Development of Artificial Gene Regulatory Networks in Saccharomyces Cerevisiae PDF Download
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Author: William Jeremy Blake
Publisher:
ISBN:
Category :
Languages : en
Pages : 244
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Book Description
Abstract: Dissecting the complex circuitry of the cell is a principal challenge in the post-genomic era. Advances in the fields of molecular biology and bioinformatics have made this problem more amenable to an engineering approach, where components are characterized and assembled to elicit predefined behaviors. In this work we implement an approach which combines computational and experimental techniques to develop artificial gene networks that are used to explore the molecular and biochemical interactions which coordinate cell function. We present the design, construction, and analysis of robust genetic switches in the single-celled eukaryote Saccharomyces cerevisiae , including a bistable switch analogous to an electrical toggle switch. The S. cerevisiae genetic toggle switch allows reversible control of target gene expression in response to an externally applied chemical signal, with up to a 100-fold difference between stable ON and OFF target gene expression states. This functional bistable switch has potential biotechnological and biomedical applications. In addition, we present an experimental and theoretical investigation of the origins and consequences of stochasticity in gene expression in eukaryootc cells. By utilizing well-characterized artificial gene networks, we modulate the expression of a single gene in S. cerevisiae and combine our results with in silico stochastic models of specific eukaryotic transcription mechanisms. A key result of our combined approach addresses the sensitivity of noise in eukaryotic gene expression to the level of transcription and proposes a possible role for the transcription apparatus assembly in modulating such noise. Further, we demonstrate a plausible mechanism for heritable changes in target gene expression whereby increased levels of noise in the expression of a regulatory protein causes a population of isogenic cells to exhibit bistable expression states. Together, our findings address the origins, propagation, and consequences of noise in the expression of eukaryotic genes, and provide insight into the biological design principles involved in noise modulation.
Author: William Jeremy Blake
Publisher:
ISBN:
Category :
Languages : en
Pages : 244
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Book Description
Abstract: Dissecting the complex circuitry of the cell is a principal challenge in the post-genomic era. Advances in the fields of molecular biology and bioinformatics have made this problem more amenable to an engineering approach, where components are characterized and assembled to elicit predefined behaviors. In this work we implement an approach which combines computational and experimental techniques to develop artificial gene networks that are used to explore the molecular and biochemical interactions which coordinate cell function. We present the design, construction, and analysis of robust genetic switches in the single-celled eukaryote Saccharomyces cerevisiae , including a bistable switch analogous to an electrical toggle switch. The S. cerevisiae genetic toggle switch allows reversible control of target gene expression in response to an externally applied chemical signal, with up to a 100-fold difference between stable ON and OFF target gene expression states. This functional bistable switch has potential biotechnological and biomedical applications. In addition, we present an experimental and theoretical investigation of the origins and consequences of stochasticity in gene expression in eukaryootc cells. By utilizing well-characterized artificial gene networks, we modulate the expression of a single gene in S. cerevisiae and combine our results with in silico stochastic models of specific eukaryotic transcription mechanisms. A key result of our combined approach addresses the sensitivity of noise in eukaryotic gene expression to the level of transcription and proposes a possible role for the transcription apparatus assembly in modulating such noise. Further, we demonstrate a plausible mechanism for heritable changes in target gene expression whereby increased levels of noise in the expression of a regulatory protein causes a population of isogenic cells to exhibit bistable expression states. Together, our findings address the origins, propagation, and consequences of noise in the expression of eukaryotic genes, and provide insight into the biological design principles involved in noise modulation.
Author: Robert Julius Gnügge
Publisher:
ISBN:
Category :
Languages : en
Pages :
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Book Description
Author: Institute of Medicine
Publisher: National Academies Press
ISBN: 0309219396
Category : Science
Languages : en
Pages : 570
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Book Description
Many potential applications of synthetic and systems biology are relevant to the challenges associated with the detection, surveillance, and responses to emerging and re-emerging infectious diseases. On March 14 and 15, 2011, the Institute of Medicine's (IOM's) Forum on Microbial Threats convened a public workshop in Washington, DC, to explore the current state of the science of synthetic biology, including its dependency on systems biology; discussed the different approaches that scientists are taking to engineer, or reengineer, biological systems; and discussed how the tools and approaches of synthetic and systems biology were being applied to mitigate the risks associated with emerging infectious diseases. The Science and Applications of Synthetic and Systems Biology is organized into sections as a topic-by-topic distillation of the presentations and discussions that took place at the workshop. Its purpose is to present information from relevant experience, to delineate a range of pivotal issues and their respective challenges, and to offer differing perspectives on the topic as discussed and described by the workshop participants. This report also includes a collection of individually authored papers and commentary.
