title: GreA and GreB Enhance Expression of Escherichia coli RNA Polymerase Promoters in a Reconstituted Transcription-Translation System.
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.
authors.affiliation: Institute of Bioengineering, School of Engineering, École Polytechnique Fédérale de Lausanne , 1015 Lausanne, Switzerland.
authors.forename: Lea L de
authors.initials: LL
authors.lastname: Maddalena
authors.affiliation: Institute of Bioengineering, School of Engineering, École Polytechnique Fédérale de Lausanne , 1015 Lausanne, Switzerland.
authors.forename: Henrike
authors.initials: H
authors.lastname: Niederholtmeyer
authors.affiliation: Department of Biochemistry, University of Turku , FI-20014 Turku, Finland.
authors.forename: Matti
authors.initials: M
authors.lastname: Turtola
authors.affiliation: Institute of Bioengineering, School of Engineering, École Polytechnique Fédérale de Lausanne , 1015 Lausanne, Switzerland.
authors.forename: Zoe N
authors.initials: ZN
authors.lastname: Swank
authors.affiliation: Department of Biochemistry, University of Turku , FI-20014 Turku, Finland.
authors.forename: Georgiy A
authors.initials: GA
authors.lastname: Belogurov
authors.affiliation: Institute of Bioengineering, School of Engineering, École Polytechnique Fédérale de Lausanne , 1015 Lausanne, Switzerland.
authors.forename: Sebastian J
authors.initials: SJ
authors.lastname: Maerkl
mesh_terms: D012321:DNA-Directed RNA Polymerases / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics; D053263:Gene Regulatory Networks / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D014176:Protein Biosynthesis / Q000235:genetics*; D014157:Transcription Factors / Q000235:genetics*; D014158:Transcription, Genetic / Q000235:genetics*; D035602:Transcriptional Elongation Factors / Q000235:genetics*
chemical_list: D029968:Escherichia coli Proteins; C079668:GreA protein, E coli; C079667:GreB protein, E coli; D014157:Transcription Factors; D035602:Transcriptional Elongation Factors; D012321:DNA-Directed RNA Polymerases
keywords: E. coli RNA polymerase; E. coli promoters; PURE system; bottom-up synthetic biology; cell-free protein synthesis; transcription