A novel 5' UTR design for improved gene/protein expression in Escherichia coli - Genetic construction, phenotypic characterization and transcript amount analysis of dualUTR constructs

Abstract

The 5' untranslated region of bacterial mRNA (UTR) play an important role in mRNA translation. It is a crucial contributor to the maintenance of a balance between transcription, transcript stability and translation. Therefore, an ideal UTR should be optimized to maximize expression at the protein level. A dualUTR has been constructed containing a transcriptional influencing 5' UTR, a spacer, and a translation stimulating 5' UTR. The XylS/Pm was used as a model system, and mCherry as reporter gene.

Phenotypic characterization of 13 dualUTR variants were performed by measuring their protein expression level. This was performed by measuring relative fluorescence levels of the protein mCherry. Transcript amounts of the dualUTR variants were measured using relative quantitative real-time reverse-transcription PCR (pPCR). Translation was blocked using the antibiotic chloramphenicol to investigate how this affects gene/protein expression in the dualUTR variants. The dualUTR lead to a synergistic effect on gene/protein expression. The highest expressing construct was r31n47, with a 137.1-fold increase of protein expression and a 103.0-fold increase in transcript amounts compared to the wildtype. The positional effects of the dualUTR variants were also investigated, concluding that the position of the transcriptional and translational influencing UTR is of great importance. The functionality of the dualUTR was demonstrated in Escherichia coli strains DH5alpha and RV308. Based on the experimental evidences demonstrated in this work the new dualUTR design principle has the potential to be applied in bacterial gene expression for maximization of gene/protein expression.