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.