Integrating Genetic Domestication and Model-Guided Kill-Switch Engineering in a Non-Model Bacterial Therapeutic
Engineered bacteria offer programmable cancer immunotherapies, but engineering tractability does not always align with therapeutic suitability. The attenuated intracellular vaccine strain Brucella melitensis {Delta}vjbR (Bm{Delta}vjbR) shows promise as an immunotherapy chassis, yet its translation requires reliable control and containment systems that remain difficult to engineer with the limited genetic tools available. Here, we establish a genetic domestication workflow that connects genetic part generation and quantitative evaluation with model-guided circuit design. Through mutagenesis and screening, we generated and characterized genetic part libraries, including promoters spanning two orders of magnitude in activity, a panel of ribosome-binding sites, and highly functional terminators. We also evaluated fluorescent reporters, plasmid compatibility, and three inducible regulatory systems. These host-specific measurements informed mathematical modeling to guide the selection of a doxycycline-inducible Cas9/gRNA kill-switch architecture before construction. Experimental validation demonstrated inducible control both in vitro and in vivo: doxycycline reduced viable Bm{Delta}vjbR below the assay detection limit in vitro and decreased bacterial burden in mouse liver by approximately 91%. To our knowledge, this work establishes the first systematic genetic domestication of Bm{Delta}vjbR and the first containment circuit in this strain. The resulting workflow provides an extensible framework for domesticating and engineering other non-model bacteria.