LAUSR

Owing to the prevalence of cadmium contamination and its serious hazards, it is important to establish an efficient and low-cost monitoring technique for the detection of the heavy metal cadmium. In this study, we first designed 30 cadmium whole-cell biosensors (WCBs) using different combinations of detection elements, reporting elements, and the host. The best performing WCB KT-5-R with Pseudomonas putida KT2440 as the host and composed of CadR and mCherry was selected for further analysis and engineering. In order to enhance its sensitivity, a positive feedback amplifier was added or the gene dosage of the reporter gene was increased. The WCB with the T7RNAP amplification module, p2T7RNAPmut-68, had the best performance and improved tolerance to cadmium with a detection limit of 0.01 μM, which is the WHO standard. It also showed excellent specificity toward cadmium when assayed with mixed metal ions. This study demonstrated the power of circuit engineering in WCB design and provided valuable insights for the development of other WCBs. KEY POINTS: • KT-5-R was selected after prescreening and engineered for better performance. • Using multi-copy reporters and the T7RNAP amplifier greatly improved the performance. • p2T7RNAPmut-68 had a detection limit of 0.01 μM and improved tolerance to cadmium.