1 Department of Microbiology & Immunology, University of British Columbia, Vancouver, BC, Canada.
2 School of Biomedical Engineering, University of British Columbia, Vancouver, BC, Canada.
3 Department of Biology, Concordia University, Montréal, QC, Canada; Department of Physics, Concordia University, Montréal, QC, Canada; Center for Applied Synthetic Biology, Concordia University, Montréal, QC, Canada.
4 Department of Microbiology & Immunology, University of British Columbia, Vancouver, BC, Canada; School of Biomedical Engineering, University of British Columbia, Vancouver, BC, Canada; Humans and the Microbiome Program, Canadian Institute for Advanced Research (CIFAR), Toronto, ON, Canada. Electronic address: carolina.tropini@ubc.ca.

The human gut is a dynamic environment, where changes in pH, oxygen, and osmolality influence microbiota composition and disease. Monitoring these environmental shifts is crucial for advancing gut health diagnostics and therapeutics, yet non-invasive monitoring tools remain limited. Genetically tractable commensals, including Bacteroides thetaiotaomicron, offer promising chassis for engineering biosensors but lack modular systems for precise sensing and reporting. Here, we developed genetic tools for B. thetaiotaomicron, including (1) repressible promoters for tunable fluorescent protein expression, (2) a DNA-based system to modulate repressor activity, (3) a modular, fluorescence-based transcriptional reporter circuit, and (4) an alternative plasmid integration mode. Using these components, we engineered biosensors to detect increased gut osmolality caused by malabsorption and validated them in vitro and in a murine model of laxative-induced osmotic diarrhea. These biosensors enabled long-term, non-invasive reporting of gut osmolality from single-cell fluorescence, demonstrating the potential of gut bacteria as monitoring platforms in gut health applications.