Genetic Systems Bioengineering for Escherichia coli

描述（由申请人提供）：将设计、构建和表征显示有趣行为的合成遗传系统，并将其用作模型系统，用于阐明负责有趣行为的系统设计原理。这项工作将促进我们对基因控制机制和系统设计原则的理解，也可能促进我们对几个自然系统的调节因子的理解。此外，我们的工作将提供高度工程化和良好表征的模块，这些模块将适用于构建复杂的合成遗传网络。在第一个具体的目标，我们提出了我们的合成遗传时钟的特性为E。杆菌该时钟提供了大细胞群体中激活子和阻遏子表达的同步但阻尼的振荡，其可用于引起报告基因的周期性表达。所提出的方法直接测试系统设计的各个方面，并且应该揭示负责振荡行为以及控制输出周期和幅度的设计特征，并导致更好时钟的开发。这些研究将推进我们对细菌基因调控基本方面的理解，并将揭示生物系统中基因表达振荡产生的设计原理。其他拟议的实验将探索遗传时钟与包含它的宿主细胞的关系，着眼于将时钟与正常的细胞生理过程隔离开来，并提高系统性能。在第二个具体的目的改进方法研究振荡系统提出。其中包括开发和分析改进的时钟功能模型的战略，以及开发进行合成遗传时钟实验的仪器和方法。在第三个具体目标中，我们建议开发更多复杂的遗传时钟。其中一些遗传时钟是基于其他实验室广泛建模的设计，但尚未构建和测试。此外，我们提出了新的遗传时钟的设计，测试的作用，各种系统的设计原则，在生产的生物振荡。

DESCRIPTION (provided by applicant): Synthetic genetic systems displaying interesting behaviors will be designed, constructed, and characterized, and used as model systems for the elucidation of the system design principles responsible for the interesting behaviors. This work will advance our understanding of gene control mechanisms and system design principles, and is also likely to advance our understanding of regulatory factors from several natural systems. In addition, our work will provide highly-engineered and well characterized modules that will be suitable for the construction of sophisticated synthetic genetic networks. In the first Specific Aim, we propose the characterization of our synthetic genetic clock for E. coli. This clock provides for synchronous but damped oscillations of activator and represser expression in large cell populations, which can be used to bring about the periodic expression of reporter genes. The proposed approaches directly test various aspects of the system design and should reveal the design features responsible for oscillatory behavior and controlling period and amplitude of the output, as well as lead to the development of a better clock. These studies will advance our understanding of fundamental aspects of gene regulation in bacteria, and will reveal design principles involved in the production of oscillations of gene expression in biological systems. Other proposed experiments will explore the relationship of the genetic clock to the host cells that contain it, with an eye towards isolating the clock from normal cellular physiological processes and improving system performance. In the second Specific Aim improved methods for studying oscillating systems are proposed. These include strategies for the development and analysis of improved models for clock function, as well as the development of instruments and methods for conducting experiments with the synthetic genetic clock. In the third Specific Aim, we propose the development of additional sophisticated genetic clocks. Some of these genetic clocks are based on designs that have been extensively modeled by other labs, but have not been constructed and tested. In addition we propose designs for novel genetic clocks that test the role of various system design principles in the production of biological oscillations.