Genetic circuit design automation for the gut resident species Bacteroides thetaiotaomicron

Bacteroides thetaiotaomicron is a human-associated bacterium that holds promise for delivery of therapies in the gut microbiome 1 . Therapeutic bacteria would benefit from the ability to turn on different programs of gene expression in response to conditions inside and outside of the gut; however, the availability of regulatory parts, and methods to combine them, have been limited in B. thetaiotaomicron 2 – 5 . We report implementation of Cello circuit design automation software 6 for this species. First, we characterize a set of genome-integrated NOT/NOR gates based on single guide RNAs (CRISPR–dCas9) to inform a Bt user constraint file (UCF) for Cello. Then, logic circuits are designed to integrate sensors that respond to bile acid and anhydrotetracycline (aTc), including one created to distinguish between environments associated with bioproduction, the human gut, and after release. This circuit was found to be stable under laboratory conditions for at least 12 days and to function in bacteria associated with a primary colonic epithelial monolayer in an in vitro human gut model system.