes of this uncultured majority. However, so far only few bacterial genomes have been obtained from metagenomic data. In this study we present a new approach to obtain individual genomes from metagenomes. We deeply...

  17. Selection for silage yield and composition did not affect genomic diversity within the Wisconsin Quality Synthetic maize population.

    Science.gov (United States)

    Lorenz, Aaron J; Beissinger, Timothy M; Silva, Renato Rodrigues; de Leon, Natalia

    2015-02-02

    Maize silage is forage of high quality and yield, and represents the second most important use of maize in the United States. The Wisconsin Quality Synthetic (WQS) maize population has undergone five cycles of recurrent selection for silage yield and composition, resulting in a genetically improved population. The application of high-density molecular markers allows breeders and geneticists to identify important loci through association analysis and selection mapping, as well as to monitor changes in the distribution of genetic diversity across the genome. The objectives of this study were to identify loci controlling variation for maize silage traits through association analysis and the assessment of selection signatures and to describe changes in the genomic distribution of gene diversity through selection and genetic drift in the WQS recurrent selection program. We failed to find any significant marker-trait associations using the historical phenotypic data from WQS breeding trials combined with 17,719 high-quality, informative single nucleotide polymorphisms. Likewise, no strong genomic signatures were left by selection on silage yield and quality in the WQS despite genetic gain for these traits. These results could be due to the genetic complexity underlying these traits, or the role of selection on standing genetic variation. Variation in loss of diversity through drift was observed across the genome. Some large regions experienced much greater loss in diversity than what is expected, suggesting limited recombination combined with small populations in recurrent selection programs could easily lead to fixation of large swaths of the genome. Copyright © 2015 Lorenz et al.

  18. From noise to synthetic nucleoli: can synthetic biology achieve new insights?

    Science.gov (United States)

    Ciechonska, Marta; Grob, Alice; Isalan, Mark

    2016-04-18

    Synthetic biology aims to re-organise and control biological components to make functional devices. Along the way, the iterative process of designing and testing gene circuits has the potential to yield many insights into the functioning of the underlying chassis of cells. Thus, synthetic biology is converging with disciplines such as systems biology and even classical cell biology, to give a new level of predictability to gene expression, cell metabolism and cellular signalling networks. This review gives an overview of the contributions that synthetic biology has made in understanding gene expression, in terms of cell heterogeneity (noise), the coupling of growth and energy usage to expression, and spatiotemporal considerations. We mainly compare progress in bacterial and mammalian systems, which have some of the most-developed engineering frameworks. Overall, one view of synthetic biology can be neatly summarised as "creating in order to understand."

  19. DNA Replication in Engineered Escherichia coli Genomes with Extra Replication Origins.

    Science.gov (United States)

    Milbredt, Sarah; Farmani, Neda; Sobetzko, Patrick; Waldminghaus, Torsten

    2016-10-21

    The standard outline of bacterial genomes is a single circular chromosome with a single replication origin. From the bioengineering perspective, it appears attractive to extend this basic setup. Bacteria with split chromosomes or multiple replication origins have been successfully constructed in the last few years. The characteristics of these engineered strains will largely depend on the respective DNA replication patterns. However, the DNA replication has not been investigated systematically in engineered bacteria with multiple origins or split replicons. Here we fill this gap by studying a set of strains consisting of (i) E. coli strains with an extra copy of the native replication origin (oriC), (ii) E. coli strains with an extra copy of the replication origin from the secondary chromosome of Vibrio cholerae (oriII), and (iii) a strain in which the E. coli chromosome is split into two linear replicons. A combination of flow cytometry, microarray-based comparative genomic hybridization (CGH), and modeling revealed silencing of extra oriC copies and differential timing of ectopic oriII copies compared to the native oriC. The results were used to derive construction rules for future multiorigin and multireplicon projects.

  20. Evaluation of synthetic zeolite as engineering passive permeable reactive barrier

    International Nuclear Information System (INIS)

    Ibrahim, O.A.A.

    2011-01-01

    The presence of toxic pollutants in groundwater brings about significant changes in the properties of water resources and has to be avoided in order to preserve the environmental quality. Heavy metals are among the most dangerous inorganic water pollutants, that related to many anthropogenic sources and their compounds are extremely toxic. The treatment of contaminated groundwater is among the most difficult and expensive environmental problems. Over the past years, permeable reactive barriers have provided an increasingly important role in the passive insitu treatment of contaminated groundwater. There are a large number of materials that are able to immobilize contaminants by sorption, including granulated active carbon, zeolite, montmorillonite, peat, compost, sawdust, etc. Zeolite X is a synthetic counterpart of the naturally occurring mineral Faujasite. It has one of the largest cavities and cavity entrances of any known zeolites. The main aim of this work is to examine the possibility of using synthetic zeolite X as an engineering permeable reactive barrier to remove heavy metals from a contaminated groundwater. Within this context, the following investigations were carried out: 1. Review on the materials most commonly used as engineered permeable reactive barriers to identify the important features to be considered in the examination of the proposed permeable reactive barrier material (zeolite X). 2. Synthesis of zeolite X and characterization of the synthesized material using different techniques. 3. Batch tests were carried out to characterize the equilibrium and kinetic sorption properties of the synthesized zeolite X towards the concerned heavy metals; zinc and cadmium ions. 4. Column tests were also performed to determine the design factors for permeable reactive barrier against zinc and cadmium ions solutions.Breakthrough curves measured in such experiments used to determine the hydrodynamic dispersion coefficients for both metal ions. 5. Analytical

  1. Varieties of noise: analogical reasoning in synthetic biology.

    Science.gov (United States)

    Knuuttila, Tarja; Loettgers, Andrea

    2014-12-01

    The picture of synthetic biology as a kind of engineering science has largely created the public understanding of this novel field, covering both its promises and risks. In this paper, we will argue that the actual situation is more nuanced and complex. Synthetic biology is a highly interdisciplinary field of research located at the interface of physics, chemistry, biology, and computational science. All of these fields provide concepts, metaphors, mathematical tools, and models, which are typically utilized by synthetic biologists by drawing analogies between the different fields of inquiry. We will study analogical reasoning in synthetic biology through the emergence of the functional meaning of noise, which marks an important shift in how engineering concepts are employed in this field. The notion of noise serves also to highlight the differences between the two branches of synthetic biology: the basic science-oriented branch and the engineering-oriented branch, which differ from each other in the way they draw analogies to various other fields of study. Moreover, we show that fixing the mapping between a source domain and the target domain seems not to be the goal of analogical reasoning in actual scientific practice.

  2. Precision control of recombinant gene transcription for CHO cell synthetic biology.

    Science.gov (United States)

    Brown, Adam J; James, David C

    2016-01-01

    The next generation of mammalian cell factories for biopharmaceutical production will be genetically engineered to possess both generic and product-specific manufacturing capabilities that may not exist naturally. Introduction of entirely new combinations of synthetic functions (e.g. novel metabolic or stress-response pathways), and retro-engineering of existing functional cell modules will drive disruptive change in cellular manufacturing performance. However, before we can apply the core concepts underpinning synthetic biology (design, build, test) to CHO cell engineering we must first develop practical and robust enabling technologies. Fundamentally, we will require the ability to precisely control the relative stoichiometry of numerous functional components we simultaneously introduce into the host cell factory. In this review we discuss how this can be achieved by design of engineered promoters that enable concerted control of recombinant gene transcription. We describe the specific mechanisms of transcriptional regulation that affect promoter function during bioproduction processes, and detail the highly-specific promoter design criteria that are required in the context of CHO cell engineering. The relative applicability of diverse promoter development strategies are discussed, including re-engineering of natural sequences, design of synthetic transcription factor-based systems, and construction of synthetic promoters. This review highlights the potential of promoter engineering to achieve precision transcriptional control for CHO cell synthetic biology. Copyright © 2015. Published by Elsevier Inc.

  3. Exploiting endogenous CRISPR-Cas system for multiplex genome editing in Clostridium tyrobutyricum and engineer the strain for high-level butanol production.

    Science.gov (United States)

    Zhang, Jie; Zong, Wenming; Hong, Wei; Zhang, Zhong-Tian; Wang, Yi

    2018-03-09

    Although CRISPR-Cas9/Cpf1 have been employed as powerful genome engineering tools, heterologous CRISPR-Cas9/Cpf1 are often difficult to introduce into bacteria and archaea due to their severe toxicity. Since most prokaryotes harbor native CRISPR-Cas systems, genome engineering can be achieved by harnessing these endogenous immune systems. Here, we report the exploitation of Type I-B CRISPR-Cas of Clostridium tyrobutyricum for genome engineering. In silico CRISPR array analysis and plasmid interference assay revealed that TCA or TCG at the 5'-end of the protospacer was the functional protospacer adjacent motif (PAM) for CRISPR targeting. With a lactose inducible promoter for CRISPR array expression, we significantly decreased the toxicity of CRISPR-Cas and enhanced the transformation efficiency, and successfully deleted spo0A with an editing efficiency of 100%. We further evaluated effects of the spacer length on genome editing efficiency. Interestingly, spacers ≤ 20 nt led to unsuccessful transformation consistently, likely due to severe off-target effects; while a spacer of 30-38 nt is most appropriate to ensure successful transformation and high genome editing efficiency. Moreover, multiplex genome editing for the deletion of spo0A and pyrF was achieved in a single transformation, with an editing efficiency of up to 100%. Finally, with the integration of the alcohol dehydrogenase gene (adhE1 or adhE2) to replace cat1 (the key gene responsible for butyrate production and previously could not be deleted), two mutants were created for n-butanol production, with the butanol titer reached historically record high of 26.2 g/L in a batch fermentation. Altogether, our results demonstrated the easy programmability and high efficiency of endogenous CRISPR-Cas. The developed protocol herein has a broader applicability to other prokaryotes containing endogenous CRISPR-Cas systems. C. tyrobutyricum could be employed as an excellent platform to be engineered for biofuel

  4. Comparative genome analysis of a thermotolerant Escherichia coli obtained by Genome Replication Engineering Assisted Continuous Evolution (GREACE) and its parent strain provides new understanding of microbial heat tolerance.

    Science.gov (United States)

    Luan, Guodong; Bao, Guanhui; Lin, Zhao; Li, Yang; Chen, Zugen; Li, Yin; Cai, Zhen

    2015-12-25

    Heat tolerance of microbes is of great importance for efficient biorefinery and bioconversion. However, engineering and understanding of microbial heat tolerance are difficult and insufficient because it is a complex physiological trait which probably correlates with all gene functions, genetic regulations, and cellular metabolisms and activities. In this work, a novel strain engineering approach named Genome Replication Engineering Assisted Continuous Evolution (GREACE) was employed to improve the heat tolerance of Escherichia coli. When the E. coli strain carrying a mutator was cultivated under gradually increasing temperature, genome-wide mutations were continuously generated during genome replication and the mutated strains with improved thermotolerance were autonomously selected. A thermotolerant strain HR50 capable of growing at 50°C on LB agar plate was obtained within two months, demonstrating the efficiency of GREACE in improving such a complex physiological trait. To understand the improved heat tolerance, genomes of HR50 and its wildtype strain DH5α were sequenced. Evenly distributed 361 mutations covering all mutation types were found in HR50. Closed material transportations, loose genome conformation, and possibly altered cell wall structure and transcription pattern were the main differences of HR50 compared with DH5α, which were speculated to be responsible for the improved heat tolerance. This work not only expanding our understanding of microbial heat tolerance, but also emphasizing that the in vivo continuous genome mutagenesis method, GREACE, is efficient in improving microbial complex physiological trait. Copyright © 2015 Elsevier B.V. All rights reserved.

  5. Libraries of Synthetic TALE-Activated Promoters: Methods and Applications.

    Science.gov (United States)

    Schreiber, T; Tissier, A

    2016-01-01

    The discovery of proteins with programmable DNA-binding specificities triggered a whole array of applications in synthetic biology, including genome editing, regulation of transcription, and epigenetic modifications. Among those, transcription activator-like effectors (TALEs) due to their natural function as transcription regulators, are especially well-suited for the development of orthogonal systems for the control of gene expression. We describe here the construction and testing of libraries of synthetic TALE-activated promoters which are under the control of a single TALE with a given DNA-binding specificity. These libraries consist of a fixed DNA-binding element for the TALE, a TATA box, and variable sequences of 19 bases upstream and 43 bases downstream of the DNA-binding element. These libraries were cloned using a Golden Gate cloning strategy making them usable as standard parts in a modular cloning system. The broad range of promoter activities detected and the versatility of these promoter libraries make them valuable tools for applications in the fine-tuning of expression in metabolic engineering projects or in the design and implementation of regulatory circuits. © 2016 Elsevier Inc. All rights reserved.

  6. Metabolic engineering of β-oxidation in Penicillium chrysogenum for improved semi-synthetic cephalosporin biosynthesis.

    Science.gov (United States)

    Veiga, Tânia; Gombert, Andreas K; Landes, Nils; Verhoeven, Maarten D; Kiel, Jan A K W; Krikken, Arjen M; Nijland, Jeroen G; Touw, Hesselien; Luttik, Marijke A H; van der Toorn, John C; Driessen, Arnold J M; Bovenberg, Roel A L; van den Berg, Marco A; van der Klei, Ida J; Pronk, Jack T; Daran, Jean-Marc

    2012-07-01

    Industrial production of semi-synthetic cephalosporins by Penicillium chrysogenum requires supplementation of the growth media with the side-chain precursor adipic acid. In glucose-limited chemostat cultures of P. chrysogenum, up to 88% of the consumed adipic acid was not recovered in cephalosporin-related products, but used as an additional carbon and energy source for growth. This low efficiency of side-chain precursor incorporation provides an economic incentive for studying and engineering the metabolism of adipic acid in P. chrysogenum. Chemostat-based transcriptome analysis in the presence and absence of adipic acid confirmed that adipic acid metabolism in this fungus occurs via β-oxidation. A set of 52 adipate-responsive genes included six putative genes for acyl-CoA oxidases and dehydrogenases, enzymes responsible for the first step of β-oxidation. Subcellular localization of the differentially expressed acyl-CoA oxidases and dehydrogenases revealed that the oxidases were exclusively targeted to peroxisomes, while the dehydrogenases were found either in peroxisomes or in mitochondria. Deletion of the genes encoding the peroxisomal acyl-CoA oxidase Pc20g01800 and the mitochondrial acyl-CoA dehydrogenase Pc20g07920 resulted in a 1.6- and 3.7-fold increase in the production of the semi-synthetic cephalosporin intermediate adipoyl-6-APA, respectively. The deletion strains also showed reduced adipate consumption compared to the reference strain, indicating that engineering of the first step of β-oxidation successfully redirected a larger fraction of adipic acid towards cephalosporin biosynthesis. Copyright © 2012 Elsevier Inc. All rights reserved.

  7. Engineering and Evolution of Saccharomyces cerevisiae to Produce Biofuels and Chemicals.

    Science.gov (United States)

    Turner, Timothy L; Kim, Heejin; Kong, In Iok; Liu, Jing-Jing; Zhang, Guo-Chang; Jin, Yong-Su

    To mitigate global climate change caused partly by the use of fossil fuels, the production of fuels and chemicals from renewable biomass has been attempted. The conversion of various sugars from renewable biomass into biofuels by engineered baker's yeast (Saccharomyces cerevisiae) is one major direction which has grown dramatically in recent years. As well as shifting away from fossil fuels, the production of commodity chemicals by engineered S. cerevisiae has also increased significantly. The traditional approaches of biochemical and metabolic engineering to develop economic bioconversion processes in laboratory and industrial settings have been accelerated by rapid advancements in the areas of yeast genomics, synthetic biology, and systems biology. Together, these innovations have resulted in rapid and efficient manipulation of S. cerevisiae to expand fermentable substrates and diversify value-added products. Here, we discuss recent and major advances in rational (relying on prior experimentally-derived knowledge) and combinatorial (relying on high-throughput screening and genomics) approaches to engineer S. cerevisiae for producing ethanol, butanol, 2,3-butanediol, fatty acid ethyl esters, isoprenoids, organic acids, rare sugars, antioxidants, and sugar alcohols from glucose, xylose, cellobiose, galactose, acetate, alginate, mannitol, arabinose, and lactose.

  8. Development of biosensors and their application in metabolic engineering

    DEFF Research Database (Denmark)

    Zhang, Jie; Jensen, Michael Krogh; Keasling, Jay

    2015-01-01

    and ease of implementation with high-throughput analysis. Here we describe recent progress in biosensor development and their applications in a metabolic engineering context. We also highlight examples of how biosensors can be integrated with synthetic circuits to exert feedback regulation...... for the desired phenotypes. However, methods available for microbial genome diversification far exceed our ability to screen and select for those variants with optimal performance. Genetically encoded biosensors have shown the potential to address this gap, given their ability to respond to small molecule binding...

  9. [Applications of synthetic biology in materials science].

    Science.gov (United States)

    Zhao, Tianxin; Zhong, Chao

    2017-03-25

    Materials are the basis for human being survival and social development. To keep abreast with the increasing needs from all aspects of human society, there are huge needs in the development of advanced materials as well as high-efficiency but low-cost manufacturing strategies that are both sustainable and tunable. Synthetic biology, a new engineering principle taking gene regulation and engineering design as the core, greatly promotes the development of life sciences. This discipline has also contributed to the development of material sciences and will continuously bring new ideas to future new material design. In this paper, we review recent advances in applications of synthetic biology in material sciences, with the focus on how synthetic biology could enable synthesis of new polymeric biomaterials and inorganic materials, phage display and directed evolution of proteins relevant to materials development, living functional materials, engineered bacteria-regulated artificial photosynthesis system as well as applications of gene circuits for material sciences.

  10. Production of Fatty Acid-Derived Valuable Chemicals in Synthetic Microbes

    International Nuclear Information System (INIS)

    Yu, Ai-Qun; Pratomo Juwono, Nina Kurniasih; Leong, Susanna Su Jan; Chang, Matthew Wook

    2014-01-01

    Fatty acid derivatives, such as hydroxy fatty acids, fatty alcohols, fatty acid methyl/ethyl esters, and fatty alka(e)nes, have a wide range of industrial applications including plastics, lubricants, and fuels. Currently, these chemicals are obtained mainly through chemical synthesis, which is complex and costly, and their availability from natural biological sources is extremely limited. Metabolic engineering of microorganisms has provided a platform for effective production of these valuable biochemicals. Notably, synthetic biology-based metabolic engineering strategies have been extensively applied to refactor microorganisms for improved biochemical production. Here, we reviewed: (i) the current status of metabolic engineering of microbes that produce fatty acid-derived valuable chemicals, and (ii) the recent progress of synthetic biology approaches that assist metabolic engineering, such as mRNA secondary structure engineering, sensor-regulator system, regulatable expression system, ultrasensitive input/output control system, and computer science-based design of complex gene circuits. Furthermore, key challenges and strategies were discussed. Finally, we concluded that synthetic biology provides useful metabolic engineering strategies for economically viable production of fatty acid-derived valuable chemicals in engineered microbes.

  11. Production of Fatty Acid-Derived Valuable Chemicals in Synthetic Microbes

    Energy Technology Data Exchange (ETDEWEB)

    Yu, Ai-Qun; Pratomo Juwono, Nina Kurniasih [Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore (Singapore); Synthetic Biology Research Program, National University of Singapore, Singapore (Singapore); Leong, Susanna Su Jan [Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore (Singapore); Synthetic Biology Research Program, National University of Singapore, Singapore (Singapore); Singapore Institute of Technology, Singapore (Singapore); Chang, Matthew Wook, E-mail: bchcmw@nus.edu.sg [Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore (Singapore); Synthetic Biology Research Program, National University of Singapore, Singapore (Singapore)

    2014-12-23

    Fatty acid derivatives, such as hydroxy fatty acids, fatty alcohols, fatty acid methyl/ethyl esters, and fatty alka(e)nes, have a wide range of industrial applications including plastics, lubricants, and fuels. Currently, these chemicals are obtained mainly through chemical synthesis, which is complex and costly, and their availability from natural biological sources is extremely limited. Metabolic engineering of microorganisms has provided a platform for effective production of these valuable biochemicals. Notably, synthetic biology-based metabolic engineering strategies have been extensively applied to refactor microorganisms for improved biochemical production. Here, we reviewed: (i) the current status of metabolic engineering of microbes that produce fatty acid-derived valuable chemicals, and (ii) the recent progress of synthetic biology approaches that assist metabolic engineering, such as mRNA secondary structure engineering, sensor-regulator system, regulatable expression system, ultrasensitive input/output control system, and computer science-based design of complex gene circuits. Furthermore, key challenges and strategies were discussed. Finally, we concluded that synthetic biology provides useful metabolic engineering strategies for economically viable production of fatty acid-derived valuable chemicals in engineered microbes.

  12. Developments in the Tools and Methodologies of Synthetic Biology

    Science.gov (United States)

    Kelwick, Richard; MacDonald, James T.; Webb, Alexander J.; Freemont, Paul

    2014-01-01

    Synthetic biology is principally concerned with the rational design and engineering of biologically based parts, devices, or systems. However, biological systems are generally complex and unpredictable, and are therefore, intrinsically difficult to engineer. In order to address these fundamental challenges, synthetic biology is aiming to unify a “body of knowledge” from several foundational scientific fields, within the context of a set of engineering principles. This shift in perspective is enabling synthetic biologists to address complexity, such that robust biological systems can be designed, assembled, and tested as part of a biological design cycle. The design cycle takes a forward-design approach in which a biological system is specified, modeled, analyzed, assembled, and its functionality tested. At each stage of the design cycle, an expanding repertoire of tools is being developed. In this review, we highlight several of these tools in terms of their applications and benefits to the synthetic biology community. PMID:25505788

  13. Developments in the tools and methodologies of synthetic biology

    Directory of Open Access Journals (Sweden)

    Richard eKelwick

    2014-11-01

    Full Text Available Synthetic biology is principally concerned with the rational design and engineering of biologically based parts, devices or systems. However, biological systems are generally complex and unpredictable and are therefore intrinsically difficult to engineer. In order to address these fundamental challenges, synthetic biology is aiming to unify a ‘body of knowledge’ from several foundational scientific fields, within the context of a set of engineering principles. This shift in perspective is enabling synthetic biologists to address complexity, such that robust biological systems can be designed, assembled and tested as part of a biological design cycle. The design cycle takes a forward-design approach in which a biological system is specified, modeled, analyzed, assembled and its functionality tested. At each stage of the design cycle an expanding repertoire of tools is being developed. In this review we highlight several of these tools in terms of their applications and benefits to the synthetic biology community.

  14. Synthetic biology for pharmaceutical drug discovery

    Directory of Open Access Journals (Sweden)

    Trosset JY

    2015-12-01

    Full Text Available Jean-Yves Trosset,1 Pablo Carbonell2,3 1Bioinformation Research Laboratory, Sup’Biotech, Villejuif, France; 2Faculty of Life Sciences, SYNBIOCHEM Centre, Manchester Institute of Biotechnology, University of Manchester, Manchester, UK; 3Department of Experimental and Health Sciences (DCEXS, Research Programme on Biomedical Informatics (GRIB, Hospital del Mar Medical Research Institute (IMIM, Universitat Pompeu Fabra (UPF, Barcelona, Spain Abstract: Synthetic biology (SB is an emerging discipline, which is slowly reorienting the field of drug discovery. For thousands of years, living organisms such as plants were the major source of human medicines. The difficulty in resynthesizing natural products, however, often turned pharmaceutical industries away from this rich source for human medicine. More recently, progress on transformation through genetic manipulation of biosynthetic units in microorganisms has opened the possibility of in-depth exploration of the large chemical space of natural products derivatives. Success of SB in drug synthesis culminated with the bioproduction of artemisinin by microorganisms, a tour de force in protein and metabolic engineering. Today, synthetic cells are not only used as biofactories but also used as cell-based screening platforms for both target-based and phenotypic-based approaches. Engineered genetic circuits in synthetic cells are also used to decipher disease mechanisms or drug mechanism of actions and to study cell–cell communication within bacteria consortia. This review presents latest developments of SB in the field of drug discovery, including some challenging issues such as drug resistance and drug toxicity. Keywords: metabolic engineering, plant synthetic biology, natural products, synthetic quorum sensing, drug resistance

  15. Computer-assisted engineering of the synthetic pathway for biodegradation of a toxic persistent pollutant.

    Science.gov (United States)

    Kurumbang, Nagendra Prasad; Dvorak, Pavel; Bendl, Jaroslav; Brezovsky, Jan; Prokop, Zbynek; Damborsky, Jiri

    2014-03-21

    Anthropogenic halogenated compounds were unknown to nature until the industrial revolution, and microorganisms have not had sufficient time to evolve enzymes for their degradation. The lack of efficient enzymes and natural pathways can be addressed through a combination of protein and metabolic engineering. We have assembled a synthetic route for conversion of the highly toxic and recalcitrant 1,2,3-trichloropropane to glycerol in Escherichia coli, and used it for a systematic study of pathway bottlenecks. Optimal ratios of enzymes for the maximal production of glycerol, and minimal toxicity of metabolites were predicted using a mathematical model. The strains containing the expected optimal ratios of enzymes were constructed and characterized for their viability and degradation efficiency. Excellent agreement between predicted and experimental data was observed. The validated model was used to quantitatively describe the kinetic limitations of currently available enzyme variants and predict improvements required for further pathway optimization. This highlights the potential of forward engineering of microorganisms for the degradation of toxic anthropogenic compounds.

  16. Persistency of Prediction Accuracy and Genetic Gain in Synthetic Populations Under Recurrent Genomic Selection.

    Science.gov (United States)

    Müller, Dominik; Schopp, Pascal; Melchinger, Albrecht E

    2017-03-10

    Recurrent selection (RS) has been used in plant breeding to successively improve synthetic and other multiparental populations. Synthetics are generated from a limited number of parents [Formula: see text] but little is known about how [Formula: see text] affects genomic selection (GS) in RS, especially the persistency of prediction accuracy ([Formula: see text]) and genetic gain. Synthetics were simulated by intermating [Formula: see text]= 2-32 parent lines from an ancestral population with short- or long-range linkage disequilibrium ([Formula: see text]) and subjected to multiple cycles of GS. We determined [Formula: see text] and genetic gain across 30 cycles for different training set ( TS ) sizes, marker densities, and generations of recombination before model training. Contributions to [Formula: see text] and genetic gain from pedigree relationships, as well as from cosegregation and [Formula: see text] between QTL and markers, were analyzed via four scenarios differing in (i) the relatedness between TS and selection candidates and (ii) whether selection was based on markers or pedigree records. Persistency of [Formula: see text] was high for small [Formula: see text] where predominantly cosegregation contributed to [Formula: see text], but also for large [Formula: see text] where [Formula: see text] replaced cosegregation as the dominant information source. Together with increasing genetic variance, this compensation resulted in relatively constant long- and short-term genetic gain for increasing [Formula: see text] > 4, given long-range LD A in the ancestral population. Although our scenarios suggest that information from pedigree relationships contributed to [Formula: see text] for only very few generations in GS, we expect a longer contribution than in pedigree BLUP, because capturing Mendelian sampling by markers reduces selective pressure on pedigree relationships. Larger TS size ([Formula: see text]) and higher marker density improved persistency of

  17. Engineering Bacteria to Search for Specific Concentrations of Molecules by a Systematic Synthetic Biology Design Method.

    Science.gov (United States)

    Tien, Shin-Ming; Hsu, Chih-Yuan; Chen, Bor-Sen

    2016-01-01

    Bacteria navigate environments full of various chemicals to seek favorable places for survival by controlling the flagella's rotation using a complicated signal transduction pathway. By influencing the pathway, bacteria can be engineered to search for specific molecules, which has great potential for application to biomedicine and bioremediation. In this study, genetic circuits were constructed to make bacteria search for a specific molecule at particular concentrations in their environment through a synthetic biology method. In addition, by replacing the "brake component" in the synthetic circuit with some specific sensitivities, the bacteria can be engineered to locate areas containing specific concentrations of the molecule. Measured by the swarm assay qualitatively and microfluidic techniques quantitatively, the characteristics of each "brake component" were identified and represented by a mathematical model. Furthermore, we established another mathematical model to anticipate the characteristics of the "brake component". Based on this model, an abundant component library can be established to provide adequate component selection for different searching conditions without identifying all components individually. Finally, a systematic design procedure was proposed. Following this systematic procedure, one can design a genetic circuit for bacteria to rapidly search for and locate different concentrations of particular molecules by selecting the most adequate "brake component" in the library. Moreover, following simple procedures, one can also establish an exclusive component library suitable for other cultivated environments, promoter systems, or bacterial strains.

  18. Engineering Bacteria to Search for Specific Concentrations of Molecules by a Systematic Synthetic Biology Design Method.

    Directory of Open Access Journals (Sweden)

    Shin-Ming Tien

    Full Text Available Bacteria navigate environments full of various chemicals to seek favorable places for survival by controlling the flagella's rotation using a complicated signal transduction pathway. By influencing the pathway, bacteria can be engineered to search for specific molecules, which has great potential for application to biomedicine and bioremediation. In this study, genetic circuits were constructed to make bacteria search for a specific molecule at particular concentrations in their environment through a synthetic biology method. In addition, by replacing the "brake component" in the synthetic circuit with some specific sensitivities, the bacteria can be engineered to locate areas containing specific concentrations of the molecule. Measured by the swarm assay qualitatively and microfluidic techniques quantitatively, the characteristics of each "brake component" were identified and represented by a mathematical model. Furthermore, we established another mathematical model to anticipate the characteristics of the "brake component". Based on this model, an abundant component library can be established to provide adequate component selection for different searching conditions without identifying all components individually. Finally, a systematic design procedure was proposed. Following this systematic procedure, one can design a genetic circuit for bacteria to rapidly search for and locate different concentrations of particular molecules by selecting the most adequate "brake component" in the library. Moreover, following simple procedures, one can also establish an exclusive component library suitable for other cultivated environments, promoter systems, or bacterial strains.

  19. In silico engineering and optimization of Transcription Activator-Like Effectors and their derivatives for improved DNA binding predictions.

    KAUST Repository

    Piatek, Marek J.

    2015-12-01

    Transcription Activator-Like Effectors (TALEs) can be used as adaptable DNAbinding modules to create site-specific chimeric nucleases or synthetic transcriptional regulators. The central repeat domain mediates specific DNA binding via hypervariable repeat di-residues (RVDs). This DNA-Binding Domain can be engineered to bind preferentially to any user-selected DNA sequence if engineered appropriately. Therefore, TALEs and their derivatives have become indispensable molecular tools in site-specific manipulation of genes and genomes. This thesis revolves around two problems: in silico design and improved binding site prediction of TALEs. In the first part, a study is shown where TALEs are successfully designed in silico and validated in laboratory to yield the anticipated effects on selected genes. Software is developed to accompany the process of designing and prediction of binding sites. I expanded the functionality of the software to be used as a more generic set of tools for the design, target and offtarget searching. Part two contributes a method and associated toolkit developed to allow users to design in silico optimized synthetic TALEs with user-defined specificities for various experimental purposes. This method is based on a mutual relationship of three consecutive tandem repeats in the DNA-binding domain. This approach revealed positional and compositional bias behind the binding of TALEs to DNA. In conclusion, I developed methods, approaches, and software to enhance the functionality of synthetic TALEs, which should improve understanding of TALEs biology and will further advance genome-engineering applications in various organisms and cell types.

  20. Engineered CRISPR Systems for Next Generation Gene Therapies.

    Science.gov (United States)

    Pineda, Michael; Moghadam, Farzaneh; Ebrahimkhani, Mo R; Kiani, Samira

    2017-09-15

    An ideal in vivo gene therapy platform provides safe, reprogrammable, and precise strategies which modulate cell and tissue gene regulatory networks with a high temporal and spatial resolution. Clustered regularly interspaced short palindromic repeats (CRISPR), a bacterial adoptive immune system, and its CRISPR-associated protein 9 (Cas9), have gained attention for the ability to target and modify DNA sequences on demand with unprecedented flexibility and precision. The precision and programmability of Cas9 is derived from its complexation with a guide-RNA (gRNA) that is complementary to a desired genomic sequence. CRISPR systems open-up widespread applications including genetic disease modeling, functional screens, and synthetic gene regulation. The plausibility of in vivo genetic engineering using CRISPR has garnered significant traction as a next generation in vivo therapeutic. However, there are hurdles that need to be addressed before CRISPR-based strategies are fully implemented. Some key issues center on the controllability of the CRISPR platform, including minimizing genomic-off target effects and maximizing in vivo gene editing efficiency, in vivo cellular delivery, and spatial-temporal regulation. The modifiable components of CRISPR systems: Cas9 protein, gRNA, delivery platform, and the form of CRISPR system delivered (DNA, RNA, or ribonucleoprotein) have recently been engineered independently to design a better genome engineering toolbox. This review focuses on evaluating CRISPR potential as a next generation in vivo gene therapy platform and discusses bioengineering advancements that can address challenges associated with clinical translation of this emerging technology.

  1. NATO Advanced Study Institute on Synthetic Membranes : Science, Engineering and Applications

    CERN Document Server

    Lonsdale, H; Pinho, M

    1986-01-01

    The chapters in this book are based upon lectures given at the NATO Advanced Study Institute on Synthetic Membranes (June 26-July 8, 1983, Alcabideche, Portugal), which provided an integrated presentation of syn­ thetic membrane science and technology in three broad areas. Currently available membrane formation mechanisms are reviewed, as well as the manner in which synthesis conditions can be controlled to achieve desired membrane structures. Membrane performance in a specific separa­ tionprocess involves complex phenomena, the understanding of which re­ quires a multidisciplinary approach encompassing polymer chemistry, phys­ ical chemistry, and chemical engineering. Progress toward a global understanding of membrane phenomena is described in chapters on the principles of membrane transport. The chapters on membrane processes and applications highlight both established and emerging membrane processes, and elucidate their myriad applications. It is our hope that this book will be an enduring, comprehensi...

