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| Construction of XylR-Pugene Lines in Escherichia coli to Detect 2,4,6-trinitrotoluene |
| WEN Guo-xia1,2, HUANG Zi-hao1,2, TAN Jun-jie3, KAN Nai-peng4, LING Jing-yi5, ZHANG Xia1,2, 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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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.
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Received: 23 January 2017
Published: 25 July 2017
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Cite this article:
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.
URL:
https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20170717 OR https://manu60.magtech.com.cn/biotech/Y2017/V37/I7/105
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