GreA and GreB enhanceEscherichia coliRNA polymerase transcription rate 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 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

A Simple, Robust, and Low-Cost Method To Produce the PURE Cell-Free System

“…Repression of the deGFP expression with zinc-finger transcription factors was measured in the reaction setup as described above and supplemented with 800 nM of E. coli core RNAP and 4 μM of σ70 factor, which were prepared as described previously. 16 One nanomolar of linear DNA template coding for deGPF and 1 nM of linear DNA template coding for ADD or CBD zinc-finger were used. The reaction was incubated at 37 °C at constant shaking for 3 h, and measured (excitation, 488 nm; emission, 507 nm) on a SynergyMX platereader (BioTek).…”

“…The PURE system has been used for genetic network engineering, 2 recombinant DNA replication, 8 molecular diagnostics, 9 therapeutics, 10 and educational kits. 11 The PURE system also represents a viable starting point for generation of an artificial cell, 12,13 and its composition has been optimized 14,15 and extended 16 to achieve higher functionality.…”