  2. Integration of multi-omics data of a genome-reduced bacterium: Prevalence of post-transcriptional regulation and its correlation with protein abundances

    Science.gov (United States)

    Chen, Wei-Hua; van Noort, Vera; Lluch-Senar, Maria; Hennrich, Marco L.; H. Wodke, Judith A.; Yus, Eva; Alibés, Andreu; Roma, Guglielmo; Mende, Daniel R.; Pesavento, Christina; Typas, Athanasios; Gavin, Anne-Claude; Serrano, Luis; Bork, Peer

    2016-01-01

    We developed a comprehensive resource for the genome-reduced bacterium Mycoplasma pneumoniae comprising 1748 consistently generated ‘-omics’ data sets, and used it to quantify the power of antisense non-coding RNAs (ncRNAs), lysine acetylation, and protein phosphorylation in predicting protein abundance (11%, 24% and 8%, respectively). These factors taken together are four times more predictive of the proteome abundance than of mRNA abundance. In bacteria, post-translational modifications (PTMs) and ncRNA transcription were both found to increase with decreasing genomic GC-content and genome size. Thus, the evolutionary forces constraining genome size and GC-content modify the relative contributions of the different regulatory layers to proteome homeostasis, and impact more genomic and genetic features than previously appreciated. Indeed, these scaling principles will enable us to develop more informed approaches when engineering minimal synthetic genomes. PMID:26773059

  3. Towards biologically relevant synthetic designer matrices in 3D bioprinting for tissue engineering and regenerative medicine

    KAUST Repository

    Costa, Rú ben M.; Rauf, Sakandar; Hauser, Charlotte

    2017-01-01

    3D bioprinting is one of the most promising technologies in tissue engineering and regenerative medicine. As new printing techniques and bioinks are getting developed, new cellular constructs with high resolution and functionality arise. Different to bioinks of animal, algal or plant origin, synthesized bioinks are proposed as superior biomaterials because their characteristics are fully under control. In this review, we will highlight the potential of synthetic biomaterials to be used as bioinks in 3D bioprinting to produce functionally enhanced matrices.

  4. Towards biologically relevant synthetic designer matrices in 3D bioprinting for tissue engineering and regenerative medicine

    KAUST Repository

    Costa, Rúben M.

    2017-05-12

    3D bioprinting is one of the most promising technologies in tissue engineering and regenerative medicine. As new printing techniques and bioinks are getting developed, new cellular constructs with high resolution and functionality arise. Different to bioinks of animal, algal or plant origin, synthesized bioinks are proposed as superior biomaterials because their characteristics are fully under control. In this review, we will highlight the potential of synthetic biomaterials to be used as bioinks in 3D bioprinting to produce functionally enhanced matrices.

  5. Genome-scale consequences of cofactor balancing in engineered pentose utilization pathways in Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Amit Ghosh

    Full Text Available Biofuels derived from lignocellulosic biomass offer promising alternative renewable energy sources for transportation fuels. Significant effort has been made to engineer Saccharomyces cerevisiae to efficiently ferment pentose sugars such as D-xylose and L-arabinose into biofuels such as ethanol through heterologous expression of the fungal D-xylose and L-arabinose pathways. However, one of the major bottlenecks in these fungal pathways is that the cofactors are not balanced, which contributes to inefficient utilization of pentose sugars. We utilized a genome-scale model of S. cerevisiae to predict the maximal achievable growth rate for cofactor balanced and imbalanced D-xylose and L-arabinose utilization pathways. Dynamic flux balance analysis (DFBA was used to simulate batch fermentation of glucose, D-xylose, and L-arabinose. The dynamic models and experimental results are in good agreement for the wild type and for the engineered D-xylose utilization pathway. Cofactor balancing the engineered D-xylose and L-arabinose utilization pathways simulated an increase in ethanol batch production of 24.7% while simultaneously reducing the predicted substrate utilization time by 70%. Furthermore, the effects of cofactor balancing the engineered pentose utilization pathways were evaluated throughout the genome-scale metabolic network. This work not only provides new insights to the global network effects of cofactor balancing but also provides useful guidelines for engineering a recombinant yeast strain with cofactor balanced engineered pathways that efficiently co-utilizes pentose and hexose sugars for biofuels production. Experimental switching of cofactor usage in enzymes has been demonstrated, but is a time-consuming effort. Therefore, systems biology models that can predict the likely outcome of such strain engineering efforts are highly useful for motivating which efforts are likely to be worth the significant time investment.

  6. Synthetic biology platform technologies for antimicrobial applications.

    Science.gov (United States)

    Braff, Dana; Shis, David; Collins, James J

    2016-10-01

    The growing prevalence of antibiotic resistance calls for new approaches in the development of antimicrobial therapeutics. Likewise, improved diagnostic measures are essential in guiding the application of targeted therapies and preventing the evolution of therapeutic resistance. Discovery platforms are also needed to form new treatment strategies and identify novel antimicrobial agents. By applying engineering principles to molecular biology, synthetic biologists have developed platforms that improve upon, supplement, and will perhaps supplant traditional broad-spectrum antibiotics. Efforts in engineering bacteriophages and synthetic probiotics demonstrate targeted antimicrobial approaches that can be fine-tuned using synthetic biology-derived principles. Further, the development of paper-based, cell-free expression systems holds promise in promoting the clinical translation of molecular biology tools for diagnostic purposes. In this review, we highlight emerging synthetic biology platform technologies that are geared toward the generation of new antimicrobial therapies, diagnostics, and discovery channels. Copyright © 2016 Elsevier B.V. All rights reserved.

  7. Synthetic biology between technoscience and thing knowledge.

    Science.gov (United States)

    Gelfert, Axel

    2013-06-01

    Synthetic biology presents a challenge to traditional accounts of biology: Whereas traditional biology emphasizes the evolvability, variability, and heterogeneity of living organisms, synthetic biology envisions a future of homogeneous, humanly engineered biological systems that may be combined in modular fashion. The present paper approaches this challenge from the perspective of the epistemology of technoscience. In particular, it is argued that synthetic-biological artifacts lend themselves to an analysis in terms of what has been called 'thing knowledge'. As such, they should neither be regarded as the simple outcome of applying theoretical knowledge and engineering principles to specific technological problems, nor should they be treated as mere sources of new evidence in the general pursuit of scientific understanding. Instead, synthetic-biological artifacts should be viewed as partly autonomous research objects which, qua their material-biological constitution, embody knowledge about the natural world-knowledge that, in turn, can be accessed via continuous experimental interrogation. Copyright © 2013 Elsevier Ltd. All rights reserved.

  8. Understanding Biological Regulation Through Synthetic Biology.

    Science.gov (United States)

    Bashor, Caleb J; Collins, James J

    2018-03-16

    Engineering synthetic gene regulatory circuits proceeds through iterative cycles of design, building, and testing. Initial circuit designs must rely on often-incomplete models of regulation established by fields of reductive inquiry-biochemistry and molecular and systems biology. As differences in designed and experimentally observed circuit behavior are inevitably encountered, investigated, and resolved, each turn of the engineering cycle can force a resynthesis in understanding of natural network function. Here, we outline research that uses the process of gene circuit engineering to advance biological discovery. Synthetic gene circuit engineering research has not only refined our understanding of cellular regulation but furnished biologists with a toolkit that can be directed at natural systems to exact precision manipulation of network structure. As we discuss, using circuit engineering to predictively reorganize, rewire, and reconstruct cellular regulation serves as the ultimate means of testing and understanding how cellular phenotype emerges from systems-level network function. Expected final online publication date for the Annual Review of Biophysics Volume 47 is May 20, 2018. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

  9. BrisSynBio: a BBSRC/EPSRC-funded Synthetic Biology Research Centre.

    Science.gov (United States)

    Sedgley, Kathleen R; Race, Paul R; Woolfson, Derek N

    2016-06-15

    BrisSynBio is the Bristol-based Biotechnology and Biological Sciences Research Council (BBSRC)/Engineering and Physical Sciences Research Council (EPSRC)-funded Synthetic Biology Research Centre. It is one of six such Centres in the U.K. BrisSynBio's emphasis is on rational and predictive bimolecular modelling, design and engineering in the context of synthetic biology. It trains the next generation of synthetic biologists in these approaches, to facilitate translation of fundamental synthetic biology research to industry and the clinic, and to do this within an innovative and responsible research framework. © 2016 The Author(s).

  10. A Transcription Activator-Like Effector (TALE) Toolbox for Genome Engineering

    Science.gov (United States)

    Sanjana, Neville E.; Cong, Le; Zhou, Yang; Cunniff, Margaret M.; Feng, Guoping; Zhang, Feng

    2013-01-01

    Transcription activator-like effectors (TALEs) are a class of naturally occurring DNA binding proteins found in the plant pathogen Xanthomonas sp. The DNA binding domain of each TALE consists of tandem 34-amino acid repeat modules that can be rearranged according to a simple cipher to target new DNA sequences. Customized TALEs can be used for a wide variety of genome engineering applications, including transcriptional modulation and genome editing. Here we describe a toolbox for rapid construction of custom TALE transcription factors (TALE-TFs) and nucleases (TALENs) using a hierarchical ligation procedure. This toolbox facilitates affordable and rapid construction of custom TALE-TFs and TALENs within one week and can be easily scaled up to construct TALEs for multiple targets in parallel. We also provide details for testing the activity in mammalian cells of custom TALE-TFs and TALENs using, respectively, qRT-PCR and Surveyor nuclease. The TALE toolbox described here will enable a broad range of biological applications. PMID:22222791

  11. [Synthetic biology and rearrangements of microbial genetic material].

    Science.gov (United States)

    Liang, Quan-Feng; Wang, Qian; Qi, Qing-Sheng

    2011-10-01

    As an emerging discipline, synthetic biology has shown great scientific values and application prospects. Although there have been many reviews of various aspects on synthetic biology over the last years, this article, for the first time, attempted to discuss the relationship and difference between microbial genetics and synthetic biology. We summarized the recent development of synthetic biology in rearranging microbial genetic materials, including synthesis, design and reduction of genetic materials, standardization of genetic parts and modularization of genetic circuits. The relationship between synthetic biology and microbial genetic engineering was also discussed in the paper.

  12. Synthetic biology approaches to engineering the nitrogen symbiosis in cereals.

    Science.gov (United States)

    Rogers, Christian; Oldroyd, Giles E D

    2014-05-01

    Nitrogen is abundant in the earth's atmosphere but, unlike carbon, cannot be directly assimilated by plants. The limitation this places on plant productivity has been circumvented in contemporary agriculture through the production and application of chemical fertilizers. The chemical reduction of nitrogen for this purpose consumes large amounts of energy and the reactive nitrogen released into the environment as a result of fertilizer application leads to greenhouse gas emissions, as well as widespread eutrophication of aquatic ecosystems. The environmental impacts are intensified by injudicious use of fertilizers in many parts of the world. Simultaneously, limitations in the production and supply of chemical fertilizers in other regions are leading to low agricultural productivity and malnutrition. Nitrogen can be directly fixed from the atmosphere by some bacteria and Archaea, which possess the enzyme nitrogenase. Some plant species, most notably legumes, have evolved close symbiotic associations with nitrogen-fixing bacteria. Engineering cereal crops with the capability to fix their own nitrogen could one day address the problems created by the over- and under-use of nitrogen fertilizers in agriculture. This could be achieved either by expression of a functional nitrogenase enzyme in the cells of the cereal crop or through transferring the capability to form a symbiotic association with nitrogen-fixing bacteria. While potentially transformative, these biotechnological approaches are challenging; however, with recent advances in synthetic biology they are viable long-term goals. This review discusses the possibility of these biotechnological solutions to the nitrogen problem, focusing on engineering the nitrogen symbiosis in cereals.

  13. Synthetic biology, metaphors and responsibility.

    Science.gov (United States)

    McLeod, Carmen; Nerlich, Brigitte

    2017-08-29

    Metaphors are not just decorative rhetorical devices that make speech pretty. They are fundamental tools for thinking about the world and acting on the world. The language we use to make a better world matters; words matter; metaphors matter. Words have consequences - ethical, social and legal ones, as well as political and economic ones. They need to be used 'responsibly'. They also need to be studied carefully - this is what we want to do through this editorial and the related thematic collection. In the context of synthetic biology, natural and social scientists have become increasingly interested in metaphors, a wave of interest that we want to exploit and amplify. We want to build on emerging articles and books on synthetic biology, metaphors of life and the ethical and moral implications of such metaphors. This editorial provides a brief introduction to synthetic biology and responsible innovation, as well as a comprehensive review of literature on the social, cultural and ethical impacts of metaphor use in genomics and synthetic biology. Our aim is to stimulate an interdisciplinary and international discussion on the impact that metaphors can have on science, policy and publics in the context of synthetic biology.

  14. The Synthetic Biology Open Language (SBOL) provides a community standard for communicating designs in synthetic biology.

    Science.gov (United States)

    Galdzicki, Michal; Clancy, Kevin P; Oberortner, Ernst; Pocock, Matthew; Quinn, Jacqueline Y; Rodriguez, Cesar A; Roehner, Nicholas; Wilson, Mandy L; Adam, Laura; Anderson, J Christopher; Bartley, Bryan A; Beal, Jacob; Chandran, Deepak; Chen, Joanna; Densmore, Douglas; Endy, Drew; Grünberg, Raik; Hallinan, Jennifer; Hillson, Nathan J; Johnson, Jeffrey D; Kuchinsky, Allan; Lux, Matthew; Misirli, Goksel; Peccoud, Jean; Plahar, Hector A; Sirin, Evren; Stan, Guy-Bart; Villalobos, Alan; Wipat, Anil; Gennari, John H; Myers, Chris J; Sauro, Herbert M

    2014-06-01

    The re-use of previously validated designs is critical to the evolution of synthetic biology from a research discipline to an engineering practice. Here we describe the Synthetic Biology Open Language (SBOL), a proposed data standard for exchanging designs within the synthetic biology community. SBOL represents synthetic biology designs in a community-driven, formalized format for exchange between software tools, research groups and commercial service providers. The SBOL Developers Group has implemented SBOL as an XML/RDF serialization and provides software libraries and specification documentation to help developers implement SBOL in their own software. We describe early successes, including a demonstration of the utility of SBOL for information exchange between several different software tools and repositories from both academic and industrial partners. As a community-driven standard, SBOL will be updated as synthetic biology evolves to provide specific capabilities for different aspects of the synthetic biology workflow.

  15. Genome-scale modeling using flux ratio constraints to enable metabolic engineering of clostridial metabolism in silico.

    Science.gov (United States)

    McAnulty, Michael J; Yen, Jiun Y; Freedman, Benjamin G; Senger, Ryan S

    2012-05-14

    Genome-scale metabolic networks and flux models are an effective platform for linking an organism genotype to its phenotype. However, few modeling approaches offer predictive capabilities to evaluate potential metabolic engineering strategies in silico. A new method called "flux balance analysis with flux ratios (FBrAtio)" was developed in this research and applied to a new genome-scale model of Clostridium acetobutylicum ATCC 824 (iCAC490) that contains 707 metabolites and 794 reactions. FBrAtio was used to model wild-type metabolism and metabolically engineered strains of C. acetobutylicum where only flux ratio constraints and thermodynamic reversibility of reactions were required. The FBrAtio approach allowed solutions to be found through standard linear programming. Five flux ratio constraints were required to achieve a qualitative picture of wild-type metabolism for C. acetobutylicum for the production of: (i) acetate, (ii) lactate, (iii) butyrate, (iv) acetone, (v) butanol, (vi) ethanol, (vii) CO2 and (viii) H2. Results of this simulation study coincide with published experimental results and show the knockdown of the acetoacetyl-CoA transferase increases butanol to acetone selectivity, while the simultaneous over-expression of the aldehyde/alcohol dehydrogenase greatly increases ethanol production. FBrAtio is a promising new method for constraining genome-scale models using internal flux ratios. The method was effective for modeling wild-type and engineered strains of C. acetobutylicum.

  16. Preparing Synthetic Biology for the World

    Directory of Open Access Journals (Sweden)

    Gerd H.G. Moe-Behrens

    2013-01-01

    Full Text Available Synthetic Biology promises low-cost, exponentially scalable products and global health solutions in the form of self-replicating organisms, or living devices. As these promises are realized, proof-of-concept systems will gradually migrate from tightly regulated laboratory or industrial environments into private spaces as, for instance, probiotic health products, food, and even do-it-yourself bioengineered systems. What additional steps, if any, should be taken before releasing engineered self-replicating organisms into a broader user space? In this review, we explain how studies of genetically modified organisms lay groundwork for the future landscape of biosafety. Early in the design process, biological engineers are anticipating potential hazards and developing innovative tools to mitigate risk. Here, we survey lessons learned, ongoing efforts to engineer intrinsic biocontainment, and how different stakeholders in synthetic biology can act to accomplish best practices for biosafety.

  17. Synthetic biology: Emerging bioengineering in Indonesia

    Science.gov (United States)

    Suhandono, Sony

    2017-05-01

    The development of synthetic biology will shape the new era of science and technology. It is an emerging bioengineering technique involving genetic engineering which can alter the phenotype and behavior of the cell or the new product. Synthetic biology may produce biomaterials, drugs, vaccines, biosensors, and even a recombinant secondary metabolite used in herbal and complementary medicine, such as artemisinin, a malaria drug which is usually extracted from the plant Artemisia annua. The power of synthetic biology has encouraged scientists in Indonesia, and is still in early development. This paper also covers some research from an Indonesian research institute in synthetic biology such as observing the production of bio surfactants and the enhanced production of artemisinin using a transient expression system. Synthetic biology development in Indonesia may also be related to the iGEM competition, a large synthetic biology research competition which was attended by several universities in Indonesia. The application of synthetic biology for drug discovery will be discussed.

  18. Computational protein design-the next generation tool to expand synthetic biology applications.

    Science.gov (United States)

    Gainza-Cirauqui, Pablo; Correia, Bruno Emanuel

    2018-05-02

    One powerful approach to engineer synthetic biology pathways is the assembly of proteins sourced from one or more natural organisms. However, synthetic pathways often require custom functions or biophysical properties not displayed by natural proteins, limitations that could be overcome through modern protein engineering techniques. Structure-based computational protein design is a powerful tool to engineer new functional capabilities in proteins, and it is beginning to have a profound impact in synthetic biology. Here, we review efforts to increase the capabilities of synthetic biology using computational protein design. We focus primarily on computationally designed proteins not only validated in vitro, but also shown to modulate different activities in living cells. Efforts made to validate computational designs in cells can illustrate both the challenges and opportunities in the intersection of protein design and synthetic biology. We also highlight protein design approaches, which although not validated as conveyors of new cellular function in situ, may have rapid and innovative applications in synthetic biology. We foresee that in the near-future, computational protein design will vastly expand the functional capabilities of synthetic cells. Copyright © 2018. Published by Elsevier Ltd.

  19. Design and construction of "synthetic species".

    Directory of Open Access Journals (Sweden)

    Eduardo Moreno

    Full Text Available Synthetic biology is an area of biological research that combines science and engineering. Here, I merge the principles of synthetic biology and regulatory evolution to create a new species with a minimal set of known elements. Using preexisting transgenes and recessive mutations of Drosophila melanogaster, a transgenic population arises with small eyes and a different venation pattern that fulfils the criteria of a new species according to Mayr's Biological Species Concept. The population described here is the first transgenic organism that cannot hybridize with the original wild type population but remains fertile when crossed with other identical transgenic animals. I therefore propose the term "synthetic species" to distinguish it from "natural species", not only because it has been created by genetic manipulation, but also because it may never be able to survive outside the laboratory environment. The use of genetic engineering to design artificial species barriers could help us understand natural speciation and may have practical applications. For instance, the transition from transgenic organisms towards synthetic species could constitute a safety mechanism to avoid the hybridization of genetically modified animals with wild type populations, preserving biodiversity.

  20. Advancing Crop Transformation in the Era of Genome Editing[OPEN

    Science.gov (United States)

    Blechl, Ann E.; Brutnell, Thomas P.; Conrad, Liza J.; Gelvin, Stanton B.; Jackson, David P.; Kausch, Albert P.; Lemaux, Peggy G.; Medford, June I.; Orozco-Cárdenas, Martha L.; Tricoli, David M.; Van Eck, Joyce; Voytas, Daniel F.

    2016-01-01

    Plant transformation has enabled fundamental insights into plant biology and revolutionized commercial agriculture. Unfortunately, for most crops, transformation and regeneration remain arduous even after more than 30 years of technological advances. Genome editing provides novel opportunities to enhance crop productivity but relies on genetic transformation and plant regeneration, which are bottlenecks in the process. Here, we review the state of plant transformation and point to innovations needed to enable genome editing in crops. Plant tissue culture methods need optimization and simplification for efficiency and minimization of time in culture. Currently, specialized facilities exist for crop transformation. Single-cell and robotic techniques should be developed for high-throughput genomic screens. Plant genes involved in developmental reprogramming, wound response, and/or homologous recombination should be used to boost the recovery of transformed plants. Engineering universal Agrobacterium tumefaciens strains and recruiting other microbes, such as Ensifer or Rhizobium, could facilitate delivery of DNA and proteins into plant cells. Synthetic biology should be employed for de novo design of transformation systems. Genome editing is a potential game-changer in crop genetics when plant transformation systems are optimized. PMID:27335450

  1. Annotating novel genes by integrating synthetic lethals and genomic information

    Directory of Open Access Journals (Sweden)

    Faty Mahamadou

    2008-01-01

    Full Text Available Abstract Background Large scale screening for synthetic lethality serves as a common tool in yeast genetics to systematically search for genes that play a role in specific biological processes. Often the amounts of data resulting from a single large scale screen far exceed the capacities of experimental characterization of every identified target. Thus, there is need for computational tools that select promising candidate genes in order to reduce the number of follow-up experiments to a manageable size. Results We analyze synthetic lethality data for arp1 and jnm1, two spindle migration genes, in order to identify novel members in this process. To this end, we use an unsupervised statistical method that integrates additional information from biological data sources, such as gene expression, phenotypic profiling, RNA degradation and sequence similarity. Different from existing methods that require large amounts of synthetic lethal data, our method merely relies on synthetic lethality information from two single screens. Using a Multivariate Gaussian Mixture Model, we determine the best subset of features that assign the target genes to two groups. The approach identifies a small group of genes as candidates involved in spindle migration. Experimental testing confirms the majority of our candidates and we present she1 (YBL031W as a novel gene involved in spindle migration. We applied the statistical methodology also to TOR2 signaling as another example. Conclusion We demonstrate the general use of Multivariate Gaussian Mixture Modeling for selecting candidate genes for experimental characterization from synthetic lethality data sets. For the given example, integration of different data sources contributes to the identification of genetic interaction partners of arp1 and jnm1 that play a role in the same biological process.

  2. Tracking the emergence of synthetic biology.

    Science.gov (United States)

    Shapira, Philip; Kwon, Seokbeom; Youtie, Jan

    2017-01-01

    Synthetic biology is an emerging domain that combines biological and engineering concepts and which has seen rapid growth in research, innovation, and policy interest in recent years. This paper contributes to efforts to delineate this emerging domain by presenting a newly constructed bibliometric definition of synthetic biology. Our approach is dimensioned from a core set of papers in synthetic biology, using procedures to obtain benchmark synthetic biology publication records, extract keywords from these benchmark records, and refine the keywords, supplemented with articles published in dedicated synthetic biology journals. We compare our search strategy with other recent bibliometric approaches to define synthetic biology, using a common source of publication data for the period from 2000 to 2015. The paper details the rapid growth and international spread of research in synthetic biology in recent years, demonstrates that diverse research disciplines are contributing to the multidisciplinary development of synthetic biology research, and visualizes this by profiling synthetic biology research on the map of science. We further show the roles of a relatively concentrated set of research sponsors in funding the growth and trajectories of synthetic biology. In addition to discussing these analyses, the paper notes limitations and suggests lines for further work.

  3. [Advances in microbial genome reduction and modification].

    Science.gov (United States)

    Wang, Jianli; Wang, Xiaoyuan

    2013-08-01

    Microbial genome reduction and modification are important strategies for constructing cellular chassis used for synthetic biology. This article summarized the essential genes and the methods to identify them in microorganisms, compared various strategies for microbial genome reduction, and analyzed the characteristics of some microorganisms with the minimized genome. This review shows the important role of genome reduction in constructing cellular chassis.

  4. Agent-based modelling in synthetic biology.

    Science.gov (United States)

    Gorochowski, Thomas E

    2016-11-30

    Biological systems exhibit complex behaviours that emerge at many different levels of organization. These span the regulation of gene expression within single cells to the use of quorum sensing to co-ordinate the action of entire bacterial colonies. Synthetic biology aims to make the engineering of biology easier, offering an opportunity to control natural systems and develop new synthetic systems with useful prescribed behaviours. However, in many cases, it is not understood how individual cells should be programmed to ensure the emergence of a required collective behaviour. Agent-based modelling aims to tackle this problem, offering a framework in which to simulate such systems and explore cellular design rules. In this article, I review the use of agent-based models in synthetic biology, outline the available computational tools, and provide details on recently engineered biological systems that are amenable to this approach. I further highlight the challenges facing this methodology and some of the potential future directions. © 2016 The Author(s).

  5. WISB: Warwick Integrative Synthetic Biology Centre.

    Science.gov (United States)

    McCarthy, John

    2016-06-15

    Synthetic biology promises to create high-impact solutions to challenges in the areas of biotechnology, human/animal health, the environment, energy, materials and food security. Equally, synthetic biologists create tools and strategies that have the potential to help us answer important fundamental questions in biology. Warwick Integrative Synthetic Biology (WISB) pursues both of these mutually complementary 'build to apply' and 'build to understand' approaches. This is reflected in our research structure, in which a core theme on predictive biosystems engineering develops underpinning understanding as well as next-generation experimental/theoretical tools, and these are then incorporated into three applied themes in which we engineer biosynthetic pathways, microbial communities and microbial effector systems in plants. WISB takes a comprehensive approach to training, education and outreach. For example, WISB is a partner in the EPSRC/BBSRC-funded U.K. Doctoral Training Centre in synthetic biology, we have developed a new undergraduate module in the subject, and we have established five WISB Research Career Development Fellowships to support young group leaders. Research in Ethical, Legal and Societal Aspects (ELSA) of synthetic biology is embedded in our centre activities. WISB has been highly proactive in building an international research and training network that includes partners in Barcelona, Boston, Copenhagen, Madrid, Marburg, São Paulo, Tartu and Valencia. © 2016 The Author(s).

  6. Misconceptions of Synthetic Biology: Lessons from an Interdisciplinary Summer School

    Science.gov (United States)

    Verseux, Cyprien; Acevedo-Rocha, Carlos G.; Chizzolini, Fabio; Rothschild, Lynn J.

    2016-01-01

    In 2014, an international group of scholars from various fields analysed the "societal dimensions" of synthetic biology in an interdisciplinary summer school. Here, we report and discuss the biologists' observations on the general perception of synthetic biology by non-biologists who took part in this event. Most attendees mainly associated synthetic biology with contributions from the best-known public figures of the field, rarely mentioning other scientists. Media extrapolations of those contributions appeared to have created unrealistic expectations and irrelevant fears that were widely disconnected from the current research in synthetic biology. Another observation was that when debating developments in synthetic biology, semantics strongly mattered: depending on the terms used to present an application of synthetic biology, attendees reacted in radically different ways. For example, using the term "GMOs" (genetically modified organisms) rather than the term "genetic engineering" led to very different reactions. Stimulating debates also happened with participants having unanticipated points of view, for instance biocentrist ethicists who argued that engineered microbes should not be used for human purposes. Another communication challenge emerged from the connotations and inaccuracies surrounding the word "life", which impaired constructive debates, thus leading to misconceptions about the abilities of scientists to engineer or even create living organisms. Finally, it appeared that synthetic biologists tend to overestimate the knowledge of non-biologists, further affecting communication. The motivation and ability of synthetic biologists to communicate their work outside their research field needs to be fostered, notably towards policymakers who need a more accurate and technical understanding of the field to make informed decisions. Interdisciplinary events gathering scholars working in and around synthetic biology are an effective tool in addressing those

  7. Surface chemical immobilization of bioactive peptides on synthetic polymers for cardiac tissue engineering.

    Science.gov (United States)

    Rosellini, Elisabetta; Cristallini, Caterina; Guerra, Giulio D; Barbani, Niccoletta

    2015-01-01

    The aim of this work was the development of new synthetic polymeric systems, functionalized by surface chemical modification with bioactive peptides, for myocardial tissue engineering. Polycaprolactone and a poly(ester-ether-ester) block copolymer synthesized in our lab, polycaprolactone-poly(ethylene oxide)-polycaprolactone (PCL-PEO-PCL), were used as the substrates to be modified. Two pentapeptides, H-Gly-Arg-Gly-Asp-Ser-OH (GRGDS) from fibronectin and H-Tyr-Ile-Gly-Ser-Arg-OH (YIGSR) from laminin, were used for the functionalization. Polymeric membranes were obtained by casting from solutions and then functionalized by means of alkaline hydrolysis and subsequent coupling of the bioactive molecules through 1-(3-dimethylaminopropyl)-3-ethylcarbodimide hydrochloride/N-hydroxysuccinimide chemistry. The hydrolysis conditions, in terms of hydrolysis time, temperature, and sodium hydroxide concentration, were optimized for the two materials. The occurrence of the coupling reaction was demonstrated by infrared spectroscopy, as the presence on the functionalized materials of the absorption peaks typical of the two peptides. The peptide surface density was determined by chromatographic analysis and the distribution was studied by infrared chemical imaging. The results showed a nearly homogeneous peptide distribution, with a density above the minimum value necessary to promote cell adhesion. Preliminary in vitro cell culture studies demonstrated that the introduction of the bioactive molecules had a positive effect on improving C2C12 myoblasts growth on the synthetic materials.

  8. pBAM1: an all-synthetic genetic tool for analysis and construction of complex bacterial phenotypes

    Directory of Open Access Journals (Sweden)

    Arévalo-Rodríguez Miguel

    2011-02-01

    Full Text Available Abstract Background Since publication in 1977 of plasmid pBR322, many breakthroughs in Biology have depended on increasingly sophisticated vector platforms for analysis and engineering of given bacterial strains. Although restriction sites impose a certain format in the procedures for assembling cloned genes, every attempt thus far to standardize vector architecture and nomenclature has ended up in failure. While this state of affairs may still be tolerable for traditional one-at-a-time studies of single genes, the onset of systems and synthetic biology calls for a simplification -along with an optimization- of the currently unwieldy pool of genetic tools. Results The functional DNA sequences present in the natural bacterial transposon Tn5 have been methodically edited and refactored for the production of a multi-purpose genetic tool named pBAM1, which allows a range of manipulations in the genome of Gram-negative bacteria. This all-synthetic construct enhances the power of mini-transposon vectors for either de-construction or re-construction of phenotypes á la carte by incorporating features inspired in systems engineering: modularity, re-usability, minimization, and compatibility with other genetic tools. pBAM1 bears an streamlined, restriction site-freed and narrow-host range replication frame bearing the sequences of R6K oriV, oriT and an ampicillin resistance marker. These go along with a business module that contains a host-independent and hyperactive transposition platform for in vivo or in vitro insertion of desired DNA into the genome of the target bacterium. All functional sequences were standardized for a straightforward replacement by equivalent counterparts, if required. pBAM1 can be delivered into recipient cells by either mating or electroporation, producing transposon insertion frequencies of 1.8 × 10-3 and 1.02 × 10-7, respectively in the soil bacterium Pseudomonas putida. Analyses of the resulting clones revealed a 100% of

  9. Synthetic Immunology: Hacking Immune Cells to Expand Their Therapeutic Capabilities.

    Science.gov (United States)

    Roybal, Kole T; Lim, Wendell A

    2017-04-26

    The ability of immune cells to survey tissues and sense pathologic insults and deviations makes them a unique platform for interfacing with the body and disease. With the rapid advancement of synthetic biology, we can now engineer and equip immune cells with new sensors and controllable therapeutic response programs to sense and treat diseases that our natural immune system cannot normally handle. Here we review the current state of engineered immune cell therapeutics and their unique capabilities compared to small molecules and biologics. We then discuss how engineered immune cells are being designed to combat cancer, focusing on how new synthetic biology tools are providing potential ways to overcome the major roadblocks for treatment. Finally, we give a long-term vision for the use of synthetic biology to engineer immune cells as a general sensor-response platform to precisely detect disease, to remodel disease microenvironments, and to treat a potentially wide range of challenging diseases.

