Cell-free protein synthesis (CFPS) is a platform biotechnology that has enabled the on-demand synthesis of proteins for a variety of applications. Numerous advances have improved the productivity of the CFPS platform to result in highyielding reactions; however, many applications remain limited due to long reaction times. To overcome this limitation, we first established the benchmarks reaction times for CFPS across inhouse E. coli extracts and commercial kits. We then set out to finetune our in-house extract systems to improve reaction times. Through the optimization of reaction composition and titration of low-cost additives, we have identified formulations that reduce reaction times by 30−50% to obtain high protein titers for biomanufacturing applications, and reduce times by more than 50% to reach the sfGFP detection limit for applications in education and diagnostics. Under optimum conditions, we report the visible observation of sfGFP signal in less than 10 min. Altogether, these advances enhance the utility of CFPS as a rapid, user-defined platform.

Fluorescent reporters have revolutionized modern applications in the fields of molecular and synthetic biology, enabling applications ranging from education to point-of-care diagnostics. Past advancements in these fields have primarily focused on improving reaction conditions, the development of new applications, and the broad dissemination of these technologies. However, field and classroom-based applications have remained limited in part due to the nature of fluorescent signal detection, which often requires the use of costly lab equipment to observe and quantify fluorescence readouts. Users without access to laboratory equipment rely on qualitative assessments of fluorescence, a process that remains highly variable from user-to-user even within the same classroom. To overcome this challenge, we have developed a foldable illuminator and incubator device to support field-applications of synthetic biology-based biosensors for education and diagnostics. The Fold-Illuminator is an affordable, portable, and recyclable device that allows for the visible detection of fluorescent biomolecules. The Fold-Illuminator's design allows for assembly in under 10 min, a user can then utilize the optional heating element to incubate biochemical reactions and visualize fluorescence outputs in a defined and light-controlled environment. Interchangeable LED strips and light-filtering screens provide modularity to pair with the fluorescence wavelengths of interest. The user can then unfold the device for convenient storage, transport, or even recycling. The cost for the Fold-Illuminator is $5.58 USD and is compatible with an optional heating element for an additional $3.98 cost, with potential for further reductions in cost for larger quantities. Open-source templates for cutting device parts from paper stock are provided for both printing and cutting by hand; cutting can also be achieved with consumer-grade smart cutting machines such as the Cricut®. Combined with the broad applications of fluorescent reporters, the Fold-Illuminator has the potential to improve access to fluorescence visualization and quantification for new users as well as emerging field applications.

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We have implemented the Genetic Code kit into classrooms at Cal Poly, San Luis Obispo for a hands‐on inquiry‐based module of transcription and translation. Our Genetic Code kit is based on cell‐free protein synthesis (CFPS) which captures the transcription and translation machinery required for protein synthesis in vitro, allowing for direct manipulation of protein production. By obviating the use of a living cell, CFPS allows early‐career STEM students to observe protein synthesis. To observe the process, superfolder green fluorescent protein (sfGFP), is used as a reporter protein. The detectable fluorescence of sfGFP gives students real‐time feedback of protein synthesis occurring. However, this process takes upwards of three hours for complete synthesis and maturation. To be more applicable in an educational setting, CFPS needs to occur in a more time‐efficient manner. Thus, this work focuses on identifying the rate‐limiting factors of CFPS. These rate‐limiting factors can be organized into two classes. The first class consists of energy sources and small molecules, and the second class consists of protein‐based translation factors. Direct titrations of these small molecules and proteins into CFPS reactions were performed. Here, we report our findings of key additives that increase the translation rate in cell‐free. Additionally, miscellaneous small molecules, such as osmolytes and detergents, have also been found to increase the reaction rate. The translation factors currently being studied are: prokaryotic initiation factors, elongation factors, release factors, ribosome recycling factor and the methionyl transferase. By optimizing the process of CFPS, the platform can become time‐efficient for a single lab setting. To date we have decreased reaction time by 30%, significantly improving the broad applicability of the Genetic Code Kit in classrooms. Support or Funding Information Our research is funded by the Center for Applications in Biotechnology, the Bill and Linda Frost Fund, and NSF‐1708919.

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