Biosensor trigger library screening in mouse gut using engineered High-Throughput Memory System

Engineering synthetic circuits into intestinal bacteria to sense, record and respond to in vivo signals is a promising new approach for the diagnosis, treatment and prevention of disease. However, because the design of disease-responsive circuits is limited by a relatively small pool of known biosensors, there is a need for expanding the capacity of engineered bacteria to sense and respond to the host environment. Here, we apply a robust genetic memory circuit in Escherichia coli to identify new bacterial biosensor triggers responding in the healthy and diseased mammalian gut, which may be used to construct diagnostic or therapeutic circuits. We develop a pipeline for rapid systems-level library construction and screening, using next-generation sequencing and computational analysis, which demonstrates remarkably reliable identification of responsive biosensor triggers from pooled libraries. By testing libraries of potential triggers—each consisting of a promoter and ribosome binding site (RBS)—and using RBS variation to augment the range of trigger sensitivity, we identify and validate triggers that selectively activate our synthetic memory circuit during transit through the gut. We further identify biosensor triggers with increased response in the inflamed gut through comparative screening of one of our libraries in healthy mice and those with intestinal inflammation. Our results demonstrate the power of systems-level screening for the identification of novel biosensor triggers in the gut and provide a platform for disease-specific screening using synthetic circuits, capable of contributing to both the understanding and clinical management of intestinal illness.