  10. SB6.0: The 6th International meeting on Synthetic Biology, July 9-11, 2013

    Energy Technology Data Exchange (ETDEWEB)

    Kahl, Linda J. [BioBricks Foundation

    2015-04-23

    The Synthetic Biology conference series (SBx.0) is the preeminent academic meeting in synthetic biology. Organized by the BioBricks Foundation, the SBx.0 conference series brings together leading researchers, students, industry executives, and policy makers from around the world to share, consider, debate, and plan efforts to make biology easier to engineer. Historically held every two years, the SBx.0 conferences are held in alternating locations in the United States, Europe, and Asia to encourage global participation and collaboration so that the ramifications of synthetic biology research and development are most likely to be safe ethical, and beneficial. On 9-11 July 2013, the 6th installment of the synthetic biology conference series (SB6.0) was held on the campus of Imperial College London (http://sb6.biobricks.org). The SB6.0 conference was attended by over 700 people, and many more were able to participate via video digital conference (http://sb6.biobricks.org/digital-conference/). Over the course of three days, the SB6.0 conference agenda included plenary sessions, workshops, and poster presentations covering topics ranging from the infrastructure needs arising when “Systematic Engineering Meets Biological Complexity” and design-led considerations for “Connecting People and Technologies” to discussions on “Engineering Biology for New Materials,” “Assessing Risk and Managing Biocontainment,” and “New Directions for Energy and Sustainability.” The $10,150 grant awarded by the U.S. Department of Energy (DE-SC0010233) to the BioBricks Foundation was used to provide partial reimbursement for the travel expenses of leading researchers from the United States to speak at the SB6.0 conference. A total of $9,450 was used to reimburse U.S. speakers for actual expenses related to the SB6.0 conference, including airfare (economy or coach only), ground transportation, hotel, and registration fees. In addition, $700 of the grant was used to offset

  11. Engineered bacterial hydrophobic oligopeptide repeats in a synthetic yeast prion, [REP-PSI+

    Directory of Open Access Journals (Sweden)

    Fátima eGasset-Rosa

    2015-04-01

    Full Text Available The yeast translation termination factor Sup35p, by aggregating as the [PSI+] prion, enables ribosomes to read-through stop codons, thus expanding the diversity of the Saccharomyces cerevisiae proteome. Yeast prions are functional amyloids that replicate by templating their conformation on native protein molecules, then assembling as large aggregates and fibers. Prions propagate epigenetically from mother to daughter cells by fragmentation of such assemblies. In the N-terminal prion-forming domain, Sup35p has glutamine/asparagine-rich oligopeptide repeats (OPRs, which enable propagation through chaperone-elicited shearing. We have engineered chimeras by replacing the polar OPRs in Sup35p by up to five repeats of a hydrophobic amyloidogenic sequence from the synthetic bacterial prionoid RepA-WH1. The resulting hybrid, [REP-PSI+], i was functional in a stop codon read-through assay in S. cerevisiae; ii generates weak phenotypic variants upon both its expression or transformation into [psi-] cells; iii these variants correlated with high molecular weight aggregates resistant to SDS during electrophoresis; and iv according to fluorescence microscopy, the fusion of the prion domains from the engineered chimeras to the reporter protein mCherry generated perivacuolar aggregate foci in yeast cells. All these are signatures of bona fide yeast prions. As assessed through biophysical approaches, the chimeras assembled as oligomers rather than as the fibers characteristic of [PSI+]. These results suggest that it is the balance between polar and hydrophobic residues in OPRs what determines prion conformational dynamics. In addition, our findings illustrate the feasibility of enabling new propagation traits in yeast prions by engineering OPRs with heterologous amyloidogenic sequence repeats.

  12. Adaptation of the Pivotal-Differential Genome Pattern for the Induction of Intergenomic Chromosome Recombination in Hybrids of Synthetic Amphidiploids within Triticeae Tribe

    Directory of Open Access Journals (Sweden)

    Michal T. Kwiatek

    2017-07-01

    Full Text Available A pivotal-differential evolution pattern is when two allopolyploids share a common genome, which is called pivotal, and differ with respect to the other genome or genomes, called differential. This feature induces the intergenomic recombination between chromosomes of differential genomes, which can lead to speciation. Our study is a cytomolecular insight into this mechanism which was adapted for the induction of intergenomic chromosome recombination in hybrids of synthetic amphidiploids Aegilops biuncialis × S. cereale (UUMMRR and triticale (AABBRR where R-genome was pivotal. We observed chromosome recombination events which were induced by both: (1 random chromosome fragmentation and non-homologous chromosome end joining at mitosis of root meristem cells and (2 intergenomic chromosome associations at meiosis of pollen mother cells (PMCs of F1 hybrids. Reciprocal chromosome translocations were identified in six F1 plants and 15 plants of F2 generation using fluorescence in situ hybridization (FISH with DNA clones (pTa-86, pTa-k374, pTa-465, pTa-535, pTa-k566, and pTa-713. We observed signals of pTa-86, pTa-535, and pTa-k566 probes in several chromosome breakpoints. The comparison of the DNA clone sequences distinguished a number of common motifs, which can be considered as characteristics of chromosome breakpoint loci. Immunodetection of synaptonemal complex proteins and genomic in situ hybridization analysis at meiosis of PMCs of F1 hybrids showed, that the homologous pairing of pivotal R—genome chromosomes is crucial for the fertility of F1 hybrids, however, these chromosomes can be also involved in the intergeneric recombination.

  13. Inhibition of Dengue Virus 3 in Mammalian Cell Culture by Synthetic ...

    African Journals Online (AJOL)

    HP

    Purpose: To evaluate the inhibition of Dengue virus 3 by synthetic siRNAs targeting the untranslated regions UTR and structural regions of DENV3 genome in Vero-81 cell line. Methods: Vero-81 cells transfected with synthetic siRNAs were challenged by DENV3. The effectiveness of siRNAs was confirmed by four ...

  14. Approaches to chemical synthetic biology.

    Science.gov (United States)

    Chiarabelli, Cristiano; Stano, Pasquale; Anella, Fabrizio; Carrara, Paolo; Luisi, Pier Luigi

    2012-07-16

    Synthetic biology is first represented in terms of two complementary aspects, the bio-engineering one, based on the genetic manipulation of extant microbial forms in order to obtain forms of life which do not exist in nature; and the chemical synthetic biology, an approach mostly based on chemical manipulation for the laboratory synthesis of biological structures that do not exist in nature. The paper is mostly devoted to shortly review chemical synthetic biology projects currently carried out in our laboratory. In particular, we describe: the minimal cell project, then the "Never Born Proteins" and lastly the Never Born RNAs. We describe and critically analyze the main results, emphasizing the possible relevance of chemical synthetic biology for the progress in basic science and biotechnology. Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  15. Decolorization of industrial synthetic dyes using engineered Pseudomonas putida cells with surface-immobilized bacterial laccase

    Directory of Open Access Journals (Sweden)

    Wang Wei

    2012-06-01

    Full Text Available Abstract Background Microbial laccases are highly useful in textile effluent dye biodegradation. However, the bioavailability of cellularly expressed or purified laccases in continuous operations is usually limited by mass transfer impediment or enzyme regeneration difficulty. Therefore, this study develops a regenerable bacterial surface-displaying system for industrial synthetic dye decolorization, and evaluates its effects on independent and continuous operations. Results A bacterial laccase (WlacD was engineered onto the cell surface of the solvent-tolerant bacterium Pseudomonas putida to construct a whole-cell biocatalyst. Ice nucleation protein (InaQ anchor was employed, and the ability of 1 to 3 tandemly aligned N-terminal repeats to direct WlacD display were compared. Immobilized WlacD was determined to be surface-displayed in functional form using Western blot analysis, immunofluorescence microscopy, flow cytometry, and whole-cell enzymatic activity assay. Engineered P. putida cells were then applied to decolorize the anthraquinone dye Acid Green (AG 25 and diazo-dye Acid Red (AR 18. The results showed that decolorization of both dyes is Cu2+- and mediator-independent, with an optimum temperature of 35°C and pH of 3.0, and can be stably performed across a temperature range of 15°C to 45°C. A high activity toward AG25 (1 g/l with relative decolorization values of 91.2% (3 h and 97.1% (18 h, as well as high activity to AR18 (1 g/l by 80.5% (3 h and 89.0% (18 h, was recorded. The engineered system exhibited a comparably high activity compared with those of separate dyes in a continuous three-round shake-flask decolorization of AG25/AR18 mixed dye (each 1 g/l. No significant decline in decolorization efficacy was noted during first two-rounds but reaction equilibriums were elongated, and the residual laccase activity eventually decreased to low levels. However, the decolorizing capacity of the system was easily retrieved

  16. Genomic composition factors affect codon usage in porcine genome ...

    African Journals Online (AJOL)

    ... be explored for designing degenerate primers, necessitate selecting appropriate hosts expression systems to manipulate the expression of target genes in vivo or in vitro and improve the accuracy of gene prediction from genomic sequences thus maximizing the effectiveness of genetic manipulations in synthetic biology.

  17. Synthetic biology for microbial production of lipid-based biofuels.

    Science.gov (United States)

    d'Espaux, Leo; Mendez-Perez, Daniel; Li, Rachel; Keasling, Jay D

    2015-12-01

    The risks of maintaining current CO2 emission trends have led to interest in producing biofuels using engineered microbes. Microbial biofuels reduce emissions because CO2 produced by fuel combustion is offset by CO2 captured by growing biomass, which is later used as feedstock for biofuel fermentation. Hydrocarbons found in petroleum fuels share striking similarity with biological lipids. Here we review synthetic metabolic pathways based on fatty acid and isoprenoid metabolism to produce alkanes and other molecules suitable as biofuels. We further discuss engineering strategies to optimize engineered biosynthetic routes, as well as the potential of synthetic biology for sustainable manufacturing. Published by Elsevier Ltd.

  18. Synthetic biology for microbial production of lipid-based biofuels

    Energy Technology Data Exchange (ETDEWEB)

    d' Espaux, L; Mendez-Perez, D; Li, R; Keasling, JD

    2015-10-23

    The risks of maintaining current CO2 emission trends have led to interest in producing biofuels using engineered microbes. Microbial biofuels reduce emissions because CO2 produced by fuel combustion is offset by CO2 captured by growing biomass, which is later used as feedstock for biofuel fermentation. Hydrocarbons found in petroleum fuels share striking similarity with biological lipids. Here in this paper we review synthetic metabolic pathways based on fatty acid and isoprenoid metabolism to produce alkanes and other molecules suitable as biofuels. Lastly, we further discuss engineering strategies to optimize engineered biosynthetic routes, as well as the potential of synthetic biology for sustainable manufacturing.

  19. [Progress of genome engineering technology via clustered regularly interspaced short palindromic repeats--a review].

    Science.gov (United States)

    Li, Hao; Qiu, Shaofu; Song, Hongbin

    2013-10-04

    In survival competition with phage, bacteria and archaea gradually evolved the acquired immune system--Clustered regularly interspaced short palindromic repeats (CRISPR), presenting the trait of transcribing the crRNA and the CRISPR-associated protein (Cas) to silence or cleaving the foreign double-stranded DNA specifically. In recent years, strong interest arises in prokaryotes primitive immune system and many in-depth researches are going on. Recently, researchers successfully repurposed CRISPR as an RNA-guided platform for sequence-specific gene expression, which provides a simple approach for selectively perturbing gene expression on a genome-wide scale. It will undoubtedly bring genome engineering into a more convenient and accurate new era.

  20. Rewiring the severe acute respiratory syndrome coronavirus (SARS-CoV) transcription circuit: Engineering a recombination-resistant genome

    Science.gov (United States)

    Yount, Boyd; Roberts, Rhonda S.; Lindesmith, Lisa; Baric, Ralph S.

    2006-08-01

    Live virus vaccines provide significant protection against many detrimental human and animal diseases, but reversion to virulence by mutation and recombination has reduced appeal. Using severe acute respiratory syndrome coronavirus as a model, we engineered a different transcription regulatory circuit and isolated recombinant viruses. The transcription network allowed for efficient expression of the viral transcripts and proteins, and the recombinant viruses replicated to WT levels. Recombinant genomes were then constructed that contained mixtures of the WT and mutant regulatory circuits, reflecting recombinant viruses that might occur in nature. Although viable viruses could readily be isolated from WT and recombinant genomes containing homogeneous transcription circuits, chimeras that contained mixed regulatory networks were invariantly lethal, because viable chimeric viruses were not isolated. Mechanistically, mixed regulatory circuits promoted inefficient subgenomic transcription from inappropriate start sites, resulting in truncated ORFs and effectively minimize viral structural protein expression. Engineering regulatory transcription circuits of intercommunicating alleles successfully introduces genetic traps into a viral genome that are lethal in RNA recombinant progeny viruses. regulation | systems biology | vaccine design

  1. Is It Time for Synthetic Biodiversity Conservation?

    Science.gov (United States)

    Piaggio, Antoinette J; Segelbacher, Gernot; Seddon, Philip J; Alphey, Luke; Bennett, Elizabeth L; Carlson, Robert H; Friedman, Robert M; Kanavy, Dona; Phelan, Ryan; Redford, Kent H; Rosales, Marina; Slobodian, Lydia; Wheeler, Keith

    2017-02-01

    Evidence indicates that, despite some critical successes, current conservation approaches are not slowing the overall rate of biodiversity loss. The field of synthetic biology, which is capable of altering natural genomes with extremely precise editing, might offer the potential to resolve some intractable conservation problems (e.g., invasive species or pathogens). However, it is our opinion that there has been insufficient engagement by the conservation community with practitioners of synthetic biology. We contend that rapid, large-scale engagement of these two communities is urgently needed to avoid unintended and deleterious ecological consequences. To this point we describe case studies where synthetic biology is currently being applied to conservation, and we highlight the benefits to conservation biologists from engaging with this emerging technology. Published by Elsevier Ltd.

  2. Accurate DNA assembly and genome engineering with optimized uracil excision cloning

    DEFF Research Database (Denmark)

    Cavaleiro, Mafalda; Kim, Se Hyeuk; Seppala, Susanna

    2015-01-01

    Simple and reliable DNA editing by uracil excision (a.k.a. USER cloning) has been described by several research groups, but the optimal design of cohesive DNA ends for multigene assembly remains elusive. Here, we use two model constructs based on expression of gfp and a four-gene pathway that pro......Simple and reliable DNA editing by uracil excision (a.k.a. USER cloning) has been described by several research groups, but the optimal design of cohesive DNA ends for multigene assembly remains elusive. Here, we use two model constructs based on expression of gfp and a four-gene pathway...... that produces β-carotene to optimize assembly junctions and the uracil excision protocol. By combining uracil excision cloning with a genomic integration technology, we demonstrate that up to six DNA fragments can be assembled in a one-tube reaction for direct genome integration with high accuracy, greatly...... facilitating the advanced engineering of robust cell factories....

  3. Accurate Dna Assembly And Direct Genome Integration With Optimized Uracil Excision Cloning To Facilitate Engineering Of Escherichia Coli As A Cell Factory

    DEFF Research Database (Denmark)

    Cavaleiro, Mafalda; Kim, Se Hyeuk; Nørholm, Morten

    2015-01-01

    Plants produce a vast diversity of valuable compounds with medical properties, but these are often difficult to purify from the natural source or produce by organic synthesis. An alternative is to transfer the biosynthetic pathways to an efficient production host like the bacterium Escherichia co......-excision-based cloning and combining it with a genome-engineering approach to allow direct integration of whole metabolic pathways into the genome of E. coli, to facilitate the advanced engineering of cell factories........ Cloning and heterologous gene expression are major bottlenecks in the metabolic engineering field. We are working on standardizing DNA vector design processes to promote automation and collaborations in early phase metabolic engineering projects. Here, we focus on optimizing the already established uracil...

  4. [Smart therapeutics based on synthetic gene circuits].

    Science.gov (United States)

    Peng, Shuguang; Xie, Zhen

    2017-03-25

    Synthetic biology has an important impact on biology research since its birth. Applying the thought and methods that reference from electrical engineering, synthetic biology uncovers many regulatory mechanisms of life systems, transforms and expands a series of biological components. Therefore, it brings a wide range of biomedical applications, including providing new ideas for disease diagnosis and treatment. This review describes the latest advances in the field of disease diagnosis and therapy based on mammalian cell or bacterial synthetic gene circuits, and provides new ideas for future smart therapy design.

  5. A multi-landing pad DNA integration platform for mammalian cell engineering

    Science.gov (United States)

    Gaidukov, Leonid; Wroblewska, Liliana; Teague, Brian; Nelson, Tom; Zhang, Xin; Liu, Yan; Jagtap, Kalpana; Mamo, Selamawit; Tseng, Wen Allen; Lowe, Alexis; Das, Jishnu; Bandara, Kalpanie; Baijuraj, Swetha; Summers, Nevin M; Zhang, Lin; Weiss, Ron

    2018-01-01

    Abstract Engineering mammalian cell lines that stably express many transgenes requires the precise insertion of large amounts of heterologous DNA into well-characterized genomic loci, but current methods are limited. To facilitate reliable large-scale engineering of CHO cells, we identified 21 novel genomic sites that supported stable long-term expression of transgenes, and then constructed cell lines containing one, two or three ‘landing pad’ recombination sites at selected loci. By using a highly efficient BxB1 recombinase along with different selection markers at each site, we directed recombinase-mediated insertion of heterologous DNA to selected sites, including targeting all three with a single transfection. We used this method to controllably integrate up to nine copies of a monoclonal antibody, representing about 100 kb of heterologous DNA in 21 transcriptional units. Because the integration was targeted to pre-validated loci, recombinant protein expression remained stable for weeks and additional copies of the antibody cassette in the integrated payload resulted in a linear increase in antibody expression. Overall, this multi-copy site-specific integration platform allows for controllable and reproducible insertion of large amounts of DNA into stable genomic sites, which has broad applications for mammalian synthetic biology, recombinant protein production and biomanufacturing. PMID:29617873

  6. Building biological foundries for next-generation synthetic biology.

    Science.gov (United States)

    Chao, Ran; Yuan, YongBo; Zhao, HuiMin

    2015-07-01

    Synthetic biology is an interdisciplinary field that takes top-down approaches to understand and engineer biological systems through design-build-test cycles. A number of advances in this relatively young field have greatly accelerated such engineering cycles. Specifically, various innovative tools were developed for in silico biosystems design, DNA de novo synthesis and assembly, construct verification, as well as metabolite analysis, which have laid a solid foundation for building biological foundries for rapid prototyping of improved or novel biosystems. This review summarizes the state-of-the-art technologies for synthetic biology and discusses the challenges to establish such biological foundries.

  7. Synthetic biology and occupational risk.

    Science.gov (United States)

    Howard, John; Murashov, Vladimir; Schulte, Paul

    2017-03-01

    Synthetic biology is an emerging interdisciplinary field of biotechnology that involves applying the principles of engineering and chemical design to biological systems. Biosafety professionals have done an excellent job in addressing research laboratory safety as synthetic biology and gene editing have emerged from the larger field of biotechnology. Despite these efforts, risks posed by synthetic biology are of increasing concern as research procedures scale up to industrial processes in the larger bioeconomy. A greater number and variety of workers will be exposed to commercial synthetic biology risks in the future, including risks to a variety of workers from the use of lentiviral vectors as gene transfer devices. There is a need to review and enhance current protection measures in the field of synthetic biology, whether in experimental laboratories where new advances are being researched, in health care settings where treatments using viral vectors as gene delivery systems are increasingly being used, or in the industrial bioeconomy. Enhanced worker protection measures should include increased injury and illness surveillance of the synthetic biology workforce; proactive risk assessment and management of synthetic biology products; research on the relative effectiveness of extrinsic and intrinsic biocontainment methods; specific safety guidance for synthetic biology industrial processes; determination of appropriate medical mitigation measures for lentiviral vector exposure incidents; and greater awareness and involvement in synthetic biology safety by the general occupational safety and health community as well as by government occupational safety and health research and regulatory agencies.

  8. Enhancement of single guide RNA transcription for efficient CRISPR/Cas-based genomic engineering.

    Science.gov (United States)

    Ui-Tei, Kumiko; Maruyama, Shohei; Nakano, Yuko

    2017-06-01

    Genomic engineering using clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) protein is a promising approach for targeting the genomic DNA of virtually any organism in a sequence-specific manner. Recent remarkable advances in CRISPR/Cas technology have made it a feasible system for use in therapeutic applications and biotechnology. In the CRISPR/Cas system, a guide RNA (gRNA), interacting with the Cas protein, recognizes a genomic region with sequence complementarity, and the double-stranded DNA at the target site is cleaved by the Cas protein. A widely used gRNA is an RNA polymerase III (pol III)-driven single gRNA (sgRNA), which is produced by artificial fusion of CRISPR RNA (crRNA) and trans-activation crRNA (tracrRNA). However, we identified a TTTT stretch, known as a termination signal of RNA pol III, in the scaffold region of the sgRNA. Here, we revealed that sgRNA carrying a TTTT stretch reduces the efficiency of sgRNA transcription due to premature transcriptional termination, and decreases the efficiency of genome editing. Unexpectedly, it was also shown that the premature terminated sgRNA may have an adverse effect of inducing RNA interference. Such disadvantageous effects were avoided by substituting one base in the TTTT stretch.

  9. Philosophy of Systems and Synthetic Biology

    DEFF Research Database (Denmark)

    Green, Sara

    2017-01-01

    This entry aims to clarify how systems and synthetic biology contribute to and extend discussions within philosophy of science. Unlike fields such as developmental biology or molecular biology, systems and synthetic biology are not easily demarcated by a focus on a specific subject area or level...... of organization. Rather, they are characterized by the development and application of mathematical, computational, and synthetic modeling strategies in response to complex problems and challenges within the life sciences. Proponents of systems and synthetic biology often stress the necessity of a perspective...... that goes beyond the scope of molecular biology and genetic engineering, respectively. With the emphasis on systems and interaction networks, the approaches explicitly engage in one of the oldest philosophical discussions on the relationship between parts and wholes, or between reductionism and holism...

  10. The logics of metabolic regulation in bacteria challenges biosensor-based metabolic engineering

    Directory of Open Access Journals (Sweden)

    Matthieu Jules

    2017-12-01

    Full Text Available Synthetic Biology (SB aims at the rational design and engineering of novel biological functions and systems. By facilitating the engineering of living organisms, SB promises to facilitate the development of many new applications for health, biomanufacturing, and the environment. Over the last decade, SB promoted the construction of libraries of components enabling the fine-tuning of genetic circuits expression and the development of novel genome engineering methodologies for many organisms of interest. SB thus opened new perspectives in the field of metabolic engineering, which was until then mainly limited to (overproducing naturally synthesized metabolic compounds. To engineer efficient cell factories, it is key to precisely reroute cellular resources from the central carbon metabolism (CCM to the synthetic circuitry. This task is however difficult as there is still significant lack of knowledge regarding both the function of several metabolic components and the regulation of the CCM fluxes for many industrially important bacteria. Pyruvate is a pivotal metabolite at the heart of the CCM and a key precursor for the synthesis of several commodity compounds and fine chemicals. Numerous bacterial species can also use it as a carbon source when present in the environment but bacterial, pyruvate-specific uptake systems were to be discovered. This is an issue for metabolic engineering as one can imagine to make use of pyruvate transport systems to replenish synthetic metabolic pathways towards the synthesis of chemicals of interest. Here we describe a recent study (MBio 8(5: e00976-17, which identified and characterized a pyruvate transport system in the Gram-positive (G+ve bacterium Bacillus subtilis, a well-established biotechnological workhorse for the production of enzymes, fine chemicals and antibiotics. This study also revealed that the activity of the two-component system (TCS responsible for its induction is retro-inhibited by the level of

  11. The emerging age of cell-free synthetic biology.

    Science.gov (United States)

    Smith, Mark Thomas; Wilding, Kristen M; Hunt, Jeremy M; Bennett, Anthony M; Bundy, Bradley C

    2014-08-25

    The engineering of and mastery over biological parts has catalyzed the emergence of synthetic biology. This field has grown exponentially in the past decade. As increasingly more applications of synthetic biology are pursued, more challenges are encountered, such as delivering genetic material into cells and optimizing genetic circuits in vivo. An in vitro or cell-free approach to synthetic biology simplifies and avoids many of the pitfalls of in vivo synthetic biology. In this review, we describe some of the innate features that make cell-free systems compelling platforms for synthetic biology and discuss emerging improvements of cell-free technologies. We also select and highlight recent and emerging applications of cell-free synthetic biology. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  12. Synthetic biology expands chemical control of microorganisms.

    Science.gov (United States)

    Ford, Tyler J; Silver, Pamela A

    2015-10-01

    The tools of synthetic biology allow researchers to change the ways engineered organisms respond to chemical stimuli. Decades of basic biology research and new efforts in computational protein and RNA design have led to the development of small molecule sensors that can be used to alter organism function. These new functions leap beyond the natural propensities of the engineered organisms. They can range from simple fluorescence or growth reporting to pathogen killing, and can involve metabolic coordination among multiple cells or organisms. Herein, we discuss how synthetic biology alters microorganisms' responses to chemical stimuli resulting in the development of microbes as toxicity sensors, disease treatments, and chemical factories. Copyright © 2015 Elsevier Ltd. All rights reserved.

  13. Industrial systems biology and its impact on synthetic biology of yeast cell factories

    DEFF Research Database (Denmark)

    Fletcher, Eugene; Krivoruchko, Anastasia; Nielsen, Jens

    2016-01-01

    Engineering industrial cell factories to effectively yield a desired product while dealing with industrially relevant stresses is usually the most challenging step in the development of industrial production of chemicals using microbial fermentation processes. Using synthetic biology tools......, microbial cell factories such as Saccharomyces cerevisiae can be engineered to express synthetic pathways for the production of fuels, biopharmaceuticals, fragrances, and food flavors. However, directing fluxes through these synthetic pathways towards the desired product can be demanding due to complex...... regulation or poor gene expression. Systems biology, which applies computational tools and mathematical modeling to understand complex biological networks, can be used to guide synthetic biology design. Here, we present our perspective on how systems biology can impact synthetic biology towards the goal...

  14. Bacterial Genome Editing Strategy for Control of Transcription and Protein Stability

    DEFF Research Database (Denmark)

    Lauritsen, Ida; Martinez, Virginia; Ronda, Carlotta

    2018-01-01

    In molecular biology and cell factory engineering, tools that enable control of protein production and stability are highly important. Here, we describe protocols for tagging genes in Escherichia coli allowing for inducible degradation and transcriptional control of any soluble protein of interest....... The underlying molecular biology is based on the two cross-kingdom tools CRISPRi and the N-end rule for protein degradation. Genome editing is performed with the CRMAGE technology and randomization of the translational initiation region minimizes the polar effects of tag insertion. The approach has previously...... been applied for targeting proteins originating from essential operon-located genes and has potential to serve as a universal synthetic biology tool....

  15. A Canadian refiner's perspective of synthetic crudes

    International Nuclear Information System (INIS)

    Halford, T.L.; McIntosh, A.P.; Rasmussen

    1997-01-01

    Some of the factors affecting a refiner's choice of crude oil include refinery hardware, particularly gas oil crackers, products slate and product specifications, crude availability, relative crude price and crude quality. An overview of synthetic crude, the use of synthetic crude combined with other crudes and a comparison of synthetic crude with conventional crude oil was given. The two main users of synthetic crude are basically two groups of refiners, those large groups who use synthetic crude combined with other crudes, and a smaller group who run synthetic crude on specially designed units as a sole feed. The effects of changes in fuel legislation were reviewed. It was predicted that the changes will have a mixed impact on the value of synthetic crude, but low sulphur diesel regulations and gasoline sulphur regulations will make current synthetic crudes attractive. The big future change with a negative impact will be diesel cetane increases to reduce engine emissions. This will reduce synthetic crude attractiveness due to distillate yields and quality and high gas oil yields. Similarly, any legislation limiting aromatics in diesel fuel will also make synthetic crudes less attractive. Problems experienced by refiners with hardware dedicated to synthetic crude (salt, naphthenic acid, fouling, quality variations) were also reviewed. 3 tabs

  16. Synthetic Biology: Mapping the Scientific Landscape

    Science.gov (United States)

    Oldham, Paul; Hall, Stephen; Burton, Geoff

    2012-01-01

    This article uses data from Thomson Reuters Web of Science to map and analyse the scientific landscape for synthetic biology. The article draws on recent advances in data visualisation and analytics with the aim of informing upcoming international policy debates on the governance of synthetic biology by the Subsidiary Body on Scientific, Technical and Technological Advice (SBSTTA) of the United Nations Convention on Biological Diversity. We use mapping techniques to identify how synthetic biology can best be understood and the range of institutions, researchers and funding agencies involved. Debates under the Convention are likely to focus on a possible moratorium on the field release of synthetic organisms, cells or genomes. Based on the empirical evidence we propose that guidance could be provided to funding agencies to respect the letter and spirit of the Convention on Biological Diversity in making research investments. Building on the recommendations of the United States Presidential Commission for the Study of Bioethical Issues we demonstrate that it is possible to promote independent and transparent monitoring of developments in synthetic biology using modern information tools. In particular, public and policy understanding and engagement with synthetic biology can be enhanced through the use of online interactive tools. As a step forward in this process we make existing data on the scientific literature on synthetic biology available in an online interactive workbook so that researchers, policy makers and civil society can explore the data and draw conclusions for themselves. PMID:22539946

  17. All-in-One CRISPR-Cas9/FokI-dCas9 Vector-Mediated Multiplex Genome Engineering in Cultured Cells.

    Science.gov (United States)

    Sakuma, Tetsushi; Sakamoto, Takuya; Yamamoto, Takashi

    2017-01-01

    CRISPR-Cas9 enables highly convenient multiplex genome engineering in cultured cells, because it utilizes generic Cas9 nuclease and an easily customizable single-guide RNA (sgRNA) for site-specific DNA double-strand break induction. We previously established a multiplex CRISPR-Cas9 assembly system for constructing an all-in-one vector simultaneously expressing multiple sgRNAs and Cas9 nuclease or other Cas9 variants including FokI-dCas9, which supersedes the wild-type Cas9 with regard to high specificity. In this chapter, we describe a streamlined protocol to design and construct multiplex CRISPR-Cas9 or FokI-dCas9 vectors, to introduce them into cultured cells by lipofection or electroporation, to enrich the genomically edited cells with a transient puromycin selection, to validate the mutation efficiency by Surveyor nuclease assay, and to perform off-target analyses. We show that our protocol enables highly efficient multiplex genome engineering even in hard-to-transfect HepG2 cells.

  18. BISRU: Synthetic Microbes for Moon, Mars and Beyond

    Science.gov (United States)

    Cumbers, J.; Rothchild, L.

    2010-04-01

    Synthetic biology and genomics will bring a new range of designer organisms into being and give us new tools for the manipulation and control of these organisms. BISRU or biological in situ resource utilization is the use of such genetically modified organisms in space.

  19. Constructs and methods for genome editing and genetic engineering of fungi and protists

    Science.gov (United States)

    Hittinger, Christopher Todd; Alexander, William Gerald

    2018-01-30

    Provided herein are constructs for genome editing or genetic engineering in fungi or protists, methods of using the constructs and media for use in selecting cells. The construct include a polynucleotide encoding a thymidine kinase operably connected to a promoter, suitably a constitutive promoter; a polynucleotide encoding an endonuclease operably connected to an inducible promoter; and a recognition site for the endonuclease. The constructs may also include selectable markers for use in selecting recombinations.

  20. Genome engineering with TALENs and ZFNs: repair pathways and donor design.

    Science.gov (United States)

    Carroll, Dana; Beumer, Kelly J

    2014-09-01

    Genome engineering with targetable nucleases depends on cellular pathways of DNA repair after target cleavage. Knowledge of how those pathways work, their requirements and their active factors, can guide experimental design and improve outcomes. While many aspects of both homologous recombination (HR) and nonhomologous end joining (NHEJ) are shared by a broad range of cells and organisms, some features are specific to individual situations. This article reviews the influence of repair mechanisms on the results of gene targeting experiments, with an emphasis on lessons learned from experiments with Drosophila. Copyright © 2014 Elsevier Inc. All rights reserved.

  1. Bottom-up synthetic biology: modular design for making artificial platelets

    Science.gov (United States)

    Majumder, Sagardip; Liu, Allen P.

    2018-01-01

    Engineering artificial cells to mimic one or multiple fundamental cell biological functions is an emerging area of synthetic biology. Reconstituting functional modules from biological components in vitro is a challenging yet an important essence of bottom-up synthetic biology. Here we describe the concept of building artificial platelets using bottom-up synthetic biology and the four functional modules that together could enable such an ambitious effort.

  2. METABOLIC MODELLING IN THE DEVELOPMENT OF CELL FACTORIES BY SYNTHETIC BIOLOGY

    Directory of Open Access Journals (Sweden)

    Paula Jouhten

    2012-10-01

    Full Text Available Cell factories are commonly microbial organisms utilized for bioconversion of renewable resources to bulk or high value chemicals. Introduction of novel production pathways in chassis strains is the core of the development of cell factories by synthetic biology. Synthetic biology aims to create novel biological functions and systems not found in nature by combining biology with engineering. The workflow of the development of novel cell factories with synthetic biology is ideally linear which will be attainable with the quantitative engineering approach, high-quality predictive models, and libraries of well-characterized parts. Different types of metabolic models, mathematical representations of metabolism and its components, enzymes and metabolites, are useful in particular phases of the synthetic biology workflow. In this minireview, the role of metabolic modelling in synthetic biology will be discussed with a review of current status of compatible methods and models for the in silico design and quantitative evaluation of a cell factory.

  3. Building synthetic cellular organization

    OpenAIRE

    Polka, Jessica K.; Silver, Pamela A.

    2013-01-01

    The elaborate spatial organization of cells enhances, restricts, and regulates protein–protein interactions. However, the biological significance of this organization has been difficult to study without ways of directly perturbing it. We highlight synthetic biology tools for engineering novel cellular organization, describing how they have been, and can be, used to advance cell biology.