Author: Nicholas J. Morse
Publisher:
ISBN:
Category :
Languages : en
Pages : 0
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Book Description
There is a current surge of interest in using synthetic biology for biotechnology applications. Metabolic engineers, for example, are interested in synthetic biology for its modular and well characterized transcriptional “parts”, such as synthetic gene promoters and terminators, which enable fine tuning in metabolic pathway optimization. Likewise, emerging gene editing methods, such as CRISPR-Cas9, are enabling quicker and more precise genomic integrations. Using both of these advances, there is an increase in the throughput for which altered pathway conditions can be screened. While some advances are being made, there are still several technological gaps, especially for eukaryotic yeast hosts. Therefore, this dissertation work focused on developing engineering methodologies for the yeast Saccharomyces cerevisiae to expand capacity in each of these areas. There were three main areas explored in this work. First, we developed a method for synthetic promoter design which establishes de novo upstream activating sequences (UAS) capable of regulating gene expression by growth phase. These UAS elements, discovered through a transcriptome mining approach, show an over 30-fold activation of a core promoter with completely synthetic designs. Secondly, we improved synthetic terminator design, whereby both minimal synthetic terminators and larger native terminators were improved by adjusting nucleosome occupancy in adjacent sequence space. Using this methodology, de novo synthetic terminators were designed for increased termination efficiency. Lastly, we developed a method for guide RNA expression in yeast organisms using T7 RNA polymerase in vivo. This method allowed guide RNA expression to be exportable across yeast hosts and enabled more complex regulation designs, such as dCas9 logic gates. Together, these approaches improved synthetic promoter design, synthetic terminator design, and guide RNA expression regulation in ways that both complement current ongoing research in S. cerevisiae and enable a generalized approach to be established for other yeast organisms
Author: Alberto Fuente
Publisher: Springer Science & Business Media
ISBN: 3642451616
Category : Science
Languages : en
Pages : 135
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Book Description
This book presents recent methods for Systems Genetics (SG) data analysis, applying them to a suite of simulated SG benchmark datasets. Each of the chapter authors received the same datasets to evaluate the performance of their method to better understand which algorithms are most useful for obtaining reliable models from SG datasets. The knowledge gained from this benchmarking study will ultimately allow these algorithms to be used with confidence for SG studies e.g. of complex human diseases or food crop improvement. The book is primarily intended for researchers with a background in the life sciences, not for computer scientists or statisticians.
Author: Yao Yan
Publisher:
ISBN:
Category :
Languages : en
Pages : 0
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Book Description
The budding yeast Saccharomyces cerevisiae is a well-studied eukaryotic model organism for investigating the fundamental aspects of molecular and cellular biology. It also can be used to evaluate the technical feasibility of new technologies in eukaryotic cells. In our lab, we aimed to use Saccharomyces cerevisiae as a model to develop CRISPR/Cas9-based gene drive biotechnology. The CRISPR/Cas9 biotechnology is a powerful gene editing tool used to modify target genomic sequences in all forms of living creatures including animals, plants, fungi, and bacteria. One potential application of this molecular method is within a "gene drive" system. This unique arrangement of CRISPR within a genome may one day allow for global control of biological populations and be used to eliminate pests, parasites, and invasive species. However, there are many concerns regarding the utilization of this technology, including gene drive design, control, and development of resistance, etc. In our study, we developed an artificial gene drive system in budding yeast, which could edit multiple loci at the same time. We demonstrated that this triple gene drive system could successfully edit three DNA targets independently with only a single copy of S. pyogenes Cas9. We also found that the occurrence of NHEJ could be repressed by modifying DNA Ligase IV. However, successful gene drives still allowed for the occurrence of a small number of "resistant" clones. We investigated potential causes of this imperfect drive activity. Our work illustrated that imperfect activation of the inducible promoter driving expression of the Cas9 nuclease or issues with multiplexing to artificial sequences may have resulted in a small percentage of resistant/inactive clones. The CRISPR/Cas9 system can also be used to regulate gene transcription. This involves a mutated Cas9 variant that has lost its nuclease activity (dCas9). We developed a CRISPR/dCas9 system by tagging dCas9 with transcriptional regulators. Our experiments demonstrated that CRISPR/dCas9 could activate target gene transcription when tagged with the transcriptional activator VPR and repress gene transcription when tagged with the transcriptional repressor Mxi1.
