Precision Expression Control in Bacteroides Thetaiotaomicron, a Human Commensal Bacterium

The human microbiota plays an integral role in diverse human physiological processes including metabolism, development and immunity. Manipulation of the native microflora using genetically engineered microbes is a promising avenue for cellular therapies and diagnostics. However, dominant members of the microbiota are difficult to robustly and reliably engineer. Here, we describe tools for engineering Bacteroides thetaiotaomicron, a relatively abundant species of bacteria that resides in the human gastrointestinal tract. We identify a library of constitutive promoters and ribosome binding sites that span a 10,000-fold range in expression.  This library is used to construct a recombinase-based memory gate that stores permanent memory on the B. thetaiotomicron chromosome. Additionally, we describe a series of fully orthogonal, sugar-inducible promoters able to conditionally elicit up to a 100-fold difference in gene expression. Finally, we demonstrate programmable knockdown of gene expression using CRISPRi targeted to both synthetic constructs and endogenous genes implicated in antimicrobial peptide resistance and carbohydrate metabolism. Together, these biological parts serve as a fundamental resource for the study and development of engineered commensal organisms that can positively impact human health.