  4. Genome editing in mouse spermatogonial stem/progenitor cells using engineered nucleases.

    Directory of Open Access Journals (Sweden)

    Danielle A Fanslow

    Full Text Available Editing the genome to create specific sequence modifications is a powerful way to study gene function and promises future applicability to gene therapy. Creation of precise modifications requires homologous recombination, a very rare event in most cell types that can be stimulated by introducing a double strand break near the target sequence. One method to create a double strand break in a particular sequence is with a custom designed nuclease. We used engineered nucleases to stimulate homologous recombination to correct a mutant gene in mouse "GS" (germline stem cells, testicular derived cell cultures containing spermatogonial stem cells and progenitor cells. We demonstrated that gene-corrected cells maintained several properties of spermatogonial stem/progenitor cells including the ability to colonize following testicular transplantation. This proof of concept for genome editing in GS cells impacts both cell therapy and basic research given the potential for GS cells to be propagated in vitro, contribute to the germline in vivo following testicular transplantation or become reprogrammed to pluripotency in vitro.

  5. Informational laws of genome structures

    Science.gov (United States)

    Bonnici, Vincenzo; Manca, Vincenzo

    2016-06-01

    In recent years, the analysis of genomes by means of strings of length k occurring in the genomes, called k-mers, has provided important insights into the basic mechanisms and design principles of genome structures. In the present study, we focus on the proper choice of the value of k for applying information theoretic concepts that express intrinsic aspects of genomes. The value k = lg2(n), where n is the genome length, is determined to be the best choice in the definition of some genomic informational indexes that are studied and computed for seventy genomes. These indexes, which are based on information entropies and on suitable comparisons with random genomes, suggest five informational laws, to which all of the considered genomes obey. Moreover, an informational genome complexity measure is proposed, which is a generalized logistic map that balances entropic and anti-entropic components of genomes and is related to their evolutionary dynamics. Finally, applications to computational synthetic biology are briefly outlined.

  6. Heterologous expression of MlcE in Saccharomyces cerevisiae provides resistance to natural and semi-synthetic statins

    Directory of Open Access Journals (Sweden)

    Ana Ley

    2015-12-01

    Full Text Available Statins are inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase, the key enzyme in cholesterol biosynthesis. Their extensive use in treatment and prevention of cardiovascular diseases placed statins among the best selling drugs. Construction of Saccharomyces cerevisiae cell factory for the production of high concentrations of natural statins will require establishment of a non-destructive self-resistance mechanism to overcome the undesirable growth inhibition effects of statins. To establish active export of statins from yeast, and thereby detoxification, we integrated a putative efflux pump-encoding gene mlcE from the mevastatin-producing Penicillium citrinum into the S. cerevisiae genome. The resulting strain showed increased resistance to both natural statins (mevastatin and lovastatin and semi-synthetic statin (simvastatin when compared to the wild type strain. Expression of RFP-tagged mlcE showed that MlcE is localized to the yeast plasma and vacuolar membranes. We provide a possible engineering strategy for improvement of future yeast based production of natural and semi-synthetic statins. Keywords: Polyketide, Statins, Saccharomyces cerevisiae, Transport, Cell factory, Resistance

  7. Engineering synthetic vaccines using cues from natural immunity

    Science.gov (United States)

    Irvine, Darrell J.; Swartz, Melody A.; Szeto, Gregory L.

    2013-11-01

    Vaccines aim to protect against or treat diseases through manipulation of the immune response, promoting either immunity or tolerance. In the former case, vaccines generate antibodies and T cells poised to protect against future pathogen encounter or attack diseased cells such as tumours; in the latter case, which is far less developed, vaccines block pathogenic autoreactive T cells and autoantibodies that target self tissue. Enormous challenges remain, however, as a consequence of our incomplete understanding of human immunity. A rapidly growing field of research is the design of vaccines based on synthetic materials to target organs, tissues, cells or intracellular compartments; to co-deliver immunomodulatory signals that control the quality of the immune response; or to act directly as immune regulators. There exists great potential for well-defined materials to further our understanding of immunity. Here we describe recent advances in the design of synthetic materials to direct immune responses, highlighting successes and challenges in prophylactic, therapeutic and tolerance-inducing vaccines.

  8. Control theory meets synthetic biology.

    Science.gov (United States)

    Del Vecchio, Domitilla; Dy, Aaron J; Qian, Yili

    2016-07-01

    The past several years have witnessed an increased presence of control theoretic concepts in synthetic biology. This review presents an organized summary of how these control design concepts have been applied to tackle a variety of problems faced when building synthetic biomolecular circuits in living cells. In particular, we describe success stories that demonstrate how simple or more elaborate control design methods can be used to make the behaviour of synthetic genetic circuits within a single cell or across a cell population more reliable, predictable and robust to perturbations. The description especially highlights technical challenges that uniquely arise from the need to implement control designs within a new hardware setting, along with implemented or proposed solutions. Some engineering solutions employing complex feedback control schemes are also described, which, however, still require a deeper theoretical analysis of stability, performance and robustness properties. Overall, this paper should help synthetic biologists become familiar with feedback control concepts as they can be used in their application area. At the same time, it should provide some domain knowledge to control theorists who wish to enter the rising and exciting field of synthetic biology. © 2016 The Author(s).

  9. [Research progress of mammalian synthetic biology in biomedical field].

    Science.gov (United States)

    Yang, Linfeng; Yin, Jianli; Wang, Meiyan; Ye, Haifeng

    2017-03-25

    Although still in its infant stage, synthetic biology has achieved remarkable development and progress during the past decade. Synthetic biology applies engineering principles to design and construct gene circuits uploaded into living cells or organisms to perform novel or improved functions, and it has been widely used in many fields. In this review, we describe the recent advances of mammalian synthetic biology for the treatment of diseases. We introduce common tools and design principles of synthetic gene circuits, and then we demonstrate open-loop gene circuits induced by different trigger molecules used in disease diagnosis and close-loop gene circuits used for biomedical applications. Finally, we discuss the perspectives and potential challenges of synthetic biology for clinical applications.

  10. Synthetic biology devices for in vitro and in vivo diagnostics.

    Science.gov (United States)

    Slomovic, Shimyn; Pardee, Keith; Collins, James J

    2015-11-24

    There is a growing need to enhance our capabilities in medical and environmental diagnostics. Synthetic biologists have begun to focus their biomolecular engineering approaches toward this goal, offering promising results that could lead to the development of new classes of inexpensive, rapidly deployable diagnostics. Many conventional diagnostics rely on antibody-based platforms that, although exquisitely sensitive, are slow and costly to generate and cannot readily confront rapidly emerging pathogens or be applied to orphan diseases. Synthetic biology, with its rational and short design-to-production cycles, has the potential to overcome many of these limitations. Synthetic biology devices, such as engineered gene circuits, bring new capabilities to molecular diagnostics, expanding the molecular detection palette, creating dynamic sensors, and untethering reactions from laboratory equipment. The field is also beginning to move toward in vivo diagnostics, which could provide near real-time surveillance of multiple pathological conditions. Here, we describe current efforts in synthetic biology, focusing on the translation of promising technologies into pragmatic diagnostic tools and platforms.

  11. Challenges for the European governance of synthetic biology for human health

    NARCIS (Netherlands)

    Stemerding, D.; Douglas, C.

    2014-01-01

    Synthetic biology is a series of scientific and technological practices involved in the application of engineering principles to the design and production of predictable and robust biological systems. While policy discussions abound in this area, emerging technologies like synthetic biology present

  12. Synthetic RNAs for Gene Regulation: Design Principles and Computational Tools

    International Nuclear Information System (INIS)

    Laganà, Alessandro; Shasha, Dennis; Croce, Carlo Maria

    2014-01-01

    The use of synthetic non-coding RNAs for post-transcriptional regulation of gene expression has not only become a standard laboratory tool for gene functional studies but it has also opened up new perspectives in the design of new and potentially promising therapeutic strategies. Bioinformatics has provided researchers with a variety of tools for the design, the analysis, and the evaluation of RNAi agents such as small-interfering RNA (siRNA), short-hairpin RNA (shRNA), artificial microRNA (a-miR), and microRNA sponges. More recently, a new system for genome engineering based on the bacterial CRISPR-Cas9 system (Clustered Regularly Interspaced Short Palindromic Repeats), was shown to have the potential to also regulate gene expression at both transcriptional and post-transcriptional level in a more specific way. In this mini review, we present RNAi and CRISPRi design principles and discuss the advantages and limitations of the current design approaches.

  13. Synthetic RNAs for Gene Regulation: Design Principles and Computational Tools

    Energy Technology Data Exchange (ETDEWEB)

    Laganà, Alessandro [Department of Molecular Virology, Immunology and Medical Genetics, Comprehensive Cancer Center, The Ohio State University, Columbus, OH (United States); Shasha, Dennis [Courant Institute of Mathematical Sciences, New York University, New York, NY (United States); Croce, Carlo Maria [Department of Molecular Virology, Immunology and Medical Genetics, Comprehensive Cancer Center, The Ohio State University, Columbus, OH (United States)

    2014-12-11

    The use of synthetic non-coding RNAs for post-transcriptional regulation of gene expression has not only become a standard laboratory tool for gene functional studies but it has also opened up new perspectives in the design of new and potentially promising therapeutic strategies. Bioinformatics has provided researchers with a variety of tools for the design, the analysis, and the evaluation of RNAi agents such as small-interfering RNA (siRNA), short-hairpin RNA (shRNA), artificial microRNA (a-miR), and microRNA sponges. More recently, a new system for genome engineering based on the bacterial CRISPR-Cas9 system (Clustered Regularly Interspaced Short Palindromic Repeats), was shown to have the potential to also regulate gene expression at both transcriptional and post-transcriptional level in a more specific way. In this mini review, we present RNAi and CRISPRi design principles and discuss the advantages and limitations of the current design approaches.

  14. Geo synthetics. a remarkable discipline with great achievements in the past and exciting challenges for a bright future

    International Nuclear Information System (INIS)

    Giroud, J. P.

    2014-01-01

    This paper presents achievements of the geo synthetics discipline and challenges facing the discipline. The paper shows that one of the main achievements of geo synthetics discipline and challenges facing the discipline. The paper shows that one of the main achievements of geo synthetics is that they have pervaded most branches of geotechnical engineering to the point where it is almost impossible to practice geotechnical engineering without geo synthetics. Then, the paper addresses the challenges facing the geo synthetics discipline. Two major types of challenges are identified: education challenges and technical challenges. Regarding technical challenges, it is recommended that researchers focus on behaviors that are not traditionally considered in geotechnical engineering in order to use geo synthetics to their full potential. Note: this is a significantly expanded version of the keynote paper presented at the 2008 GeoAmericas Conferences. (Author)

  15. Synthetic Biology: game changer in intelectual property

    Directory of Open Access Journals (Sweden)

    Laurens Landeweerd

    2016-12-01

    Full Text Available Synthetic biology can be considered a game changer that plays an important role in the current NBIC, or BINC convergence of nano-, bio-, info and cognitive sciences. Although most synthetic biology experts are unaware of it, the field appeals to the imagination in its adherence to targets that were usually associated with premodern alchemist science. This paper elaborates several aspects of synthetic biology as well as its consequences for long held notions of intellectual property and the ontological categories of scientific discovery on the one hand and engineering on the other, the distinction between natural and artificial, the grown and the made.

  16. Synthetic multicellular oscillatory systems: controlling protein dynamics with genetic circuits

    International Nuclear Information System (INIS)

    Koseska, Aneta; Volkov, Evgenii; Kurths, Juergen

    2011-01-01

    Synthetic biology is a relatively new research discipline that combines standard biology approaches with the constructive nature of engineering. Thus, recent efforts in the field of synthetic biology have given a perspective to consider cells as 'programmable matter'. Here, we address the possibility of using synthetic circuits to control protein dynamics. In particular, we show how intercellular communication and stochasticity can be used to manipulate the dynamical behavior of a population of coupled synthetic units and, in this manner, finely tune the expression of specific proteins of interest, e.g. in large bioreactors.

  17. Improvement of Synthetic Biology Tools for DNA Editing

    DEFF Research Database (Denmark)

    Cavaleiro, Mafalda

    with the development and improvement of DNA editing strategies,compatible with other DNA assembly methodologies, genome engineering and,eventually, automation processes. Expanding and optimizing the synbio toolkit has important applications in pathway optimization for metabolic engineering, design and characterization...... of gene circuits, synthesis of whole genomes and natural product discovery. In line with this, it is also described in this thesis how discovery of new cytochromes P450 (CYPs) from marine bacteria could benefit industrial processes....

  18. Mutagenesis and Genome Engineering of Epstein-Barr Virus in Cultured Human Cells by CRISPR/Cas9.

    Science.gov (United States)

    Yuen, Kit-San; Chan, Chi-Ping; Kok, Kin-Hang; Jin, Dong-Yan

    2017-01-01

    The clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein 9 nuclease (Cas9) system is a powerful genome-editing tool for both chromosomal and extrachromosomal DNA. DNA viruses such as Epstein-Barr virus (EBV), which undergoes episomal replication in human cells, can be effectively edited by CRISPR/Cas9. We have demonstrated targeted editing of the EBV genome by CRISPR/Cas9 in several lines of EBV-infected cells. CRISPR/Cas9-based mutagenesis and genome engineering of EBV provides a new method for genetic analysis, which has some advantages over bacterial artificial chromosome-based recombineering. This approach might also prove useful in the cure of EBV infection. In this chapter, we use the knockout of the BART promoter as an example to detail the experimental procedures for construction of recombinant EBV in human cells.

  19. RNAslider: a faster engine for consecutive windows folding and its application to the analysis of genomic folding asymmetry

    Directory of Open Access Journals (Sweden)

    Ziv-Ukelson Michal

    2009-03-01

    Full Text Available Abstract Background Scanning large genomes with a sliding window in search of locally stable RNA structures is a well motivated problem in bioinformatics. Given a predefined window size L and an RNA sequence S of size N (L 3 by applying any of the classical cubic-time RNA folding algorithms to each of the N-L windows of size L. Recently an O(NL2 solution for this problem has been described. Results Here, we describe and implement an O(NLψ(L engine for the consecutive windows folding problem, where ψ(L is shown to converge to O(1 under the assumption of a standard probabilistic polymer folding model, yielding an O(L speedup which is experimentally confirmed. Using this tool, we note an intriguing directionality (5'-3' vs. 3'-5' folding bias, i.e. that the minimal free energy (MFE of folding is higher in the native direction of the DNA than in the reverse direction of various genomic regions in several organisms including regions of the genomes that do not encode proteins or ncRNA. This bias largely emerges from the genomic dinucleotide bias which affects the MFE, however we see some variations in the folding bias in the different genomic regions when normalized to the dinucleotide bias. We also present results from calculating the MFE landscape of a mouse chromosome 1, characterizing the MFE of the long ncRNA molecules that reside in this chromosome. Conclusion The efficient consecutive windows folding engine described in this paper allows for genome wide scans for ncRNA molecules as well as large-scale statistics. This is implemented here as a software tool, called RNAslider, and applied to the scanning of long chromosomes, leading to the observation of features that are visible only on a large scale.

  20. Engineering Molecular Immunity Against Plant Viruses

    KAUST Repository

    Zaidi, Syed Shan-e-Ali; Tashkandi, Manal; Mahfouz, Magdy M.

    2017-01-01

    Genomic engineering has been used to precisely alter eukaryotic genomes at the single-base level for targeted gene editing, replacement, fusion, and mutagenesis, and plant viruses such as Tobacco rattle virus have been developed into efficient vectors for delivering genome-engineering reagents. In addition to altering the host genome, these methods can target pathogens to engineer molecular immunity. Indeed, recent studies have shown that clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) systems that target the genomes of DNA viruses can interfere with viral activity and limit viral symptoms in planta, demonstrating the utility of this system for engineering molecular immunity in plants. CRISPR/Cas9 can efficiently target single and multiple viral infections and confer plant immunity. Here, we discuss the use of site-specific nucleases to engineer molecular immunity against DNA and RNA viruses in plants. We also explore how to address the potential challenges encountered when producing plants with engineered resistance to single and mixed viral infections.

  1. Engineering Molecular Immunity Against Plant Viruses

    KAUST Repository

    Zaidi, Syed Shan-e-Ali

    2017-04-26

    Genomic engineering has been used to precisely alter eukaryotic genomes at the single-base level for targeted gene editing, replacement, fusion, and mutagenesis, and plant viruses such as Tobacco rattle virus have been developed into efficient vectors for delivering genome-engineering reagents. In addition to altering the host genome, these methods can target pathogens to engineer molecular immunity. Indeed, recent studies have shown that clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) systems that target the genomes of DNA viruses can interfere with viral activity and limit viral symptoms in planta, demonstrating the utility of this system for engineering molecular immunity in plants. CRISPR/Cas9 can efficiently target single and multiple viral infections and confer plant immunity. Here, we discuss the use of site-specific nucleases to engineer molecular immunity against DNA and RNA viruses in plants. We also explore how to address the potential challenges encountered when producing plants with engineered resistance to single and mixed viral infections.

  2. Synthetic Biology: Knowledge Accessed by Everyone (Open Sources)

    Science.gov (United States)

    Sánchez Reyes, Patricia Margarita

    2016-01-01

    Using the principles of biology, along with engineering and with the help of computer, scientists manage to copy. DNA sequences from nature and use them to create new organisms. DNA is created through engineering and computer science managing to create life inside a laboratory. We cannot dismiss the role that synthetic biology could lead in…

  3. Cell-type-specific genome editing with a microRNA-responsive CRISPR–Cas9 switch

    Science.gov (United States)

    Hirosawa, Moe; Fujita, Yoshihiko; Parr, Callum J. C.; Hayashi, Karin; Kashida, Shunnichi; Hotta, Akitsu; Woltjen, Knut

    2017-01-01

    Abstract The CRISPR–Cas9 system is a powerful genome-editing tool useful in a variety of biotechnology and biomedical applications. Here we developed a synthetic RNA-based, microRNA (miRNA)-responsive CRISPR–Cas9 system (miR-Cas9 switch) in which the genome editing activity of Cas9 can be modulated through endogenous miRNA signatures in mammalian cells. We created miR-Cas9 switches by using a miRNA-complementary sequence in the 5΄-UTR of mRNA encoding Streptococcus pyogenes Cas9. The miR-21-Cas9 or miR-302-Cas9 switches selectively and efficiently responded to miR-21-5p in HeLa cells or miR-302a-5p in human induced pluripotent stem cells, and post-transcriptionally attenuated the Cas9 activity only in the target cells. Moreover, the miR-Cas9 switches could differentially control the genome editing by sensing endogenous miRNA activities within a heterogeneous cell population. Our miR-Cas9 switch system provides a promising framework for cell-type selective genome editing and cell engineering based on intracellular miRNA information. PMID:28525578

  4. BuD, a helix–loop–helix DNA-binding domain for genome modification

    Energy Technology Data Exchange (ETDEWEB)

    Stella, Stefano [Spanish National Cancer Research Centre (CNIO), Calle de Melchor Fernández Almagro 3, 28029 Madrid (Spain); University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen (Denmark); Molina, Rafael; López-Méndez, Blanca [Spanish National Cancer Research Centre (CNIO), Calle de Melchor Fernández Almagro 3, 28029 Madrid (Spain); Juillerat, Alexandre; Bertonati, Claudia; Daboussi, Fayza [Cellectis, 8 Rue de la Croix Jarry, 75013 Paris (France); Campos-Olivas, Ramon [Spanish National Cancer Research Centre (CNIO), Calle de Melchor Fernández Almagro 3, 28029 Madrid (Spain); Duchateau, Phillippe [Cellectis, 8 Rue de la Croix Jarry, 75013 Paris (France); Montoya, Guillermo, E-mail: guillermo.montoya@cpr.ku.dk [Spanish National Cancer Research Centre (CNIO), Calle de Melchor Fernández Almagro 3, 28029 Madrid (Spain); University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen (Denmark)

    2014-07-01

    Crystal structures of BurrH and the BurrH–DNA complex are reported. DNA editing offers new possibilities in synthetic biology and biomedicine for modulation or modification of cellular functions to organisms. However, inaccuracy in this process may lead to genome damage. To address this important problem, a strategy allowing specific gene modification has been achieved through the addition, removal or exchange of DNA sequences using customized proteins and the endogenous DNA-repair machinery. Therefore, the engineering of specific protein–DNA interactions in protein scaffolds is key to providing ‘toolkits’ for precise genome modification or regulation of gene expression. In a search for putative DNA-binding domains, BurrH, a protein that recognizes a 19 bp DNA target, was identified. Here, its apo and DNA-bound crystal structures are reported, revealing a central region containing 19 repeats of a helix–loop–helix modular domain (BurrH domain; BuD), which identifies the DNA target by a single residue-to-nucleotide code, thus facilitating its redesign for gene targeting. New DNA-binding specificities have been engineered in this template, showing that BuD-derived nucleases (BuDNs) induce high levels of gene targeting in a locus of the human haemoglobin β (HBB) gene close to mutations responsible for sickle-cell anaemia. Hence, the unique combination of high efficiency and specificity of the BuD arrays can push forward diverse genome-modification approaches for cell or organism redesign, opening new avenues for gene editing.

  5. The PLOS ONE Synthetic Biology Collection: Six Years and Counting

    Science.gov (United States)

    Peccoud, Jean; Isalan, Mark

    2012-01-01

    Since it was launched in 2006, PLOS ONE has published over fifty articles illustrating the many facets of the emerging field of synthetic biology. This article reviews these publications by organizing them into broad categories focused on DNA synthesis and assembly techniques, the development of libraries of biological parts, the use of synthetic biology in protein engineering applications, and the engineering of gene regulatory networks and metabolic pathways. Finally, we review articles that describe enabling technologies such as software and modeling, along with new instrumentation. In order to increase the visibility of this body of work, the papers have been assembled into the PLOS ONE Synthetic Biology Collection (www.ploscollections.org/synbio). Many of the innovative features of the PLOS ONE web site will help make this collection a resource that will support a lively dialogue between readers and authors of PLOS ONE synthetic biology papers. The content of the collection will be updated periodically by including relevant articles as they are published by the journal. Thus, we hope that this collection will continue to meet the publishing needs of the synthetic biology community. PMID:22916228

  6. Space Synthetic Biology Project

    Science.gov (United States)

    Howard, David; Roman, Monsi; Mansell, James (Matt)

    2015-01-01

    Synthetic biology is an effort to make genetic engineering more useful by standardizing sections of genetic code. By standardizing genetic components, biological engineering will become much more similar to traditional fields of engineering, in which well-defined components and subsystems are readily available in markets. Specifications of the behavior of those components and subsystems can be used to model a system which incorporates them. Then, the behavior of the novel system can be simulated and optimized. Finally, the components and subsystems can be purchased and assembled to create the optimized system, which most often will exhibit behavior similar to that indicated by the model. The Space Synthetic Biology project began in 2012 as a multi-Center effort. The purpose of this project was to harness Synthetic Biology principals to enable NASA's missions. A central target for application was to Environmental Control & Life Support (ECLS). Engineers from NASA Marshall Space Flight Center's (MSFC's) ECLS Systems Development Branch (ES62) were brought into the project to contribute expertise in operational ECLS systems. Project lead scientists chose to pursue the development of bioelectrochemical technologies to spacecraft life support. Therefore, the ECLS element of the project became essentially an effort to develop a bioelectrochemical ECLS subsystem. Bioelectrochemical systems exploit the ability of many microorganisms to drive their metabolisms by direct or indirect utilization of electrical potential gradients. Whereas many microorganisms are capable of deriving the energy required for the processes of interest (such as carbon dioxide (CO2) fixation) from sunlight, it is believed that subsystems utilizing electrotrophs will exhibit smaller mass, volume, and power requirements than those that derive their energy from sunlight. In the first 2 years of the project, MSFC personnel conducted modeling, simulation, and conceptual design efforts to assist the

  7. The CRISPR-Cas9 technology: Closer to the ultimate toolkit for targeted genome editing.

    Science.gov (United States)

    Quétier, Francis

    2016-01-01

    The first period of plant genome editing was based on Agrobacterium; chemical mutagenesis by EMS (ethyl methanesulfonate) and ionizing radiations; each of these technologies led to randomly distributed genome modifications. The second period is associated with the discoveries of homing and meganuclease enzymes during the 80s and 90s, which were then engineered to provide efficient tools for targeted editing. From 2006 to 2012, a few crop plants were successfully and precisely modified using zinc-finger nucleases. A third wave of improvement in genome editing, which led to a dramatic decrease in off-target events, was achieved in 2009-2011 with the TALEN technology. The latest revolution surfaced in 2013 with the CRISPR-Cas9 system, whose high efficiency and technical ease of use is really impressive; scientists can use in-house kits or commercially available kits; the only two requirements are to carefully choose the location of the DNA double strand breaks to be induced and then to order an oligonucleotide. While this close-to- ultimate toolkit for targeted editing of genomes represents dramatic scientific progress which allows the development of more complex useful agronomic traits through synthetic biology, the social acceptance of genome editing remains regularly questioned by anti-GMO citizens and organizations. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  8. Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex.

    Science.gov (United States)

    Konermann, Silvana; Brigham, Mark D; Trevino, Alexandro E; Joung, Julia; Abudayyeh, Omar O; Barcena, Clea; Hsu, Patrick D; Habib, Naomi; Gootenberg, Jonathan S; Nishimasu, Hiroshi; Nureki, Osamu; Zhang, Feng

    2015-01-29

    Systematic interrogation of gene function requires the ability to perturb gene expression in a robust and generalizable manner. Here we describe structure-guided engineering of a CRISPR-Cas9 complex to mediate efficient transcriptional activation at endogenous genomic loci. We used these engineered Cas9 activation complexes to investigate single-guide RNA (sgRNA) targeting rules for effective transcriptional activation, to demonstrate multiplexed activation of ten genes simultaneously, and to upregulate long intergenic non-coding RNA (lincRNA) transcripts. We also synthesized a library consisting of 70,290 guides targeting all human RefSeq coding isoforms to screen for genes that, upon activation, confer resistance to a BRAF inhibitor. The top hits included genes previously shown to be able to confer resistance, and novel candidates were validated using individual sgRNA and complementary DNA overexpression. A gene expression signature based on the top screening hits correlated with markers of BRAF inhibitor resistance in cell lines and patient-derived samples. These results collectively demonstrate the potential of Cas9-based activators as a powerful genetic perturbation technology.

  9. Industrial systems biology and its impact on synthetic biology of yeast cell factories.

    Science.gov (United States)

    Fletcher, Eugene; Krivoruchko, Anastasia; Nielsen, Jens

    2016-06-01

    Engineering industrial cell factories to effectively yield a desired product while dealing with industrially relevant stresses is usually the most challenging step in the development of industrial production of chemicals using microbial fermentation processes. Using synthetic biology tools, microbial cell factories such as Saccharomyces cerevisiae can be engineered to express synthetic pathways for the production of fuels, biopharmaceuticals, fragrances, and food flavors. However, directing fluxes through these synthetic pathways towards the desired product can be demanding due to complex regulation or poor gene expression. Systems biology, which applies computational tools and mathematical modeling to understand complex biological networks, can be used to guide synthetic biology design. Here, we present our perspective on how systems biology can impact synthetic biology towards the goal of developing improved yeast cell factories. Biotechnol. Bioeng. 2016;113: 1164-1170. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.

  10. Synthetic Biology: Engineering, Evolution and Design (SEED) Conference 2015

    Science.gov (United States)

    2015-12-01

    yield stability. Transgenic approach plays an important role in crop improvement. Currently, transgenes are randomly inserted into maize genome...Improve Microbial Biofuel Production - Mary Dunlop, University of Vermont 2:30-3:00 PM Targeted Integration of Genes into the Maize Genome Facilitated...highlights gaps in the present understanding of M13 biology. Understanding the subtleties of regulation will be important for maximally ex- ploiting the

  11. Development of Genome Engineering Tools from Plant-Specific PPR Proteins Using Animal Cultured Cells.

    Science.gov (United States)

    Kobayashi, Takehito; Yagi, Yusuke; Nakamura, Takahiro

    2016-01-01

    The pentatricopeptide repeat (PPR) motif is a sequence-specific RNA/DNA-binding module. Elucidation of the RNA/DNA recognition mechanism has enabled engineering of PPR motifs as new RNA/DNA manipulation tools in living cells, including for genome editing. However, the biochemical characteristics of PPR proteins remain unknown, mostly due to the instability and/or unfolding propensities of PPR proteins in heterologous expression systems such as bacteria and yeast. To overcome this issue, we constructed reporter systems using animal cultured cells. The cell-based system has highly attractive features for PPR engineering: robust eukaryotic gene expression; availability of various vectors, reagents, and antibodies; highly efficient DNA delivery ratio (>80 %); and rapid, high-throughput data production. In this chapter, we introduce an example of such reporter systems: a PPR-based sequence-specific translational activation system. The cell-based reporter system can be applied to characterize plant genes of interested and to PPR engineering.

  12. Synthetic Phage for Tissue Regeneration

    Directory of Open Access Journals (Sweden)

    So Young Yoo

    2014-01-01

    Full Text Available Controlling structural organization and signaling motif display is of great importance to design the functional tissue regenerating materials. Synthetic phage, genetically engineered M13 bacteriophage has been recently introduced as novel tissue regeneration materials to display a high density of cell-signaling peptides on their major coat proteins for tissue regeneration purposes. Structural advantages of their long-rod shape and monodispersity can be taken together to construct nanofibrous scaffolds which support cell proliferation and differentiation as well as direct orientation of their growth in two or three dimensions. This review demonstrated how functional synthetic phage is designed and subsequently utilized for tissue regeneration that offers potential cell therapy.

  13. Synthetic Biology and the U.S. Biotechnology Regulatory System: Challenges and Options

    Energy Technology Data Exchange (ETDEWEB)

    Carter, Sarah R. [J. Craig Venter Inst., Rockville, MD (United States); Rodemeyer, Michael [Univ. of Virginia, Charlottesville, VA (United States); Garfinkel, Michele S. [EMBO, Heidelberg (Germany); Friedman, Robert M. [J. Craig Venter Inst., Rockville, MD (United States)

    2014-05-01

    Synthetic Biology and the U.S. Biotechnology Regulatory System: Challenges and Options Sarah R. Carter, Ph.D., J. Craig Venter Institute; Michael Rodemeyer, J.D., University of Virginia; Michele S. Garfinkel, Ph.D., EMBO; Robert M. Friedman, Ph.D., J. Craig Venter Institute In recent years, a range of genetic engineering techniques referred to as “synthetic biology” has significantly expanded the tool kit available to scientists and engineers, providing them with far greater capabilities to engineer organisms than previous techniques allowed. The field of synthetic biology includes the relatively new ability to synthesize long pieces of DNA from chemicals, as well as improved methods for genetic manipulation and design of genetic pathways to achieve more precise control of biological systems. These advances will help usher in a new generation of genetically engineered microbes, plants, and animals. The JCVI Policy Center team, along with researchers at the University of Virginia and EMBO, examined how well the current U.S. regulatory system for genetically engineered products will handle the near-term introduction of organisms engineered using synthetic biology. In particular, the focus was on those organisms intended to be used or grown directly in the environment, outside of a contained facility. The study concludes that the U.S. regulatory agencies have adequate legal authority to address most, but not all, potential environmental, health and safety concerns posed by these organisms. Such near-term products are likely to represent incremental changes rather than a marked departure from previous genetically engineered organisms. However, the study also identified two key challenges for the regulatory system, which are detailed in the report. First, USDA’s authority over genetically engineered plants depends on the use of an older engineering technique that is no longer necessary for many applications. The shift to synthetic biology and other newer genetic

  14. CSBB: synthetic biology research at Newcastle University.

    Science.gov (United States)

    Goñi-Moreno, Angel; Wipat, Anil; Krasnogor, Natalio

    2017-06-15

    The Centre for Synthetic Biology and the Bioeconomy (CSBB) brings together a far-reaching multidisciplinary community across all Newcastle University's faculties - Medical Sciences, Science, Agriculture and Engineering, and Humanities, Arts and Social Sciences. The CSBB focuses on many different areas of Synthetic Biology, including bioprocessing, computational design and in vivo computation, as well as improving understanding of basic molecular machinery. Such breadth is supported by major national and international research funding, a range of industrial partners in the North East of England and beyond, as well as a large number of doctoral and post-doctoral researchers. The CSBB trains the next generation of scientists through a 1-year MSc in Synthetic Biology. © 2017 The Author(s).

  15. Yeast synthetic biology toolbox and applications for biofuel production.

    Science.gov (United States)

    Tsai, Ching-Sung; Kwak, Suryang; Turner, Timothy L; Jin, Yong-Su

    2015-02-01

    Yeasts are efficient biofuel producers with numerous advantages outcompeting bacterial counterparts. While most synthetic biology tools have been developed and customized for bacteria especially for Escherichia coli, yeast synthetic biological tools have been exploited for improving yeast to produce fuels and chemicals from renewable biomass. Here we review the current status of synthetic biological tools and their applications for biofuel production, focusing on the model strain Saccharomyces cerevisiae We describe assembly techniques that have been developed for constructing genes, pathways, and genomes in yeast. Moreover, we discuss synthetic parts for allowing precise control of gene expression at both transcriptional and translational levels. Applications of these synthetic biological approaches have led to identification of effective gene targets that are responsible for desirable traits, such as cellulosic sugar utilization, advanced biofuel production, and enhanced tolerance against toxic products for biofuel production from renewable biomass. Although an array of synthetic biology tools and devices are available, we observed some gaps existing in tool development to achieve industrial utilization. Looking forward, future tool development should focus on industrial cultivation conditions utilizing industrial strains. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.

