GreA and GreB Enhance Expression of Escherichia coli RNA Polymerase Promoters in a Reconstituted Transcription-Translation System

Lea L.De Maddalena, Henrike Niederholtmeyer, Matti Turtola, Zoe N. Swank, Georgiy A. Belogurov, Sebastian J. Maerkl

Research output: Contribution to journalArticlepeer-review

13 Scopus citations

Abstract

Cell-free environments are becoming viable alternatives for implementing biological networks in synthetic biology. The reconstituted cell-free expression system (PURE) allows characterization of genetic networks under defined conditions but its applicability to native bacterial promoters and endogenous genetic networks is limited due to the poor transcription rate of Escherichia coli RNA polymerase in this minimal system. We found that addition of transcription elongation factors GreA and GreB to the PURE system increased transcription rates of E. coli RNA polymerase from sigma factor 70 promoters up to 6-fold and enhanced the performance of a genetic network. Furthermore, we reconstituted activation of natural E. coli promoters controlling flagella biosynthesis by the transcriptional activator FlhDC and sigma factor 28. Addition of GreA/GreB to the PURE system allows efficient expression from natural and synthetic E. coli promoters and characterization of their regulation in minimal and defined reaction conditions, making the PURE system more broadly applicable to study genetic networks and bottom-up synthetic biology.

Original languageEnglish
Pages (from-to)929-935
Number of pages7
JournalACS Synthetic Biology
Volume5
Issue number9
DOIs
StatePublished - 16 Sep 2016
Externally publishedYes

Keywords

  • E. coli RNA polymerase
  • E. coli promoters
  • PURE system
  • bottom-up synthetic biology
  • cell-free protein synthesis
  • transcription

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