BIOCOMPLEXITY:ANALYSIS, DESIGN, EVOLUTION-COMPLEX GENES

我们建议设计和实现一种协议，用于正向设计复杂的遗传电路，以精确控制特定细胞类型中受一组环境或其他细胞输入影响的基因表达。高效而准确地设计和制造这种电路的能力将促进一些核心生物技术目标的实现。例如，电路可以被设计成：1)“仪器”细胞以读出复杂的状态，2)在不同的代谢条件下最大限度地表达蛋白质，3)在满足特定条件时执行特定的动作(蛋白质的合成，宿主细胞过程的启动)，或4)提供一种可控的机制，通过该机制，特定的细胞系统可能会以一种设计的方式被扰动，以了解细胞的功能。这种技术的应用范围包括工业蛋白质生产的微生物设计，以及基因治疗载体的精确设计。在项目过程中，将制定一个理论和实验框架，以表征自然产生的遗传控制电路，并从表征的部分组装新的遗传控制电路，以满足特定的控制策略。具体目标可以简明扼要地表述如下：1)创建和实施一项实验方案，旨在快速表征和调整生物部分(启动子、终止子、转录本等)。为了充分详细地说明，可以为每个电路的动力学推导出准确的数学模型，2)创建非常详细的、经过实验验证的细胞环境传感网络的模型，其中有不同程度的先前知识来磨练电路分析技术并为未来的电路设计提供概念模型；3)使用这些结果，开发用于计算机辅助基因表达设计的简化协议，随后实施许多“规范”电路设计。作为范例，我们将从数学、计算和实验三个方面研究大肠杆菌中基因表达开关的三个“正交”例子：化学敏感阿拉伯糖启动子系统、1C型菌毛相位变异控制网络和OmpR介导的渗透压调节系统。

We propose the design and implementation of a protocol for the forward design of complex genetic circuitry for precise control of gene expression in a given cell type subject to a set of environmental or other cellular inputs. The ability to efficiently and accurately design and build such circuitry will facilitate a number of central biotechnological goals. For example, circuits might be designed to: 1) "instrument" cells to read out complex states, 2) maximize protein expression under different metabolic conditions, 3) perform a particular action (synthesis of a protein, initiation of a host-cell process) when particular conditions are met, or 4) provide a controllable mechanism by which a particular cellular system may be perturbed in a designed way to understand cellular function. Applications for such technology span the design of microorganisms for industrial protein production, to precise design of vectors for gene therapy. During the course of the project a theoretical and experimental framework to characterize naturally occurring genetic control circuits and to assemble novel genetic control circuits from the characterized parts to meet a particular control strategy will be developed. The specific aims may be concisely stated as: 1) to create and implement an experimental protocol designed to rapidly characterize and tune biological parts (promoters, terminators, transcripts, etc.) to sufficient detail that accurate mathematical models may be derived for the kinetics of each, 2) to create very detailed, experimentally validated models of cellular environmental sensing networks in which there are varying degrees of previous knowledge to hone the circuit analysis technology and provide conceptual models for future circuit designs; 3) using these results, to develop a streamlined protocol for computer-aided design of gene expression followed by implementation of a number of "canonical" circuit designs. As exemplars, we will study mathematically, computationally and experimentally, three "orthogonal" examples of genetic expression switches in E coli: the chemosensing arabinose promoter system, the type-1C pili phase variation control network, and the OmpR-mediated osmoregulatory system.