  16. Genomics:GTL Contractor-Grantee Workshop IV and Metabolic Engineering Working Group Inter-Agency Conference on Metabolic Engineering 2006

    Energy Technology Data Exchange (ETDEWEB)

    Mansfield, Betty Kay [ORNL; Martin, Sheryl A [ORNL

    2006-02-01

    Welcome to the 2006 joint meeting of the fourth Genomics:GTL Contractor-Grantee Workshop and the six Metabolic Engineering Working Group Inter-Agency Conference. The vision and scope of the Genomics:GTL program continue to expand and encompass research and technology issues from diverse scientific disciplines, attracting broad interest and support from researchers at universities, DOE national laboratories, and industry. Metabolic engineering's vision is the targeted and purposeful alteration of metabolic pathways to improve the understanding and use of cellular pathways for chemical transformation, energy transduction, and supramolecular assembly. These two programs have much complementarity in both vision and technological approaches, as reflected in this joint workshop. GLT's challenge to the scientific community remains the further development and use of a broad array of innovative technologies and computational tools to systematically leverage the knowledge and capabilities brought to us by DNA sequencing projects. The goal is to seek a broad and predictive understanding of the functioning and control of complex systems--individual microbes, microbial communities, and plants. GTL's prominent position at the interface of the physical, computational, and biological sciences is both a strength and challenge. Microbes remain GTL's principal biological focus. In the complex 'simplicity' of microbes, they find capabilities needed by DOE and the nation for clean and secure energy, cleanup of environmental contamination, and sequestration of atmospheric carbon dioxide that contributes to global warming. An ongoing challenge for the entire GTL community is to demonstrate that the fundamental science conducted in each of your research projects brings us a step closer to biology-based solutions for these important national energy and environmental needs.

  17. Synthetic Biology: Tools to Design, Build, and Optimize Cellular Processes

    Science.gov (United States)

    Young, Eric; Alper, Hal

    2010-01-01

    The general central dogma frames the emergent properties of life, which make biology both necessary and difficult to engineer. In a process engineering paradigm, each biological process stream and process unit is heavily influenced by regulatory interactions and interactions with the surrounding environment. Synthetic biology is developing the tools and methods that will increase control over these interactions, eventually resulting in an integrative synthetic biology that will allow ground-up cellular optimization. In this review, we attempt to contextualize the areas of synthetic biology into three tiers: (1) the process units and associated streams of the central dogma, (2) the intrinsic regulatory mechanisms, and (3) the extrinsic physical and chemical environment. Efforts at each of these three tiers attempt to control cellular systems and take advantage of emerging tools and approaches. Ultimately, it will be possible to integrate these approaches and realize the vision of integrative synthetic biology when cells are completely rewired for biotechnological goals. This review will highlight progress towards this goal as well as areas requiring further research. PMID:20150964

  18. Synthetic Biology: Tools to Design, Build, and Optimize Cellular Processes

    Directory of Open Access Journals (Sweden)

    Eric Young

    2010-01-01

    Full Text Available The general central dogma frames the emergent properties of life, which make biology both necessary and difficult to engineer. In a process engineering paradigm, each biological process stream and process unit is heavily influenced by regulatory interactions and interactions with the surrounding environment. Synthetic biology is developing the tools and methods that will increase control over these interactions, eventually resulting in an integrative synthetic biology that will allow ground-up cellular optimization. In this review, we attempt to contextualize the areas of synthetic biology into three tiers: (1 the process units and associated streams of the central dogma, (2 the intrinsic regulatory mechanisms, and (3 the extrinsic physical and chemical environment. Efforts at each of these three tiers attempt to control cellular systems and take advantage of emerging tools and approaches. Ultimately, it will be possible to integrate these approaches and realize the vision of integrative synthetic biology when cells are completely rewired for biotechnological goals. This review will highlight progress towards this goal as well as areas requiring further research.

  19. Synthetic biology: tools to design, build, and optimize cellular processes.

    Science.gov (United States)

    Young, Eric; Alper, Hal

    2010-01-01

    The general central dogma frames the emergent properties of life, which make biology both necessary and difficult to engineer. In a process engineering paradigm, each biological process stream and process unit is heavily influenced by regulatory interactions and interactions with the surrounding environment. Synthetic biology is developing the tools and methods that will increase control over these interactions, eventually resulting in an integrative synthetic biology that will allow ground-up cellular optimization. In this review, we attempt to contextualize the areas of synthetic biology into three tiers: (1) the process units and associated streams of the central dogma, (2) the intrinsic regulatory mechanisms, and (3) the extrinsic physical and chemical environment. Efforts at each of these three tiers attempt to control cellular systems and take advantage of emerging tools and approaches. Ultimately, it will be possible to integrate these approaches and realize the vision of integrative synthetic biology when cells are completely rewired for biotechnological goals. This review will highlight progress towards this goal as well as areas requiring further research.

  20. Molecular Cloning Designer Simulator (MCDS): All-in-one molecular cloning and genetic engineering design, simulation and management software for complex synthetic biology and metabolic engineering projects.

    Science.gov (United States)

    Shi, Zhenyu; Vickers, Claudia E

    2016-12-01

    Molecular Cloning Designer Simulator (MCDS) is a powerful new all-in-one cloning and genetic engineering design, simulation and management software platform developed for complex synthetic biology and metabolic engineering projects. In addition to standard functions, it has a number of features that are either unique, or are not found in combination in any one software package: (1) it has a novel interactive flow-chart user interface for complex multi-step processes, allowing an integrated overview of the whole project; (2) it can perform a user-defined workflow of cloning steps in a single execution of the software; (3) it can handle multiple types of genetic recombineering, a technique that is rapidly replacing classical cloning for many applications; (4) it includes experimental information to conveniently guide wet lab work; and (5) it can store results and comments to allow the tracking and management of the whole project in one platform. MCDS is freely available from https://mcds.codeplex.com.

  1. Synthetic biology: a challenge to mechanical explanations in biology?

    Science.gov (United States)

    Morange, Michel

    2012-01-01

    In their plans to modify organisms, synthetic biologists have contrasted engineering and tinkering. By drawing this contrast between their endeavors and what has happened during the evolution of organisms by natural selection, they underline the novelty of their projects and justify their ambitions. Synthetic biologists are at odds with a long tradition that has considered organisms as "perfect machines." This tradition had already been questioned by Stephen Jay Gould in the 1970s and received a major blow with the comparison made by François Jacob between organisms and the results of "bricolage" (tinkering). These contrasts between engineering and tinkering, synthetic biology and evolution, have no raison d'être. Machines built by humans are increasingly inspired by observations made on organisms. This is not a simple reversal of the previous trend-the mechanical conception of organisms-in which the characteristics of the latter were explained by comparison with human-built machines. Relations between organisms and machines have always been complex and ambiguous.

  2. Regenesis how synthetic biology will reinvent nature and ourselves

    CERN Document Server

    Church, George M

    2012-01-01

    Imagine a future in which human beings have become immune to all viruses, in which bacteria can custom-produce everyday items, like a drinking cup, or generate enough electricity to end oil dependency. Building a house would entail no more work than planting a seed in the ground. These scenarios may seem far-fetched, but pioneering geneticist George Church and science writer Ed Regis show that synthetic biology is bringing us ever closer to making such visions a reality. In "Regenesis," Church and Regis explorethe possibilities--and perils--of the emerging field of synthetic biology. Synthetic biology, in which living organisms are selectively altered by modifying substantial portions of their genomes, allows for the creation of entirely new species of organisms. Until now, nature has been the exclusive arbiter of life, death, and evolution; with synthetic biology, we now have the potential to write our own biological future. Indeed, as Church and Regis show, it even enables us to revisit crucial points in th...

  3. Genome-based Modeling and Design of Metabolic Interactions in Microbial Communities.

    Science.gov (United States)

    Mahadevan, Radhakrishnan; Henson, Michael A

    2012-01-01

    Biotechnology research is traditionally focused on individual microbial strains that are perceived to have the necessary metabolic functions, or the capability to have these functions introduced, to achieve a particular task. For many important applications, the development of such omnipotent microbes is an extremely challenging if not impossible task. By contrast, nature employs a radically different strategy based on synergistic combinations of different microbial species that collectively achieve the desired task. These natural communities have evolved to exploit the native metabolic capabilities of each species and are highly adaptive to changes in their environments. However, microbial communities have proven difficult to study due to a lack of suitable experimental and computational tools. With the advent of genome sequencing, omics technologies, bioinformatics and genome-scale modeling, researchers now have unprecedented capabilities to analyze and engineer the metabolism of microbial communities. The goal of this review is to summarize recent applications of genome-scale metabolic modeling to microbial communities. A brief introduction to lumped community models is used to motivate the need for genome-level descriptions of individual species and their metabolic interactions. The review of genome-scale models begins with static modeling approaches, which are appropriate for communities where the extracellular environment can be assumed to be time invariant or slowly varying. Dynamic extensions of the static modeling approach are described, and then applications of genome-scale models for design of synthetic microbial communities are reviewed. The review concludes with a summary of metagenomic tools for analyzing community metabolism and an outlook for future research.

  4. Streamlining genomes: toward the generation of simplified and stabilized microbial systems

    NARCIS (Netherlands)

    Leprince, A.; Passel, van M.W.J.; Martins Dos Santos, V.A.P.

    2012-01-01

    At the junction between systems and synthetic biology, genome streamlining provides a solid foundation both for increased understanding of cellular circuitry, and for the tailoring of microbial chassis towards innovative biotechnological applications. Iterative genomic deletions (targeted and

  5. Combinatorial metabolic engineering of Pseudomonas putida KT2440 for efficient mineralization of 1,2,3-trichloropropane.

    Science.gov (United States)

    Gong, Ting; Xu, Xiaoqing; Che, You; Liu, Ruihua; Gao, Weixia; Zhao, Fengjie; Yu, Huilei; Liang, Jingnan; Xu, Ping; Song, Cunjiang; Yang, Chao

    2017-08-01

    An industrial waste, 1,2,3-trichloropropane (TCP), is toxic and extremely recalcitrant to biodegradation. To date, no natural TCP degraders able to mineralize TCP aerobically have been isolated. In this work, we engineered a biosafety Pseudomonas putida strain KT2440 for aerobic mineralization of TCP by implantation of a synthetic biodegradation pathway into the chromosome and further improved TCP mineralization using combinatorial engineering strategies. Initially, a synthetic pathway composed of haloalkane dehalogenase, haloalcohol dehalogenase and epoxide hydrolase was functionally assembled for the conversion of TCP into glycerol in P. putida KT2440. Then, the growth lag-phase of using glycerol as a growth precursor was eliminated by deleting the glpR gene, significantly enhancing the flux of carbon through the pathway. Subsequently, we improved the oxygen sequestering capacity of this strain through the heterologous expression of Vitreoscilla hemoglobin, which makes this strain able to mineralize TCP under oxygen-limited conditions. Lastly, we further improved intracellular energy charge (ATP/ADP ratio) and reducing power (NADPH/NADP + ratio) by deleting flagella-related genes in the genome of P. putida KT2440. The resulting strain (named KTU-TGVF) could efficiently utilize TCP as the sole source of carbon for growth. Degradation studies in a bioreactor highlight the value of this engineered strain for TCP bioremediation.

  6. Paper-based synthetic gene networks.

    Science.gov (United States)

    Pardee, Keith; Green, Alexander A; Ferrante, Tom; Cameron, D Ewen; DaleyKeyser, Ajay; Yin, Peng; Collins, James J

    2014-11-06

    Synthetic gene networks have wide-ranging uses in reprogramming and rewiring organisms. To date, there has not been a way to harness the vast potential of these networks beyond the constraints of a laboratory or in vivo environment. Here, we present an in vitro paper-based platform that provides an alternate, versatile venue for synthetic biologists to operate and a much-needed medium for the safe deployment of engineered gene circuits beyond the lab. Commercially available cell-free systems are freeze dried onto paper, enabling the inexpensive, sterile, and abiotic distribution of synthetic-biology-based technologies for the clinic, global health, industry, research, and education. For field use, we create circuits with colorimetric outputs for detection by eye and fabricate a low-cost, electronic optical interface. We demonstrate this technology with small-molecule and RNA actuation of genetic switches, rapid prototyping of complex gene circuits, and programmable in vitro diagnostics, including glucose sensors and strain-specific Ebola virus sensors.

  7. Paper-based Synthetic Gene Networks

    Science.gov (United States)

    Pardee, Keith; Green, Alexander A.; Ferrante, Tom; Cameron, D. Ewen; DaleyKeyser, Ajay; Yin, Peng; Collins, James J.

    2014-01-01

    Synthetic gene networks have wide-ranging uses in reprogramming and rewiring organisms. To date, there has not been a way to harness the vast potential of these networks beyond the constraints of a laboratory or in vivo environment. Here, we present an in vitro paper-based platform that provides a new venue for synthetic biologists to operate, and a much-needed medium for the safe deployment of engineered gene circuits beyond the lab. Commercially available cell-free systems are freeze-dried onto paper, enabling the inexpensive, sterile and abiotic distribution of synthetic biology-based technologies for the clinic, global health, industry, research and education. For field use, we create circuits with colorimetric outputs for detection by eye, and fabricate a low-cost, electronic optical interface. We demonstrate this technology with small molecule and RNA actuation of genetic switches, rapid prototyping of complex gene circuits, and programmable in vitro diagnostics, including glucose sensors and strain-specific Ebola virus sensors. PMID:25417167

  8. Synergistic Synthetic Biology: Units in Concert

    Science.gov (United States)

    Trosset, Jean-Yves; Carbonell, Pablo

    2013-01-01

    Synthetic biology aims at translating the methods and strategies from engineering into biology in order to streamline the design and construction of biological devices through standardized parts. Modular synthetic biology devices are designed by means of an adequate elimination of cross-talk that makes circuits orthogonal and specific. To that end, synthetic constructs need to be adequately optimized through in silico modeling by choosing the right complement of genetic parts and by experimental tuning through directed evolution and craftsmanship. In this review, we consider an additional and complementary tool available to the synthetic biologist for innovative design and successful construction of desired circuit functionalities: biological synergies. Synergy is a prevalent emergent property in biological systems that arises from the concerted action of multiple factors producing an amplification or cancelation effect compared with individual actions alone. Synergies appear in domains as diverse as those involved in chemical and protein activity, polypharmacology, and metabolic pathway complementarity. In conventional synthetic biology designs, synergistic cross-talk between parts and modules is generally attenuated in order to verify their orthogonality. Synergistic interactions, however, can induce emergent behavior that might prove useful for synthetic biology applications, like in functional circuit design, multi-drug treatment, or in sensing and delivery devices. Synergistic design principles are therefore complementary to those coming from orthogonal design and may provide added value to synthetic biology applications. The appropriate modeling, characterization, and design of synergies between biological parts and units will allow the discovery of yet unforeseeable, novel synthetic biology applications. PMID:25022769

  9. Synergistic Synthetic Biology: Units in Concert

    International Nuclear Information System (INIS)

    Trosset, Jean-Yves; Carbonell, Pablo

    2013-01-01

    Synthetic biology aims at translating the methods and strategies from engineering into biology in order to streamline the design and construction of biological devices through standardized parts. Modular synthetic biology devices are designed by means of an adequate elimination of cross-talk that makes circuits orthogonal and specific. To that end, synthetic constructs need to be adequately optimized through in silico modeling by choosing the right complement of genetic parts and by experimental tuning through directed evolution and craftsmanship. In this review, we consider an additional and complementary tool available to the synthetic biologist for innovative design and successful construction of desired circuit functionalities: biological synergies. Synergy is a prevalent emergent property in biological systems that arises from the concerted action of multiple factors producing an amplification or cancelation effect compared with individual actions alone. Synergies appear in domains as diverse as those involved in chemical and protein activity, polypharmacology, and metabolic pathway complementarity. In conventional synthetic biology designs, synergistic cross-talk between parts and modules is generally attenuated in order to verify their orthogonality. Synergistic interactions, however, can induce emergent behavior that might prove useful for synthetic biology applications, like in functional circuit design, multi-drug treatment, or in sensing and delivery devices. Synergistic design principles are therefore complementary to those coming from orthogonal design and may provide added value to synthetic biology applications. The appropriate modeling, characterization, and design of synergies between biological parts and units will allow the discovery of yet unforeseeable, novel synthetic biology applications.

  10. RNAslider: a faster engine for consecutive windows folding and its application to the analysis of genomic folding asymmetry.

    Science.gov (United States)

    Horesh, Yair; Wexler, Ydo; Lebenthal, Ilana; Ziv-Ukelson, Michal; Unger, Ron

    2009-03-04

    Scanning large genomes with a sliding window in search of locally stable RNA structures is a well motivated problem in bioinformatics. Given a predefined window size L and an RNA sequence S of size N (L free energy (MFE) for the folding of each of the L-sized substrings of S. The consecutive windows folding problem can be naively solved in O(NL3) by applying any of the classical cubic-time RNA folding algorithms to each of the N-L windows of size L. Recently an O(NL2) solution for this problem has been described. Here, we describe and implement an O(NLpsi(L)) engine for the consecutive windows folding problem, where psi(L) is shown to converge to O(1) under the assumption of a standard probabilistic polymer folding model, yielding an O(L) speedup which is experimentally confirmed. Using this tool, we note an intriguing directionality (5'-3' vs. 3'-5') folding bias, i.e. that the minimal free energy (MFE) of folding is higher in the native direction of the DNA than in the reverse direction of various genomic regions in several organisms including regions of the genomes that do not encode proteins or ncRNA. This bias largely emerges from the genomic dinucleotide bias which affects the MFE, however we see some variations in the folding bias in the different genomic regions when normalized to the dinucleotide bias. We also present results from calculating the MFE landscape of a mouse chromosome 1, characterizing the MFE of the long ncRNA molecules that reside in this chromosome. The efficient consecutive windows folding engine described in this paper allows for genome wide scans for ncRNA molecules as well as large-scale statistics. This is implemented here as a software tool, called RNAslider, and applied to the scanning of long chromosomes, leading to the observation of features that are visible only on a large scale.

  11. Synthetic Biology Guides Biofuel Production

    Directory of Open Access Journals (Sweden)

    Michael R. Connor

    2010-01-01

    Full Text Available The advancement of microbial processes for the production of renewable liquid fuels has increased with concerns about the current fuel economy. The development of advanced biofuels in particular has risen to address some of the shortcomings of ethanol. These advanced fuels have chemical properties similar to petroleum-based liquid fuels, thus removing the need for engine modification or infrastructure redesign. While the productivity and titers of each of these processes remains to be improved, progress in synthetic biology has provided tools to guide the engineering of these processes through present and future challenges.

  12. A combinatorial approach to synthetic transcription factor-promoter combinations for yeast strain engineering

    DEFF Research Database (Denmark)

    Dossani, Zain Y.; Apel, Amanda Reider; Szmidt-Middleton, Heather

    2018-01-01

    regions, we have built a library of hybrid promoters that are regulated by a synthetic transcription factor. The hybrid promoters consist of native S. cerevisiae promoters, in which the operator regions have been replaced with sequences that are recognized by the bacterial LexA DNA binding protein....... Correspondingly, the synthetic transcription factor (TF) consists of the DNA binding domain of the LexA protein, fused with the human estrogen binding domain and the viral activator domain, VP16. The resulting system with a bacterial DNA binding domain avoids the transcription of native S. cerevisiae genes...... levels, using the same synthetic TF and a given estradiol. This set of promoters, in combination with our synthetic TF, has the potential to regulate numerous genes or pathways simultaneously, to multiple desired levels, in a single strain....

  13. News: Synthetic biology leading to specialty chemicals

    Science.gov (United States)

    Synthetic biology can combine the disciplines of biology, engineering, and chemistry productively to form molecules of great scientific and commercial value. Recent advances in the new field are explored for their connection to new tools that have been used to elucidate productio...

  14. Robust synthetic biology design: stochastic game theory approach.

    Science.gov (United States)

    Chen, Bor-Sen; Chang, Chia-Hung; Lee, Hsiao-Ching

    2009-07-15

    Synthetic biology is to engineer artificial biological systems to investigate natural biological phenomena and for a variety of applications. However, the development of synthetic gene networks is still difficult and most newly created gene networks are non-functioning due to uncertain initial conditions and disturbances of extra-cellular environments on the host cell. At present, how to design a robust synthetic gene network to work properly under these uncertain factors is the most important topic of synthetic biology. A robust regulation design is proposed for a stochastic synthetic gene network to achieve the prescribed steady states under these uncertain factors from the minimax regulation perspective. This minimax regulation design problem can be transformed to an equivalent stochastic game problem. Since it is not easy to solve the robust regulation design problem of synthetic gene networks by non-linear stochastic game method directly, the Takagi-Sugeno (T-S) fuzzy model is proposed to approximate the non-linear synthetic gene network via the linear matrix inequality (LMI) technique through the Robust Control Toolbox in Matlab. Finally, an in silico example is given to illustrate the design procedure and to confirm the efficiency and efficacy of the proposed robust gene design method. http://www.ee.nthu.edu.tw/bschen/SyntheticBioDesign_supplement.pdf.

  15. Synthetic Biology and the Translational Imperative.

    Science.gov (United States)

    Heidari Feidt, Raheleh; Ienca, Marcello; Elger, Bernice Simone; Folcher, Marc

    2017-12-18

    Advances at the interface between the biological sciences and engineering are giving rise to emerging research fields such as synthetic biology. Harnessing the potential of synthetic biology requires timely and adequate translation into clinical practice. However, the translational research enterprise is currently facing fundamental obstacles that slow down the transition of scientific discoveries from the laboratory to the patient bedside. These obstacles including scarce financial resources and deficiency of organizational and logistic settings are widely discussed as primary impediments to translational research. In addition, a number of socio-ethical considerations inherent in translational research need to be addressed. As the translational capacity of synthetic biology is tightly linked to its social acceptance and ethical approval, ethical limitations may-together with financial and organizational problems-be co-determinants of suboptimal translation. Therefore, an early assessment of such limitations will contribute to proactively favor successful translation and prevent the promising potential of synthetic biology from remaining under-expressed. Through the discussion of two case-specific inventions in synthetic biology and their associated ethical implications, we illustrate the socio-ethical challenges ahead in the process of implementing synthetic biology into clinical practice. Since reducing the translational lag is essential for delivering the benefits of basic biomedical research to society at large and promoting global health, we advocate a moral obligation to accelerating translational research: the "translational imperative."

  16. Synthetic biology era: Improving antibiotic's world.

    Science.gov (United States)

    Guzmán-Trampe, Silvia; Ceapa, Corina D; Manzo-Ruiz, Monserrat; Sánchez, Sergio

    2017-06-15

    The emergence of antibiotic-resistant pathogen microorganisms is problematic in the context of the current spectrum of available medication. The poor specificity and the high toxicity of some available molecules have made imperative the search for new strategies to improve the specificity and to pursue the discovery of novel compounds with increased bioactivity. Using living cells as platforms, synthetic biology has counteracted this problem by offering novel pathways to create synthetic systems with improved and desired functions. Among many other biotechnological approaches, the advances in synthetic biology have made it possible to design and construct novel biological systems in order to look for new drugs with increased bioactivity. Advancements have also been made in the redesigning of RNA and DNA molecules in order to engineer antibiotic clusters for antibiotic overexpression. As for the production of these antibacterial compounds, yeasts and filamentous fungi as well as gene therapy are utilized to enhance protein solubility. Specific delivery is achieved by creating chimeras using plant genes into bacterial hosts. Some of these synthetic systems are currently in clinical trials, proving the proficiency of synthetic biology in terms of both pharmacological activities as well as an increase in the biosafety of treatments. It is possible that we may just be seeing the tip of the iceberg, and synthetic biology applications will overpass expectations beyond our present knowledge. Copyright © 2017. Published by Elsevier Inc.

  17. A Critical Perspective on Synthetic Biology

    OpenAIRE

    Michel Morange

    2009-01-01

    Synthetic biology emerged around 2000 as a new biological discipline. It shares with systems biology the same modular vision of organisms, but is more concerned with applications than with a better understanding of the functioning of organisms. A herald of this new discipline is Craig Venter who aims to create an artificial microorganism with the minimal genome compatible with life and to implement into it different 'functional modules' to generate new micro-organisms adapted to specific task...

  18. Cell-type-specific genome editing with a microRNA-responsive CRISPR-Cas9 switch.

    Science.gov (United States)

    Hirosawa, Moe; Fujita, Yoshihiko; Parr, Callum J C; Hayashi, Karin; Kashida, Shunnichi; Hotta, Akitsu; Woltjen, Knut; Saito, Hirohide

    2017-07-27

    The CRISPR-Cas9 system is a powerful genome-editing tool useful in a variety of biotechnology and biomedical applications. Here we developed a synthetic RNA-based, microRNA (miRNA)-responsive CRISPR-Cas9 system (miR-Cas9 switch) in which the genome editing activity of Cas9 can be modulated through endogenous miRNA signatures in mammalian cells. We created miR-Cas9 switches by using a miRNA-complementary sequence in the 5΄-UTR of mRNA encoding Streptococcus pyogenes Cas9. The miR-21-Cas9 or miR-302-Cas9 switches selectively and efficiently responded to miR-21-5p in HeLa cells or miR-302a-5p in human induced pluripotent stem cells, and post-transcriptionally attenuated the Cas9 activity only in the target cells. Moreover, the miR-Cas9 switches could differentially control the genome editing by sensing endogenous miRNA activities within a heterogeneous cell population. Our miR-Cas9 switch system provides a promising framework for cell-type selective genome editing and cell engineering based on intracellular miRNA information. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  19. A Powerful Toolkit for Synthetic Biology: Over 3.8 Billion Years of Evolution

    Science.gov (United States)

    Rothschild, Lynn J.

    2010-01-01

    The combination of evolutionary with engineering principles will enhance synthetic biology. Conversely, synthetic biology has the potential to enrich evolutionary biology by explaining why some adaptive space is empty, on Earth or elsewhere. Synthetic biology, the design and construction of artificial biological systems, substitutes bio-engineering for evolution, which is seen as an obstacle. But because evolution has produced the complexity and diversity of life, it provides a proven toolkit of genetic materials and principles available to synthetic biology. Evolution operates on the population level, with the populations composed of unique individuals that are historical entities. The source of genetic novelty includes mutation, gene regulation, sex, symbiosis, and interspecies gene transfer. At a phenotypic level, variation derives from regulatory control, replication and diversification of components, compartmentalization, sexual selection and speciation, among others. Variation is limited by physical constraints such as diffusion, and chemical constraints such as reaction rates and membrane fluidity. While some of these tools of evolution are currently in use in synthetic biology, all ought to be examined for utility. A hybrid approach of synthetic biology coupled with fine-tuning through evolution is suggested

  20. Synthetic biology as red herring.

    Science.gov (United States)

    Preston, Beth

    2013-12-01

    It has become commonplace to say that with the advent of technologies like synthetic biology the line between artifacts and living organisms, policed by metaphysicians since antiquity, is beginning to blur. But that line began to blur 10,000 years ago when plants and animals were first domesticated; and has been thoroughly blurred at least since agriculture became the dominant human subsistence pattern many millennia ago. Synthetic biology is ultimately only a late and unexceptional offshoot of this prehistoric development. From this perspective, then, synthetic biology is a red herring, distracting us from more thorough philosophical consideration of the most truly revolutionary human practice-agriculture. In the first section of this paper I will make this case with regard to ontology, arguing that synthetic biology crosses no ontological lines that were not crossed already in the Neolithic. In the second section I will construct a parallel case with regard to cognition, arguing that synthetic biology as biological engineering represents no cognitive advance over what was required for domestication and the new agricultural subsistence pattern it grounds. In the final section I will make the case with regard to human existence, arguing that synthetic biology, even if wildly successful, is not in a position to cause significant existential change in what it is to be human over and above the massive existential change caused by the transition to agriculture. I conclude that a longer historical perspective casts new light on some important issues in philosophy of technology and environmental philosophy. Copyright © 2013 Elsevier Ltd. All rights reserved.

  1. US Competitiveness in Synthetic Biology.

    Science.gov (United States)

    Gronvall, Gigi Kwik

    2015-01-01

    Synthetic biology is an emerging technical field that aims to make biology easier to engineer; the field has applications in strategically important sectors for the US economy. While the United States currently leads in synthetic biology R&D, other nations are heavily investing in order to boost their economies, which will inevitably diminish the US leadership position. This outcome is not entirely negative--additional investments will expand markets--but it is critical that the US government take steps to remain competitive: There are applications from which the US population and economy may benefit; there are specific applications with importance for national defense; and US technical leadership will ensure that US experts have a leading role in synthetic biology governance, regulation, and oversight. Measures to increase competitiveness in S&T generally are broadly applicable for synthetic biology and should be pursued. However, the US government will also need to take action on fundamental issues that will affect the field's development, such as countering anti-GMO (genetically modified organism) sentiments and anti-GMO legislation. The United States should maintain its regulatory approach so that it is the product that is regulated, not the method used to create a product. At the same time, the United States needs to ensure that the regulatory framework is updated so that synthetic biology products do not fall into regulatory gaps. Finally, the United States needs to pay close attention to how synthetic biology applications may be governed internationally, such as through the Nagoya Protocol of the Convention on Biological Diversity, so that beneficial applications may be realized.

  2. Synthetic control of a fitness tradeoff in yeast nitrogen metabolism

    Directory of Open Access Journals (Sweden)

    Lee Jack J

    2009-01-01

    Full Text Available Abstract Background Microbial communities are involved in many processes relevant to industrial and medical biotechnology, such as the formation of biofilms, lignocellulosic degradation, and hydrogen production. The manipulation of synthetic and natural microbial communities and their underlying ecological parameters, such as fitness, evolvability, and variation, is an increasingly important area of research for synthetic biology. Results Here, we explored how synthetic control of an endogenous circuit can be used to regulate a tradeoff between fitness in resource abundant and resource limited environments in a population of Saccharomyces cerevisiae. We found that noise in the expression of a key enzyme in ammonia assimilation, Gdh1p, mediated a tradeoff between growth in low nitrogen environments and stress resistance in high ammonia environments. We implemented synthetic control of an endogenous Gdh1p regulatory network to construct an engineered strain in which the fitness of the population was tunable in response to an exogenously-added small molecule across a range of ammonia environments. Conclusion The ability to tune fitness and biological tradeoffs will be important components of future efforts to engineer microbial communities.

  3. CRISPR-Cas9-Mediated Genome Editing and Transcriptional Control in Yarrowia lipolytica.

    Science.gov (United States)

    Schwartz, Cory; Wheeldon, Ian

    2018-01-01

    The discovery and adaptation of RNA-guided nucleases has resulted in the rapid development of efficient, scalable, and easily accessible synthetic biology tools for targeted genome editing and transcriptional control. In these systems, for example CRISPR-Cas9 from Streptococcus pyogenes, a protein with nuclease activity is targeted to a specific nucleotide sequence by a short RNA molecule, whereupon binding it cleaves the targeted nucleotide strand. To extend this genome-editing ability to the industrially important oleaginous yeast Yarrowia lipolytica, we developed a set of easily usable and effective CRISPR-Cas9 episomal vectors. In this protocols chapter, we first present a method by which arbitrary protein-coding genes can be disrupted via indel formation after CRISPR-Cas9 targeting. A second method demonstrates how the same CRISPR-Cas9 system can be used to induce markerless gene cassette integration into the genome by inducing homologous recombination after DNA cleavage by Cas9. Finally, we describe how a catalytically inactive form of Cas9 fused to a transcriptional repressor can be used to control transcription of native genes in Y. lipolytica. The CRISPR-Cas9 tools and strategies described here greatly increase the types of genome editing and transcriptional control that can be achieved in Y. lipolytica, and promise to facilitate more advanced engineering of this important oleaginous host.

  4. Synthetic Botany.

    Science.gov (United States)

    Boehm, Christian R; Pollak, Bernardo; Purswani, Nuri; Patron, Nicola; Haseloff, Jim

    2017-07-05

    Plants are attractive platforms for synthetic biology and metabolic engineering. Plants' modular and plastic body plans, capacity for photosynthesis, extensive secondary metabolism, and agronomic systems for large-scale production make them ideal targets for genetic reprogramming. However, efforts in this area have been constrained by slow growth, long life cycles, the requirement for specialized facilities, a paucity of efficient tools for genetic manipulation, and the complexity of multicellularity. There is a need for better experimental and theoretical frameworks to understand the way genetic networks, cellular populations, and tissue-wide physical processes interact at different scales. We highlight new approaches to the DNA-based manipulation of plants and the use of advanced quantitative imaging techniques in simple plant models such as Marchantia polymorpha. These offer the prospects of improved understanding of plant dynamics and new approaches to rational engineering of plant traits. Copyright © 2017 Cold Spring Harbor Laboratory Press; all rights reserved.

  5. Application of synthetic biology for production of chemicals in yeast Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Borodina, Irina; Li, Mingji

    2015-01-01

    Synthetic biology and metabolic engineering enable generation of novel cell factories that efficiently convert renewable feedstocks into biofuels, bulk, and fine chemicals, thus creating the basis for biosustainable economy independent on fossil resources. While over a hundred proof...... biology has the potential to bring down this cost by improving our ability to predictably engineer biological systems. This review highlights synthetic biology applications for design, assembly, and optimization of non-native biochemical pathways in baker's yeast Saccharomyces cerevisiae. We describe......-of-concept chemicals have been made in yeast, only a very small fraction of those has reached commercial-scale production so far. The limiting factor is the high research cost associated with the development of a robust cell factory that can produce the desired chemical at high titer, rate, and yield. Synthetic...

