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 transcription elongation factors GreA and GreB increased transcription rates of E. coli RNA polymerase from sigma factor 70 promoters up to 6-fold in an enhanced PURE system (ePURE). 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. Graphical abstract 70 DNA mRNA E. coli RNAP DNA mRNA Protein GreA, GreB
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