⚠️ Preprint – Noch nicht peer-reviewed
Dieser Artikel wurde noch nicht von unabhängigen Experten begutachtet. Die Ergebnisse sind vorläufig und sollten mit Vorsicht interpretiert werden.
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.