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

Yeonhwee JeongiD, Hari R. NamboothiriiD, Oluwatola Femi-OlatunjiiD, Paul de FigueiredoiD, Cansu AgcaiD, Chelsea Y. HuiD

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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Claude Sonnet 4.6 · xhigh effort · 7 Nov 2026 · from the full text of version 1 (20 Oct 2026) · CC BY 4.0

Host-measured parts and inducible systems for Brucella melitensis ΔvjbR guided a modeled Cas9 kill-switch, which cut mouse liver bacterial burden by about 91% at 30 mg/kg doxycycline.

The paper argues that genetic accessibility alone does not give a basis for quantitative circuit design in BmΔvjbR, and that safe use needs control and containment circuits. Its new element is a workflow that links host-specific part libraries and measurements to a mathematical screen of circuit designs before anything is built.

Key results

  1. ShownMutagenesis libraries gave promoter and RBS variants with graded strength and a terminator set with high efficiency in BmΔvjbR. Two of three common E. coli terminators performed poorly in this host.

    RBS and promoter variants spanned about two orders of magnitude in reporter output; minimal pTrcD gave about 5-fold higher expression than minimal pGroE; terminator variants reached up to 99% efficiency. · Creating a toolbox for gene expression…

  2. ShownThree inducible systems (LacI/IPTG, TetR/aTc, TlpA temperature switch) work in BmΔvjbR, with fitted dose-response parameters for the two chemical inducers.

    IPTG gave about 170-180-fold activation (K about 0.105 mM, Hill n about 1.46); aTc gave >200-fold (K about 1.02 ng/mL, n about 2.4) with growth loss at the top dose; TlpA was near background at 30 C and strongly on at 37 C. · Establishing inducible gene regulation in…; Inducer dose-response fitting

  3. SuggestedA computational screen ranked kill-switch designs by a viability score, found the score peaked at intermediate killing sensitivity (Kvia), and led to a chosen architecture and a gRNA targeting six genomic loci.

    480 circuit variants varied module order, orientation, promoter and RBS strength. The chosen design has both gRNA and Cas9 under pTet, with Cas9 on a weak RBS. Only this selected design was built, so model predictions were not tested against other designs. · Modeled guided design of doxycycline-inducible…; Computational identification of gRNA targets

  4. ShownIn vitro, doxycycline induction eliminated recoverable kill-switch cells, with basal toxicity comparable to the parental strain.

    CFUs fell from about 10^9 CFU/mL to below the limit of detection at 40 ng/mL doxycycline, while parental BmΔvjbR stayed viable; without doxycycline, CFUs were comparable to parental. · In vitro and in vivo evaluation of the…; In vitro kill-switch evaluation

  5. SuggestedIn mice, a single doxycycline dose reduced liver bacterial burden of the kill-switch strain compared with parental BmΔvjbR, but less completely than in vitro.

    Significant reductions at 25 and 30 mg/kg; about 91% reduction at 30 mg/kg versus the parental control; n = 2 mice per group; liver harvested 24 h after dosing. · In vitro and in vivo evaluation of the…; In vivo kill-switch evaluation; Figure legends

Methods, data and reproducibility

Fluorescent reporter assays in BmΔvjbR were used to characterize promoter, RBS and terminator libraries and three inducible systems. Custom simulation scripts then screened 480 Cas9/gRNA/TetR circuit variants, and the selected kill-switch was tested by CFU counts in culture and in mouse liver.

  • Reporter characterization of fluorescent proteins, plasmid compatibility, promoter/RBS/terminator libraries and inducible systems in BmΔvjbR · Brucella melitensis 16M ΔvjbR · n = 3 to 9 biological replicates per assay
  • In silico screen of kill-switch circuit designs · n = 480 circuit variants
  • In vitro CFU kill-switch assay with 40 ng/mL doxycycline · Brucella melitensis 16M ΔvjbR · n = 9 biological replicates
  • In vivo liver burden after intraperitoneal inoculation (1.07 x 10^9 CFU) and doxycycline at 0, 20, 25 or 30 mg/kg · mouse (C57BL/6) · n = n = 2 mice per group
Methods & tools
Kazusa Codon Usage Database, MoClo Golden Gate-Gibson (3G) assembly, Benchling, BioTek Synergy H1 plate reader, NIS-Elements, Microsoft Excel Solver (Hill fitting), Cas9/gRNA, TetR/pTet, LacI/pLac, TlpA
Reproducibility gaps
Simulation code is described as Supplementary Code 1 in the Supporting Information, with no repository URL. Data are stated to be in the article and Supporting Information.
Limitations (4)
  • The in vivo effect was only partial, which the authors attribute partly to plasmid loss during colonization. The circuit is plasmid-borne, and genomic integration is proposed as future work. · stated by the authors
  • Parts were characterized under selected laboratory conditions. Genetic stability, escape frequency, dose timing, tissue-specific efficacy and payload compatibility of the kill-switch remain to be evaluated. · stated by the authors
  • The in vivo test used n = 2 mice per dose, one organ and one time point, with a single dosing regimen, so the 91% estimate is imprecise. · noted in this summary
  • Only the model-selected circuit was built, so the model's ranking of designs was not tested against experimentally built alternatives. · noted in this summary

Who should read:Read this if you want a template for domesticating a non-model bacterial chassis, from part libraries to a model-ranked CRISPR kill-switch tested in mice.

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