A Bacteroides synthetic biology toolkit to build an in vivo malabsorption biosensor

The bacterial gut microbiota represents a powerful platform for continuous, non-invasive biosensing technologies for the gut environment, with genetically tractable commensal species like Bacteroides thetaiotaomicron (B. theta) emerging as promising hosts for engineered biosensors. However, the availability of genetic tools for precise, modular environmental sensing and reporting control in B. theta remains limited. Here, we present an expanded genetic engineering toolkit for B. theta that enables precise, fluorescence-based environmental sensing of the gut environment.

This toolkit includes (i) three libraries of orthogonally inducible promoters capable of driving fluorescence expression, (ii) a DNA-based system to tune repressor activity in B. theta, (iii) a resulting modular transcriptional reporter circuit that integrates native promoter activation with fluorescent outputs, and (iv) characterization of a novel plasmid integration mode in B. theta. To demonstrate its utility, we engineered biosensors for gut malabsorption, a condition characterized by increased luminal osmolality.

Using identified osmolality-responsive native promoters from B. theta, we made biosensors capable of detecting changes in gut physiology through graded fluorescent outputs. These biosensors were validated both in vitro and in vivo using a murine model of laxative-induced malabsorption, where they enabled near real-time, non-invasive monitoring of single-cell response from fecal samples with sensitivity to subclinical malabsorption levels.

By expanding the genetic toolkit for B. theta and demonstrating its use in a physiologically relevant context, this approach highlights the potential of engineered gut bacteria as a monitoring platform for diverse gut health applications. This work advances strategies for microbial biosensing and positions gut commensals as key players in next-generation diagnostic methods.