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

Abstract Whole-cell biosensors provide a cost-effective and sensitive approach for real-time monitoring of toxic metals in environmental samples. In this study, a bacterial whole-cell biosensor was engineered using Escherichia coli BL21(DE3) by integrating the cadC regulatory gene from Bacillus megaterium TWSL_4 with a green fluorescent protein (GFP) reporter to enable fluorescence-based detection of heavy metals. The biosensor gene cassette (Pcad + cadC + gfp) was first cloned into the pUC19 vector and then subcloned into the pET28a(+) expression vector to generate the recombinant plasmid pETCG28. Functional characterization showed that the engineered strain E. coli BL21/pETCG28 exhibited enhanced tolerance to heavy metals, sustaining growth at Pb²⁺ concentrations up to 1600 ppm, Cd²⁺ up to 200 ppm, and Zn²⁺ up to 60 ppm, significantly higher than the wild-type strain. Fluorescence analyses demonstrated strong concentration-dependent responses to heavy metal exposure. Corrected total cell fluorescence increased nearly fourfold between 1 ppb and 10 ppb of Pb²⁺ (R² = 0.95, p &amp;lt; 0.0001). Cd²⁺ exposure produced an approximately threefold increase (R² = 0.96, p &amp;lt; 0.0001), while Zn²⁺ generated a moderate twofold response (R² = 0.94, p &amp;lt; 0.0001). Optimal biosensor performance occurred at pH 7.0 and 37°C, demonstrating potential for portable environmental monitoring applications.