We report on and provide a detailed characterization of the performance and properties of a recently developed, all Escherichia coli, cell-free transcription and translation system. Gene expression is entirely based on the endogenous translation components and transcription machinery provided by an E. coli cytoplasmic extract, thus expanding the repertoire of regulatory parts to hundreds of elements. We use a powerful metabolism for ATP regeneration to achieve more than 2 mg/mL of protein synthesis in batch mode reactions, and more than 6 mg/mL in semicontinuous mode. While the strength of cell-free expression is increased by a factor of 3 on average, the output signal of simple gene circuits and the synthesis of entire bacteriophages are increased by orders of magnitude compared to previous results. Messenger RNAs and protein degradation, respectively tuned using E. coli MazF interferase and ClpXP AAA+ proteases, are characterized over a much wider range of rates than the first version of the cell-free toolbox. This system is a highly versatile cell-free platform to construct complex biological systems through the execution of DNA programs composed of synthetic and natural bacterial regulatory parts.
Keywords: biosynthesis; cell-free transcription−translation; gene circuits prototyping; minimal cell; self-assembly.