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目的:恶臭假单胞菌中的TOL质粒上的XylR-Pu是经典的甲苯代谢通路,在甲苯类化合物存在时,调控蛋白XylR可以特异性的激活Pu启动子,进而启动相应甲苯代谢通路的表达。基于合成生物学的思想,优化设计此通路并导入遗传背景清楚、操作简单的大肠杆菌中构建全细胞生物传感器,用于检测环境污染物2,4,6-三硝基甲苯(TNT)。方法:以pETDuet-1为载体骨架,构建了XylR-Pu基因线路,并以绿色荧光蛋白GFP为报告分子,GFP的荧光值可以指征结合诱导剂后的XylR蛋白对Pu启动子的诱导强度,并在基因线路中加入四串联终止序列来降低背景值。最后对XylR蛋白的信号识别区进行连续易错PCR,构建随机突变体文库,从中筛选具有更高感应强度、更好灵敏度及特异性的调控蛋白。结果:四串联终止序列可有效降低XylR-Pu通路的背景值,随机突变体文库中筛选出的生物元件eX0-4,对TNT表现出良好的感应强度、灵敏度及特异性。结论:XylR蛋白在大肠杆菌中对硝基甲苯响应不明显,但筛选得到的突变蛋白eX0-4,为后续生物传感器的更深层次地开发提供了优良的元件储备。另外,利用易错PCR构建随机突变体文库从中筛选发挥预定功能的突变蛋白质也可成为挖掘生物元件的一种通用方法。
OBJECTIVE: XylR-Pu on the TOL plasmid in Pseudomonas putida is a classical toluene metabolic pathway. In the presence of toluene compounds, the regulatory protein XylR specifically activates the Pu promoter, which in turn initiates the expression of the corresponding toluene metabolic pathway . Based on the idea of synthetic biology, we optimized this pathway and introduced whole cell biosensors into E. coli with a clear genetic background and easy operation to detect environmental pollutants such as 2,4,6-trinitrotoluene (TNT). Methods: The XylR-Pu gene was constructed by using pETDuet-1 as a carrier, and the green fluorescent protein GFP was used as the reporter. The fluorescence value of GFP indicated the induction intensity of Pu promoter by the XylR protein combined with the inducer, And add four tandem termination sequences to the gene circuit to reduce the background value. Finally, the signal recognition region of XylR protein was subjected to continuous error-prone PCR, and a random mutant library was constructed to screen for regulatory proteins with higher sensitivity, better sensitivity and specificity. Results: Four tandem termination sequences could effectively reduce the background value of XylR-Pu pathway. The eX0-4, a biological element screened from a random mutant library, showed good sensitivity, specificity and sensitivity to TNT. CONCLUSION: The response of XylR protein to nitrotoluene in E. coli is not obvious. However, the mutant protein eX0-4 screened out provides an excellent component reserve for the further development of subsequent biosensors. In addition, the use of error-prone PCR to construct a library of random mutants to screen for mutant proteins that perform their intended function can also be a versatile method for mining biological elements.