A discovery platform to identify inducible synthetic circuitry from varied microbial sources

Abstract

AbstractGut-resident microbes encode a variety of systems capable of sophisticated sensing and conditional gene expression control within the mammalian gut during health and disease. Bacterial synthetic biology approaches, such as whole-cell biosensing and ‘sense-and-respond’ therapeutics, aim to re-purpose such systems for research and clinical applications; however, the toolbox of well characterisedin vivoand disease-related inducible systems remains limited. Here, we dramatically expand the flexibility and power of a high-throughput approach to biosensor evaluation using bacterial memory circuits encoded in a gut commensalE. colispecies. We construct a novel library of bacterial two-component system (TCS) based biosensors sourced from gut pathogens, tagged with unique DNA barcodes using a single-pot construction method. Using our pipeline, we evaluate sensor activity and performance heterogeneity acrossin vitroandin vivoconditions leading to novel sensor discovery. In the process, we optimise library construction, sample quality thresholds, and parameters for engineered bacterial biosensor screening in the murine gut. This approach can be applied to transcriptionally activated sensing elements of any type and will allow rapid development of new biosensors that can advance synthetic biology approaches for complex environments, such as the gut.