Directed evolution of a transcription factor PbrR to improve lead selectivity and reduce zinc interference through dual selection.

Directed evolution has been proven as a powerful tool for developing proteins and strains with novel or enhanced features. In this study, a dual selection system was designed to tune the binding specificity of a transcription factor to a particular ligand with the ampicillin resistance gene amp (ON selection) as the positive selection marker and the levansucrase gene sacB (OFF selection) as the negative selection marker. It was applied to the lead responsive transcription factor PbrR in a whole-cell lead biosensor previously constructed in our lab (Jia et al.

Sensitive and Specific Whole-Cell Biosensor for Arsenic Detection.

Whole-cell biosensors (WCBs) have been designed to detect As(III), but most suffer from poor sensitivity and specificity. In this paper, we developed an arsenic WCB with a positive feedback amplifier in DH5α. The output signal from the reporter mCherry was significantly enhanced by the positive feedback amplifier.

Gene circuit engineering to improve the performance of a whole-cell lead biosensor.

To improve the performance of a whole-cell biosensor for lead detection, we designed six gene circuits by re-configuring the regulatory elements and incorporating positive feedback loops to the circuits. The lead resistance operon pbr encodes six genes with pbrRT on one side of the promoter and pbrABCD on the other side. PbrR, the divergent promoter it regulates, and GFP were used to design the lead biosensors.