Development of a Sensitive Bioreporter Without Antibiotic Markers for Detecting Bioavailable Copper in Water Environments.

The whole-cell bioreporters based on the -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 △△△ mutant with three copper transport genes knocked out was constructed. Then, the sensing element was inserted into the chromosome of △△△ by gene knock-in method to obtain the bioreporter strain WMC-007. In optimized assay conditions, the linear detection range of Cu was 0.025-5 mg/L (0.39-78.68 μM) after incubating WMC-007 in Luria-Bertani medium for 5 h. The limit of detection of Cu 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 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-(-Morpholino)propanesulfonic acid buffer to 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 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.