1,381 research outputs found

    Designer Gene Networks: Towards Fundamental Cellular Control

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    The engineered control of cellular function through the design of synthetic genetic networks is becoming plausible. Here we show how a naturally occurring network can be used as a parts list for artificial network design, and how model formulation leads to computational and analytical approaches relevant to nonlinear dynamics and statistical physics.Comment: 35 pages, 8 figure

    A Forward-Genetic Screen and Dynamic Analysis of Lambda Phage Host-Dependencies Reveals an Extensive Interaction Network and a New Anti-Viral Strategy

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    Latently infecting viruses are an important class of virus that plays a key role in viral evolution and human health. Here we report a genome-scale forward-genetics screen for host-dependencies of the latently-infecting bacteriophage lambda. This screen identified 57 Escherichia coli (E. coli) genes—over half of which have not been previously associated with infection—that when knocked out inhibited lambda phage's ability to replicate. Our results demonstrate a highly integrated network between lambda and its host, in striking contrast to the results from a similar screen using the lytic-only infecting T7 virus. We then measured the growth of E. coli under normal and infected conditions, using wild-type and knockout strains deficient in one of the identified host genes, and found that genes from the same pathway often exhibited similar growth dynamics. This observation, combined with further computational and experimental analysis, led us to identify a previously unannotated gene, yneJ, as a novel regulator of lamB gene expression. A surprising result of this work was the identification of two highly conserved pathways involved in tRNA thiolation—one pathway is required for efficient lambda replication, while the other has anti-viral properties inhibiting lambda replication. Based on our data, it appears that 2-thiouridine modification of tRNAGlu, tRNAGln, and tRNALys is particularly important for the efficient production of infectious lambda phage particles

    Gene Regulation in the Pi Calculus: Simulating Cooperativity at the Lambda Switch

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    Part of the Lecture Notes in Computer Science book series (LNCS, volume 4230).Also part of the Lecture Notes in Bioinformatics book sub series (volume 4230).International audienceWe propose to model the dynamics of gene regulatory networks as concurrent processes in the stochastic pi calculus. As a first case study, we show how to express the control of transcription initiation at the lambda switch, a prototypical example where cooperative enhancement is crucial. This requires concurrent programming techniques that are new to systems biology, and necessitates stochastic parameters that we derive from the literature. We test all components of our model by exhaustive stochastic simulations. A comparison with previous results reported in the literature, experimental and simulation based, confirms the appropriateness of our modeling approach

    Programming Synthetic Microbial Communities for Coexistence, Coordination, and Information Processing

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    Synthetic microbial communities offer a variety of potential advantages over single species approaches for many medical, industrial, and environmental applications. At the cellular level, metabolic pathways can be distributed amongst several community residents to lower the metabolic burden on individual cells and to enable optimization of reaction conditions for different parts of metabolic pathways. At the population level, diverse microbial communities in different natural contexts have been shown to be more productive, efficient, stable, and resistant to invasion by foreign agents. Along with these potential advantages, however, come a variety of new challenges as well. First, different species or cell types of interest must be able to coexist. Additionally, in many scenarios the relative abundance of each resident can impact the overall property of the community. Beyond coexistence and community composition, information processing and sharing is often essential to the types of complex, coordinated behavior that is required for many desired medical, industrial, and environmental applications. My dissertation has centered around the design and implementation of two novel systems which address some of the challenges discussed above that must be overcome to realize the potential of synthetic microbial communities for use in technological applications. In the first system our goal was to develop a tool that can be used to enable coexistence and program community composition within a synthetic microbial community. We use xvi temperature as a modality to enable coexistence of two microorganisms, Escherichia coli and Pseudomonas putida, with different thermal niches and to further program the composition of this model synthetic bi-culture. Specifically, I developed two different approaches, referred to as a constant temperature regime and a cycling temperature regime. Employing a combination of wet-lab experiments and mathematical modeling, I showed that a variety of parameters such as temperature, cycle duration, etc. can be manipulated to achieve desired community compositions. Building on this work, I then used a mathematical framework developed by ecologists to explore design principles and specific mechanisms underlying the observed relationship between culture temperature and coexistence. In the second system, I designed a novel synthetic microbial community with a distributed sensing and centralized reporting architecture that is enabled by what we have termed bacteriophage-mediated information transfer. Our goal is to explore a novel distributed sensing with centralized memory system architecture that is capable of addressing limitations of previously developed systems. A modular genetic circuit was developed that connects the input of an environmental signal of interest to activation of a lysogenic lambda bacteriophage which is used to transfer information about the sensing event from the sensor cell population to a reporter cell population. A variety of different ways to encode and store information were explored. While seemingly different, the lines of work described above are connected by a common thread of developing generalizable and modular approaches for engineering synthetic microbial communities to deliver the potential advantages they offer in a variety of medical, industrial, and environmental applications. Synthetic microbial communities are capable of xvii performing complex and varied functions within these contexts and this dissertation is contributing to the rapidly growing body of research work for addressing the challenges that must be overcome to realize that potential.PHDCellular & Molecular BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/163080/1/agkrieg_1.pd

    Engineering Genetic Networks to Measure Real Time Repression by λ Cro in vivo

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    The field of systems biology seeks to engineer systems in order to explore specific dynamic properties as they act in vivo. Cellular activities have been proven to be regulated by a range of DNA binding proteins that bind to specific sequences that activate or repress transcription. Certain phage and bacterial repressor proteins were among the first of these DNA binding proteins to be characterized structurally and functionally. The demand for study of key steps in regulation of protein expression, folding, and repression has created multiple studies into systems of varying complexity. The data in this report explores the lambda system, and more specifically the kinetics of the repressor protein, Cro. The network of regulatory proteins and DNA sites that control a simple developmental switch in bacteriophage lambda have been extensively studied and modeled mathematically.Previous work in our laboratory has shown folding of Cro is very slow in vitro, and this phenomenon has not been included in models to date. The goal is to measure the time of Cro folding and repression in vivo. In order to examine the kinetic properties of the Cro protein, a reporter plasmid containing the gene for the green fluorescent protein is fused to a promoter that is repressible by both Cro and lacR. Several strains of bacteria have been engineered containing lacR, the reporter plasmid, and optionally variants of Cro. Initially GFP and Cro are expressed at a low level. After addition of IPTG, a compound that inactivates lacR, GFP accumulates and the fluorescence of the cell increases. In strains that contain the Cro gene, GFP accumulation is halted after enough active Cro has accumulated to repress transcription of GFP. Several Cro variants have been investigated. Initial observations of fluorescence spectra and induction kinetics of living bacterial cultures are reported

    Forces During Bacteriophage DNA Packaging and Ejection

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    The conjunction of insights from structural biology, solution biochemistry, genetics and single molecule biophysics has provided a renewed impetus for the construction of quantitative models of biological processes. One area that has been a beneficiary of these experimental techniques is the study of viruses. In this paper we describe how the insights obtained from such experiments can be utilized to construct physical models of processes in the viral life cycle. We focus on dsDNA bacteriophages and show that the bending elasticity of DNA and its electrostatics in solution can be combined to determine the forces experienced during packaging and ejection of the viral genome. Furthermore, we quantitatively analyze the effect of fluid viscosity and capsid expansion on the forces experienced during packaging. Finally, we present a model for DNA ejection from bacteriophages based on the hypothesis that the energy stored in the tightly packed genome within the capsid leads to its forceful ejection. The predictions of our model can be tested through experiments in vitro where DNA ejection is inhibited by the application of external osmotic pressure
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