BIOCOMPLEXITY:ANALYSIS, DESIGN, EVOLUTION-COMPLEX GENES

我们提出设计和实现一种方案，用于复杂遗传电路的正向设计，以精确控制给定细胞类型中受一系列环境或其他细胞输入的基因表达。有效而准确地设计和建造这种电路的能力将促进许多核心生物技术目标的实现。例如，电路可能被设计为：1)“仪器”细胞读取复杂状态，2)在不同代谢条件下最大化蛋白质表达，3)在满足特定条件时执行特定动作（蛋白质合成，启动宿主细胞过程），或4)提供一种可控机制，通过这种机制，特定的细胞系统可能会以一种设计的方式受到干扰，以了解细胞功能。这种技术的应用范围涵盖了用于工业蛋白质生产的微生物设计，以及用于基因治疗的载体精确设计。在项目过程中，将开发一个理论和实验框架，以表征自然发生的遗传控制电路，并从表征部分组装新的遗传控制电路，以满足特定的控制策略。具体目标可以简洁地表述为：1)创建并实施一项实验方案，旨在快速表征和调整生物部分（启动子、终止子、转录本等），使其足够详细，从而可以为每个部分的动力学推导出准确的数学模型；实验验证的细胞环境感知网络模型，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)利用这些结果，开发一种简化的基因表达计算机辅助设计方案，随后实施一些“规范”电路设计。作为例子，我们将从数学、计算和实验上研究大肠杆菌中基因表达开关的三个“正交”例子：化学感应阿拉伯糖启动子系统、1c型菌毛期变化控制网络和ompr介导的渗透调节系统。

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.