Abstract
SummarySOS box of the recA promoter, PVRecA 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 PVRecA. The redesigned PVRecA‐AT promoter showed a sensitive and robust response to DNA damage induced by UV and genotoxic compounds. Rational design of PVRecA 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 PVRecA‐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 PVRecA‐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 PVRecA‐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.
Highlights
Occurring microorganisms living on and in the human body present an opportunity for in situ bacterialSome carcinogenic or DNA damaging compounds generated in the human body are responsible for cancers (Seitz and Becker, 2007; Ridlon et al, 2016; Nguyen et al, 2018)
We developed a toolbox for SOS gene circuit for the detection of DNA damage, which can detect a range of disease biomarker molecules and carcinogenic antibiotics in a sensitive and robust manner
The results demonstrated that the sensitivity to mitomycin C has been increased (Fig. 2A and B), and the baseline has been significantly repressed due to the expression of extra lexA on PVRecA-AT -superfolder GFP (sfGFP) compared to the cases without (Fig. 2 D)
Summary
SOS box of the recA promoter, PVRecA from Vibrio natriegens was characterized, cloned and expressed in a probiotic strain E. coli Nissle 1917. This promoter was rationally engineered according to predicted interactions between LexA repressor and PVRecA. The redesigned PVRecA-AT promoter showed a sensitive and robust response to DNA damage induced by UV and genotoxic compounds. Rational design of PVRecA coupled to an amplification gene circuit increased circuit output amplitude 4.3-fold in response to a DNA damaging compound mitomycin C. We found that E. coli Nissle 1917 with optimized PVRecA-AT adapted to UV exposure and increased SOS response 128-fold over 40 h cultivation in turbidostat mini-reactor. The ultrasensitive SOS gene circuits in probiotic E. coli Nissle 1917 would be potentially useful for bacterial diagnosis
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