以大肠杆菌为底盘细胞构建XylR-Pu线路检测2,4,6-三硝基甲苯
发布时间：2018-09-19 09:59
【摘要】：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。
[Abstract]:Aim: the XylR-Pu on TOL plasmid in Pseudomonas stenosensis is a classic toluene metabolic pathway. In the presence of toluene compounds, the regulatory protein XylR can specifically activate the Pu promoter and then initiate the expression of the corresponding toluene metabolic pathway. Based on the idea of synthetic biology, this pathway was optimized and introduced into Escherichia coli with a clear genetic background. A whole cell biosensor was constructed in E. coli, which was simple to operate, and used for the detection of environmental pollutant 2O4O4-trinitrotoluene (TNT). Methods: the XylR-Pu gene line was constructed using pETDuet-1 as the carrier skeleton. The fluorescence value of the green fluorescent protein GFP as a reporter molecule could indicate the intensity of Pu promoter induced by the XylR protein combined with the inducer. Four series terminations were added to the gene line to reduce the background value. Finally, the random mutants library was constructed by continuous error-prone PCR, in the signal recognition region of XylR protein, and the regulatory proteins with higher sensitivity, higher sensitivity and specificity were screened from the library. Results: Four-series termination sequence could effectively reduce the background value of XylR-Pu pathway. EX0-4, a biological element screened from random mutants library, showed good sensitivity, sensitivity and specificity to TNT. Conclusion the reaction of the protein to nitrotoluene is not obvious in Escherichia coli, but the screened mutant protein eX0-4, provides a good component reserve for the further development of biosensor. In addition, using error-prone PCR to construct random mutants library and screen mutant proteins with predetermined function can also become a general method for mining biological components.
【作者单位】： 安徽大学健康科学研究院;军事医学科学院;成都军区总医院;福建省疾病预防控制中心;南京军区福州总医院;
【基金】：国家“863”计划重点专项子课题资助项目(2016YFC1202403)
【分类号】：Q78;TP212.3
[Abstract]:Aim: the XylR-Pu on TOL plasmid in Pseudomonas stenosensis is a classic toluene metabolic pathway. In the presence of toluene compounds, the regulatory protein XylR can specifically activate the Pu promoter and then initiate the expression of the corresponding toluene metabolic pathway. Based on the idea of synthetic biology, this pathway was optimized and introduced into Escherichia coli with a clear genetic background. A whole cell biosensor was constructed in E. coli, which was simple to operate, and used for the detection of environmental pollutant 2O4O4-trinitrotoluene (TNT). Methods: the XylR-Pu gene line was constructed using pETDuet-1 as the carrier skeleton. The fluorescence value of the green fluorescent protein GFP as a reporter molecule could indicate the intensity of Pu promoter induced by the XylR protein combined with the inducer. Four series terminations were added to the gene line to reduce the background value. Finally, the random mutants library was constructed by continuous error-prone PCR, in the signal recognition region of XylR protein, and the regulatory proteins with higher sensitivity, higher sensitivity and specificity were screened from the library. Results: Four-series termination sequence could effectively reduce the background value of XylR-Pu pathway. EX0-4, a biological element screened from random mutants library, showed good sensitivity, sensitivity and specificity to TNT. Conclusion the reaction of the protein to nitrotoluene is not obvious in Escherichia coli, but the screened mutant protein eX0-4, provides a good component reserve for the further development of biosensor. In addition, using error-prone PCR to construct random mutants library and screen mutant proteins with predetermined function can also become a general method for mining biological components.
【作者单位】： 安徽大学健康科学研究院;军事医学科学院;成都军区总医院;福建省疾病预防控制中心;南京军区福州总医院;
【基金】：国家“863”计划重点专项子课题资助项目(2016YFC1202403)
【分类号】：Q78;TP212.3
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