High-throughput visualization mutation screening technology to enhance the specificity of CadR based whole-cell...

As a heavy metal pollutant, Cd²⁺ often enters the human body through the food chain causing great harm to human health. Whole-cell biosensor is an emerging technology for rapid on-site detection of heavy metals with the advantages of inexpensive, fast to mass-produce, and strong in anti-interference resistance, but suffering from insatisfactory specificity. In this study, a strategy of Adjacent Site Saturation Mutation (ASSM) was designed to improve the specificity of transcription factor CadR, which acted as the recognition element and determined the specificity of whole cell Cd²⁺ biosensors. A specific saturated library was constructed using the strategy of adjacent mutation. After two rounds of high-throughput visual screening, a whole-cell biosensor with good response to Cd²⁺, and with significant weakened Hg²⁺ interference was obtained. The optimized whole-cell biosensor showed a linear dynamic concentration range from 500 nM to 100 μM, a detection limit of 0.079 μM, and has satisfactory specificity and anti-interference. The ASSM strategy proposed in this study can provide a new method for the application of synthetic biology in food safety detection, indicating the importance of whole-cell biosensors for the detection of heavy metals.