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.

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 a platform biotechnology that enables a breadth of applications, from expressing traditionally intractable proteins to metabolic engineering and point‐of‐care diagnostics. However, field applications have remained limited due to the poor shelf stability of aqueous E. coli cell‐free extracts, which contain a complex mixture of cellular machinery. Lyophilization of the E. coli extract improves shelf‐life, but remains insufficient for maintaining extract productivity over extended storage periods at room temperature. For many pure protein‐based biologics, a variety of additives have been identified that help to stabilize the protein for lyophilization, transportation, and storage. However, the advances made for pure proteins cannot be directly applied to the complex cell extract, and current investigations into improving cell extract stability via additives has been limited. Thus, we have addressed the cold chain limitation in cell‐free by mapping the chemical space of ten low‐cost additives with four distinct mechanisms of action in a combinatorial manner to elucidate their capacity to further stabilize lyophilized cell extract. We report three key findings: 1) unique additive formulations that maintain full productivity of the cell extract at 4°C and 23°C; 2) additive formulations that enhance extract productivity up to 195%; 3) a machine learning algorithm that provides predictive capacity for the stabilizing effects of additive formulations that were not tested experimentally. The identified additive formulations provide a simple and low‐cost advance toward making CFPS field‐ready. Support or Funding Information Bill and Linda Frost Fund, Center for Applications in Biotechnology’s Chevron Biotechnology Applied Research Endowment Grant, Cal Poly Research, Scholarly, and Creative Activities Grant Program (RSCA 2017), and the National Science Foundation (NSF‐1708919) Comparison of traditional cellfree extract production and reaction setup with our modified workflow for improved stability and productivity to enable field applications.

Hands‐on learning of transcription and translation remains limited due to a lack of classroom modules for early‐career students. Currently, classrooms implement animations, simulations, games, and bacterial transformation/protein expression to teach the genetic code. The cell wall poses the primary barrier for students to directly observe and manipulate the intracellular environment, limiting inquiry‐based learning of the genetic code. We have overcome this limit by levering transcription and translation in vitro. The open nature of cell‐free protein synthesis (CFPS) is achieved by removing the cell wall and genomic DNA, while isolating the cellular machinery involved in transcription and translation. This allows experimenter to harness the genetic code in a test tube and directly manipulate the reaction conditions for hands‐on learning. While reconstitution of cellular machinery in vitro for CFPS has been transformative for biotechnology, CFPS has not been accessible to early‐career students due to the complexity in reagent preparation and reaction set‐up. We have successfully adapted the CFPS biotechnology to develop a learning module accessible to early‐career STEM students focusing on learning objectives involving the genetic code. Here we report the reformulation of the CFPS reaction setup for simplicity and improved reagent shelf life in order to adapt CFPS for the classroom. First, our simplified platform requires the pipetting of only 3 pre‐mixed reagents, an 80% reduction in setup steps, making it more accessible to an early‐career student with little‐to‐no laboratory experience. Second, our simplified platform is shelf‐stable at −20°C rather than −80°C, making it accessible for storage and implementation in undergraduate and high school classrooms. Longer‐term experiments are in progress to validate shelf‐life at 4°C and room temperature. Our data show that these improvements are achieved without sacrificing CFPS performance in reaction kinetics or total protein yield. Third, our analysis of green fluorescent protein synthesis kinetics shows that 90% of maximum protein yields can be achieved within 3 hours at 37°C, making this platform compatible with most college laboratory courses. We believe that this educational technology will be transformative to the way the genetic code is taught in classrooms today. Toward this end, we have developed a CFPS classroom ‘kit’ for broad dissemination.Support or Funding InformationOur research is funded by the Center for Applications in Biotechnology @ Cal Poly SLO, Bill and Linda Frost Fund, and NSF‐1708919.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

E. coli‐based cell‐free protein synthesis (CFPS) is a flexible platform technology for on‐demand protein production that allows users to express traditionally intractable proteins, perform high‐throughput screening, and, with augmentation, supports applications such as metabolic engineering and genetic code expansion. The broad utility of the CFPS platform arises from the elimination of the cellular membrane and capture of transcription and translation machinery in vitro. This obviates the need for living cells and creates an open system for direct manipulation of the environment of protein production. However, broad dissemination to field and classroom applications remains limited, as CFPS is dependent on technical scientific expertise for proper reaction setup and laboratory infrastructure for proper storage of reagents. Here, we report our efforts to improve broad accessibility of CFPS by 1) simplifying reaction setup and 2) improving shelf stability of the cell‐extract at various storage conditions. In order to simplify reaction setup, which currently requires precise pipetting of 10 reagents, we have combined the CFPS reagents into two stable premixes. These premixes can be stored at −20 °C for at least 4 months and allow the user to pipette fewer times with larger volumes for more accurate reaction setup by non‐experts. To improve the shelf stability of cell extract, which contains the sensitive transcription and translation machinery and is traditionally stored at −80 °C, we have characterized the functional effects of 10 protein stabilizing additives such as sugars, osmolytes, surfactants, and molecular crowding agents. Tests of single additives and combinations of two or three have allowed us to identify the landscape of small molecule additives that can support a variety of storage conditions ranging from −80 °C to room temperature to suit a variety of applications. When combined, these advances to reaction setup and cell extract composition provide the foundation for broad implementation of CFPS. Improved ease of reaction setup enables the use of CFPS by non‐experts, such as early career students, and improves efficiency for researchers and field applications. Additionally, increased storage stability diminishes reliance on the cold chain, making CFPS reagents easier to disseminate, commercialize, and use in the field. Together, these modifications provide a foundation for democratization of the CFPS platform.Support or Funding InformationOur research is funded by the Center for Applications in Biotechnology, Bill and Linda Frost Fund, and NSF‐1708919.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.