Rapid detection of genotoxicity employing an engineered high-sensitivity and tight SOS-response biosensor in Escherichia coli MG1655.

Industrial wastewater and pesticide residues contain numerous genotoxic compounds that pose serious health risks by causing DNA damage and potentially triggering carcinogenesis. Conventional analytical methods such as chromatography and mass spectrometry are limited by high costs, technical complexity, and lengthy procedures, making them unsuitable for rapid, large-scale screening. To address this need, we developed a fluorescence-based SOS-responsive whole-cell biosensor (SRWCB) by integrating the P sulA promoter with superfolder GFP (sfGFP). Through directed evolution of the lexA binding motif in P sulA , we obtained an optimized variant (P55) with significantly enhanced sensitivity-being 8.5 times more sensitive than the commonly used P umuDC system-while maintaining rapid response kinetics. Further engineering via antisense transcription improved biosensor tightness without compromising induction efficiency. The final construct (SRWCB-55-J23119) successfully detected genotoxicants in industrial wastewater, demonstrating its practical applicability. This study presents a sensitive, rapid, and robust biosensor platform for high-throughput environmental genotoxicity monitoring.