Construction of a RFP-lacZ alpha bicistronic reporter system and its application in lead biosensing

The combination of a fluorescent reporter and enzymatic reporter provides a flexible and versatile way for the study of diverse biological processes, such as the detection of transcription and translation. Thus, there is an urgent need to develop this novel bifunctional reporter system. This study reports the design, construction, and validation of a new dicistronic mCherry-lacZ alpha reporter system by artificial lac operon and pbr operon models in lacZM15-producing E. coli. It allows two reporter genes to be co-transcribed into a dicistronic mRNA strand, followed by coupled expression of mCherry and lacZ alpha. In artificial lac operons, expression of the downstream lacZ alpha was demonstrated to be positively related to expression of the upstream ORF. In artificial pbr operons, compared with the insertion of downstream full-length lacZ, the insertion of downstream lacZ alpha exerted a slight effect on the response from the upstream mCherry. Furthermore, the downstream lacZ alpha reporter showed stronger response to Pb(II) than the downstream full-length lacZ. Importantly, the response sensitivity of downstream lacZ alpha was still higher than that of upstream mCherry in a dual RFP-lacZ alpha reporter construct. The highly efficient expression profile of the reporter lacZ alpha peptide makes it a preferred downstream reporter in polycistronic constructs. This novel bifunctional reporter system offers a robust tool for biological studies.