Author: Miles W. Gander
Publisher:
ISBN:
Category :
Languages : en
Pages : 88
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Book Description
Biology is capable of a wide range of amazing functions, from complex pathway synthesis of high value chemicals to embryonic development. Synthetic biology seeks to harness and control the incredible potential of living systems for the betterment of society. These incredible biological functions are governed by the interaction of complex networks of interacting genes. In principle, the underlying circuitry that control biology function can be recapitulated synthetically, allowing for precise control of biology. In this dissertation, we describe our efforts to rationally design synthetic gene circuits in Saccharomyces cerevisiae. The main thrust of this work is the development, analysis and demonstration of a set of single gene NOR gates based on the CRISPR/Cas9 system. Using these NOR gates we constructed logic circuits with up to seven gRNAs, including repression cascades with up to seven layers. Our NOR gates allowed for the construction of the largest eukaryotic gene circuits to date. We detail the process of development of the NOR gates, their limitations and considerations for future use and improvement of the single gene circuit components. The NOR gate technology represents an advancement of the state of the art in synthetic gene circuit design can in principle be used to implement arbitrary internal logic for a variety of synthetic cellular decision making systems, such as those being explored for diagnostics, therapeutics, and development.
Author: Wilfried Weber
Publisher: Humana Press
ISBN: 9781617794117
Category : Science
Languages : en
Pages : 393
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Book Description
The rapid expansion of synthetic biology is due to the design and construction of synthetic gene networks that have opened many new avenues in fundamental and applied research. Synthetic Gene Networks: Methods and Protocols provides the necessary information to design and construct synthetic gene networks in different host backgrounds. Divided into four convenient sections, this volume focuses on design concepts to devise synthetic gene networks and how mathematical models can be applied to the predictable engineering of desired network features. The volume continues by highlighting the construction and validation of biologic tools, describing strategies to optimize and streamline the host cell for optimized network performance, and covering how optimally designed gene networks can be implemented in a large variety of host cells ranging from bacteria over yeast and insect cells to plant and mammalian cell culture. Written in the successful Methods in Molecular BiologyTM series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible protocols, and notes on troubleshooting and avoiding known pitfalls. Authoritative and easily accessible, Synthetic Gene Networks: Methods and Protocols serves as an invaluable resource for established biologists, engineers, and computer scientists or novices just entering into the rapidly growing field of synthetic biology
Author: Ilya Shmulevich
Publisher: SIAM
ISBN: 0898716926
Category : Mathematics
Languages : en
Pages : 276
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Book Description
The first comprehensive treatment of probabilistic Boolean networks, unifying different strands of current research and addressing emerging issues.
Author: Edgar Wingender
Publisher: Wiley-Blackwell
ISBN:
Category : Science
Languages : en
Pages : 452
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Book Description
A much-needed guide through the overwhelming amount of literature in the field. Comprehensive and detailed, this book combines background information with the most recentinsights. It introduces current concepts, emphasizing the transcriptional control of genetic information. Moreover, it links data on the structure of regulatory proteins with basic cellular processes. Both advanced students and experts will find answers to such intriguing questions as: - How are programs of specific gene repertoires activated and controlled? - Which genes drive and control morphogenesis? - Which genes govern tissue-specific tasks? - How do hormones control gene expression in coordinating the activities of different tissues? An abundant number of clearly presented glossary terms facilitates understanding of the biological background. Speacial feature: over 2200 (!) literature references.
