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. in Fems Microbiol Lett 365:fny157, 2018). After multiple rounds of ON-OFF selection, two mutants with higher specificity for lead were selected. Structural analysis revealed that the mutation C134 located on the metal-binding loop at the C-terminal of PbrR is likely associated with the enhanced binding to both lead and cadmium. The double mutations D64A and L68S close to the metal-binding residue C79 may lead to the reduced binding specificity toward zinc ions. This dual selection system can be applied to engineer the specificity of other transcription factors and provide fine-tuned tools to synthetic biology.

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 Escherichia coli DH5α. The output signal from the reporter mCherry was significantly enhanced by the positive feedback amplifier. The sensitivity of the WCB with positive feedback is about 1 order of magnitude higher than that without positive feedback when evaluated using a half-saturation As(III) concentration. The minimum detection limit for As(III) was reduced by 1 order of magnitude to 0.1 µM, lower than the World Health Organization standard for the arsenic level in drinking water, 0.01 mg/liter or 0.13 µM. Due to the amplification of the output signal, the WCB was able to give detectable signals within a shorter period, and a fast response is essential for in situ operations. Moreover, the WCB with the positive feedback amplifier showed exceptionally high specificity toward As(III) when compared with other metal ions. Collectively, the designed positive feedback amplifier WCB meets the requirements for As(III) detection with high sensitivity and specificity. This work also demonstrates the importance of genetic circuit engineering in designing WCBs, and the use of genetic positive feedback amplifiers is a good strategy to improve the performance of WCBs.IMPORTANCE Arsenic poisoning is a severe public health issue. Rapid and simple methods for the sensitive and specific monitoring of arsenic concentration in drinking water are needed. In this study, we designed an arsenic WCB with a positive feedback amplifier. It is highly sensitive and able to detect arsenic below the WHO limit level. In addition, it also significantly improves the specificity of the biosensor toward arsenic, giving a signal that is about 10 to 20 times stronger in response to As(III) than to other metals. This work not only provides simple but effective arsenic biosensors but also demonstrates the importance of genetic engineering, particularly the use of positive feedback amplifiers, in designing WCBs.

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. One has pbrR and gfp on opposite sides of the promoter mimicking the native operon. We re-configured it by placing pbrR and gfp on the same side or under two separate promoters. The one with pbrR and gfp on the same side demonstrated lead sensitivity 10 times higher than the others. Positive feedback loop was introduced to these circuits. The strength of the output signal from the designs with positive feedback loop was 1.5-2 times stronger than those without positive feedback. This study demonstrates the importance of configuration and positive feedback as effective strategies to improve the performance of lead biosensors and they can be extended to the design of other whole-cell biosensors.