Integrating Genetic Domestication and Model-Guided Kill-Switch Engineering in a Non-Model Bacterial Therapeutic
Year: 2026
Type: preprint
Abstract: Engineered bacteria offer programmable cancer immunotherapies, but engineering tractability does not always align with therapeutic suitability. The attenuated intracellular vaccine strain Brucella melitensis Δ vjbR (BmΔ 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 BmΔ 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 BmΔ vjbR and the first containment circuit in this strain. The resulting workflow provides an extensible framework for domesticating and engineering other non-model bacteria.
Institutions: Texas A&M University, University of Missouri
Language: English
Topic: Bacterial Cancer Therapy
Subfield: Biotechnology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W7223064285