Development and optimisation of mammalian cell-free systems for synthetic biology applications
Creators: name: Canavan, Caoimhe
Publisher: name: Imperial College London
Year: 2022

description: Cell-free systems are an in vitro tool for synthesising proteins outside of a living cell. In recent years they have been used as a synthetic biology tool to produce a variety of proteins including those that are traditionally difficult to express such as toxic, membrane and viral proteins. Cell-free reactions can be scaled down to very small volumes making them ideal for use with automated liquid handling platforms, allowing for low cost, high-throughput screening of large DNA libraries in one run. The lack of membrane barriers in cell-free allows for their continuous measurement and easy manipulation enabling the optimisation of experimental runs for chosen applications. One of the major drawbacks of cell-free systems is the problem of batch-to-batch variability which limits the reliability and predictability of performance, restricting their widespread adoption. Cell-free systems could benefit from the development of improved measurement techniques and standards to identify the causes of variation and in turn minimise them. Much of the research into cell-free systems has been based on prokaryotic cell-line; however, in recent years the use of eukaryotic cell-free systems has been expanding. Mammalian cell-free systems including HeLa, CHO and RRL have the unique capacity to produce therapeutic proteins containing post-translation modifications. HEK-293 is a mammalian cell-line broadly used for industrial applications including the synthesis of vaccines and the development of biotherapeutics. In this study, a HEK-293 cell-free system for synthetic biology applications was developed, including a lysate preparation protocol with minimal manual elements to minimise batch-to-batch variation and enable future scale up. Using the Echo acoustic liquid handler and DOE, screening experiments of up to 15 reaction mix components were conducted to determine the optimum energy mix composition. From this, HEPES, magnesium glutamate and creatine phosphate were identified as significant factors to improving reaction yield. Cell-free reactions ran using the optimum energy mix composition and HEK-293 lysates made in house produced up to 6.4 µg/ml of GFP. The commercial HeLa IVT energy mix was shown to have improved yields when supplemented with 40 mM of HEPES, with GFP yields of up to 25.4 µg/ml observed when ran using HEK-293 lysates made in-house. A systematic investigation into the causes of batch-to-batch variation was conducted to identify parameter candidates that could predict extract performance. This could eventually be used as part of a toolkit of measurements to facilitate production of cell-free lysates with minimal batch-to-batch variability. Using the optimised HEK 293 cell free system, self-amplifying RNA vaccine constructs were synthesised, demonstrating the compatibility of this system with high-throughput prototyping platforms. Ultimately, the development of a highly active standardised HEK-293 lysate, with minimal variation and predictable performance, enables the vast numbers of applications that mammalian cell-free systems hold to be realised, from high-throughput prototyping screening studies to fast synthesis of biotherapeutics for personalised medicine applications.