Design, mutate, screen: Multiplexed creation and arrayed screening of synchronized genetic clocks

Abstract

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.

Keywords: directed mutagenesis; dynamic phenotype screening; genetic circuit modeling; genetic oscillator; microfluidics; mutant library; synthetic biology.