Engineering Insulation Between Synthetic Bacterial Signaling Pathways

Two component systems (TCS) are the primary family of sensors in prokaryotes and are popular tools for synthetic biology. In response to environmental stimuli, a sensor histidine kinase (HK) phosphorylates a downstream response regulator (RR), eliciting changes in gene expression. A given bacterium uses 10-200 different HK/RR pairs to sense the various elements of its environment. TCSs are attractive sensors for engineered systems because of their natural abundance, their diverse inputs, and domain modularity that permits protein engineering. However, because the HK/RR interaction is mediated by highly conserved domains, engineered TCS sensors must avoid interactions with tens or hundreds of endogenous TCS proteins. To generate new orthogonal TCS pairs and learn design principles of TCS insulation, we mutated the specificity-determining regions of HKs and RRs and built large mutant libraries. We are leveraging cell sorting and deep-sequencing to quantify the compatibility of mutant HKs and RRs in E. coli, which has 32 endogenous TCS. Furthermore, we’re submitting these libraries to in vitro selections to test how the function of these pathways is affected by the presence or absence of paralogous pathways. In addition to generating toolkits of insulated sensors, this work will provide tools for integrating or propagating signals, interfacing with host physiology and monitoring cell states.