一种检测生活饮用水和污水中生物有效性铜离子的微生物报告菌株的构建

doi:10.3969/j.issn.2095-9400.2020.05.001

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Abstract Objective: To construct a microbial reporter strain that can detect bioavailable copper ions in drinking water and wastewater. Methods: Firstly, the gene sequence encoding enhanced green fluorescent protein was fused with the promoter region of copper ion “efflux pump” gene copA by recombinant PCR. The obtained PCR product was subcloned to pET28a plasmid to generate the copper-responsive vector CopAp::gfpmut2pET28a. Next, the genes responsible for the copper resistance in wild-type E. coli cell (MC4100) were deleted using the Red recombination method, before the CopAp::gfpmut2-pET28a vector was transformed into the engineered E. coli cell to accomplish the construction of the copper-sensing E. coli strain. Finally, the optimal conditions for copper detection were established and the copper-detecting strain was applied to detect copper ions in water. Results: Compared with wild-type strain, the sensitivity of the E. coli ΔcusA ΔcopA ΔcueO mutant to copper was enhanced by 64-fold, thus higher fluorescence signal to copper ions was successfully achieved. Furthermore, the fluorescence intensity of the biosensor also had dose-dependent relation with copper concentration. In optimized assay conditions, the linear detection range of Cu2+ was 0.05-2.5 μmol/L (0.0032-0.16 mg/L). In comparison with atomic absorption spectrometry, the recovery rate of 0.05 mg/L and 1 mg/L copper ions in drinking water was 87.90% and 104.37% respectively. Conclusion: According to the national standard, the linear detection range of the bioreporter strain constructed in this study can fully meet the demand for effective copper detection in drinking water and wastewater, which may provide this whole-cell bioreporter strain with technical feasibility for practical application.

Key words： Escherichia coli whole-cell bioreporter detection method of copper ion enhanced green fluorescent protein Red recombination

Received: 23 May 2019

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	ZHOU Huaibin
	WANG Juanjuan
	ZHAO Jianguo
	HONG Xufen
	PANG Yilin
	LI Jianghui
	TAN Guoqiang.

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https://xb.wmu.edu.cn/EN/10.3969/j.issn.2095-9400.2020.05.001 OR https://xb.wmu.edu.cn/EN/Y2020/V50/I5/345

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