Journal
FRONTIERS IN BIOENGINEERING AND BIOTECHNOLOGY
Volume 11, Issue -, Pages -Publisher
FRONTIERS MEDIA SA
DOI: 10.3389/fbioe.2023.1201580
Keywords
cell-free system; gene expression regulation; protein engineering; synthetic biology; E. coli
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Strict control of protein synthesis is crucial in synthetic biology. In this study, the consistency of 5'-UTR function in different bacterial cells and in vitro systems was systematically evaluated. The results showed a divergence between in vivo and in vitro protein translation, contrary to the strong correlation between two cellular systems. It was also found that the absence of nucleotide C and complex secondary structure in the 5'-UTR significantly improved protein translation efficiency.
Strict on-demand control of protein synthesis is a crucial aspect of synthetic biology. The 5'-terminal untranslated region (5'-UTR) is an essential bacterial genetic element that can be designed for the regulation of translation initiation. However, there is insufficient systematical data on the consistency of 5'-UTR function among various bacterial cells and in vitro protein synthesis systems, which is crucial for the standardization and modularization of genetic elements in synthetic biology. Here, more than 400 expression cassettes comprising the GFP gene under the regulation of various 5'-UTRs were systematically characterized to evaluate the protein translation consistency in the two popular Escherichia coli strains of JM109 and BL21, as well as an in vitro protein expression system based on cell lysate. In contrast to the very strong correlation between the two cellular systems, the consistency between in vivo and in vitro protein translation was lost, whereby both in vivo and in vitro translation evidently deviated from the estimation of the standard statistical thermodynamic model. Finally, we found that the absence of nucleotide C and complex secondary structure in the 5'-UTR significantly improve the efficiency of protein translation, both in vitro and in vivo.
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