Computational Design and Biosecurity Assessment of a Dual-Function E. coli Circuit for S. aureus Sensing and Targeted Lysostaphin Therapy

Engineered therapeutic microbes represent a promising strategy to combat antibiotic-resistant pathogens such as Staphylococcus aureus; however, the absence of predictive biosafety frameworks remains a major barrier to their clinical translation. Here, we present an in silico design of a dual-function Escherichia coli genetic circuit integrating pathogen sensing via the AgrC/AgrA system, targeted lysostaphin-mediated killing, and a tightly regulated MazE/MazF kill switch for biocontainment. A hybrid computational framework was developed to systematically evaluate functional performance and biosafety risks, combining ordinary differential equations (ODEs) for deterministic dynamics with Gillespie-based stochastic simulations to capture intrinsic molecular noise. The model predicts robust and responsive bacteriolytic activity upon detection of S. aureus signaling molecules, while maintaining controlled system behavior under varying conditions. Additionally, critical thresholds for kill-switch leakiness were identified, and evolutionary stability was assessed through failure-time analysis. Collectively, this study provides a quantitative framework for integrating circuit design with biosafety evaluation, offering practical guidance for the development and regulatory assessment of engineered therapeutic microbes. However, these computational predictions require future experimental validation to confirm circuit efficacy and safety.