Cell-free gene expression (CFE) emerged as an alternative approach to living cells for specific applications in protein synthesis and labelling for structural biology and proteomics studies. CFE has since been repurposed as a versatile technology for synthetic biology and bioengineering. However, taking full advantage of this technology requires in-depth understanding of its fundamental workflow beyond existing protocols. This Primer provides new practitioners with a comprehensive, detailed and actionable guide to best practices in CFE, to inform research in the laboratory at the state of the art. We focus on Escherichia coli-based CFE systems, which remain the primary platform for efficient CFE. Producing proteins, biomanufacturing therapeutics, developing sensors and prototyping genetic circuits illustrate the broader utility and opportunities provided by this practical introduction to CFE. With its extensive functionality and portability, CFE is becoming a powerful and enabling research tool for biotechnology.
Bibliographical noteFunding Information:
This work was supported by the National Science Foundation (NSF) (MCB-1844152, EF-1934496, CBET-1916030), the Human Frontier Science Program (HFSP) (RGP0037/2015) and the Israel Binational Science Foundation (2018208) (V.N. and D.G.). P.F. and M.C.H. are supported by UKRI Engineering and the Physical Sciences Research Council (EPSRC) (EP/T013788/1).
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