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bioRxiv · 10.64898/2026.10.18.759933

Integrating Genetic Domestication and Model-Guided Kill-Switch Engineering in a Non-Model Bacterial Therapeutic

Jeong, Y.·Namboothiri, H. R.·Femi-Olatunji, O.·de Figueiredo, P.·Agca, C.·Hu, C. Y.
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Abstract

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.

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Jeong, Y., Namboothiri, H. R., Femi-Olatunji, O., de Figueiredo, P., Agca, C., Hu, C. Y.. 2026-10-20. Integrating Genetic Domestication and Model-Guided Kill-Switch Engineering in a Non-Model Bacterial Therapeutic. https://doi.org/10.64898/2026.10.18.759933

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