重新设计超灵敏和稳健的RecA基因回路来感知DNA损伤

IF 4.8 2区 生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY
Jack X. Chen, Boon Lim, Harrison Steel, Yizhi Song, Mengmeng Ji, Wei E. Huang
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引用次数: 2

摘要

对产自营养弧菌的recA启动子PVRecA的SOS box进行了鉴定、克隆并在大肠杆菌Nissle 1917中表达。然后根据预测的LexA抑制因子和PVRecA之间的相互作用合理地设计该启动子。重新设计的PVRecA-AT启动子对紫外线和基因毒性化合物诱导的DNA损伤表现出敏感和强大的反应。PVRecA与扩增基因电路的合理设计使电路输出振幅增加了4.3倍,以响应DNA损伤化合物丝裂霉素c。在PVRecA- at放大器中添加了一个基于tetra的负反馈回路,实现了一个强大的SOS系统,可以抵抗pH、温度、氧气和营养条件等环境参数的波动。结果发现,经优化PVRecA-AT的e.c oli Nissle 1917能适应紫外线照射,在浊化器微型反应器中培养40小时后,SOS响应提高了128倍。我们还展示了PVRecA-AT系统作为光致动器的潜力,该系统可以通过紫外线辐射进行空间控制。我们证明,优化的SOS响应基因回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中超灵敏的SOS基因回路可能对细菌诊断有潜在的应用价值。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Redesign of ultrasensitive and robust RecA gene circuit to sense DNA damage

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.

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来源期刊
Microbial Biotechnology
Microbial Biotechnology BIOTECHNOLOGY & APPLIED MICROBIOLOGY-MICROBIOLOGY
CiteScore
9.80
自引率
3.50%
发文量
162
审稿时长
6-12 weeks
期刊介绍: Microbial Biotechnology publishes papers of original research reporting significant advances in any aspect of microbial applications, including, but not limited to biotechnologies related to: Green chemistry; Primary metabolites; Food, beverages and supplements; Secondary metabolites and natural products; Pharmaceuticals; Diagnostics; Agriculture; Bioenergy; Biomining, including oil recovery and processing; Bioremediation; Biopolymers, biomaterials; Bionanotechnology; Biosurfactants and bioemulsifiers; Compatible solutes and bioprotectants; Biosensors, monitoring systems, quantitative microbial risk assessment; Technology development; Protein engineering; Functional genomics; Metabolic engineering; Metabolic design; Systems analysis, modelling; Process engineering; Biologically-based analytical methods; Microbially-based strategies in public health; Microbially-based strategies to influence global processes
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