Modulating Heterologous Gene Expression with Portable mRNA-Stabilizing 5′-UTR Sequences

Sandra C. Viegas, Patrícia Apura, Esteban Martínez-García, Víctor De Lorenzo, Cecília M. Arraiano

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

RNA half-lives are frequently perceived as depending on too many variables, and transcript stability is generally missed as a checkpoint amenable to manipulation in synthetic designs. In this work, the contribution of mRNA stability to heterologous protein production levels in E. coli has been inspected. To this end, we capitalized on the wealth of information available on intrinsic mRNA stability determinants, four of which were formatted as portable modules consisting of 5′-untranslated regions (UTRs). The cognate DNA sequences were then assembled in a genetic frame in which mRNA stability endowed by the UTRs was the only variable to run expression of sfGFP. Reporter output and Northern blot-based measurements of absolute mRNA half-lives revealed that such UTRs were found to keep intact their ability to modulate transcript stability when excised from their natural context and placed as the upstream region of the reporter gene. By keeping transcription fixed and combining different UTRs with a constant ribosomal binding site, we showed that mRNA decay can be made the limiting constituent of the overall gene expression flow. Moreover, the data indicated that manipulating mRNA stability had little effect on expression noise in the corresponding population. Finally, augmented heterologous expression brought about by mRNA stability did not make cells more vulnerable to resource-consuming stresses. The tangible result of this work was a collection of well-characterized mRNA-stabilizing sequences that can be composed along with other expression signals in any construct following the assembly rules of the Standard European Vector Architecture (SEVA) format.

Original languageEnglish
Pages (from-to)2177-2188
Number of pages12
JournalACS Synthetic Biology
Volume7
Issue number9
DOIs
Publication statusPublished - 21 Sep 2018

Keywords

  • 5′-UTRs
  • heterologous expression
  • mRNA decay
  • pSEVA
  • sfGFP

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