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，以阐明负责有趣行为的系统设计原则。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，也可能促进我们对来自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，将适用于复杂的合成遗传网络的建设。在第一个特异性目标中，我们提出了我们的大肠杆菌合成遗传时钟的特征。这个时钟在大细胞群中提供了激活子和抑制子表达的同步但有阻尼的振荡，这可以用来实现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，并应揭示负责振荡行为和控制输出的周期和幅度的设计特征，以及导致开发更好的时钟。这些研究将促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则。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，着眼于将时钟从正常的细胞生理过程中分离出来并提高系统性能。在第二个特定目标中，提出了研究振荡系统的改进方法。其中包括开发和分析时钟功能改进模型的策略，以及开发用合成遗传时钟进行实验的仪器和方法。在第三个具体目标中，我们建议开发更多复杂的遗传时钟。其中一些遗传时钟的设计已经被其他实验室广泛建模，但尚未被构建和测试。此外，我们提出了新型遗传时钟的设计，以测试各种系统设计原则在生物振荡产生中的作用。

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.