Design and construct modular transcriptional repressors to facilitate the development of living diagnostics

项目摘要/摘要 到目前为止，开发多输入、多输出的遗传电路一直是一个巨大的挑战，因为 自然遗传系统只能将一种单一的化学输入连接到一种特定的启动子，从而 控制基因表达，这对创造具有复杂信号的工程生物构成了主要障碍 生物医学应用的响应行为。这个研究团队的长期目标是建立 构建遗传电路的生物部分的稳健策略，并使用这些部分实现新的 实际应用中的蜂窝功能。作为实现这些目标的重要步骤，第一个目标是确定 TetR家族同系物中用于小分子传感和DNA识别的功能模块。中环 假设TetR家族阻遏物是由离散的功能模块组成的，用于检测配体 分子，并与启动子相互作用，控制基因表达，在其中交换这些模块 导致具有变构和DNA结合特性的新组合的杂交抑制物。该战略旨在 实现目标#1是使用生物信息学方法分析TetR的序列和结构信息 同源物，预测功能蛋白质模块，并通过评估来实验验证预测 这些杂交抑制子在体内转录调控过程中的表现。对于目标2，研究 该团队提议创建一个基因电路平台，便于使用各种生物进行监测 多种生理参数。工作假说是模块抑制因子能够起到 生物传感器在多种生物体中实现多个同时检测的遗传电路设计 生理变化。这个目标与目标1无关，因为我们可以通过使用 以前从另一个蛋白质家族发展而来的模块化阻遏物。实现目标2的策略是 一系列生物体中的工程启动子，其中产生的启动子可以由 令人向往的抑制者。这些工程设计的可诱导表达系统然后可以用来实现 多路信号检测电路设计。预计该项目的贡献将是建立 从TetR同源基因创建模块化部件的设计原理，以及遗传电路的创建 一种利用各种生物监测多种生理参数的平台。这一贡献将是 意义重大，因为它有望为工程中的电路拓扑带来许多新的可能性 生物医学用途的生物，包括本项目中开发的生物监测平台，该平台具有 在生物医学条件的诊断方面有很大的潜力。因此，拟议的工作预计将 在基本级别和应用级别垂直移动该字段。

PROJECT SUMMARY/ABSTRACT Developing multi-input, multi-output genetic circuits has to date been a significant challenge due to the fact that natural genetic systems are only capable to connect one single chemical input to one specific promoter to control gene expression, which poses a major barrier to creating engineered organisms with complex signal response behavior for biomedical applications. The long-term goals of this research team are to establish robust strategies for constructing biological parts of genetic circuits, and to use these parts to implement new cellular functions for practical applications. As important steps toward these goals, Aim #1 is to identify functional modules among TetR family homologs for small molecule sensing and DNA recognition. The central hypothesis is that TetR family repressors are composed of discrete and functional modules for detecting ligand molecules and for interacting with promoters to control gene expression, in which swapping these modules leads to hybrid repressors with new combinations of allosteric and DNA-binding properties. The strategy to achieve Aim #1 is to use bioinformatics approaches to analyze sequence and structure information of TetR homologs, predicting functional protein modules, and to experimentally validate the predictions by assessing the performance of these hybrid repressors during in vivo transcription regulation. For Aim #2, the research team proposes to create a genetic circuit platform that facilitates the use of various organisms for monitoring multiple physiological parameters. The working hypothesis is that modular repressors are able to serve as biosensors in many types of organisms to realize a genetic circuit design for simultaneous detection of multiple physiological changes. This aim is independent from Aim #1 because we can achieve the goal by using modular repressors developed previously from another protein family. The strategy to achieve Aim #2 is to engineer promoters in a range of organisms, in which the resulting promoters can be controlled by the desirable repressors. These engineered inducible expression systems can then be used to implement the circuit design for multiple signal detection. The contribution of this project is expected to be the establishment of a design principle for creating modular parts from TetR homologs and also, the creation of a genetic circuit platform for monitoring multiple physiological parameters by using various organisms. This contribution will be significant because it is expected to release many new possibilities in circuit topologies for engineering organisms for biomedical uses, including the biomonitoring platform developed in this project, which has a great potential to be used for diagnosis of biomedical conditions. Therefore, the proposed work is expected to move the field vertically at both the basic and applied levels.