  6. Promoting microbiology education through the iGEM synthetic biology competition.

    Science.gov (United States)

    Kelwick, Richard; Bowater, Laura; Yeoman, Kay H; Bowater, Richard P

    2015-08-01

    Synthetic biology has developed rapidly in the 21st century. It covers a range of scientific disciplines that incorporate principles from engineering to take advantage of and improve biological systems, often applied to specific problems. Methods important in this subject area include the systematic design and testing of biological systems and, here, we describe how synthetic biology projects frequently develop microbiology skills and education. Synthetic biology research has huge potential in biotechnology and medicine, which brings important ethical and moral issues to address, offering learning opportunities about the wider impact of microbiological research. Synthetic biology projects have developed into wide-ranging training and educational experiences through iGEM, the International Genetically Engineered Machines competition. Elements of the competition are judged against specific criteria and teams can win medals and prizes across several categories. Collaboration is an important element of iGEM, and all DNA constructs synthesized by iGEM teams are made available to all researchers through the Registry for Standard Biological Parts. An overview of microbiological developments in the iGEM competition is provided. This review is targeted at educators that focus on microbiology and synthetic biology, but will also be of value to undergraduate and postgraduate students with an interest in this exciting subject area. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  7. Synthetic Biology Open Language (SBOL) Version 2.2.0.

    Science.gov (United States)

    Cox, Robert Sidney; Madsen, Curtis; McLaughlin, James Alastair; Nguyen, Tramy; Roehner, Nicholas; Bartley, Bryan; Beal, Jacob; Bissell, Michael; Choi, Kiri; Clancy, Kevin; Grünberg, Raik; Macklin, Chris; Misirli, Goksel; Oberortner, Ernst; Pocock, Matthew; Samineni, Meher; Zhang, Michael; Zhang, Zhen; Zundel, Zach; Gennari, John H; Myers, Chris; Sauro, Herbert; Wipat, Anil

    2018-04-02

    Synthetic biology builds upon the techniques and successes of genetics, molecular biology, and metabolic engineering by applying engineering principles to the design of biological systems. The field still faces substantial challenges, including long development times, high rates of failure, and poor reproducibility. One method to ameliorate these problems would be to improve the exchange of information about designed systems between laboratories. The synthetic biology open language (SBOL) has been developed as a standard to support the specification and exchange of biological design information in synthetic biology, filling a need not satisfied by other pre-existing standards. This document details version 2.2.0 of SBOL that builds upon version 2.1.0 published in last year's JIB special issue. In particular, SBOL 2.2.0 includes improved description and validation rules for genetic design provenance, an extension to support combinatorial genetic designs, a new class to add non-SBOL data as attachments, a new class for genetic design implementations, and a description of a methodology to describe the entire design-build-test-learn cycle within the SBOL data model.

  8. Erasing Borders: A Brief Chronicle of Early Synthetic Biology.

    Science.gov (United States)

    Peretó, Juli

    2016-12-01

    Synthetic Biology is currently presented as an emergent field involving the application of engineering principles to living matter. However, the scientific pursuit of making life in a laboratory is not new and has been the ultimate, if somewhat distant, aim of the origin-of-life research program for many years. Actually, over a century ago, the idea that the synthesis of life was indispensable to fully understand its nature already appealed to material scientists and evolutionists alike. Jacques Loeb proposed a research program from an engineering standpoint, following a synthetic method (experimental abiogenesis) and based on his mechanist vision of living beings, which he considered true chemical machines. Early synthetic biology endeavors, such as the premature experiments by Alfonso L. Herrera in Mexico, Stéphane Leduc in France, and John B. Burke in United Kingdom, were easily ridiculed on both scientific and ideological grounds. However, in retrospect, all those attempts should be considered as legitimate and sincere anti-vitalistic efforts to cross the apparent border between inert and living matter.

  9. Synthetic Biology Open Language (SBOL) Version 2.2.0

    KAUST Repository

    Cox, Robert Sidney

    2018-04-04

    Synthetic biology builds upon the techniques and successes of genetics, molecular biology, and metabolic engineering by applying engineering principles to the design of biological systems. The field still faces substantial challenges, including long development times, high rates of failure, and poor reproducibility. One method to ameliorate these problems would be to improve the exchange of information about designed systems between laboratories. The synthetic biology open language (SBOL) has been developed as a standard to support the specification and exchange of biological design information in synthetic biology, filling a need not satisfied by other pre-existing standards. This document details version 2.2.0 of SBOL that builds upon version 2.1.0 published in last year\\'s JIB special issue. In particular, SBOL 2.2.0 includes improved description and validation rules for genetic design provenance, an extension to support combinatorial genetic designs, a new class to add non-SBOL data as attachments, a new class for genetic design implementations, and a description of a methodology to describe the entire design-build-test-learn cycle within the SBOL data model.

  10. Synthetic Biology Open Language (SBOL Version 2.2.0

    Directory of Open Access Journals (Sweden)

    Cox Robert Sidney

    2018-04-01

    Full Text Available Synthetic biology builds upon the techniques and successes of genetics, molecular biology, and metabolic engineering by applying engineering principles to the design of biological systems. The field still faces substantial challenges, including long development times, high rates of failure, and poor reproducibility. One method to ameliorate these problems would be to improve the exchange of information about designed systems between laboratories. The synthetic biology open language (SBOL has been developed as a standard to support the specification and exchange of biological design information in synthetic biology, filling a need not satisfied by other pre-existing standards. This document details version 2.2.0 of SBOL that builds upon version 2.1.0 published in last year’s JIB special issue. In particular, SBOL 2.2.0 includes improved description and validation rules for genetic design provenance, an extension to support combinatorial genetic designs, a new class to add non-SBOL data as attachments, a new class for genetic design implementations, and a description of a methodology to describe the entire design-build-test-learn cycle within the SBOL data model.

  11. Synthetic Biology Open Language (SBOL) Version 2.2.0

    KAUST Repository

    Cox, Robert Sidney; Madsen, Curtis; McLaughlin, James Alastair; Nguyen, Tramy; Roehner, Nicholas; Bartley, Bryan; Beal, Jacob; Bissell, Michael; Choi, Kiri; Clancy, Kevin; Grunberg, Raik; Macklin, Chris; Misirli, Goksel; Oberortner, Ernst; Pocock, Matthew; Samineni, Meher; Zhang, Michael; Zhang, Zhen; Zundel, Zach; Gennari, John H.; Myers, Chris; Sauro, Herbert; Wipat, Anil

    2018-01-01

    Synthetic biology builds upon the techniques and successes of genetics, molecular biology, and metabolic engineering by applying engineering principles to the design of biological systems. The field still faces substantial challenges, including long development times, high rates of failure, and poor reproducibility. One method to ameliorate these problems would be to improve the exchange of information about designed systems between laboratories. The synthetic biology open language (SBOL) has been developed as a standard to support the specification and exchange of biological design information in synthetic biology, filling a need not satisfied by other pre-existing standards. This document details version 2.2.0 of SBOL that builds upon version 2.1.0 published in last year's JIB special issue. In particular, SBOL 2.2.0 includes improved description and validation rules for genetic design provenance, an extension to support combinatorial genetic designs, a new class to add non-SBOL data as attachments, a new class for genetic design implementations, and a description of a methodology to describe the entire design-build-test-learn cycle within the SBOL data model.

  12. Mutanome Engineered RNA Immunotherapy: Towards Patient-Centered Tumor Vaccination

    Directory of Open Access Journals (Sweden)

    Mathias Vormehr

    2015-01-01

    Full Text Available Advances in nucleic acid sequencing technologies have revolutionized the field of genomics, allowing the efficient targeting of mutated neoantigens for personalized cancer vaccination. Due to their absence during negative selection of T cells and their lack of expression in healthy tissue, tumor mutations are considered as optimal targets for cancer immunotherapy. Preclinical and early clinical data suggest that synthetic mRNA can serve as potent drug format allowing the cost efficient production of highly efficient vaccines in a timely manner. In this review, we describe a process, which integrates next generation sequencing based cancer mutanome mapping, in silico target selection and prioritization approaches, and mRNA vaccine manufacturing and delivery into a process we refer to as MERIT (mutanome engineered RNA immunotherapy.

  13. Genomic resources for identification of the minimal N2 -fixing symbiotic genome.

    Science.gov (United States)

    diCenzo, George C; Zamani, Maryam; Milunovic, Branislava; Finan, Turlough M

    2016-09-01

    The lack of an appropriate genomic platform has precluded the use of gain-of-function approaches to study the rhizobium-legume symbiosis, preventing the establishment of the genes necessary and sufficient for symbiotic nitrogen fixation (SNF) and potentially hindering synthetic biology approaches aimed at engineering this process. Here, we describe the development of an appropriate system by reverse engineering Sinorhizobium meliloti. Using a novel in vivo cloning procedure, the engA-tRNA-rmlC (ETR) region, essential for cell viability and symbiosis, was transferred from Sinorhizobium fredii to the ancestral location on the S. meliloti chromosome, rendering the ETR region on pSymB redundant. A derivative of this strain lacking both the large symbiotic replicons (pSymA and pSymB) was constructed. Transfer of pSymA and pSymB back into this strain restored symbiotic capabilities with alfalfa. To delineate the location of the single-copy genes essential for SNF on these replicons, we screened a S. meliloti deletion library, representing > 95% of the 2900 genes of the symbiotic replicons, for their phenotypes with alfalfa. Only four loci, accounting for < 12% of pSymA and pSymB, were essential for SNF. These regions will serve as our preliminary target of the minimal set of horizontally acquired genes necessary and sufficient for SNF. © 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.

  14. Synthetic algae and cyanobacteria: Great potential but what is the exposure risk?

    Science.gov (United States)

    Green algae and cyanobacteria (hereafter, algae) have the attractive properties of relatively simple genomes, rapid growth rates, and an ability to synthesize useful compounds using solar energy and carbon dioxide. They are attractive targets for applications of synthetic biology...

  15. From pathways to genomes and beyond. The metabolic engineering toolbox and its place in biofuels production

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Leqian; Reed, Ben; Alper, Hal [Texas Univ., Austin, TX (United States). Dept. of Chemical Engineering

    2011-07-01

    Concerns about the availability of petroleum-derived fuels and chemicals have led to the exploration of metabolically engineered organisms as novel hosts for biofuels and chemicals production. However, the complexity inherent in metabolic and regulatory networks makes this undertaking a complex task. To address these limitations, metabolic engineering has adapted a wide-variety of tools for altering phenotypes. In this review, we will highlight traditional and recent metabolic engineering tools for optimizing cells including pathway-based, global, and genomic-enabled approaches. Specifically, we describe these tools as well as provide demonstrations of their effectiveness in optimizing biofuels production. However, each of these tools provides stepping stones towards the grand goal of biofuels production. Thus, developing methods for large-scale cellular optimization and integrative approaches are invaluable for further cell optimization. This review highlights the challenges that still must be met to accomplish this goal. (orig.)

  16. Applications of computational modeling in metabolic engineering of yeast.

    Science.gov (United States)

    Kerkhoven, Eduard J; Lahtvee, Petri-Jaan; Nielsen, Jens

    2015-02-01

    Generally, a microorganism's phenotype can be described by its pattern of metabolic fluxes. Although fluxes cannot be measured directly, inference of fluxes is well established. In biotechnology the aim is often to increase the capacity of specific fluxes. For this, metabolic engineering methods have been developed and applied extensively. Many of these rely on balancing of intracellular metabolites, redox, and energy fluxes, using genome-scale models (GEMs) that in combination with appropriate objective functions and constraints can be used to predict potential gene targets for obtaining a preferred flux distribution. These methods point to strategies for altering gene expression; however, fluxes are often controlled by post-transcriptional events. Moreover, GEMs are usually not taking into account metabolic regulation, thermodynamics and enzyme kinetics. To facilitate metabolic engineering, tools from synthetic biology have emerged, enabling integration and assembly of naturally nonexistent, but well-characterized components into a living organism. To describe these systems kinetic models are often used and to integrate these systems with the standard metabolic engineering approach, it is necessary to expand the modeling of metabolism to consider kinetics of individual processes. This review will give an overview about models available for metabolic engineering of yeast and discusses their applications. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.

  17. Future combustion technology for synthetic and renewable fuels in compression ignition engines (REFUEL). Final report

    Energy Technology Data Exchange (ETDEWEB)

    Aakko-Saksa, P.; Brink, A.; Happonen, M. [and others

    2012-07-01

    This domestic project, Future Combustion Technology for Synthetic and Renewable Fuels in Compression Ignition Engines (ReFuel), was part of a Collaborative Task 'Future Combustion Technology for Synthetic and Renewable Fuels in Transport' of International Energy Agency (IEA) Combustion Agreement. This international Collaborative Task is coordinated by Finland. The three-year (2009-2011) prooject was a joint research project with Aalto University (Aalto), Tampere University of Technology (TUT), Technical Research Centre of Finland (VTT) and Aabo Akademi University (AAU). The project was funded by TEKES, Waertsilae Oyj, Agro Sisu Power, Aker Arctic Technology Oy and the research partners listed above. Modern renewable diesel fuels have excellent physical and chemical properties, in comparison to traditional crude oil based fuels. Purely paraffinic fuels do not contain aromatic compounds and they are totally sulphur free. Hydrotreated Vegetable Oil (HVO) was studied as an example of paraffinic high cetane number (CN) diesel fuels. HVO has no storage and low temperature problems like the fatty acid methyl esters (FAMEs) have. The combustion properties are better than those of crude oil based fuels and FAME, because they have very high cetane numbers and contain no polyaromatic hydrocarbons (PAH). With low HVO density, viscosity and distillation temperatures, these advantageous properties allow far more advanced combustion strategies, such as very high exhaust gas recirculation (EGR) rates or extreme Miller timings, than has been possible with current fossil fuels. The implementation of these advanced combustion technologies, together with the novel renewable diesel fuel, brought significant nitrogen oxides (NO{sub x}), particulate matter (PM) emission reductions with no efficiency losses. (orig.)

  18. Targeting the Synthetic Essential Kinases of Breast Cancers

    Science.gov (United States)

    2016-05-01

    engineering via Cas9. Science 2013, 339(6121):823-826. 31. Koike-Yusa H, Li Y, Tan EP, Velasco- Herrera MD, Yusa K: Genome-wide recessive genetic screening in...Yusa H, Li Y, Tan EP, Velasco- Herrera Mdel C, Yusa K: Genome-wide recessive genetic screening in mammalian cells with a lentiviral CRISPR-guide RNA

  19. Original Research doi:10.4102/http://www.indieskriflig.org.za ids.v48i2.722 An exploration of synthetic biology: A preliminary Christian ethical assessment of the advantages and disadvantages of synthetic biology

    Directory of Open Access Journals (Sweden)

    Riaan A.L. Rheeder

    2014-10-01

    Full Text Available On 20 May 2010, the Venter Institute in America announced that they have fully synthesised the genome of the organism Mycoplasma mycoides whilst in vitro by using a computer connected to a machine that synthesises genes. Thereafter, the genome was placed back into the casing of another organism (Mycoplasma capricolum and it was reported that the synthesised organism and the genome functioned normally. This synthesised organism was reconstructed to function as a minute little factory with the aim of producing and secreting fuel and medicine − something that is not the natural function of this organism. There are certain potential dangers inherent in this kind of technology. Scientists fear that this technology may contaminate or infect humans, animals or the environment, and that it can as such be extremely harmful, or even lead to the destruction of humans. Other scientists are concerned that terrorists can use this technology to kill innocent citizens. Some ethicists are of the opinion that the consequences of synthetic biology is currently unpredictable and that it is therefore risky. In opposition to the potential dangers, one has to mention that synthetic biology indeed can result in far-reaching positive outcomes such as the manufacturing of biofuel and medication. Most scientists and ethicists are of the opinion that the potential dangers involved in synthetic biology should be evaluated in light of the fact that genetic manipulation has not caused any biological devastation over the last 30 years. From a Christian point of departure, the opinion is currently that synthetic biology is not an irresponsible science and technology.

  20. Engineering Plant Architecture via CRISPR/Cas9-mediated Alteration of Strigolactone Biosynthesis

    KAUST Repository

    Butt, Haroon

    2018-01-28

    Precision plant genome engineering holds much promise for targeted improvement of crop traits via unprecedented single-base level control over the genetic material. Strigolactones (SLs) are a key determinant of plant architecture, known for their role in inhibiting shoot branching (tillering). Here, we used CRISPR/Cas9 in rice (Oryza sativa) for targeted disruption of CAROTENOID CLEAVAGE DIOXYGENASE 7 (CCD7), which controls a key step in SL biosynthesis. The ccd7 mutants exhibited a striking increase in tillering, combined with a dwarf phenotype, which could be rescued by application of the synthetic SL analog GR24. Striga germination assays and liquid chromatography mass spectrometry analysis showed that root exudates of ccd7 mutants were also SL deficient. Taken together, our results show the power of CRISPR/Cas9 for targeted engineering of plant architecture and for elucidating the molecular underpinnings of architecture-related traits.

  1. Engineering Plant Architecture via CRISPR/Cas9-mediated Alteration of Strigolactone Biosynthesis

    KAUST Repository

    Butt, Haroon; Jamil, Muhammad; Wang, Jian You; Al-Babili, Salim; Mahfouz, Magdy M.

    2018-01-01

    Precision plant genome engineering holds much promise for targeted improvement of crop traits via unprecedented single-base level control over the genetic material. Strigolactones (SLs) are a key determinant of plant architecture, known for their role in inhibiting shoot branching (tillering). Here, we used CRISPR/Cas9 in rice (Oryza sativa) for targeted disruption of CAROTENOID CLEAVAGE DIOXYGENASE 7 (CCD7), which controls a key step in SL biosynthesis. The ccd7 mutants exhibited a striking increase in tillering, combined with a dwarf phenotype, which could be rescued by application of the synthetic SL analog GR24. Striga germination assays and liquid chromatography mass spectrometry analysis showed that root exudates of ccd7 mutants were also SL deficient. Taken together, our results show the power of CRISPR/Cas9 for targeted engineering of plant architecture and for elucidating the molecular underpinnings of architecture-related traits.

  2. Synthetic biology advances for pharmaceutical production

    OpenAIRE

    Breitling, Rainer; Takano, Eriko

    2015-01-01

    Synthetic biology enables a new generation of microbial engineering for the biotechnological production of pharmaceuticals and other high-value chemicals. This review presents an overview of recent advances in the field, describing new computational and experimental tools for the discovery, optimization and production of bioactive molecules, and outlining progress towards the application of these tools to pharmaceutical production systems.

  3. Creating to understand – developmental biology meets engineering in Paris

    NARCIS (Netherlands)

    Kicheva, Anna; Rivron, Nicolas C.

    2017-01-01

    In November 2016, developmental biologists, synthetic biologists and engineers gathered in Paris for a meeting called ‘Engineering the embryo’. The participants shared an interest in exploring how synthetic systems can reveal new principles of embryonic development, and how the in vitro manipulation

  4. Exploring Networks at the genome scale

    NARCIS (Netherlands)

    Lam, M.C.; Puchalka, J.; Diez, M.S.; Martins Dos Santos, V.A.P.

    2010-01-01

    Systems biology is aimed at achieving a holistic understanding of living organisms, while synthetic biology seeks to design and construct new living organisms with targeted functionalities. Genome sequencing and the fields of ‘omics’ technology have proven a goldmine of information for scientists

  5. Engineering microbial consortia for controllable outputs

    Energy Technology Data Exchange (ETDEWEB)

    Lindemann, Stephen R.; Bernstein, Hans C.; Song, Hyun-Seob; Fredrickson, Jim K.; Fields, Matthew W.; Shou, Wenying; Johnson, David R.; Beliaev, Alexander S.

    2016-03-11

    Much research has been invested into engineering microorganisms to perform desired biotransformations; nonetheless, these efforts frequently fall short of expected results due to the unforeseen effects of biofeedback regulation and functional incompatibility. In nature, metabolic function is compartmentalized into diverse organisms assembled into resilient consortia, in which the division of labor is thought to lead to increased community efficiency and productivity. Here, we consider whether and how consortia can be designed to perform bioprocesses of interest beyond the metabolic flexibility limitations of a single organism. Advances in post-genomic analysis of microbial consortia and application of high-resolution global measurements now offer the promise of systems-level understanding of how microbial consortia adapt to changes in environmental variables and inputs of carbon and energy. We argue that when combined with appropriate modeling framework that predictive knowledge generates testable hypotheses and orthogonal synthetic biology tools, such understanding can dramatically improve our ability to control the fate and functioning of consortia. In this article, we articulate our collective perspective on the current and future state of microbial community engineering and control while placing specific emphasis on ecological principles that promote control over community function and emergent properties.

  6. Synthetic biology approaches to fluorinated polyketides.

    Science.gov (United States)

    Thuronyi, Benjamin W; Chang, Michelle C Y

    2015-03-17

    The catalytic diversity of living systems offers a broad range of opportunities for developing new methods to produce small molecule targets such as fuels, materials, and pharmaceuticals. In addition to providing cost-effective and renewable methods for large-scale commercial processes, the exploration of the unusual chemical phenotypes found in living organisms can also enable the expansion of chemical space for discovery of novel function by combining orthogonal attributes from both synthetic and biological chemistry. In this context, we have focused on the development of new fluorine chemistry using synthetic biology approaches. While fluorine has become an important feature in compounds of synthetic origin, the scope of biological fluorine chemistry in living systems is limited, with fewer than 20 organofluorine natural products identified to date. In order to expand the diversity of biosynthetically accessible organofluorines, we have begun to develop methods for the site-selective introduction of fluorine into complex natural products by engineering biosynthetic machinery to incorporate fluorinated building blocks. To gain insight into how both enzyme active sites and metabolic pathways can be evolved to manage and select for fluorinated compounds, we have studied one of the only characterized natural hosts for organofluorine biosynthesis, the soil microbe Streptomyces cattleya. This information provides a template for designing engineered organofluorine enzymes, pathways, and hosts and has allowed us to initiate construction of enzymatic and cellular pathways for the production of fluorinated polyketides.

  7. Tunable promoters in synthetic and systems biology

    DEFF Research Database (Denmark)

    Dehli, Tore; Solem, Christian; Jensen, Peter Ruhdal

    2012-01-01

    in synthetic biology. A number of tools exist to manipulate the steps in between gene sequence and functional protein in living cells, but out of these the most straight-forward approach is to alter the gene expression level by manipulating the promoter sequence. Some of the promoter tuning tools available......Synthetic and systems biologists need standardized, modular and orthogonal tools yielding predictable functions in vivo. In systems biology such tools are needed to quantitatively analyze the behavior of biological systems while the efficient engineering of artificial gene networks is central...... for accomplishing such altered gene expression levels are discussed here along with examples of their use, and ideas for new tools are described. The road ahead looks very promising for synthetic and systems biologists as tools to achieve just about anything in terms of tuning and timing multiple gene expression...

  8. Micro flow chemistry : new possibilities for synthetic chemists

    NARCIS (Netherlands)

    Noel, T.; Pignataro, B.

    2014-01-01

    The use of microreactor technology for continuous-flow chemistry has received a significant amount of attention in recent years. In this chapter, we discuss the important aspects of this technology and its impact on synthetic organic chemistry. Basic engineering principles, unusual reaction

  9. Reprint of Design of synthetic microbial communities for biotechnological production processes.

    Science.gov (United States)

    Jagmann, Nina; Philipp, Bodo

    2014-12-20

    In their natural habitats microorganisms live in multi-species communities, in which the community members exhibit complex metabolic interactions. In contrast, biotechnological production processes catalyzed by microorganisms are usually carried out with single strains in pure cultures. A number of production processes, however, may be more efficiently catalyzed by the concerted action of microbial communities. This review will give an overview of organismic interactions between microbial cells and of biotechnological applications of microbial communities. It focuses on synthetic microbial communities that consist of microorganisms that have been genetically engineered. Design principles for such synthetic communities will be exemplified based on plausible scenarios for biotechnological production processes. These design principles comprise interspecific metabolic interactions via cross-feeding, regulation by interspecific signaling processes via metabolites and autoinducing signal molecules, and spatial structuring of synthetic microbial communities. In particular, the implementation of metabolic interdependencies, of positive feedback regulation and of inducible cell aggregation and biofilm formation will be outlined. Synthetic microbial communities constitute a viable extension of the biotechnological application of metabolically engineered single strains and enlarge the scope of microbial production processes. Copyright © 2014 Elsevier B.V. All rights reserved.

  10. GLIDERS - A web-based search engine for genome-wide linkage disequilibrium between HapMap SNPs

    Directory of Open Access Journals (Sweden)

    Broxholme John

    2009-10-01

    Full Text Available Abstract Background A number of tools for the examination of linkage disequilibrium (LD patterns between nearby alleles exist, but none are available for quickly and easily investigating LD at longer ranges (>500 kb. We have developed a web-based query tool (GLIDERS: Genome-wide LInkage DisEquilibrium Repository and Search engine that enables the retrieval of pairwise associations with r2 ≥ 0.3 across the human genome for any SNP genotyped within HapMap phase 2 and 3, regardless of distance between the markers. Description GLIDERS is an easy to use web tool that only requires the user to enter rs numbers of SNPs they want to retrieve genome-wide LD for (both nearby and long-range. The intuitive web interface handles both manual entry of SNP IDs as well as allowing users to upload files of SNP IDs. The user can limit the resulting inter SNP associations with easy to use menu options. These include MAF limit (5-45%, distance limits between SNPs (minimum and maximum, r2 (0.3 to 1, HapMap population sample (CEU, YRI and JPT+CHB combined and HapMap build/release. All resulting genome-wide inter-SNP associations are displayed on a single output page, which has a link to a downloadable tab delimited text file. Conclusion GLIDERS is a quick and easy way to retrieve genome-wide inter-SNP associations and to explore LD patterns for any number of SNPs of interest. GLIDERS can be useful in identifying SNPs with long-range LD. This can highlight mis-mapping or other potential association signal localisation problems.

  11. Engineering synergy in biotechnology

    DEFF Research Database (Denmark)

    Nielsen, Jens; Fussenegger, Martin; Keasling, Jay

    2014-01-01

    In this article, the author focuses on approaches in metabolic engineering and synthetic biology for the creation of efficient cell factories, which can be bused to convert biomass and other feedstocks for the generation of chemicals. Topics discussed include development of restriction enzymes......, engineering plasmids and recyclable markers, production of 1,3-propanediol using a metabolically engineered Escherichia coli and production of isobutanol by using metabolically engineered yeast....

  12. Protein engineering techniques gateways to synthetic protein universe

    CERN Document Server

    Poluri, Krishna Mohan

    2017-01-01

    This brief provides a broad overview of protein-engineering research, offering a glimpse of the most common experimental methods. It also presents various computational programs with applications that are widely used in directed evolution, computational and de novo protein design. Further, it sheds light on the advantages and pitfalls of existing methodologies and future perspectives of protein engineering techniques.

  13. Leveraging knowledge engineering and machine learning for microbial bio-manufacturing.

    Science.gov (United States)

    Oyetunde, Tolutola; Bao, Forrest Sheng; Chen, Jiung-Wen; Martin, Hector Garcia; Tang, Yinjie J

    2018-05-03

    Genome scale modeling (GSM) predicts the performance of microbial workhorses and helps identify beneficial gene targets. GSM integrated with intracellular flux dynamics, omics, and thermodynamics have shown remarkable progress in both elucidating complex cellular phenomena and computational strain design (CSD). Nonetheless, these models still show high uncertainty due to a poor understanding of innate pathway regulations, metabolic burdens, and other factors (such as stress tolerance and metabolite channeling). Besides, the engineered hosts may have genetic mutations or non-genetic variations in bioreactor conditions and thus CSD rarely foresees fermentation rate and titer. Metabolic models play important role in design-build-test-learn cycles for strain improvement, and machine learning (ML) may provide a viable complementary approach for driving strain design and deciphering cellular processes. In order to develop quality ML models, knowledge engineering leverages and standardizes the wealth of information in literature (e.g., genomic/phenomic data, synthetic biology strategies, and bioprocess variables). Data driven frameworks can offer new constraints for mechanistic models to describe cellular regulations, to design pathways, to search gene targets, and to estimate fermentation titer/rate/yield under specified growth conditions (e.g., mixing, nutrients, and O 2 ). This review highlights the scope of information collections, database constructions, and machine learning techniques (such as deep learning and transfer learning), which may facilitate "Learn and Design" for strain development. Copyright © 2018. Published by Elsevier Inc.

  14. Synchronous long-term oscillations in a synthetic gene circuit.

    Science.gov (United States)

    Potvin-Trottier, Laurent; Lord, Nathan D; Vinnicombe, Glenn; Paulsson, Johan

    2016-10-27

    Synthetically engineered genetic circuits can perform a wide variety of tasks but are generally less accurate than natural systems. Here we revisit the first synthetic genetic oscillator, the repressilator, and modify it using principles from stochastic chemistry in single cells. Specifically, we sought to reduce error propagation and information losses, not by adding control loops, but by simply removing existing features. We show that this modification created highly regular and robust oscillations. Furthermore, some streamlined circuits kept 14 generation periods over a range of growth conditions and kept phase for hundreds of generations in single cells, allowing cells in flasks and colonies to oscillate synchronously without any coupling between them. Our results suggest that even the simplest synthetic genetic networks can achieve a precision that rivals natural systems, and emphasize the importance of noise analyses for circuit design in synthetic biology.

  15. Design and construction of synthetic microbial consortia in China

    Directory of Open Access Journals (Sweden)

    Ming-Zhu Ding

    2016-12-01

    Full Text Available The rapid development of synthetic biology enables the design, construction and optimization of synthetic microbial consortia to achieve specific functions. In China, the “973” project-“Design and Construction of Microbial Consortia” was funded by the National Basic Research Program of China in January 2014. It was proposed to address the fundamental challenges in engineering natural microbial consortia and reconstructing microbial consortia to meet industrial demands. In this review, we will introduce this “973” project, including the significance of microbial consortia, the fundamental scientific issues, the recent research progresses, and some case studies about synthetic microbial consortia in the past two and a half years.

  16. Engineered Cpf1 variants with altered PAM specificities increase genome targeting range

    Science.gov (United States)

    Gao, Linyi; Cox, David B.T.; Yan, Winston X.; Manteiga, John C.; Schneider, Martin W.; Yamano, Takashi; Nishimasu, Hiroshi; Nureki, Osamu; Crosetto, Nicola; Zhang, Feng

    2017-01-01

    The RNA-guided endonuclease Cpf1 is a promising tool for genome editing in eukaryotic cells1–7. However, the utility of the commonly used Acidaminococcus sp. BV3L6 Cpf1 (AsCpf1) and Lachnospiraceae bacterium ND2006 Cpf1 (LbCpf1) is limited by their requirement of a TTTV protospacer adjacent motif (PAM) in the DNA substrate. To address this limitation, we performed a structure-guided mutagenesis screen to increase the targeting range of Cpf1. We engineered two AsCpf1 variants carrying the mutations S542R/K607R and S542R/K548V/N552R, which recognize TYCV and TATV PAMs, respectively, with enhanced activities in vitro and in human cells. Genome-wide assessment of off-target activity using BLISS7 assay indicated that these variants retain high DNA targeting specificity, which we further improved by introducing an additional non-PAM-interacting mutation. Introducing the identified mutations at their corresponding positions in LbCpf1 similarly altered its PAM specificity. Together, these variants increase the targeting range of Cpf1 by approximately three-fold in human coding sequences to one cleavage site per ~11 bp. PMID:28581492

  17. "Knight in shining armour" or "Frankenstein's creation"? The coverage of synthetic biology in German-language media.

    Science.gov (United States)

    Gschmeidler, Brigitte; Seiringer, Alexandra

    2012-02-01

    Although still a side issue in the German-language media, attention towards synthetic biology has risen clearly during the last years, in line with the first applications being presented. This paper presents findings from a content analysis of synthetic biology coverage in German-language media over the years 2004-2009. In the media, synthetic biology is not clearly separated from gene technology. News value is attributed to established categories such as persons and events. Many metaphors and analogies used in describing gene technology can also be found in the coverage of synthetic biology; however, engineering metaphors are more prominent. In addition, playfulness constitutes an aspect rarely found in genetic engineering coverage. Overall, the picture emerging is ambivalent, which leaves prospects for the further development of public debate ambiguous.

