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

1目前设计和构建合成基因电路的方法已经达到了一个新的水平。 2由于电路-主机交互驱动的实质性异质性而导致的困境，特别是对于 3大规模基因回路。合成基因的传统试错迭代法 4电路开发被认为是低效的，因为组装的基因电路通常是 5、对实验条件敏感。一个根本原因是， 6电路-主机的相互作用变得显着的组件的数量增加， 7基因电路，但往往被忽视。此外，由于缺乏量化框架， 8表征和控制宿主感知合成基因回路中的异质性， 九是在实地工作。我的实验室一直致力于研究 电路与宿主的相互作用影响基因电路的功能和发育控制 11种针对电路-宿主相互作用的策略，以优化工程合成基因电路。 12最近，我们发现了一个拓扑依赖的干扰合成基因电路功能的生长 13反馈，发表在《自然化学生物学》上。我们还发现赢家通吃 14资源竞争，重定向级联细胞命运转变，这是在自然修订 15.通信。在建议的项目中，我们将建立实验和计算 16个框架来量化，表征和控制宿主中的基因表达异质性- 17个感知合成基因回路这种异质性可能是随机的细胞资源造成的 18分配，基因电路中的随机生化反应，以及随机细胞分裂。这些 19异质性是交织在一起，由于基因电路之间的复杂相互作用， 20种宿主生物，为工程化强大的基因创造了另一层挑战和复杂性 21个电路我们将整合一个用于延时活细胞分析的微流体系统， 22平台，基于Python的易于使用的Web界面，用于精确的增长率控制， 23自动但可远程控制的原位荧光测量，以及基于混合试剂的 24个建模算法，用于随机模拟细菌群落中的所有单细胞， 25表征来自宿主感知合成基因中的各种噪声源的异质性 26个电路。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， 27、完成项目建设。这项工作将提供一个系统深入的机械 28理解由电路-主机交互驱动的异质性，将极大地帮助我们 29合理设计和控制合成基因电路，用于复杂的临床应用， 30真实世界的环境，如细菌感染和肿瘤微环境。

1 Current approaches to designing and constructing synthetic gene circuits have reached a 2 dilemma due to the substantial heterogeneity driven by circuit-host interactions, especially for 3 large-scale gene circuits. The conventional trial-and-error iteration approach on synthetic gene 4 circuit development is regarded as inefficient since the assembled gene circuits often are 5 susceptible to experimental conditions. One fundamental reason is that the heterogeneity driven 6 by circuit-host interactions become significant with the increase of the number of components in 7 gene circuits but are often neglected. Moreover, the lack of quantitative frameworks for quantifying, 8 characterizing, and controlling heterogeneity in the host-aware synthetic gene circuits impedes 9 the progress in the field. My laboratory has been focusing on dissecting the mechanisms of how 10 the circuit-host mutual interactions affect the gene circuit functions and developing control 11 strategies targeting circuit-host interactions to optimize engineered synthetic gene circuits. 12 Recently we found a topology-dependent interference of synthetic gene circuit function by growth 13 feedback, which was published in Nature Chemical Biology. We also found winner-takes-all 14 resource competition that redirected cascading cell fate transitions, which is in revision to Nature 15 Communication. In the proposed projects, we will establish experimental and computational 16 frameworks to quantify, characterize, and control the gene expression heterogeneity in the host- 17 aware synthetic gene circuits. The heterogeneity can result from stochastic cellular resource 18 allocation, stochastic biochemical reactions in gene circuits, and stochastic cell divisions. These 19 heterogeneities are intertwined due to the complex interactions between the gene circuits and the 20 host organisms, creating another layer of challenge and complexity to engineering robust gene 21 circuits. We will integrate a microfluidics system for time-lapse live-cell analysis, a Turbidostat 22 platform with Python-based easy-to-use web interface for accurate growth rate control and 23 automatic yet remotely-controllable in-situ fluorescence measurement, and hybrid agent-based 24 modeling algorithms for stochastic simulation of all the single cells in the bacterial community to 25 characterize the heterogeneity from various noise sources in the host-aware synthetic gene 26 circuits. I have built up my research group with all the necessary expertise and capabilities to 27 complete the proposed projects. This work will provide a systematic in-depth mechanical 28 understanding of the heterogeneity driven by circuit-host interactions, and will greatly help us to 29 rationally design and control the synthetic gene circuits for sophisticated clinical applications in a 30 real-world environment, such as bacterial infection and tumor microenvironments.