Validation and calibration of a novel GEM biosensor for specific detection of Cd2+, Zn2+, and Pb2

Abstract

Background: In this study, we designed a novel genetic circuit sensitive to Cd²⁺, Zn²⁺ and Pb²⁺ by mimicking the CadA/CadR operon system mediated heavy metal homeostasis mechanism of Pseudomonas aeruginosa. The regular DNA motifs on natural operon were reconfigured and coupled with the enhanced Green Fluorescent Protein (eGFP) reporter to develop a novel basic NOT type logic gate CadA/CadR-eGFP to respond metal ions mentioned above. A Genetically Engineered Microbial (GEM)-based biosensor (E.coli-BL21:pJET1.2-CadA/CadR-eGFP) was developed by cloning the chemically synthesised CadA/CadR-eGFP gene circuit into pJET1.2-plasmid and transforming into Escherichia coli (E. coli)-BL21 bacterial cells.

Results: The GEM-based biosensor cells indicated the reporter gene expression in the presence of Cd²⁺, Zn²⁺ and Pb²⁺ either singly or in combination. Further, the same biosensor cells calibrated for fluorescent intensity against heavy metal concentration generated linear graphs for Cd²⁺, Zn²⁺ and Pb²⁺ with the R² values of 0.9809, 0.9761 and 0.9758, respectively as compared to non-specific metals, Fe³⁺ (0.0373), AsO₄^3- (0.3825) and Ni²⁺ (0.8498) making our biosensor suitable for the detection of low concentration of the former metal ions in the range of 1-6 ppb. Furthermore, the GEM based biosensor cells were growing naturally within the concentration range of heavy metals, at 37 °C and optimum pH = 7.0 in the medium, resembling the characteristics of wildtype E.coli.

Conclusion: Finally, the novel GEM based biosensor cells developed in this study can be applied for detection of targeted heavy metals in low concentration ranges (1-6 ppb) at normal bacterial physiological conditions.

Keywords: Cadmium; Genetic circuits; Heavy metal detection; Lead; Microbial biosensors; Zinc.