Cell-free protein synthesis (CFPS) platforms have undergone numerous workflow improvements to enable diverse applications in research, biomanufacturing, point-ofcare 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 longlasting autoinduction media formulation for cell-free that obviates human intervention between inoculation and harvest. CelI-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 that allows 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 will improve 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.

Cell-free protein synthesis (CFPS) platforms have undergone numerous workflow improvements to enable diverse applications in research, biomanufacturing, point-ofcare 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 longlasting autoinduction media formulation for cell-free that obviates human intervention between inoculation and harvest. CelI-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 that allows 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 will improve 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. Cell-free protein synthesis (CFPS) platforms have provided a robust, flexible, and accessible strategy to express high titers of proteins for the scientific community [1]. The open nature of the platform enables researchers to monitor protein expression in real time, to alter reaction conditions, and to produce traditionally intractable proteins ondemand. CFPS systems have undergone numerous and significant developments over the last 50 years, resulting in long-lived reactions with improved yields at lower costs [2]. The Escherichia coli-based CFPS platform in particular has gained traction over the last 30 years and has surpassed the Wheat Germ and Rabbit Reticulocyte platforms in cumulative publications [1]. The broad adoption of the E. coli-based crude extracts for CFPS is in part a function of consistent effort by the scientific community to enhance robustness of the platform, streamline the workflow of generating and utilizing cell extracts, and expand the utility and accessibility for new users. From its inception in...

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