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

doi:10.13523/j.cb.20170717

中国生物工程杂志

2017, Vol. 37

Issue (7): 105-114 DOI: 10.13523/j.cb.20170717

技术与方法

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

温国霞^1,2, 黄子豪^1,2, 谭俊杰³, 阚乃鹏⁴, 凌婧怡⁵, 张霞^1,2, 刘刚², 陈惠鹏²

1. 安徽大学健康科学研究院合肥 230601;
2. 军事医学科学院北京 100071;
3. 成都军区总医院成都 610083;
4. 福建省疾病预防控制中心福州 350001;
5. 南京军区福州总医院福州 350001

Construction of XylR-Pugene Lines in Escherichia coli to Detect 2,4,6-trinitrotoluene

WEN Guo-xia^1,2, HUANG Zi-hao^1,2, TAN Jun-jie³, KAN Nai-peng⁴, LING Jing-yi⁵, ZHANG Xia^1,2, LIU Gang², CHEN Hui-peng²

1. Institute of Health Sciences, Anhui University, Hefei 230601, China;
2. Academy of Military Medical Sciences, Beijing 100071, China;
3. Chengdu Military General Hospital, Chengdu 610083, China;
4. Fujian CenterFor Disease Control & Prevention, Fuzhou 350001, China;
5. Fuzhou General Hospital of Nanjing Military Command, Fuzhou 350001, China

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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。

关键词： 合成生物学; TOL质粒; 生物传感器; 基因线路; 6-三硝基甲苯; 2; 4

Abstract: Objective: The XylR-Pu is a classic toluene catabolic pathway, which is from TOL plasmid of Pseudomonas putida. In the presence of toluene, the XylR regulatory protein can activate Pu promoter and thus induce expression of corresponding metabolic genes. To detect 2,4,6-trinitrotoluene (TNT),the significant environmental pollutant, the pathway was optimized and put into Escherichia coli to construct whole-cell biosensor, which was based on the idea of synthetic biology. E.coliwas chosen as chassis cell due to its genetic background was clear and it was simple to operate. Methods: pETDuet-1 was used as backbone to construct gene circuit of XylR-Pu, XylR was inserted in first multi cloning site. The second T7 promoter was substituted by Pu promoter and reporter gene of green fluorescent protein was under the control of Pupromoter. The fluorescent values can indicate the strength of the activation of XylR protein to Pu promoter. Then four series terminator was inserted between XylR and Pu to minimize background expression. Finally, the receptor domain of XylR protein was randomly mutated using sequential error prone PCR to construct a mutant library and to identify XylR mutants, which can be more sensitive and specific to TNT. Results: The four series terminator can effectively prevent read-through and decrease background fluorescent. After selection, one mutant protein named eX0-4 displayed better induction intensity, sensitivity and specificity to TNT. Conclusions:As Nitrobenzene was not XylR natural inducer, so XylR showed no obvious response to TNT. But the method is feasible to modify the A domain of XylR protein to obtain non-natural but better protein components. The mutant of eX0-4 enriched the reservoir of TNT-sensing elements, and provided a more applicable toolkit to be applied in genetic routes and live systems of biosensors in future. It can be a common method to identify biological elements to use error prone PCR to construct mutant library.

Key words: 2,4,6-trinitrotoluene Biosensor Synthetic biology Gene line TOL plasmid

收稿日期: 2017-01-23 出版日期: 2017-07-25

ZTFLH:

Q815

基金资助: 国家"863"计划重点专项子课题资助项目（2016YFC1202403）

通讯作者: 刘刚 E-mail: jueliu@sohu.com

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引用本文:

温国霞, 黄子豪, 谭俊杰, 阚乃鹏, 凌婧怡, 张霞, 刘刚, 陈惠鹏. 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[J]. 中国生物工程杂志, 2017, 37(7): 105-114.

WEN Guo-xia, HUANG Zi-hao, TAN Jun-jie, KAN Nai-peng, LING Jing-yi, ZHANG Xia, LIU Gang, CHEN Hui-peng. Construction of XylR-Pugene Lines in Escherichia coli to Detect 2,4,6-trinitrotoluene. China Biotechnology, 2017, 37(7): 105-114.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20170717 或 https://manu60.magtech.com.cn/biotech/CN/Y2017/V37/I7/105

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