Department of Bioengineering, University of California, San Diego, La Jolla, CA, USA.
Department of Bioengineering, University of California, San Diego, La Jolla, CA, USA; Molecular Biology Section, Division of Biological Sciences, University of California, San Diego, La Jolla, CA, USA; BioCircuits Institute, University of California, San Diego, La Jolla, CA, USA.
Cell Syst. 2022 May 18;13(5):365-375.e5. doi: 10.1016/j.cels.2022.02.005. Epub 2022 Mar 22.
A major goal in synthetic biology is coordinating cellular behavior using cell-cell interactions; however, designing and testing complex genetic circuits that function only in large populations remains challenging. Although directed evolution has commonly supplemented rational design methods for synthetic gene circuits, this method relies on the efficient screening of mutant libraries for desired phenotypes. Recently, multiple techniques have been developed for identifying dynamic phenotypes from large, pooled libraries. These technologies have advanced library screening for single-cell, time-varying phenotypes but are currently incompatible with population-level phenotypes dependent on cell-cell communication. Here, we utilize directed mutagenesis and multiplexed microfluidics to develop an arrayed-screening workflow for dynamic, population-level genetic circuits. Specifically, we create a mutant library of an existing oscillator, the synchronized lysis circuit, and discover variants with different period-amplitude characteristics. Lastly, we utilize our screening workflow to construct a transcriptionally regulated synchronized oscillator that functions over long timescales. A record of this paper's transparent peer review process is included in the supplemental information.
合成生物学的一个主要目标是利用细胞间相互作用来协调细胞行为；然而，设计和测试仅在大群体中起作用的复杂遗传电路仍然具有挑战性。尽管定向进化通常为合成基因电路的合理设计方法提供了补充，但该方法依赖于对所需表型的突变文库进行有效的筛选。最近，已经开发出多种从大型混合文库中鉴定动态表型的技术。这些技术已经推进了单细胞、时变表型的文库筛选，但目前与依赖细胞间通讯的群体水平表型不兼容。在这里，我们利用定向诱变和多重微流控技术，开发了用于动态群体水平遗传电路的阵列筛选工作流程。具体来说，我们创建了现有振荡器（同步裂解电路）的突变文库，并发现了具有不同周期-幅度特征的变体。最后，我们利用我们的筛选工作流程构建了一个转录调控的同步振荡器，它可以在很长时间内工作。本论文的透明同行评审过程记录包含在补充信息中。
