Refinement of OnePot PURE and Crude Ribosome Production for Reproducible Cell-free Protein Synthesis

Recent advances have enabled the Protein synthesis Using Recombinant Elements (PURE) cell-free system to be produced in individual laboratories economically and with reduced labor burden. However, the preparation of the 36 protein components and ribosome, which make up PURE, is still a complex undertaking, with much scope for variation and error. We present a detailed and updated procedure to manufacture PURE based on the recently published OnePot protocol, which involves regulating a number of key steps, in particular, the inoculation of cultures using optical density (OD)-normalized glycerol stocks, careful monitoring of cell growth, and controlling final glycerol concentrations. Using this approach, we report reproducible preparations of PURE from multiple biological replicates and users. This protocol serves as a reference and starting point for other laboratories to manufacture and troubleshoot their own PURE systems, thus increasing democratization of the many applications that PURE enables, such as the bottom-up construction of biomolecular systems and the development of synthetic cells.