Redesign of ultrasensitive and robust RecA gene circuit to sense DNA damage.
Redesign of ultrasensitive and robust RecA gene circuit to sense DNA damage.
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DOI：
10.1111/1751-7915.13767
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发表时间：
2021-11
影响因子：
5.7
通讯作者：
Huang WE
中科院分区：
文献类型：
--
作者：
Chen JX;Lim B;Steel H;Song Y;Ji M;Huang WE
来自产钠弧菌的recA启动子PVRecA的SOS盒在益生菌菌株大肠杆菌Nissle 1917中进行了表征、克隆和表达。然后根据预测的LexA阻遏物和PVRecA之间的相互作用对该启动子进行了合理的改造。重新设计的 PVRecA-AT 启动子对紫外线和基因毒性化合物引起的 DNA 损伤表现出敏感而强大的反应。 PVRecA 的合理设计与放大基因电路相结合，响应 DNA 损伤化合物丝裂霉素 C，电路输出幅度增加了 4.3 倍。在 PVRecA-AT 放大器中添加了基于 TetR 的负反馈回路，以实现强大的 SOS 系统，可抵抗 pH、温度、氧气和营养条件等参数的环境波动。我们发现，经过优化的 PVRecA-AT 的大肠杆菌 Nissle 1917 能够适应紫外线照射，并且在恒浊器微型反应器中培养 40 小时后，SOS 响应增加了 128 倍。我们还展示了该 PVRecA-AT 系统作为光遗传学执行器的潜力，可以通过紫外线辐射进行空间控制。我们证明，优化的 SOS 响应基因电路能够检测具有临床相关浓度的致癌生物标志物分子。益生菌大肠杆菌 Nissle 1917 中的超灵敏 SOS 基因电路可能对细菌诊断有用。合理设计SOS启动子以创建超灵敏且强大的recA基因电路来感知DNA损伤。益生菌大肠杆菌 Nissle 1917 中的超灵敏 SOS 基因电路可能对细菌诊断有用。
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 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. Rational design of SOS promoter to create ultra‐sensitive and robust recA gene circuit to sense DNA damage. The ultrasensitive SOS gene circuits in probiotic E. coli Nissle 1917 would be potentially useful for bacterial diagnosis.
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