A flexible ‘plug and play’ bicistronic construct and its application in the screening of protein expression system in Escherichia coli

• Published in: Journal of microbiological methods 2024-06, Vol.221, p.106928, Article 106928
• Main Authors: Feng, Binbin, Hu, Weiwei, Duan, Yingyi, Li, Zhaopeng
• Format: Article
• Language: English
• Subjects: Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Bicistronic construct; Cysteine Endopeptidases - genetics; Cysteine Endopeptidases - metabolism; Escherichia coli; Escherichia coli - genetics; Escherichia coli - metabolism; Escherichia coli Proteins - genetics; Escherichia coli Proteins - metabolism; Fluorescent protein; Gene Expression; Gene Expression Regulation, Bacterial; Genes, Reporter; Genetic Vectors; Luminescent Proteins - genetics; Open Reading Frames; Plasmids - genetics; Promoter Regions, Genetic; Protein expression; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Red Fluorescent Protein; Removability; Replicon - genetics
• ISSN: 0167-7012; 1872-8359; 1872-8359
• DOI: 10.1016/j.mimet.2024.106928

The bicistronic expression system that utilizes fluorescent reporters has been demonstrated to be a straightforward method for detecting recombinant protein expression levels, particularly when compared to polyacrylamide gel electrophoresis and immunoblot analysis, which are tedious and labor-intensive. However, existing bicistronic reporter systems are less capable of quantitative measurement due to the lag in reporter expression and its negative impact on target protein. In this work, a plug and play bicistronic construct using mCherry as reporter was applied in the screening of optimal replicon and promoter for Sortase expression in Escherichia coli (E. coli). The bicistronic construct allowed the reporter gene and target open reading frame (ORF) to be co-transcribed under the same promoter, resulting in a highly positive quantitative correlation between the expression titer of Sortase and the fluorescent intensity (R2 > 0.97). With the correlation model, the titer of target protein can be quantified by noninvasively measuring the fluorescent intensity. On top of this, the expression of reporter has no significant effect on the yield of target protein, thus favoring a plug and play design for removing reporter gene to generate a plain plasmid for industrial use. •A plug and play design is applied to get a plasmid without the reporter gene affecting the the target protein expression.•The bicistronic reporter system enables the tight co-expression of the target gene (srtA) and the reporter (mCherry).•The linear regression model demonstrated that the protein expression can be quantified by reading the fluorescent intensity.