Development of a Sensitive Escherichia coli Bioreporter Without Antibiotic Markers for Detecting Bioavailable Copper in Water Environments
Development of a Sensitive Escherichia coli Bioreporter Without Antibiotic Markers for Detecting Bioavailable Copper in Water Environments
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开发无抗生素标记的灵敏大肠杆菌生物报告仪，用于检测水环境中的生物可利用铜
DOI：
10.3389/fmicb.2019.03031
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发表时间：
2020-01
影响因子：
5.2
通讯作者：
Tan Guoqiang
中科院分区：
文献类型：
--
作者：
Pang Yilin;Ren Xiaojun;Li Jianghui;Liang Feng;Rao Xiaoyu;Gao Yang;Wu Wenhe;Li Dong;Wang Juanjuan;Zhao Jianguo;Hong Xufen;Jiang Fengying;Wang Wu;Zhou Huaibin;Lyu Jianxin;Tan Guoqiang
基于CoP操纵子传感元件的全细胞生物记录仪已被证明在水环境中生物有效铜离子的评估中特别有用。在本研究中，为进一步提高生物记者的敏感性和稳健性，进行了一系列实验。首先，构建了三个铜转运基因缺失的大肠杆菌Copa cueO CUSA突变体。然后，利用基因敲入的方法将CopAp：：gfpmu2敏感元件插入到大肠杆菌Copa cueO CUSA的染色体中，获得了生物报告菌株E.coliWMC-007。在优化的检测条件下，铜离子的线性范围为0.025~5 mg/L(0.39~78.68μM)，检出限为0.0157 mg/L(0.25μM)。此外，荧光光谱分析和流式细胞仪实验表明，与基于质粒的传感器元件相比，该传感器元件具有更强的环境稳定性和更低的背景荧光信号。此外，我们还发现GFPmu2在大肠杆菌WMC-007中的表达是由游离铜离子诱导的，而不是由络合结合铜诱导的，且呈剂量依赖关系。特别是，在大肠杆菌WMC-007培养中加入40 mM的3-(N-吗啉)丙磺酸缓冲液，可以在pH从0.87到12.84的范围内准确地定量水溶液样品中的生物有效铜含量。铜回收率为95.88-113.40%。这些结果证明了大肠杆菌WMC-007作为生物报告器在监测酸性矿山废水、工业废水和饮用水中铜污染的潜在应用。由于全细胞生物记录仪相对便宜且易于操作，这种方法与其他物理化学技术相结合，将反过来提供关于水环境中毒性程度的更具体信息。
The whole-cell bioreporters based on the cop-operon sensing elements have been proven specifically useful in the assessment of bioavailable copper ions in water environments. In this study, a series of experiments was conducted to further improve the sensitivity and robustness of bioreporters. First, an Escherichia coli △copA△cueO△cusA mutant with three copper transport genes knocked out was constructed. Then, the copAp::gfpmut2 sensing element was inserted into the chromosome of E. coli △copA△cueO△cusA by gene knock-in method to obtain the bioreporter strain E. coli WMC-007. In optimized assay conditions, the linear detection range of Cu2+ was 0.025–5 mg/L (0.39–78.68 μM) after incubating E. coli WMC-007 in Luria–Bertani medium for 5 h. The limit of detection of Cu2+ was 0.0157 mg/L (0.25 μM). Moreover, fluorescence spectrometry and flow cytometry experiments showed more environmental robustness and lower background fluorescence signal than those of the sensor element based on plasmids. In addition, we found that the expression of GFPmut2 in E. coli WMC-007 was induced by free copper ions, rather than complex-bound copper, in a dose-dependent manner. Particularly, the addition of 40 mM 3-(N-Morpholino)propanesulfonic acid buffer to E. coli WMC-007 culture enabled accurate quantification of bioavailable copper content in aqueous solution samples within a pH range from 0.87 to 12.84. The copper recovery rate was about 95.88–113.40%. These results demonstrate potential applications of E. coli WMC-007 as a bioreporter to monitor copper contamination in acidic mine drainage, industrial wastewater, and drinking water. Since whole-cell bioreporters are relatively inexpensive and easy to operate, the combination of this method with other physicochemical techniques will in turn provide more specific information on the degree of toxicity in water environments.
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DOI：
10.1007/s00128-013-1143-z
发表时间：
2014-02
影响因子：
2.7
作者：
A. Peters;G. Merrington;D. Leverett;B. Ellor;S. Lofts;A. Gravell
通讯作者：
A. Peters;G. Merrington;D. Leverett;B. Ellor;S. Lofts;A. Gravell
影响因子：
5.6
作者：
David H. Ezekiel;Herma Brockman
通讯作者：
David H. Ezekiel;Herma Brockman
影响因子：
4.4
作者：
Y. Goh;B. Ho;J. Ding
通讯作者：
Y. Goh;B. Ho;J. Ding
影响因子：
4.2
作者：
Andreas Tom-Petersen;Carsten Hosbond;O. Nybroe
通讯作者：
Andreas Tom-Petersen;Carsten Hosbond;O. Nybroe
影响因子：
2.8
作者：
Chen Zhao;Chongchen Wang;Jun-qi Li;Peng Wang;Jiangbo Ou;Jingna Cui
通讯作者：
Chen Zhao;Chongchen Wang;Jun-qi Li;Peng Wang;Jiangbo Ou;Jingna Cui