Integrating Genetic Domestication and Model-Guided Kill-Switch Engineering in a Non-Model Bacterial Therapeutic
- 1Texas A&M University
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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BibTeX
@misc{jeong2026integrating,
title = {{Integrating Genetic Domestication and Model-Guided Kill-Switch Engineering in a Non-Model Bacterial Therapeutic}},
author = {Y. Jeong and H. R. Namboothiri and O. Femi-Olatunji and P. de Figueiredo and C. Agca and C. Y. Hu},
howpublished = {bioRxiv},
year = {2026},
month = oct,
doi = {10.64898/2026.10.18.759933},
url = {https://www.biorxiv.org/content/10.64898/2026.10.18.759933v1},
note = {CC BY},
}
RIS
TY - UNPB
TI - Integrating Genetic Domestication and Model-Guided Kill-Switch Engineering in a Non-Model Bacterial Therapeutic
AU - Y. Jeong
AU - H. R. Namboothiri
AU - O. Femi-Olatunji
AU - P. de Figueiredo
AU - C. Agca
AU - C. Y. Hu
PY - 2026
DA - 2026/10/18
PB - bioRxiv
DO - 10.64898/2026.10.18.759933
UR - https://www.biorxiv.org/content/10.64898/2026.10.18.759933v1
AB - 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.
ER -