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...

Teaching the processes of transcription and translation is challenging due to the intangibility of these concepts and a lack of instructional, laboratory-based, active learning modules. Harnessing the genetic code in vitro with cell-free protein synthesis (CFPS) provides an open platform that allows for the direct manipulation of reaction conditions and biological machinery to enable inquiry-based learning. Here, we report our efforts to transform the research-based CFPS biotechnology into a hands-on module called the "Genetic Code Kit" for implementation into teaching laboratories. The Genetic Code Kit includes all reagents necessary for CFPS, as well as a laboratory manual, student worksheet, and augmented reality activity. This module allows students to actively explore transcription and translation while gaining exposure to an emerging research technology. In our testing of this module, undergraduate students who used the Genetic Code Kit in a teaching laboratory showed significant score increases on transcription and translation questions in a post-lab questionnaire compared with students who did not participate in the activity. Students also demonstrated an increase in self-reported confidence in laboratory methods and comfort with CFPS, indicating that this module helps prepare students for careers in laboratory research. Importantly, the Genetic Code Kit can accommodate a variety of learning objectives beyond transcription and translation and enables hypothesis-driven science. This opens the possibility of developing Course-Based Undergraduate Research Experiences (CUREs) based on the Genetic Code Kit, as well as supporting next-generation science standards in 8-12th grade science courses. Keywords: biochemical education, learn by doing, cell-free protein synthesis (CFPS), in vitro transcription and translation, synthetic biology (synbio), central dogma of molecular biology (CDMB), chemical education and teaching, augmented reality (AR) Abbreviations: CFPS, cell-free protein synthesis; CUREs, course-based undergraduate research experiences; sfGFP, superfolder green fluorescent protein.

Cell-free protein synthesis (CFPS) is an in vitro process that enables diverse applications in research, biomanufacturing, point-of-care diagnostics, therapeutics, and education using minimal laboratory equipment and reagents. One of the major limitations of CFPS implementation is its sensitivity to plasmid type. Specifically, plasmid templates based on commonly used vector backbones such as the pET series of bacterial expression vectors result in the inferior production of proteins. To overcome this limitation, we have evaluated the effect of expression cassette elements present in the pET30 vector on protein production across three different CFPS systems: NEBExpress, PURExpress, and CFAI-based E. coli extracts. Through the systematic elimination of genetic elements within the pET30 vector, we have identified elements that are responsible for the poor performance of pET30 vectors in the various CFPS systems. As a result, we demonstrate that through the removal of the lac operator (lacO) and N-terminal tags included in the vector backbone sequence, a pET vector can support high titers of protein expression when using extract-based CFPS systems. This work provides two key advances for the research community: 1) identification of vector sequence elements that affect robust production of proteins; 2) evaluation of expression across three unique CFPS systems including CFAI extracts, NEBexpress, and PURExpress. We anticipate that this work will improve access to CFPS by enabling researchers to choose the correct expression backbone within the context of their preferred expression system.

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