Abstract
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-lacZα 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 lacZα. In artificial lac operons, expression of the downstream lacZα 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 lacZα exerted a slight effect on the response from the upstream mCherry. Furthermore, the downstream lacZα reporter showed stronger response to Pb(II) than the downstream full-length lacZ. Importantly, the response sensitivity of downstream lacZα was still higher than that of upstream mCherry in a dual RFP-lacZα reporter construct. The highly efficient expression profile of the reporter lacZα peptide makes it a preferred downstream reporter in polycistronic constructs. This novel bifunctional reporter system offers a robust tool for biological studies.
Highlights
Recombinant production of enzymatic and fluorescent reporters has greatly improved our ability to explore the potential molecular processes underlying gene expression and regulation in various living organisms [1, 2]
The upstream open reading frame (ORF) is composed of a multiple cloning site (MCS) used for the insertion of another reporter gene and a His Tag used for the blot assays
One, two, and three consecutive rare codons were introduced into the upstream ORF to facilitate the detection of response change from the downstream lacZα reporter system
Summary
Recombinant production of enzymatic and fluorescent reporters has greatly improved our ability to explore the potential molecular processes underlying gene expression and regulation in various living organisms [1, 2]. The fluorescent proteins have been widely used to study promoter activity and regulation, protein function and localization, and non-invasive imaging over many years [3, 4]. Since their discovery, fluorescent proteins have been extensively modified to improve stability, brightness, and oligomerization state [5]. Fluorescent proteins have been extensively modified to improve stability, brightness, and oligomerization state [5] Reporter enzymes, such as β-galactosidase, hydrolyze externally supplied substrates, and produce detectable signals. They have been widely used for promoter identification and gene.
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