Multi-Scale Engineering of Heterogeneity in the Host-Aware Synthetic Gene Circuits

项目摘要 目前设计和构建合成基因电路的方法已经达到了 由电路-主机交互驱动的实质上的异质性造成的困境，尤其是对于 大规模基因电路。合成基因的传统试错迭代法 电路开发被认为是低效的，因为组装的基因电路通常 易受实验条件影响的。一个根本原因是，异质性推动了 随着组件数量的增加，电路与主机的相互作用变得显著 但经常被忽视的是基因回路。此外，缺乏量化的框架， 表征和控制寄主感知的合成基因电路中的异质性 这一领域的进展。我的实验室一直致力于剖析 电路-宿主的相互作用影响基因的电路功能和发育控制 针对电路-宿主相互作用的策略，以优化工程合成基因电路。 最近，我们发现了生长对合成基因回路功能的拓扑依赖性干扰 反馈，发表在《自然化学生物学》上。我们还发现了赢家通吃 资源竞争改变了级联细胞命运的转变，这是对自然的修订 沟通。在拟议的项目中，我们将建立实验和计算 用于量化、表征和控制宿主中的基因表达异质性的框架- 感知合成基因电路。随机蜂窝资源可能导致异构性 基因电路中的随机生化反应和随机细胞分裂。这些 异质性由于基因电路和基因序列之间复杂的相互作用而相互交织 宿主生物，为设计健壮的基因创造了另一层挑战和复杂性 电路。我们将集成一个用于延时活细胞分析的微流体系统，一个涡流调节器 具有基于Python的易于使用的Web界面的平台，可实现准确的增长率控制和 自动但远程控制的原位荧光测量和基于混合试剂的 细菌群落中所有单细胞随机模拟的建模算法 表征宿主感知合成基因中来自不同噪声源的异质性 电路。我已经建立了我的研究小组，拥有所有必要的专业知识和能力，以 完成拟建项目。这项工作将为我们提供系统深入的机械 了解由电路-主机相互作用驱动的异构性，将极大地帮助我们 合理设计和控制合成基因电路，用于复杂的临床应用 真实环境，如细菌感染和肿瘤微环境。

Project Summary Current approaches to designing and constructing synthetic gene circuits have reached a dilemma due to the substantial heterogeneity driven by circuit-host interactions, especially for large-scale gene circuits. The conventional trial-and-error iteration approach on synthetic gene circuit development is regarded as inefficient since the assembled gene circuits often are susceptible to experimental conditions. One fundamental reason is that the heterogeneity driven by circuit-host interactions become significant with the increase of the number of components in gene circuits but are often neglected. Moreover, the lack of quantitative frameworks for quantifying, characterizing, and controlling heterogeneity in the host-aware synthetic gene circuits impedes the progress in the field. My laboratory has been focusing on dissecting the mechanisms of how the circuit-host mutual interactions affect the gene circuit functions and developing control strategies targeting circuit-host interactions to optimize engineered synthetic gene circuits. Recently we found a topology-dependent interference of synthetic gene circuit function by growth feedback, which was published in Nature Chemical Biology. We also found winner-takes-all resource competition that redirected cascading cell fate transitions, which is in revision to Nature Communication. In the proposed projects, we will establish experimental and computational frameworks to quantify, characterize, and control the gene expression heterogeneity in the host- aware synthetic gene circuits. The heterogeneity can result from stochastic cellular resource allocation, stochastic biochemical reactions in gene circuits, and stochastic cell divisions. These heterogeneities are intertwined due to the complex interactions between the gene circuits and the host organisms, creating another layer of challenge and complexity to engineering robust gene circuits. We will integrate a microfluidics system for time-lapse live-cell analysis, a Turbidostat platform with Python-based easy-to-use web interface for accurate growth rate control and automatic yet remotely-controllable in-situ fluorescence measurement, and hybrid agent-based modeling algorithms for stochastic simulation of all the single cells in the bacterial community to characterize the heterogeneity from various noise sources in the host-aware synthetic gene circuits. I have built up my research group with all the necessary expertise and capabilities to complete the proposed projects. This work will provide a systematic in-depth mechanical understanding of the heterogeneity driven by circuit-host interactions, and will greatly help us to rationally design and control the synthetic gene circuits for sophisticated clinical applications in a real-world environment, such as bacterial infection and tumor microenvironments.