  18. Synthetic biology advances for pharmaceutical production

    Science.gov (United States)

    Breitling, Rainer; Takano, Eriko

    2015-01-01

    Synthetic biology enables a new generation of microbial engineering for the biotechnological production of pharmaceuticals and other high-value chemicals. This review presents an overview of recent advances in the field, describing new computational and experimental tools for the discovery, optimization and production of bioactive molecules, and outlining progress towards the application of these tools to pharmaceutical production systems. PMID:25744872

  19. TinkerCell: modular CAD tool for synthetic biology

    Science.gov (United States)

    Chandran, Deepak; Bergmann, Frank T; Sauro, Herbert M

    2009-01-01

    Background Synthetic biology brings together concepts and techniques from engineering and biology. In this field, computer-aided design (CAD) is necessary in order to bridge the gap between computational modeling and biological data. Using a CAD application, it would be possible to construct models using available biological "parts" and directly generate the DNA sequence that represents the model, thus increasing the efficiency of design and construction of synthetic networks. Results An application named TinkerCell has been developed in order to serve as a CAD tool for synthetic biology. TinkerCell is a visual modeling tool that supports a hierarchy of biological parts. Each part in this hierarchy consists of a set of attributes that define the part, such as sequence or rate constants. Models that are constructed using these parts can be analyzed using various third-party C and Python programs that are hosted by TinkerCell via an extensive C and Python application programming interface (API). TinkerCell supports the notion of a module, which are networks with interfaces. Such modules can be connected to each other, forming larger modular networks. TinkerCell is a free and open-source project under the Berkeley Software Distribution license. Downloads, documentation, and tutorials are available at . Conclusion An ideal CAD application for engineering biological systems would provide features such as: building and simulating networks, analyzing robustness of networks, and searching databases for components that meet the design criteria. At the current state of synthetic biology, there are no established methods for measuring robustness or identifying components that fit a design. The same is true for databases of biological parts. TinkerCell's flexible modeling framework allows it to cope with changes in the field. Such changes may involve the way parts are characterized or the way synthetic networks are modeled and analyzed computationally. TinkerCell can readily

  20. TinkerCell: modular CAD tool for synthetic biology

    Directory of Open Access Journals (Sweden)

    Bergmann Frank T

    2009-10-01

    Full Text Available Abstract Background Synthetic biology brings together concepts and techniques from engineering and biology. In this field, computer-aided design (CAD is necessary in order to bridge the gap between computational modeling and biological data. Using a CAD application, it would be possible to construct models using available biological "parts" and directly generate the DNA sequence that represents the model, thus increasing the efficiency of design and construction of synthetic networks. Results An application named TinkerCell has been developed in order to serve as a CAD tool for synthetic biology. TinkerCell is a visual modeling tool that supports a hierarchy of biological parts. Each part in this hierarchy consists of a set of attributes that define the part, such as sequence or rate constants. Models that are constructed using these parts can be analyzed using various third-party C and Python programs that are hosted by TinkerCell via an extensive C and Python application programming interface (API. TinkerCell supports the notion of a module, which are networks with interfaces. Such modules can be connected to each other, forming larger modular networks. TinkerCell is a free and open-source project under the Berkeley Software Distribution license. Downloads, documentation, and tutorials are available at http://www.tinkercell.com. Conclusion An ideal CAD application for engineering biological systems would provide features such as: building and simulating networks, analyzing robustness of networks, and searching databases for components that meet the design criteria. At the current state of synthetic biology, there are no established methods for measuring robustness or identifying components that fit a design. The same is true for databases of biological parts. TinkerCell's flexible modeling framework allows it to cope with changes in the field. Such changes may involve the way parts are characterized or the way synthetic networks are modeled

  1. Selection platforms for directed evolution in synthetic biology.

    Science.gov (United States)

    Tizei, Pedro A G; Csibra, Eszter; Torres, Leticia; Pinheiro, Vitor B

    2016-08-15

    Life on Earth is incredibly diverse. Yet, underneath that diversity, there are a number of constants and highly conserved processes: all life is based on DNA and RNA; the genetic code is universal; biology is limited to a small subset of potential chemistries. A vast amount of knowledge has been accrued through describing and characterizing enzymes, biological processes and organisms. Nevertheless, much remains to be understood about the natural world. One of the goals in Synthetic Biology is to recapitulate biological complexity from simple systems made from biological molecules-gaining a deeper understanding of life in the process. Directed evolution is a powerful tool in Synthetic Biology, able to bypass gaps in knowledge and capable of engineering even the most highly conserved biological processes. It encompasses a range of methodologies to create variation in a population and to select individual variants with the desired function-be it a ligand, enzyme, pathway or even whole organisms. Here, we present some of the basic frameworks that underpin all evolution platforms and review some of the recent contributions from directed evolution to synthetic biology, in particular methods that have been used to engineer the Central Dogma and the genetic code. © 2016 The Author(s).

  2. Synthetic biology of antimicrobial discovery.

    Science.gov (United States)

    Zakeri, Bijan; Lu, Timothy K

    2013-07-19

    Antibiotic discovery has a storied history. From the discovery of penicillin by Sir Alexander Fleming to the relentless quest for antibiotics by Selman Waksman, the stories have become like folklore used to inspire future generations of scientists. However, recent discovery pipelines have run dry at a time when multidrug-resistant pathogens are on the rise. Nature has proven to be a valuable reservoir of antimicrobial agents, which are primarily produced by modularized biochemical pathways. Such modularization is well suited to remodeling by an interdisciplinary approach that spans science and engineering. Herein, we discuss the biological engineering of small molecules, peptides, and non-traditional antimicrobials and provide an overview of the growing applicability of synthetic biology to antimicrobials discovery.

  3. Synthetic biology and molecular genetics in non-conventional yeasts: Current tools and future advances.

    Science.gov (United States)

    Wagner, James M; Alper, Hal S

    2016-04-01

    Coupling the tools of synthetic biology with traditional molecular genetic techniques can enable the rapid prototyping and optimization of yeast strains. While the era of yeast synthetic biology began in the well-characterized model organism Saccharomyces cerevisiae, it is swiftly expanding to include non-conventional yeast production systems such as Hansenula polymorpha, Kluyveromyces lactis, Pichia pastoris, and Yarrowia lipolytica. These yeasts already have roles in the manufacture of vaccines, therapeutic proteins, food additives, and biorenewable chemicals, but recent synthetic biology advances have the potential to greatly expand and diversify their impact on biotechnology. In this review, we summarize the development of synthetic biological tools (including promoters and terminators) and enabling molecular genetics approaches that have been applied in these four promising alternative biomanufacturing platforms. An emphasis is placed on synthetic parts and genome editing tools. Finally, we discuss examples of synthetic tools developed in other organisms that can be adapted or optimized for these hosts in the near future. Copyright © 2015 Elsevier Inc. All rights reserved.

  4. A low cost, customizable turbidostat for use in synthetic circuit characterization.

    Science.gov (United States)

    Takahashi, Chris N; Miller, Aaron W; Ekness, Felix; Dunham, Maitreya J; Klavins, Eric

    2015-01-16

    Engineered biological circuits are often disturbed by a variety of environmental factors. In batch culture, where the majority of synthetic circuit characterization occurs, environmental conditions vary as the culture matures. Turbidostats are powerful characterization tools that provide static culture environments; however, they are often expensive, especially when purchased in custom configurations, and are difficult to design and construct in a lab. Here, we present a low cost, open source multiplexed turbidostat that can be manufactured and used with minimal experience in electrical or software engineering. We demonstrate the utility of this system to profile synthetic circuit behavior in S. cerevisiae. We also demonstrate the flexibility of the design by showing that a fluorometer can be easily integrated.

  5. Genomic signal processing

    CERN Document Server

    Shmulevich, Ilya

    2007-01-01

    Genomic signal processing (GSP) can be defined as the analysis, processing, and use of genomic signals to gain biological knowledge, and the translation of that knowledge into systems-based applications that can be used to diagnose and treat genetic diseases. Situated at the crossroads of engineering, biology, mathematics, statistics, and computer science, GSP requires the development of both nonlinear dynamical models that adequately represent genomic regulation, and diagnostic and therapeutic tools based on these models. This book facilitates these developments by providing rigorous mathema

  6. TeachEnG: a Teaching Engine for Genomics.

    Science.gov (United States)

    Kim, Minji; Kim, Yeonsung; Qian, Lei; Song, Jun S

    2017-10-15

    Bioinformatics is a rapidly growing field that has emerged from the synergy of computer science, statistics and biology. Given the interdisciplinary nature of bioinformatics, many students from diverse fields struggle with grasping bioinformatic concepts only from classroom lectures. Interactive tools for helping students reinforce their learning would be thus desirable. Here, we present an interactive online educational tool called TeachEnG (acronym for Teaching Engine for Genomics) for reinforcing key concepts in sequence alignment and phylogenetic tree reconstruction. Our instructional games allow students to align sequences by hand, fill out the dynamic programming matrix in the Needleman-Wunsch global sequence alignment algorithm, and reconstruct phylogenetic trees via the maximum parsimony, Unweighted Pair Group Method with Arithmetic mean (UPGMA) and Neighbor-Joining algorithms. With an easily accessible interface and instant visual feedback, TeachEnG will help promote active learning in bioinformatics. TeachEnG is freely available at http://teacheng.illinois.edu. The source code is available from https://github.com/KnowEnG/TeachEnG under the Artistic License 2.0. It is written in JavaScript and compatible with Firefox, Safari, Chrome and Microsoft Edge. songj@illinois.edu. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com

  7. News: Synthetic biology leading to specialty chemicals ...

    Science.gov (United States)

    Synthetic biology can combine the disciplines of biology, engineering, and chemistry productively to form molecules of great scientific and commercial value. Recent advances in the new field are explored for their connection to new tools that have been used to elucidate production pathways to a wide variety of chemicals generated by microorganisms. The selection and enhancement of microbiological strains through the practice of strain engineering enables targets of design, construction, and optimization. This news column aspires to cover recent literature relating to the development and understanding of clean technology.

  8. Molecular analysis of genetic diversity in elite II synthetic hexaploid ...

    African Journals Online (AJOL)

    The present study was conducted to assess the genetic diversity of Elite-II synthetic hexaploid (SH) wheat by genome DNA fingerprinting as revealed by random amplified polymorphic DNA (RAPD) analysis. Ten decamer RAPD primers (OPG-1, OPG-2, OPG-3, OPG-4, OPG-5, OPA-3, OPA-4, OPA-5, OPA-8, and OPA-15) ...

  9. Metabolic 'engines' of flight drive genome size reduction in birds.

    Science.gov (United States)

    Wright, Natalie A; Gregory, T Ryan; Witt, Christopher C

    2014-03-22

    The tendency for flying organisms to possess small genomes has been interpreted as evidence of natural selection acting on the physical size of the genome. Nonetheless, the flight-genome link and its mechanistic basis have yet to be well established by comparative studies within a volant clade. Is there a particular functional aspect of flight such as brisk metabolism, lift production or maneuverability that impinges on the physical genome? We measured genome sizes, wing dimensions and heart, flight muscle and body masses from a phylogenetically diverse set of bird species. In phylogenetically controlled analyses, we found that genome size was negatively correlated with relative flight muscle size and heart index (i.e. ratio of heart to body mass), but positively correlated with body mass and wing loading. The proportional masses of the flight muscles and heart were the most important parameters explaining variation in genome size in multivariate models. Hence, the metabolic intensity of powered flight appears to have driven genome size reduction in birds.

  10. Cfd modeling of a synthetic jet actuator

    International Nuclear Information System (INIS)

    Dghim, Marouane; Ben Chiekh, Maher; Ben Nasrallah, Sassi

    2009-01-01

    Synthetic jet actuators show good promise as an enabling technology for innovative boundary layer flow control applied to external surfaces, like airplane wings, and to internal flows, like those occurring in a curved engine inlet. The appealing characteristics of a synthetic jet are zero-net-mass flux operation and an efficient control effect that takes advantages of unsteady fluid phenomena. The formation of a synthetic jet in a quiescent external air flow is only beginning to be understood and a rational understanding of these devices is necessary before they can be applied to the control of flows outside of the laboratory. The synthetic jet flow generated by a planar orifice is investigated here using computational approach. Computations of the 2D synthetic jet are performed with unsteady RANS modeled with the Realizable κ - ε turbulence model available in FLUENT environment. In this present work, the ability of the first order turbulence model, employed in our computations, to model the formation of the counter-rotating-vortex pair (CVP) that appears in the flow-field was investigated. Computational results were in good agreement with experimental measurements. The effectiveness of such control actuator was tested on separated boundary layer. Preliminary investigation were presented and discussed

  11. Terpene metabolic engineering via nuclear or chloroplast genomes profoundly and globally impacts off-target pathways through metabolite signalling.

    Science.gov (United States)

    Pasoreck, Elise K; Su, Jin; Silverman, Ian M; Gosai, Sager J; Gregory, Brian D; Yuan, Joshua S; Daniell, Henry

    2016-09-01

    The impact of metabolic engineering on nontarget pathways and outcomes of metabolic engineering from different genomes are poorly understood questions. Therefore, squalene biosynthesis genes FARNESYL DIPHOSPHATE SYNTHASE (FPS) and SQUALENE SYNTHASE (SQS) were engineered via the Nicotiana tabacum chloroplast (C), nuclear (N) or both (CN) genomes to promote squalene biosynthesis. SQS levels were ~4300-fold higher in C and CN lines than in N, but all accumulated ~150-fold higher squalene due to substrate or storage limitations. Abnormal leaf and flower phenotypes, including lower pollen production and reduced fertility, were observed regardless of the compartment or level of transgene expression. Substantial changes in metabolomes of all lines were observed: levels of 65-120 unrelated metabolites, including the toxic alkaloid nicotine, changed by as much as 32-fold. Profound effects of transgenesis on nontarget gene expression included changes in the abundance of 19 076 transcripts by up to 2000-fold in CN; 7784 transcripts by up to 1400-fold in N; and 5224 transcripts by as much as 2200-fold in C. Transporter-related transcripts were induced, and cell cycle-associated transcripts were disproportionally repressed in all three lines. Transcriptome changes were validated by qRT-PCR. The mechanism underlying these large changes likely involves metabolite-mediated anterograde and/or retrograde signalling irrespective of the level of transgene expression or end product, due to imbalance of metabolic pools, offering new insight into both anticipated and unanticipated consequences of metabolic engineering. © 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

  12. Critical mm-wave components for synthetic automatic test systems

    CERN Document Server

    Hrobak, Michael

    2015-01-01

    Michael Hrobak studied hybrid integrated front end modules for high frequency measurement equipment and especially for synthetic automatic test systems. Recent developments of innovative, critical millimeter-wave components like frequency multipliers, directional couplers, filters, triple balanced mixers and power detectors are illustrated by the author separately and in combination.  Contents Synthetic Instruments Resistive Diode Frequency Multipliers Planar Directional Couplers and Filters Triple Balanced Mixers Zero Bias Schottky Power Detectors Integrated Front End Assemblies  Target Groups Scientists and students in the field of electrical engineering with main emphasis on high frequency technology Engineers and Practitioners dealing with the development of micro- and millimeter-wave measurement instruments  About the Author Dr. Michael Hrobak is with the Microwave Department of the Ferdinand-Braun-Institut (FBH), Berlin, Germany, where he is involved in the development and measurement of monolithic i...

  13. Genome editing and genetic engineering in livestock for advancing agricultural and biomedical applications.

    Science.gov (United States)

    Telugu, Bhanu P; Park, Ki-Eun; Park, Chi-Hun

    2017-08-01

    Genetic modification of livestock has a longstanding and successful history, starting with domestication several thousand years ago. Modern animal breeding strategies predominantly based on marker-assisted and genomic selection, artificial insemination, and embryo transfer have led to significant improvement in the performance of domestic animals, and are the basis for regular supply of high quality animal derived food. However, the current strategy of breeding animals over multiple generations to introduce novel traits is not realistic in responding to the unprecedented challenges such as changing climate, pandemic diseases, and feeding an anticipated 3 billion increase in global population in the next three decades. Consequently, sophisticated genetic modifications that allow for seamless introgression of novel alleles or traits and introduction of precise modifications without affecting the overall genetic merit of the animal are required for addressing these pressing challenges. The requirement for precise modifications is especially important in the context of modeling human diseases for the development of therapeutic interventions. The animal science community envisions the genome editors as essential tools in addressing these critical priorities in agriculture and biomedicine, and for advancing livestock genetic engineering for agriculture, biomedical as well as "dual purpose" applications.

  14. Modular design of artificial tissue homeostasis: robust control through synthetic cellular heterogeneity.

    Directory of Open Access Journals (Sweden)

    Miles Miller

    Full Text Available Synthetic biology efforts have largely focused on small engineered gene networks, yet understanding how to integrate multiple synthetic modules and interface them with endogenous pathways remains a challenge. Here we present the design, system integration, and analysis of several large scale synthetic gene circuits for artificial tissue homeostasis. Diabetes therapy represents a possible application for engineered homeostasis, where genetically programmed stem cells maintain a steady population of β-cells despite continuous turnover. We develop a new iterative process that incorporates modular design principles with hierarchical performance optimization targeted for environments with uncertainty and incomplete information. We employ theoretical analysis and computational simulations of multicellular reaction/diffusion models to design and understand system behavior, and find that certain features often associated with robustness (e.g., multicellular synchronization and noise attenuation are actually detrimental for tissue homeostasis. We overcome these problems by engineering a new class of genetic modules for 'synthetic cellular heterogeneity' that function to generate beneficial population diversity. We design two such modules (an asynchronous genetic oscillator and a signaling throttle mechanism, demonstrate their capacity for enhancing robust control, and provide guidance for experimental implementation with various computational techniques. We found that designing modules for synthetic heterogeneity can be complex, and in general requires a framework for non-linear and multifactorial analysis. Consequently, we adapt a 'phenotypic sensitivity analysis' method to determine how functional module behaviors combine to achieve optimal system performance. We ultimately combine this analysis with Bayesian network inference to extract critical, causal relationships between a module's biochemical rate-constants, its high level functional behavior in

  15. Modular design of artificial tissue homeostasis: robust control through synthetic cellular heterogeneity.

    Science.gov (United States)

    Miller, Miles; Hafner, Marc; Sontag, Eduardo; Davidsohn, Noah; Subramanian, Sairam; Purnick, Priscilla E M; Lauffenburger, Douglas; Weiss, Ron

    2012-01-01

    Synthetic biology efforts have largely focused on small engineered gene networks, yet understanding how to integrate multiple synthetic modules and interface them with endogenous pathways remains a challenge. Here we present the design, system integration, and analysis of several large scale synthetic gene circuits for artificial tissue homeostasis. Diabetes therapy represents a possible application for engineered homeostasis, where genetically programmed stem cells maintain a steady population of β-cells despite continuous turnover. We develop a new iterative process that incorporates modular design principles with hierarchical performance optimization targeted for environments with uncertainty and incomplete information. We employ theoretical analysis and computational simulations of multicellular reaction/diffusion models to design and understand system behavior, and find that certain features often associated with robustness (e.g., multicellular synchronization and noise attenuation) are actually detrimental for tissue homeostasis. We overcome these problems by engineering a new class of genetic modules for 'synthetic cellular heterogeneity' that function to generate beneficial population diversity. We design two such modules (an asynchronous genetic oscillator and a signaling throttle mechanism), demonstrate their capacity for enhancing robust control, and provide guidance for experimental implementation with various computational techniques. We found that designing modules for synthetic heterogeneity can be complex, and in general requires a framework for non-linear and multifactorial analysis. Consequently, we adapt a 'phenotypic sensitivity analysis' method to determine how functional module behaviors combine to achieve optimal system performance. We ultimately combine this analysis with Bayesian network inference to extract critical, causal relationships between a module's biochemical rate-constants, its high level functional behavior in isolation, and

  16. Mammalian synthetic biology for studying the cell.

    Science.gov (United States)

    Mathur, Melina; Xiang, Joy S; Smolke, Christina D

    2017-01-02

    Synthetic biology is advancing the design of genetic devices that enable the study of cellular and molecular biology in mammalian cells. These genetic devices use diverse regulatory mechanisms to both examine cellular processes and achieve precise and dynamic control of cellular phenotype. Synthetic biology tools provide novel functionality to complement the examination of natural cell systems, including engineered molecules with specific activities and model systems that mimic complex regulatory processes. Continued development of quantitative standards and computational tools will expand capacities to probe cellular mechanisms with genetic devices to achieve a more comprehensive understanding of the cell. In this study, we review synthetic biology tools that are being applied to effectively investigate diverse cellular processes, regulatory networks, and multicellular interactions. We also discuss current challenges and future developments in the field that may transform the types of investigation possible in cell biology. © 2017 Mathur et al.

  17. From essential to persistent genes: a functional approach to constructing synthetic life

    DEFF Research Database (Denmark)

    Acevedo-Rocha, Carlos G.; Fang, Gang; Schmidt, Markus

    2013-01-01

    A central undertaking in synthetic biology (SB) is the quest for the ‘minimal genome’. However, ‘minimal sets’ of essential genes are strongly context-dependent and, in all prokaryotic genomes sequenced to date, not a single protein-coding gene is entirely conserved. Furthermore, a lack...

  18. The Next Generation of Synthetic Biology Chassis: Moving Synthetic Biology from the Laboratory to the Field.

    Science.gov (United States)

    Adams, Bryn L

    2016-12-16

    Escherichia coli (E. coli) has played a pivotal role in the development of genetics and molecular biology as scientific fields. It is therefore not surprising that synthetic biology (SB) was built upon E. coli and continues to dominate the field. However, scientific capabilities have advanced from simple gene mutations to the insertion of rationally designed, complex synthetic circuits and creation of entirely synthetic genomes. The point is rapidly approaching where E. coli is no longer an adequate host for the increasingly sophisticated genetic designs of SB. It is time to develop the next generation of SB chassis; robust organisms that can provide the advanced physiology novel synthetic circuits will require to move SB from the laboratory into fieldable technologies. This can be accomplished by developing chassis-specific genetic toolkits that are as extensive as those for E. coli. However, the holy grail of SB would be the development of a universal toolkit that can be ported into any chassis. This viewpoint article underscores the need for new bacterial chassis, as well as discusses some of the important considerations in their selection. It also highlights a few examples of robust, tractable bacterial species that can meet the demands of tomorrow's state-of-the-art in SB. Significant advances have been made in the first 15 years since this field has emerged. However, the advances over the next 15 years will occur not in laboratory organisms, but in fieldable species where the potential of SB can be fully realized in game changing technology.

  19. Generalized schemes for high throughput manipulation of the Desulfovibrio vulgaris Hildenborough genome

    Energy Technology Data Exchange (ETDEWEB)

    Chhabra, S.R.; Butland, G.; Elias, D.; Chandonia, J.-M.; Fok, V.; Juba, T.; Gorur, A.; Allen, S.; Leung, C.-M.; Keller, K.; Reveco, S.; Zane, G.; Semkiw, E.; Prathapam, R.; Gold, B.; Singer, M.; Ouellet, M.; Sazakal, E.; Jorgens, D.; Price, M.; Witkowska, E.; Beller, H.; Hazen, T.C.; Biggin, M.; Auer, M.; Wall, J.; Keasling, J.

    2011-07-15

    The ability to conduct advanced functional genomic studies of the thousands of sequenced bacteria has been hampered by the lack of available tools for making high- throughput chromosomal manipulations in a systematic manner that can be applied across diverse species. In this work, we highlight the use of synthetic biological tools to assemble custom suicide vectors with reusable and interchangeable DNA “parts” to facilitate chromosomal modification at designated loci. These constructs enable an array of downstream applications including gene replacement and creation of gene fusions with affinity purification or localization tags. We employed this approach to engineer chromosomal modifications in a bacterium that has previously proven difficult to manipulate genetically, Desulfovibrio vulgaris Hildenborough, to generate a library of over 700 strains. Furthermore, we demonstrate how these modifications can be used for examining metabolic pathways, protein-protein interactions, and protein localization. The ubiquity of suicide constructs in gene replacement throughout biology suggests that this approach can be applied to engineer a broad range of species for a diverse array of systems biological applications and is amenable to high-throughput implementation.

  20. Plant synthetic biology: a new platform for industrial biotechnology.

    Science.gov (United States)

    Fesenko, Elena; Edwards, Robert

    2014-05-01

    Thirty years after the production of the first generation of genetically modified plants we are now set to move into a new era of recombinant crop technology through the application of synthetic biology to engineer new and complex input and output traits. The use of synthetic biology technologies will represent more than incremental additions of transgenes, but rather the directed design of completely new metabolic pathways, physiological traits, and developmental control strategies. The need to enhance our ability to improve crops through new engineering capability is now increasingly pressing as we turn to plants not just for food, but as a source of renewable feedstocks for industry. These accelerating and diversifying demands for new output traits coincide with a need to reduce inputs and improve agricultural sustainability. Faced with such challenges, existing technologies will need to be supplemented with new and far-more-directed approaches to turn valuable resources more efficiently into usable agricultural products. While these objectives are challenging enough, the use of synthetic biology in crop improvement will face public acceptance issues as a legacy of genetically modified technologies in many countries. Here we review some of the potential benefits of adopting synthetic biology approaches in improving plant input and output traits for their use as industrial chemical feedstocks, as linked to the rapidly developing biorefining industry. Several promising technologies and biotechnological targets are identified along with some of the key regulatory and societal challenges in the safe and acceptable introduction of such technology.

  1. Synthetic analog computation in living cells.

    Science.gov (United States)

    Daniel, Ramiz; Rubens, Jacob R; Sarpeshkar, Rahul; Lu, Timothy K

    2013-05-30

    A central goal of synthetic biology is to achieve multi-signal integration and processing in living cells for diagnostic, therapeutic and biotechnology applications. Digital logic has been used to build small-scale circuits, but other frameworks may be needed for efficient computation in the resource-limited environments of cells. Here we demonstrate that synthetic analog gene circuits can be engineered to execute sophisticated computational functions in living cells using just three transcription factors. Such synthetic analog gene circuits exploit feedback to implement logarithmically linear sensing, addition, ratiometric and power-law computations. The circuits exhibit Weber's law behaviour as in natural biological systems, operate over a wide dynamic range of up to four orders of magnitude and can be designed to have tunable transfer functions. Our circuits can be composed to implement higher-order functions that are well described by both intricate biochemical models and simple mathematical functions. By exploiting analog building-block functions that are already naturally present in cells, this approach efficiently implements arithmetic operations and complex functions in the logarithmic domain. Such circuits may lead to new applications for synthetic biology and biotechnology that require complex computations with limited parts, need wide-dynamic-range biosensing or would benefit from the fine control of gene expression.

  2. The Pseudomonas aeruginosa pirA gene encodes a second receptor for ferrienterobactin and synthetic catecholate analogues.

    Science.gov (United States)

    Ghysels, Bart; Ochsner, Urs; Möllman, Ute; Heinisch, Lothar; Vasil, Michael; Cornelis, Pierre; Matthijs, Sandra

    2005-05-15

    Actively secreted iron chelating agents termed siderophores play an important role in the virulence and rhizosphere competence of fluorescent pseudomonads, including Pseudomonas aeruginosa which secretes a high affinity siderophore, pyoverdine, and the low affinity siderophore, pyochelin. Uptake of the iron-siderophore complexes is an active process that requires specific outer membrane located receptors, which are dependent of the inner membrane-associated protein TonB and two other inner membrane proteins, ExbB and ExbC. P. aeruginosa is also capable of using a remarkable variety of heterologous siderophores as sources of iron, apparently by expressing their cognate receptors. Illustrative of this feature are the 32 (of which 28 putative) siderophore receptor genes observed in the P. aeruginosa PAO1 genome. However, except for a few (pyoverdine, pyochelin, enterobactin), the vast majority of P. aeruginosa siderophore receptor genes still remain to be characterized. Ten synthetic iron chelators of catecholate type stimulated growth of a pyoverdine/pyochelin deficient P. aeruginosa PAO1 mutant under condition of severe iron limitation. Null mutants of the 32 putative TonB-dependent siderophore receptor encoding genes engineered in the same genetic background were screened for obvious deficiencies in uptake of the synthetic siderophores, but none showed decreased growth stimulation in the presence of the different siderophores. However, a double knock-out mutant of ferrienterobactin receptor encoding gene pfeA (PA 2688) and pirA (PA0931) failed to be stimulated by 4 of the tested synthetic catecholate siderophores whose chemical structures resemble enterobactin. Ferric-enterobactin also failed to stimulate growth of the double pfeA-pirA mutant although, like its synthetic analogues, it stimulated growth of the corresponding single mutants. Hence, we confirmed that pirA represents a second P. aeruginosa ferric-enterobactin receptor. The example of these two

  3. DFAST: a flexible prokaryotic genome annotation pipeline for faster genome publication.

    Science.gov (United States)

    Tanizawa, Yasuhiro; Fujisawa, Takatomo; Nakamura, Yasukazu

    2018-03-15

    We developed a prokaryotic genome annotation pipeline, DFAST, that also supports genome submission to public sequence databases. DFAST was originally started as an on-line annotation server, and to date, over 7000 jobs have been processed since its first launch in 2016. Here, we present a newly implemented background annotation engine for DFAST, which is also available as a standalone command-line program. The new engine can annotate a typical-sized bacterial genome within 10 min, with rich information such as pseudogenes, translation exceptions and orthologous gene assignment between given reference genomes. In addition, the modular framework of DFAST allows users to customize the annotation workflow easily and will also facilitate extensions for new functions and incorporation of new tools in the future. The software is implemented in Python 3 and runs in both Python 2.7 and 3.4-on Macintosh and Linux systems. It is freely available at https://github.com/nigyta/dfast_core/under the GPLv3 license with external binaries bundled in the software distribution. An on-line version is also available at https://dfast.nig.ac.jp/. yn@nig.ac.jp. Supplementary data are available at Bioinformatics online.

  4. [How to be prudent with synthetic biology. Synthetic Biology and the precautionary principle].

    Science.gov (United States)

    Rodríguez López, Blanca

    2014-01-01

    Synthetic biology is a new discipline that is twofold: firstly it offers the promise to pay benefits that can alleviate some of the ills that plague mankind; On the other hand, like all technologies, holds risks. Given these, the most critical and concerned about the risks, invoke the application of the precautionary principle, common in cases where an activity or new technology creates risks to the environment and/or human health, but far from universally accepted happens to be currently one of the most controversial principles. In this paper the question of the risks and benefits of synthetic biology and the relevance of applying the precautionary principle are analyzed. To do this we proceed as follows. The first part focuses on synthetic biology. At first, this discipline is characterized, with special attention to what is novel compared to the known as "genetic engineering". In the second stage both the benefits and the risks associated with it are discussed. The first part concludes with a review of the efforts currently being made to control or minimize the risks. The second part aims to analyze the precautionary principle and its possible relevance to the case of Synthetic Biology. At first, the different versions and interpretations of the principle and the various criticisms of which has been the subject are reviewed. Finally, after discarding the Precautionary Principle as an useful tool, it is seen as more appropriate some recent proposals to treat technologies that take into account not only risks but also their benefits.

  5. SBOLme: a Repository of SBOL Parts for Metabolic Engineering

    KAUST Repository

    Kuwahara, Hiroyuki

    2017-01-12

    The Synthetic Biology Open Language (SBOL) is a community-driven open language to promote standardization in synthetic biology. To support the use of SBOL in metabolic engineering, we developed SBOLme, the first open-access repository of SBOL 2-compliant biochemical parts for a wide range of metabolic engineering applications. The URL of our repository is http://www.cbrc.kaust.edu.sa/sbolme.

  6. Development of a broad-host synthetic biology toolbox for Ralstonia eutropha and its application to engineering hydrocarbon biofuel production.

    Science.gov (United States)

    Bi, Changhao; Su, Peter; Müller, Jana; Yeh, Yi-Chun; Chhabra, Swapnil R; Beller, Harry R; Singer, Steven W; Hillson, Nathan J

    2013-11-13

    The chemoautotrophic bacterium Ralstonia eutropha can utilize H2/CO2 for growth under aerobic conditions. While this microbial host has great potential to be engineered to produce desired compounds (beyond polyhydroxybutyrate) directly from CO2, little work has been done to develop genetic part libraries to enable such endeavors. We report the development of a toolbox for the metabolic engineering of Ralstonia eutropha H16. We have constructed a set of broad-host-range plasmids bearing a variety of origins of replication, promoters, 5' mRNA stem-loop structures, and ribosomal binding sites. Specifically, we analyzed the origins of replication pCM62 (IncP), pBBR1, pKT (IncQ), and their variants. We tested the promoters P(BAD), T7, P(xyls/PM), P(lacUV5), and variants thereof for inducible expression. We also evaluated a T7 mRNA stem-loop structure sequence and compared a set of ribosomal binding site (RBS) sequences derived from Escherichia coli, R. eutropha, and a computational RBS design tool. Finally, we employed the toolbox to optimize hydrocarbon production in R. eutropha and demonstrated a 6-fold titer improvement using the appropriate combination of parts. We constructed and evaluated a versatile synthetic biology toolbox for Ralstonia eutropha metabolic engineering that could apply to other microbial hosts as well.

  7. Genome-derived vaccines.

    Science.gov (United States)

    De Groot, Anne S; Rappuoli, Rino

    2004-02-01

    Vaccine research entered a new era when the complete genome of a pathogenic bacterium was published in 1995. Since then, more than 97 bacterial pathogens have been sequenced and at least 110 additional projects are now in progress. Genome sequencing has also dramatically accelerated: high-throughput facilities can draft the sequence of an entire microbe (two to four megabases) in 1 to 2 days. Vaccine developers are using microarrays, immunoinformatics, proteomics and high-throughput immunology assays to reduce the truly unmanageable volume of information available in genome databases to a manageable size. Vaccines composed by novel antigens discovered from genome mining are already in clinical trials. Within 5 years we can expect to see a novel class of vaccines composed by genome-predicted, assembled and engineered T- and Bcell epitopes. This article addresses the convergence of three forces--microbial genome sequencing, computational immunology and new vaccine technologies--that are shifting genome mining for vaccines onto the forefront of immunology research.

  8. Synthetic biology of antimicrobial discovery

    Science.gov (United States)

    Zakeri, Bijan; Lu, Timothy K.

    2012-01-01

    Antibiotic discovery has a storied history. From the discovery of penicillin by Sir Alexander Fleming to the relentless quest for antibiotics by Selman Waksman, the stories have become like folklore, used to inspire future generations of scientists. However, recent discovery pipelines have run dry at a time when multidrug resistant pathogens are on the rise. Nature has proven to be a valuable reservoir of antimicrobial agents, which are primarily produced by modularized biochemical pathways. Such modularization is well suited to remodeling by an interdisciplinary approach that spans science and engineering. Herein, we discuss the biological engineering of small molecules, peptides, and non-traditional antimicrobials and provide an overview of the growing applicability of synthetic biology to antimicrobials discovery. PMID:23654251

  9. Biomimetic engineering of colloidal nanoarchitectures with "in vitro" and "in vivo" functionality

    OpenAIRE

    Einfalt, Tomaž

    2017-01-01

    Biomimetic engineering opens unprecedented possibilities of combining biomolecules (i.e. proteins, DNA, polysaccharides) with synthetic materials (i.e. synthetic polymers). This combination results in unique hybrid systems with functionalities that mimic processes in living organisms. While the translational value of functional biomimetically engineered structures is of exceptional importance in fields such as technology, engineering, chemistry, biology and medicine, due to the properties the...

