(610f) Building a Synthetic Biology Toolbox for a Non-Model Bacterial Chassis for Cancer Therapeutics

The next generation of bacterial cancer therapeutics will rely on non-model organisms with intrinsic immunological and tissue-targeting advantages. Brucella melitensis ΔvjbR, the chassis of our SPIKE (Synthetic Programmable bacteria for Immune-directed Killing in tumor Environments) system, offers a compelling platform due to its tumor-colonizing capabilities, immune stealth, safety in non-human primates, and genetic tractability. However, like many members of the Brucella genus, it has historically been difficult to engineer due to low transformation efficiency and a lack of characterized molecular tools. Unlocking its full therapeutic potential requires a dedicated synthetic biology toolkit to enable programmable control of gene expression, growth, and immune engagement in vivo.

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.