Christopher J. Waite, Matthew Haines, Marko Storch, Paul Freemont.
The London Biofoundry, Imperial College London, United Kingdom
Cell-free expression (CFE) systems, consisting of the molecular machinery required to reconstitute coupled transcription-translation in vitro, are an invaluable technology across the synthetic biology spectrum, from prototyping genetic parts and circuits to small-scale biomanufacturing. In contrast to traditional cell-based systems, their ease of genetic programmability and suitability to low-volume and high-throughput workflows enables both scaling and streamlining of the design–build–test–learn (DBTL) cycle. E. coli lysate-derived CFE systems are accessible, versatile and high yielding, making them the ideal workhorse for many applications. The recombinant PURE system (1), consisting of <40 purified transcription-translation factors from E. coli, is ideal for applications that benefit from minimal contaminating factors and activities such as incorporation of unnatural amino acids, in vitro directed evolution and direct assay of products without purification. The high cost of commercial PURE systems limits their widespread use, especially at the scale and throughput required for meaningful proof-of-concept studies at the junction between research and commercial enterprise. We have validated the robustness of a homemade ‘one-pot’ preparation method of PURE (2) and are further optimizing its composition for various applications in the Biofoundry setting. Being able to prototype designs, de-risk workflows and fail quickly is as critical to the success of small biotech enterprises as securing lab space and talent. Having embedded a portfolio of CFE systems into our synthetic biology stack at the London Biofoundry, we describe successful case-studies for their use in accelerating proof of concept projects and explore advanced applications for future implementation.
(1) Shimizu, Y., et al. (2001) Nat. Biotechnol. 19, 751–755.
(2) Lavickova, B. et al. (2019) ACS Synth Biol. 8(2):455-462
