Cell-free protein synthesis (CFPS) is a platform technology that provides new opportunities for protein expression, metabolic engineering, therapeutic development, education, and more. The advantages of CFPS over in vivo protein expression include its open system, the elimination of reliance on living cells, and the ability to focus all system energy on production of the protein of interest. Over the last 60 years, the CFPS platform has grown and diversified greatly, and it continues to evolve today. Both new applications and new types of extracts based on a variety of organisms are current areas of development. However, new users interested in CFPS may find it challenging to implement a cell-free platform in their laboratory due to the technical and functional considerations involved in choosing and executing a platform that best suits their needs. Here we hope to reduce this barrier to implementing CFPS by clarifying the similarities and differences amongst cell-free platforms, highlighting the various applications that have been accomplished in each of them, and detailing the main methodological and instrumental requirement for their preparation. Additionally, this review will help to contextualize the landscape of work that has been done using CFPS and showcase the diversity of applications that it enables.

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Cell-free protein synthesis (CFPS) is a platform biotechnology that enables a breadth of applications. However, field applications remain limited due to the poor shelf-stability of aqueous cell extracts required for CFPS. Lyophilization of E. coli extracts improves shelf life but remains insufficient for extended storage at room temperature. To address this limitation, we mapped the chemical space of ten low-cost additives with four distinct mechanisms of action in a combinatorial manner to identify formulations capable of stabilizing lyophilized cell extract. We report three key findings: (1) unique additive formulations that maintain full productivity of cell extracts stored at 4 °C and 23 °C; (2) additive formulations that enhance extract productivity by nearly 2-fold; (3) a machine learning algorithm that provides predictive capacity for the stabilizing effects of additive formulations that were not tested experimentally. These findings provide a simple and low-cost advance toward making CFPS field-ready and cost-competitive for biomanufacturing.

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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.