  10. A Synthetic Biology Framework for Programming Eukaryotic Transcription Functions

    Science.gov (United States)

    Khalil, Ahmad S.; Lu, Timothy K.; Bashor, Caleb J.; Ramirez, Cherie L.; Pyenson, Nora C.; Joung, J. Keith; Collins, James J.

    2013-01-01

    SUMMARY Eukaryotic transcription factors (TFs) perform complex and combinatorial functions within transcriptional networks. Here, we present a synthetic framework for systematically constructing eukaryotic transcription functions using artificial zinc fingers, modular DNA-binding domains found within many eukaryotic TFs. Utilizing this platform, we construct a library of orthogonal synthetic transcription factors (sTFs) and use these to wire synthetic transcriptional circuits in yeast. We engineer complex functions, such as tunable output strength and transcriptional cooperativity, by rationally adjusting a decomposed set of key component properties, e.g., DNA specificity, affinity, promoter design, protein-protein interactions. We show that subtle perturbations to these properties can transform an individual sTF between distinct roles (activator, cooperative factor, inhibitory factor) within a transcriptional complex, thus drastically altering the signal processing behavior of multi-input systems. This platform provides new genetic components for synthetic biology and enables bottom-up approaches to understanding the design principles of eukaryotic transcriptional complexes and networks. PMID:22863014

  11. Membrane transporter engineering in industrial biotechnology and whole cell biocatalysis.

    Science.gov (United States)

    Kell, Douglas B; Swainston, Neil; Pir, Pınar; Oliver, Stephen G

    2015-04-01

    Because they mainly do not involve chemical changes, membrane transporters have been a Cinderella subject in the biotechnology of small molecule production, but this is a serious oversight. Influx transporters contribute significantly to the flux towards product, and efflux transporters ensure the accumulation of product in the much greater extracellular space of fermentors. Programmes for improving biotechnological processes might therefore give greater consideration to transporters than may have been commonplace. Strategies for identifying important transporters include expression profiling, genome-wide knockout studies, stress-based selection, and the use of inhibitors. In addition, modern methods of directed evolution and synthetic biology, especially those effecting changes in energy coupling, offer huge opportunities for increasing the flux towards extracellular product formation by transporter engineering. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  12. Leaner and meaner genomes in Escherichia coli

    DEFF Research Database (Denmark)

    Ussery, David

    2006-01-01

    A 'better' Escherichia coli K-12 genome has recently been engineered in which about 15% of the genome has been removed by planned deletions. Comparison with related bacterial genomes that have undergone a natural reduction in size suggests that there is plenty of scope for yet more deletions....

  13. Integrating biological redesign: where synthetic biology came from and where it needs to go.

    Science.gov (United States)

    Way, Jeffrey C; Collins, James J; Keasling, Jay D; Silver, Pamela A

    2014-03-27

    Synthetic biology seeks to extend approaches from engineering and computation to redesign of biology, with goals such as generating new chemicals, improving human health, and addressing environmental issues. Early on, several guiding principles of synthetic biology were articulated, including design according to specification, separation of design from fabrication, use of standardized biological parts and organisms, and abstraction. We review the utility of these principles over the past decade in light of the field's accomplishments in building complex systems based on microbial transcription and metabolism and describe the progress in mammalian cell engineering. Copyright © 2014 Elsevier Inc. All rights reserved.

  14. Synthetic biology as it relates to CAM photosynthesis: challenges and opportunities.

    Science.gov (United States)

    DePaoli, Henrique C; Borland, Anne M; Tuskan, Gerald A; Cushman, John C; Yang, Xiaohan

    2014-07-01

    To meet future food and energy security needs, which are amplified by increasing population growth and reduced natural resource availability, metabolic engineering efforts have moved from manipulating single genes/proteins to introducing multiple genes and novel pathways to improve photosynthetic efficiency in a more comprehensive manner. Biochemical carbon-concentrating mechanisms such as crassulacean acid metabolism (CAM), which improves photosynthetic, water-use, and possibly nutrient-use efficiency, represent a strategic target for synthetic biology to engineer more productive C3 crops for a warmer and drier world. One key challenge for introducing multigene traits like CAM onto a background of C3 photosynthesis is to gain a better understanding of the dynamic spatial and temporal regulatory events that underpin photosynthetic metabolism. With the aid of systems and computational biology, vast amounts of experimental data encompassing transcriptomics, proteomics, and metabolomics can be related in a network to create dynamic models. Such models can undergo simulations to discover key regulatory elements in metabolism and suggest strategic substitution or augmentation by synthetic components to improve photosynthetic performance and water-use efficiency in C3 crops. Another key challenge in the application of synthetic biology to photosynthesis research is to develop efficient systems for multigene assembly and stacking. Here, we review recent progress in computational modelling as applied to plant photosynthesis, with attention to the requirements for CAM, and recent advances in synthetic biology tool development. Lastly, we discuss possible options for multigene pathway construction in plants with an emphasis on CAM-into-C3 engineering. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  15. Synthetic biology and biomimetic chemistry as converging technologies fostering a new generation of smart biosensors.

    Science.gov (United States)

    Scognamiglio, Viviana; Antonacci, Amina; Lambreva, Maya D; Litescu, Simona C; Rea, Giuseppina

    2015-12-15

    Biosensors are powerful tunable systems able to switch between an ON/OFF status in response to an external stimulus. This extraordinary property could be engineered by adopting synthetic biology or biomimetic chemistry to obtain tailor-made biosensors having the desired requirements of robustness, sensitivity and detection range. Recent advances in both disciplines, in fact, allow to re-design the configuration of the sensing elements - either by modifying toggle switches and gene networks, or by producing synthetic entities mimicking key properties of natural molecules. The present review considered the role of synthetic biology in sustaining biosensor technology, reporting examples from the literature and reflecting on the features that make it a useful tool for designing and constructing engineered biological systems for sensing application. Besides, a section dedicated to bioinspired synthetic molecules as powerful tools to enhance biosensor potential is reported, and treated as an extension of the concept of biomimetic chemistry, where organic synthesis is used to generate artificial molecules that mimic natural molecules. Thus, the design of synthetic molecules, such as aptamers, biomimetics, molecular imprinting polymers, peptide nucleic acids, and ribozymes were encompassed as "products" of biomimetic chemistry. Copyright © 2015 Elsevier B.V. All rights reserved.

  16. Germline Cas9 expression yields highly efficient genome engineering in a major worldwide disease vector, Aedes aegypti.

    Science.gov (United States)

    Li, Ming; Bui, Michelle; Yang, Ting; Bowman, Christian S; White, Bradley J; Akbari, Omar S

    2017-12-05

    The development of CRISPR/Cas9 technologies has dramatically increased the accessibility and efficiency of genome editing in many organisms. In general, in vivo germline expression of Cas9 results in substantially higher activity than embryonic injection. However, no transgenic lines expressing Cas9 have been developed for the major mosquito disease vector Aedes aegypti Here, we describe the generation of multiple stable, transgenic Ae. aegypti strains expressing Cas9 in the germline, resulting in dramatic improvements in both the consistency and efficiency of genome modifications using CRISPR. Using these strains, we disrupted numerous genes important for normal morphological development, and even generated triple mutants from a single injection. We have also managed to increase the rates of homology-directed repair by more than an order of magnitude. Given the exceptional mutagenic efficiency and specificity of the Cas9 strains we engineered, they can be used for high-throughput reverse genetic screens to help functionally annotate the Ae. aegypti genome. Additionally, these strains represent a step toward the development of novel population control technologies targeting Ae. aegypti that rely on Cas9-based gene drives. Copyright © 2017 the Author(s). Published by PNAS.

  17. Multiplex metabolic pathway engineering using CRISPR/Cas9 in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Jakociunas, Tadas; Bonde, Ida; Herrgard, Markus

    2015-01-01

    CRISPR/Cas9 is a simple and efficient tool for targeted and marker-free genome engineering. Here, we report the development and successful application of a multiplex CRISPR/Cas9 system for genome engineering of up to 5 different genomic loci in one transformation step in baker's yeast Saccharomyces...... cerevisiae. To assess the specificity of the tool we employed genome re-sequencing to screen for off-target sites in all single knock-out strains targeted by different gRNAs. This extensive analysis identified no more genome variants in CRISPR/Cas9 engineered strains compared to wild-type reference strains...

  18. CRISPR/Cas9 advances engineering of microbial cell factories

    DEFF Research Database (Denmark)

    Jakociunas, Tadas; Jensen, Michael Krogh; Keasling, Jay D.

    2016-01-01

    interspaced palindromic repeats (CRISPR) and its associated proteins (Cas) have become the method of choice for precision genome engineering in many organisms due to their orthogonality, versatility and efficacy. Here we review the strategies adopted for implementation of RNA-guided CRISPR/Cas9 genome editing......-RNAs will be highlighted. Finally, this review will provide a perspective on the immediate challenges and opportunities foreseen by the use of CRISPR/Cas9 genome engineering and regulation in the context of metabolic engineering....

  19. Synthetic Control of Exciton Behavior in Colloidal Quantum Dots.

    Science.gov (United States)

    Pu, Chaodan; Qin, Haiyan; Gao, Yuan; Zhou, Jianhai; Wang, Peng; Peng, Xiaogang

    2017-03-08

    Colloidal quantum dots are promising optical and optoelectronic materials for various applications, whose performance is dominated by their excited-state properties. This article illustrates synthetic control of their excited states. Description of the excited states of quantum-dot emitters can be centered around exciton. We shall discuss that, different from conventional molecular emitters, ground-state structures of quantum dots are not necessarily correlated with their excited states. Synthetic control of exciton behavior heavily relies on convenient and affordable monitoring tools. For synthetic development of ideal optical and optoelectronic emitters, the key process is decay of band-edge excitons, which renders transient photoluminescence as important monitoring tool. On the basis of extensive synthetic developments in the past 20-30 years, synthetic control of exciton behavior implies surface engineering of quantum dots, including surface cation/anion stoichiometry, organic ligands, inorganic epitaxial shells, etc. For phosphors based on quantum dots doped with transition metal ions, concentration and location of the dopant ions within a nanocrystal lattice are found to be as important as control of the surface states in order to obtain bright dopant emission with monoexponential yet tunable photoluminescence decay dynamics.

  20. Exploring Synthetic and Systems Biology at the University of Edinburgh.

    Science.gov (United States)

    Fletcher, Liz; Rosser, Susan; Elfick, Alistair

    2016-06-15

    The Centre for Synthetic and Systems Biology ('SynthSys') was originally established in 2007 as the Centre for Integrative Systems Biology, funded by the Biotechnology and Biological Sciences Research Council (BBSRC) and the Engineering and Physical Sciences Research Council (EPSRC). Today, SynthSys embraces an extensive multidisciplinary community of more than 200 researchers from across the University with a common interest in synthetic and systems biology. Our research is broad and deep, addressing a diversity of scientific questions, with wide ranging impact. We bring together the power of synthetic biology and systems approaches to focus on three core thematic areas: industrial biotechnology, agriculture and the environment, and medicine and healthcare. In October 2015, we opened a newly refurbished building as a physical hub for our new U.K. Centre for Mammalian Synthetic Biology funded by the BBSRC/EPSRC/MRC as part of the U.K. Research Councils' Synthetic Biology for Growth programme. © 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.

  1. Complete genome sequence of Nakamurella multipartita type strain (Y-104).

    Science.gov (United States)

    Tice, Hope; Mayilraj, Shanmugam; Sims, David; Lapidus, Alla; Nolan, Matt; Lucas, Susan; Glavina Del Rio, Tijana; Copeland, Alex; Cheng, Jan-Fang; Meincke, Linda; Bruce, David; Goodwin, Lynne; Pitluck, Sam; Ivanova, Natalia; Mavromatis, Konstantinos; Ovchinnikova, Galina; Pati, Amrita; Chen, Amy; Palaniappan, Krishna; Land, Miriam; Hauser, Loren; Chang, Yun-Juan; Jeffries, Cynthia D; Detter, John C; Brettin, Thomas; Rohde, Manfred; Göker, Markus; Bristow, Jim; Eisen, Jonathan A; Markowitz, Victor; Hugenholtz, Philip; Kyrpides, Nikos C; Klenk, Hans-Peter; Chen, Feng

    2010-03-30

    Nakamurella multipartita (Yoshimi et al. 1996) Tao et al. 2004 is the type species of the monospecific genus Nakamurella in the actinobacterial suborder Frankineae. The nonmotile, coccus-shaped strain was isolated from activated sludge acclimated with sugar-containing synthetic wastewater, and is capable of accumulating large amounts of polysaccharides in its cells. Here we describe the features of the organism, together with the complete genome sequence and annotation. This is the first complete genome sequence of a member of the family Nakamurellaceae. The 6,060,298 bp long single replicon genome with its 5415 protein-coding and 56 RNA genes is part of the Genomic Encyclopedia of Bacteria and Archaea project.

  2. Synthetic Plasma Liquid Based Electronic Circuits Realization-A Novel Concept.

    Science.gov (United States)

    Pandya, Killol V; Kosta, ShivPrasad

    2016-09-01

    Biomedical research is contributing significant role in the field of biomedical engineering and applied science. It brings research and innovations to a different level. This study investigated artificial human blood -synthetic plasma liquid as conductive medium. Keeping in mind the conductivity of synthetic plasma, astable multivibrator as well as differential amplifier circuit were demonstrated. The circuits were given normal input voltages at regular temperature and ideal conditions. The result shows desired response which supports the novel concept. For both the circuits, phase shift of 180° achieved by analysing biological electronic circuits.

  3. Experience with synthetic fluorinated fluid lubricants

    Science.gov (United States)

    Conley, Peter L.; Bohner, John J.

    1990-01-01

    Since the late 1970's, the wet lubricant of choice for space mechanisms has been one of the family of synthetic perfluoro polyalkylether (PFPE) compounds, namely Fomblin Z-25 (Bray-815Z) or DuPont's Krytox 143xx series. While offering the advantages of extremely low vapor pressures and wide temperature ranges, these oils and derived greases have a complex chemistry compared to the more familiar natural and synthetic hydrocarbons. Many aerospace companies have conducted test programs to characterize the behavior of these compounds in a space environment, resulting in a large body of hard knowledge as well as considerable space lore concerning the suitability of the lubricants for particular applications and techniques for successful application. The facts are summarized and a few myths about the compounds are dispelled, and some performance guidelines for the mechanism design engineer are provided.

  4. Application of synthetic biology for production of chemicals in yeast Saccharomyces cerevisiae.

    Science.gov (United States)

    Li, Mingji; Borodina, Irina

    2015-02-01

    Synthetic biology and metabolic engineering enable generation of novel cell factories that efficiently convert renewable feedstocks into biofuels, bulk, and fine chemicals, thus creating the basis for biosustainable economy independent on fossil resources. While over a hundred proof-of-concept chemicals have been made in yeast, only a very small fraction of those has reached commercial-scale production so far. The limiting factor is the high research cost associated with the development of a robust cell factory that can produce the desired chemical at high titer, rate, and yield. Synthetic biology has the potential to bring down this cost by improving our ability to predictably engineer biological systems. This review highlights synthetic biology applications for design, assembly, and optimization of non-native biochemical pathways in baker's yeast Saccharomyces cerevisiae We describe computational tools for the prediction of biochemical pathways, molecular biology methods for assembly of DNA parts into pathways, and for introducing the pathways into the host, and finally approaches for optimizing performance of the introduced pathways. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.

  5. Production of Medium Chain Fatty Acids by Yarrowia lipolytica: Combining Molecular Design and TALEN to Engineer the Fatty Acid Synthase.

    Science.gov (United States)

    Rigouin, Coraline; Gueroult, Marc; Croux, Christian; Dubois, Gwendoline; Borsenberger, Vinciane; Barbe, Sophie; Marty, Alain; Daboussi, Fayza; André, Isabelle; Bordes, Florence

    2017-10-20

    Yarrowia lipolytica is a promising organism for the production of lipids of biotechnological interest and particularly for biofuel. In this study, we engineered the key enzyme involved in lipid biosynthesis, the giant multifunctional fatty acid synthase (FAS), to shorten chain length of the synthesized fatty acids. Taking as starting point that the ketoacyl synthase (KS) domain of Yarrowia lipolytica FAS is directly involved in chain length specificity, we used molecular modeling to investigate molecular recognition of palmitic acid (C16 fatty acid) by the KS. This enabled to point out the key role of an isoleucine residue, I1220, from the fatty acid binding site, which could be targeted by mutagenesis. To address this challenge, TALEN (transcription activator-like effector nucleases)-based genome editing technology was applied for the first time to Yarrowia lipolytica and proved to be very efficient for inducing targeted genome modifications. Among the generated FAS mutants, those having a bulky aromatic amino acid residue in place of the native isoleucine at position 1220 led to a significant increase of myristic acid (C14) production compared to parental wild-type KS. Particularly, the best performing mutant, I1220W, accumulates C14 at a level of 11.6% total fatty acids. Overall, this work illustrates how a combination of molecular modeling and genome-editing technology can offer novel opportunities to rationally engineer complex systems for synthetic biology.

  6. CRISPR/Cas9 for Human Genome Engineering and Disease Research.

    Science.gov (United States)

    Xiong, Xin; Chen, Meng; Lim, Wendell A; Zhao, Dehua; Qi, Lei S

    2016-08-31

    The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) system, a versatile RNA-guided DNA targeting platform, has been revolutionizing our ability to modify, manipulate, and visualize the human genome, which greatly advances both biological research and therapeutics development. Here, we review the current development of CRISPR/Cas9 technologies for gene editing, transcription regulation, genome imaging, and epigenetic modification. We discuss the broad application of this system to the study of functional genomics, especially genome-wide genetic screening, and to therapeutics development, including establishing disease models, correcting defective genetic mutations, and treating diseases.

  7. Rewiring protein synthesis: From natural to synthetic amino acids.

    Science.gov (United States)

    Fan, Yongqiang; Evans, Christopher R; Ling, Jiqiang

    2017-11-01

    The protein synthesis machinery uses 22 natural amino acids as building blocks that faithfully decode the genetic information. Such fidelity is controlled at multiple steps and can be compromised in nature and in the laboratory to rewire protein synthesis with natural and synthetic amino acids. This review summarizes the major quality control mechanisms during protein synthesis, including aminoacyl-tRNA synthetases, elongation factors, and the ribosome. We will discuss evolution and engineering of such components that allow incorporation of natural and synthetic amino acids at positions that deviate from the standard genetic code. The protein synthesis machinery is highly selective, yet not fixed, for the correct amino acids that match the mRNA codons. Ambiguous translation of a codon with multiple amino acids or complete reassignment of a codon with a synthetic amino acid diversifies the proteome. Expanding the genetic code with synthetic amino acids through rewiring protein synthesis has broad applications in synthetic biology and chemical biology. Biochemical, structural, and genetic studies of the translational quality control mechanisms are not only crucial to understand the physiological role of translational fidelity and evolution of the genetic code, but also enable us to better design biological parts to expand the proteomes of synthetic organisms. This article is part of a Special Issue entitled "Biochemistry of Synthetic Biology - Recent Developments" Guest Editor: Dr. Ilka Heinemann and Dr. Patrick O'Donoghue. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. Computational and Genomic Analysis of Mycobacteriophage: A Longitudinal Study of Technology Engineered Biology Courses That Implemented an Inquiry Based Laboratory Practice Designed to Enhance, Encourage, and Empower Student Learning

    Science.gov (United States)

    Hollowell, Gail P.; Osler, James E.; Hester, April L.

    2015-01-01

    This paper provides an applied research rational for a longitudinal investigation that involved teaching a "Technology Engineered Science Education Course" via an Interactive Laboratory Based Genomics Curriculum. The Technology st Engineering [TE] methodology was first introduced at the SAPES: South Atlantic Philosophy of Education…

  9. Synthetic Biology with Cytochromes P450 Using Photosynthetic Chassis

    DEFF Research Database (Denmark)

    Gnanasekaran, Thiyagarajan

    , this modern field of synthetic biology is completely dependent on the nature of the chassis - the host organisms - for its endeavor. Of all the chassis, photosynthetic organisms such as cyanobacteria and plants gains special attention due to the remarkable amount of sunlight that is striking the Earth...... in cyanobacteria and plant chloroplasts for the purpose of light driven synthesis of bioactive compounds by using synthetic biology approaches. As model pathways, in this thesis, the pathway involved in the synthesis of the cyanogenic glucoside dhurrin from Sorghum bicolor, and the pathway involved......Synthetic biology is a rapidly growing engineering discipline in biology. It aims at building novel biological systems that do not exist in nature by selecting the interchangeable standardized biological parts that are already available in the nature, and assembling them in a specific order. Today...

  10. Synthetic Biology: Putting Synthesis into Biology

    Science.gov (United States)

    Liang, Jing; Luo, Yunzi; Zhao, Huimin

    2010-01-01

    The ability to manipulate living organisms is at the heart of a range of emerging technologies that serve to address important and current problems in environment, energy, and health. However, with all its complexity and interconnectivity, biology has for many years been recalcitrant to engineering manipulations. The recent advances in synthesis, analysis, and modeling methods have finally provided the tools necessary to manipulate living systems in meaningful ways, and have led to the coining of a field named synthetic biology. The scope of synthetic biology is as complicated as life itself – encompassing many branches of science, and across many scales of application. New DNA synthesis and assembly techniques have made routine the customization of very large DNA molecules. This in turn has allowed the incorporation of multiple genes and pathways. By coupling these with techniques that allow for the modeling and design of protein functions, scientists have now gained the tools to create completely novel biological machineries. Even the ultimate biological machinery – a self-replicating organism – is being pursued at this moment. It is the purpose of this review to dissect and organize these various components of synthetic biology into a coherent picture. PMID:21064036

  11. Study of mechanical properties and fracture mechanisms of synthetic fibers nylon-and-polyester type, used in engineering products

    International Nuclear Information System (INIS)

    Cardoso, Sergio Gomes

    2009-01-01

    Fibers are groups formed by molecular-chain-oriented filaments. Fibers play a fundamental role in human being's daily life and they can be found in several forms and geometries, such as filaments, yarns, beams, rope, fabric, composite, coatings, others. They are used in various segments such as civil, mechanical, electrical, electronics, military, naval, nautical, aviation, health, medicine, environment, communications, safety, space, others. Fibers are divided into two distinct classes: natural and chemical ones, which cover synthetic and man-made sub-classes. They can be produced from several materials, such as wool, cotton, rayon, flax, silk, rock, nylon, polyester, polyethylene, poly-propylene, aramid, glass, carbon, steel, ceramic, others. Globally, the participation of chemical fibers corresponds to approximately 59,9%, and the synthetic fiber polyester, the most used one, represents approximately 63% of the world market. Vital needs have led to the development of multi-function fibers and the focus has changed in the last 10 years with the use of nano technology for environmental responsibility and smart fibers. The study of mechanical properties and fracture mechanisms of fibers is of great relevance for characterization and understanding of causes as consequence of failures. For such reason, it was selected technical fabrics made of high performance synthetic fiber nylon-and-polyester type, used in engineered products such as tires, belts, hoses and pneumatic springs, which have been analyzed in each processing phase. Fiber samples were extracted after each processing phase to be analyzed, by traction destructive tests and scanning electron microscopy. The results of analysis of mechanical properties showed loss of resistance to temperature and multi axial stress during fiber processing phase. Through microscopy tests, it was possible to find contamination, surface stains, plastic deformations, scaling, variations in the fracture faces of the filaments and

  12. Multiplex Genome Editing in Escherichia coli

    DEFF Research Database (Denmark)

    Ingemann Jensen, Sheila; Nielsen, Alex Toftgaard

    2018-01-01

    Lambda Red recombineering is an easy and efficient method for generating genetic modifications in Escherichia coli. For gene deletions, lambda Red recombineering is combined with the use of selectable markers, which are removed through the action of, e.g., flippase (Flp) recombinase. This PCR......-based engineering method has also been applied to a number of other bacteria. In this chapter, we describe a recently developed one plasmid-based method as well as the use of a strain with genomically integrated recombineering genes, which significantly speeds up the engineering of strains with multiple genomic...

  13. How to turn a genetic circuit into a synthetic tunable oscillator, or a bistable switch.

    Directory of Open Access Journals (Sweden)

    Lucia Marucci

    2009-12-01

    Full Text Available Systems and Synthetic Biology use computational models of biological pathways in order to study in silico the behaviour of biological pathways. Mathematical models allow to verify biological hypotheses and to predict new possible dynamical behaviours. Here we use the tools of non-linear analysis to understand how to change the dynamics of the genes composing a novel synthetic network recently constructed in the yeast Saccharomyces cerevisiae for In-vivo Reverse-engineering and Modelling Assessment (IRMA. Guided by previous theoretical results that make the dynamics of a biological network depend on its topological properties, through the use of simulation and continuation techniques, we found that the network can be easily turned into a robust and tunable synthetic oscillator or a bistable switch. Our results provide guidelines to properly re-engineering in vivo the network in order to tune its dynamics.

  14. Integrated In Silico Analysis of Pathway Designs for Synthetic Photo-Electro-Autotrophy.

    Directory of Open Access Journals (Sweden)

    Michael Volpers

    Full Text Available The strong advances in synthetic biology enable the engineering of novel functions and complex biological features in unprecedented ways, such as implementing synthetic autotrophic metabolism into heterotrophic hosts. A key challenge for the sustainable production of fuels and chemicals entails the engineering of synthetic autotrophic organisms that can effectively and efficiently fix carbon dioxide by using sustainable energy sources. This challenge involves the integration of carbon fixation and energy uptake systems. A variety of carbon fixation pathways and several types of photosystems and other energy uptake systems can be chosen and, potentially, modularly combined to design synthetic autotrophic metabolism. Prior to implementation, these designs can be evaluated by the combination of several computational pathway analysis techniques. Here we present a systematic, integrated in silico analysis of photo-electro-autotrophic pathway designs, consisting of natural and synthetic carbon fixation pathways, a proton-pumping rhodopsin photosystem for ATP regeneration and an electron uptake pathway. We integrated Flux Balance Analysis of the heterotrophic chassis Escherichia coli with kinetic pathway analysis and thermodynamic pathway analysis (Max-min Driving Force. The photo-electro-autotrophic designs are predicted to have a limited potential for anaerobic, autotrophic growth of E. coli, given the relatively low ATP regenerating capacity of the proton pumping rhodopsin photosystems and the high ATP maintenance of E. coli. If these factors can be tackled, our analysis indicates the highest growth potential for the natural reductive tricarboxylic acid cycle and the synthetic pyruvate synthase-pyruvate carboxylate -glyoxylate bicycle. Both carbon fixation cycles are very ATP efficient, while maintaining fast kinetics, which also results in relatively low estimated protein costs for these pathways. Furthermore, the synthetic bicycles are highly

  15. Targeted viral-mediated plant genome editing using crispr/cas9

    KAUST Repository

    Mahfouz, Magdy M.; Ali, Zahir

    2015-01-01

    The present disclosure provides a viral-mediated genome-editing platform that facilitates multiplexing, obviates stable transformation, and is applicable across plant species. The RNA2 genome of the tobacco rattle virus (TRV) was engineered to carry and systemically deliver a guide RNA molecules into plants overexpressing Cas9 endonuclease. High genomic modification frequencies were observed in inoculated as well as systemic leaves including the plant growing points. This system facilitates multiplexing and can lead to germinal transmission of the genomic modifications in the progeny, thereby obviating the requirements of repeated transformations and tissue culture. The editing platform of the disclosure is useful in plant genome engineering and applicable across plant species amenable to viral infections for agricultural biotechnology applications.

  16. Targeted viral-mediated plant genome editing using crispr/cas9

    KAUST Repository

    Mahfouz, Magdy M.

    2015-12-17

    The present disclosure provides a viral-mediated genome-editing platform that facilitates multiplexing, obviates stable transformation, and is applicable across plant species. The RNA2 genome of the tobacco rattle virus (TRV) was engineered to carry and systemically deliver a guide RNA molecules into plants overexpressing Cas9 endonuclease. High genomic modification frequencies were observed in inoculated as well as systemic leaves including the plant growing points. This system facilitates multiplexing and can lead to germinal transmission of the genomic modifications in the progeny, thereby obviating the requirements of repeated transformations and tissue culture. The editing platform of the disclosure is useful in plant genome engineering and applicable across plant species amenable to viral infections for agricultural biotechnology applications.

  17. Targeted Genome Regulation and Editing in Plants

    KAUST Repository

    Piatek, Agnieszka

    2016-03-01

    The ability to precisely regulate gene expression patterns and to modify genome sequence in a site-specific manner holds much promise in determining gene function and linking genotype to phenotype. DNA-binding modules have been harnessed to generate customizable and programmable chimeric proteins capable of binding to site-specific DNA sequences and regulating the genome and epigenome. Modular DNA-binding domains from zinc fingers (ZFs) and transcriptional activator-like effectors (TALEs) are amenable to engineering to bind any DNA target sequence of interest. Deciphering the code of TALE repeat binding to DNA has helped to engineer customizable TALE proteins capable of binding to any sequence of interest. Therefore TALE repeats provide a rich resource for bioengineering applications. However, the TALE system is limited by the requirement to re-engineer one or two proteins for each new target sequence. Recently, the clustered regularly interspaced palindromic repeats (CRISPR)/ CRISPR associated 9 (Cas9) has been used as a versatile genome editing tool. This machinery has been also repurposed for targeted transcriptional regulation. Due to the facile engineering, simplicity and precision, the CRISPR/Cas9 system is poised to revolutionize the functional genomics studies across diverse eukaryotic species. In this dissertation I employed transcription activator-like effectors and CRISPR/Cas9 systems for targeted genome regulation and editing and my achievements include: 1) I deciphered and extended the DNA-binding code of Ralstonia TAL effectors providing new opportunities for bioengineering of customizable proteins; 2) I repurposed the CRISPR/Cas9 system for site-specific regulation of genes in plant genome; 3) I harnessed the power of CRISPR/Cas9 gene editing tool to study the function of the serine/arginine-rich (SR) proteins.

  18. Genome-scale modeling for metabolic engineering.

    Science.gov (United States)

    Simeonidis, Evangelos; Price, Nathan D

    2015-03-01

    We focus on the application of constraint-based methodologies and, more specifically, flux balance analysis in the field of metabolic engineering, and enumerate recent developments and successes of the field. We also review computational frameworks that have been developed with the express purpose of automatically selecting optimal gene deletions for achieving improved production of a chemical of interest. The application of flux balance analysis methods in rational metabolic engineering requires a metabolic network reconstruction and a corresponding in silico metabolic model for the microorganism in question. For this reason, we additionally present a brief overview of automated reconstruction techniques. Finally, we emphasize the importance of integrating metabolic networks with regulatory information-an area which we expect will become increasingly important for metabolic engineering-and present recent developments in the field of metabolic and regulatory integration.

  19. Engineering Robustness of Microbial Cell Factories.

    Science.gov (United States)

    Gong, Zhiwei; Nielsen, Jens; Zhou, Yongjin J

    2017-10-01

    Metabolic engineering and synthetic biology offer great prospects in developing microbial cell factories capable of converting renewable feedstocks into fuels, chemicals, food ingredients, and pharmaceuticals. However, prohibitively low production rate and mass concentration remain the major hurdles in industrial processes even though the biosynthetic pathways are comprehensively optimized. These limitations are caused by a variety of factors unamenable for host cell survival, such as harsh industrial conditions, fermentation inhibitors from biomass hydrolysates, and toxic compounds including metabolic intermediates and valuable target products. Therefore, engineered microbes with robust phenotypes is essential for achieving higher yield and productivity. In this review, the recent advances in engineering robustness and tolerance of cell factories is described to cope with these issues and briefly introduce novel strategies with great potential to enhance the robustness of cell factories, including metabolic pathway balancing, transporter engineering, and adaptive laboratory evolution. This review also highlights the integration of advanced systems and synthetic biology principles toward engineering the harmony of overall cell function, more than the specific pathways or enzymes. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. Rational Diversification of a Promoter Providing Fine-Tuned Expression and Orthogonal Regulation for Synthetic Biology

    Science.gov (United States)

    Blount, Benjamin A.; Weenink, Tim; Vasylechko, Serge; Ellis, Tom

    2012-01-01

    Yeast is an ideal organism for the development and application of synthetic biology, yet there remain relatively few well-characterised biological parts suitable for precise engineering of this chassis. In order to address this current need, we present here a strategy that takes a single biological part, a promoter, and re-engineers it to produce a fine-graded output range promoter library and new regulated promoters desirable for orthogonal synthetic biology applications. A highly constitutive Saccharomyces cerevisiae promoter, PFY1p, was identified by bioinformatic approaches, characterised in vivo and diversified at its core sequence to create a 36-member promoter library. TetR regulation was introduced into PFY1p to create a synthetic inducible promoter (iPFY1p) that functions in an inverter device. Orthogonal and scalable regulation of synthetic promoters was then demonstrated for the first time using customisable Transcription Activator-Like Effectors (TALEs) modified and designed to act as orthogonal repressors for specific PFY1-based promoters. The ability to diversify a promoter at its core sequences and then independently target Transcription Activator-Like Orthogonal Repressors (TALORs) to virtually any of these sequences shows great promise toward the design and construction of future synthetic gene networks that encode complex “multi-wire” logic functions. PMID:22442681