Construction and Validation of an E. coli Whole-Cell Biosensor Based on CadC and GFP for Heavy Metal Detection

Heavy metal contamination is a serious environmental and public health concern that demands rapid and reliable detection methods. Whole-cell biosensors offer a cost-effective and sensitive approach for real-time monitoring. In this study, a biosensor based on Escherichia coli BL21(DE3) was constructed by incorporating the cadC regulatory gene from Bacillus megaterium TWSL_4 and a green fluorescent protein (GFP) reporter, enabling fluorescence-based detection of heavy metals. The biosensor cassette (Pcad+cadC+gfp) was first cloned into pUC19 and then subcloned into the pET28a(+) expression vector to generate the recombinant plasmid pETCG28. Functional characterization demonstrated that E. coli BL21/pETCG28 tolerated Pb²⁺ concentrations up to 1600 ppm, Cd²⁺ up to 200 ppm, and Zn²⁺ up to 60 ppm, showing markedly higher resistance than the wild-type strain. Fluorescence analyses revealed strong concentration-dependent responses, with corrected total cell fluorescence (CTCF) increasing nearly fourfold between 1 ppb and 10 ppb of Pb²⁺ (R² = 0.95, p < 0.0001). Similarly, Cd²⁺ exposure induced a threefold increase in CTCF over the same concentration range (R² = 0.96, p < 0.0001), while Zn²⁺ caused a comparatively moderate twofold increase (R² = 0.94, p < 0.0001). Optimal biosensing performance was achieved at a pH of 7.0 and 37 °C. These findings establish E. coli BL21/pETCG28 as a sensitive and specific biosensor for heavy metal detection, providing a foundation for the development of field-deployable environmental detection systems.