Cell-free systems that mimic essential cell functions, such as gene expression, have dramatically expanded in recent years, both in terms of applications and widespread adoption. Here we provide a review of cell-extract methods, with a specific focus on prokaryotic systems. Firstly, we describe the diversity of Escherichia coli genetic strains available and their corresponding utility. We then trace the history of cell-extract methodology over the past 20 years, showing key improvements that lower the entry level for new researchers. Next, we survey the rise of new prokaryotic cell-free systems, with associated methods, and the opportunities provided. Finally, we use this historical perspective to comment on the role of methodology improvements and highlight where further improvements may be possible.
Keywords: CFE, Cell-Free Expression; CFPS, Cell-Free Protein Synthesis; Cell-free expression; Cell-free extract; Cell-free systems; GFP, Green Fluorescent Protein; Methods; OD, Optical Density; Synthetic biology; TFF, Tangential Flow Filtration; TXTL, Transcription and Translation.