Constructing cell-free expression systems for low-cost access

Cell-free systems for gene expression have gained attention as platforms for the facile study of genetic circuits and as highly effective tools for teaching. Despite recent progress, the technology remains inaccessible for many in low and middle income countries due to the expensive reagents required for its manufacturing, as well as specialized equipment required for distribution and storage. To address these challenges, we deconstructed processes required for cell-free mixture preparation and developed a set of alternative low-cost strategies for easy production and sharing of extracts. First, we explored the stability of cell-free reactions dried through a low-cost device based on silica beads, as an alternative to commercial automated freeze dryers. Second, we report the positive effect of lactose as an additive for increasing protein synthesis in maltodextrin-based cell-free using either circular or linear DNA templates. The modifications were used to produce active amounts of two high- value reagents: the isothermal polymerase Bst and the restriction enzyme BsaI. Third, we demonstrated the endogenous regeneration of nucleoside triphosphates and synthesis of pyruvate in CFS based on PEP and MDX. We exploited this novel finding to demonstrate use of a cell-free mixture completely free of any exogenous NTPs to generate high yields of sfGFP expression. Together, these modifications can produce desiccated extracts that are 203-424-fold cheaper than commercial versions. These improvements will facilitate wider use of CFS for research and education purposes.The authors acknowledge the funding support from EPRSC (EP/R014000/1, LCVD: Low-cost Cell- extract Viral Diagnostics, FGC was supported by LCVD project; BBSRC/EPSRC OpenPlant Synthetic Biology Research Centre Grant BB/ L014130/1 to JH. The authors acknowledge Dr. Khalid K. Alam, former postdoc at Northwestern University who shared tips and protocols on preservation strategies after lyophilization. FF was funded by Fondo de Desarrollo de Áreas Prioritarias - Center for Genome Regulation (ANID/FONDAP/ 15090007). FF and AA were founded by Proyecto Investigación Interdisciplinaria 2018 VRI. AA was funded by ANID National Doctoral Scholarship ID:21140714 , and ANID - Millennium Science Initiative Program - ICN17_022, ICGEB CRP/CHL19-01. AAdhikari and SV were supported by the Center on the Physics of Cancer Metabolism at Cornell University through Award Number 1U54CA210184-01 from the National Cancer Institute. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Cancer Institute or the National Institutes of Health. Funding from CONACyT A1-S-17269, DGAPA- PAPIIT IN225220 to SSN