Redesigned Upstream Processing Enables a 24-Hour Workflow From� E. coli� Cells to Cell-Free Protein Synthesis

Cell-free protein synthesis (CFPS) platforms have undergone numerous workflow improvements to enable diverse applications in research, biomanufacturing, point-of-care detection, therapeutics, and education using affordable laboratory equipment and reagents. The Escherichia coli cell extract-based platform, being one of the most affordable and versatile CFPS platforms, has been broadly adopted. In spite of the promise of simplicity, the cell-free platform remains technically nuanced, posing challenges to reproducible implementation and broad adoption. Additionally, while the CFPS reaction itself can be implemented on-demand, the upstream processing of cells to generate crude cell lysate remains time-intensive, representing one of the largest sources of cost associated with the biotechnology. To circumvent the lengthy and tedious upstream workflow, we have redesigned the processes by developing a long-lasting autoinduction media formulation for cell-free that obviates human intervention between inoculation and harvest. Cell-free autoinduction (CFAI) media supports these advantages through the production of highly robust cell extracts from high cell density cultures nearing stationary phase of growth. Growth of cells to high density and autoinduction of T7 RNAP expression can be achieved by incubation overnight, eliminating the need for user intervention for the entirety of the process. The total mass of cells obtained is substantially increased, which directly results in a 400% increase in total extract volume obtained compared to past workflows. Based on these advances, we outline a new upstream processing workflow allowing researchers to go from cells on a streak plate to completing CFPS reactions within 24 hours while maintaining robust reaction yields of sfGFP (>1 mg/ml). We hope this advance improves the time and cost-efficiency for existing CFPS researchers, increase the simplicity and reproducibility, and reduce the barrier-to-entry for new researchers interested in implementing CFPS. Lastly, we have completed preliminary metabolomics analysis to elucidate the mechanisms by which CFAI-grown cells are capable of producing robust extracts.