To address this need, we developed a comprehensive synthetic biology toolbox for B. melitensis ΔvjbR. We first established a generalizable transformation method that increased efficiency by over 1000-fold and shortened the process from one week to a single day. Building on this, we constructed a modular promoter library spanning several orders of magnitude in expression strength and characterized six fluorescent proteins covering the blue-to-red spectrum for gene expression monitoring. To enable genome-level modifications, we implemented a robust editing pipeline based on CRISPR-associated transposase (CAST) systems and generated targeted auxotrophic mutants to support biocontainment strategies. Finally, we engineered intracellular biosensors responsive to tumor-associated metabolic cues, enabling tumor-specific activation of therapeutic programs.
Together, these tools enable precise and programmable control over gene expression, growth, and therapeutic output in a clinically relevant, immune-evasive chassis. Our work establishes Brucella melitensis ΔvjbR as a next-generation platform for live bacterial cancer therapies and highlights the broader potential of engineering non-model organisms for synthetic biology applications in medicine.