Engineering the Ultrasensitive Visual Whole-Cell Biosensors by Evolved MerR and 5' UTR for Detection of Ultratrace...

The existing mercury whole-cell biosensors (WCBs, parts per billion range) are not able to meet the real-world requirements due to their lack of sensitivity for the detection of ultratrace mercury in the environment. Ultratrace mercury is a potential threat to human health via the food chain. Here, we developed an ultrasensitive mercury WCB by directed evolution of the mercury-responsive transcriptional activator (MerR) sensing module to detect ultratrace mercury. Subsequently, the mutant WCB (m4-1) responding to mercury in the parts per trillion range after 1 h of induction was obtained. Its detection limit (LOD) was 0.313 ng/L, comparable to those of some analytical instruments. Surprisingly, the m4-1 WCB also responded to methylmercury (LOD = 98 ng/L), which is far more toxic than inorganic mercury. For more convenient detection, we have increased another green fluorescent protein reporter module with an optimized 5' untranslated region (5' UTR) sequence. This yields two visual WCBs with an enhanced fluorescence output. At a concentration of 2.5 ng/L, the fluorescence signals can be directly observed by the naked eye. With the combination of mobile phone imaging and image processing software, the 2GC WCB provided simple, rapid, and reliable quantitative and qualitative analysis of real samples (LOD = 0.307 ng/L). Taken together, these results indicate that the ultrasensitive visual whole-cell biosensors for ultratrace mercury detection are successfully designed using a combination of directed evolution and synthetic biotechnology.