Subjects: Biology >> Bioengineering submitted time 2017-07-24 Cooperative journals: 《中国生物工程杂志》
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.coli was 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 Pu promoter . 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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