Redesign of ultrasensitive and robust RecA gene circuit to sense DNA damage

Abstract

SOS box of the recA promoter, P_VRecA from Vibrio natriegens was characterized, cloned and expressed in a probiotic strain E. coli Nissle 1917. This promoter was then rationally engineered according to predicted interactions between LexA repressor and P_VRecA. The redesigned P_VRecA-AT promoter showed a sensitive and robust response to DNA damage induced by UV and genotoxic compounds. Rational design of P_VRecA coupled to an amplification gene circuit increased circuit output amplitude 4.3-fold in response to a DNA damaging compound mitomycin C. A TetR-based negative feedback loop was added to the P_VRecA-AT amplifier to achieve a robust SOS system, resistant to environmental fluctuations in parameters including pH, temperature, oxygen and nutrient conditions. We found that E. coli Nissle 1917 with optimized P_VRecA-AT adapted to UV exposure and increased SOS response 128-fold over 40 h cultivation in turbidostat mini-reactor. We also showed the potential of this P_VRecA-AT system as an optogenetic actuator, which can be controlled spatially through UV radiation. We demonstrated that the optimized SOS responding gene circuits were able to detect carcinogenic biomarker molecules with clinically relevant concentrations. The ultrasensitive SOS gene circuits in probiotic E. coli Nissle 1917 would be potentially useful for bacterial diagnosis.