以大腸桿菌為底盤細胞構(gòu)建XylR-Pu線路檢測2,4,6-三硝基甲苯
發(fā)布時間：2018-09-19 09:59
【摘要】：目的:惡臭假單胞菌中的TOL質(zhì)粒上的XylR-Pu是經(jīng)典的甲苯代謝通路,在甲苯類化合物存在時,調(diào)控蛋白XylR可以特異性的激活Pu啟動子,進而啟動相應甲苯代謝通路的表達�；诤铣缮飳W的思想,優(yōu)化設(shè)計此通路并導入遺傳背景清楚、操作簡單的大腸桿菌中構(gòu)建全細胞生物傳感器,用于檢測環(huán)境污染物2,4,6-三硝基甲苯(TNT)。方法:以pETDuet-1為載體骨架,構(gòu)建了XylR-Pu基因線路,并以綠色熒光蛋白GFP為報告分子,GFP的熒光值可以指征結(jié)合誘導劑后的XylR蛋白對Pu啟動子的誘導強度,并在基因線路中加入四串聯(lián)終止序列來降低背景值。最后對XylR蛋白的信號識別區(qū)進行連續(xù)易錯PCR,構(gòu)建隨機突變體文庫,從中篩選具有更高感應強度、更好靈敏度及特異性的調(diào)控蛋白。結(jié)果:四串聯(lián)終止序列可有效降低XylR-Pu通路的背景值,隨機突變體文庫中篩選出的生物元件eX0-4,對TNT表現(xiàn)出良好的感應強度、靈敏度及特異性。結(jié)論:XylR蛋白在大腸桿菌中對硝基甲苯響應不明顯,但篩選得到的突變蛋白eX0-4,為后續(xù)生物傳感器的更深層次地開發(fā)提供了優(yōu)良的元件儲備。另外,利用易錯PCR構(gòu)建隨機突變體文庫從中篩選發(fā)揮預定功能的突變蛋白質(zhì)也可成為挖掘生物元件的一種通用方法。
[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.
【作者單位】： 安徽大學健康科學研究院;軍事醫(yī)學科學院;成都軍區(qū)總醫(yī)院;福建省疾病預防控制中心;南京軍區(qū)福州總醫(yī)院;
【基金】：國家“863”計劃重點專項子課題資助項目(2016YFC1202403)
【分類號】：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.
【作者單位】： 安徽大學健康科學研究院;軍事醫(yī)學科學院;成都軍區(qū)總醫(yī)院;福建省疾病預防控制中心;南京軍區(qū)福州總醫(yī)院;
【基金】：國家“863”計劃重點專項子課題資助項目(2016YFC1202403)
【分類號】：Q78;TP212.3
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