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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摘要目的：构建一种能够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。方法：首先采用交叉PCR将增强型绿色荧光蛋白与铜离子“外排泵”基因 copA 的启动子融合构建响应铜离子的 CopAp::gfpmut2 -pET28a感应元件。然后利用Red重组系统分别敲除野生型大肠杆菌（ E . c o l i ）MC4100体内负责外排转运铜离子的基因，并将感应元件转化至突变菌中完成菌株的构建。最后优化出最佳检测条件，并将其初步应用于检测水环境中铜离子。结果：相对野生型菌株， E . c o l i Δ cusA -Δ copA -Δ cueO 突变菌对铜离子的敏感性提高了64倍，从而使报告菌株的荧光信号显著增强，且荧光信号强度与铜离子浓度呈剂量依赖关系；报告菌株的线性检测范围为0.05～2.5 μmol/L（0.0032～0.16 mg/L）Cu2+；与原子吸收光谱法相比，其对生活饮用水中0.05 mg/L和1 mg/L铜离子的回收率分别为87.90%和104.37%。结论：本研究构建报告菌株的灵敏度能够有效覆盖国标中针对生活饮用水和污水中铜离子的限制，从而为报告菌株的应用奠定了技术基础。

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	周怀彬
	王娟娟
	赵建国
	洪旭芬
	庞一林
	李江辉
	谭国强

关键词 ：大肠杆菌全细胞报告菌株, 铜离子检测, 增强型绿色荧光蛋白, Red重组系统

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

收稿日期: 2019-05-23

基金资助:国家自然科学基金资助项目（81500440）；国家高技术研究发展计划（863计划）项目（2014AA06A514）。

通讯作者: 谭国强，副研究员，Email：tgq@wmu.edu.cn。

作者简介: 周怀彬（1981-），男，浙江永嘉人，实验师，硕士。

链接本文:

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

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