Nuclera (United Kingdom)

Protein reagents are indispensable for most molecular and synthetic biology procedures. Most conventional protocols rely on highly purified protein reagents that require considerable expertise, time, and infrastructure to produce. In consequence, most proteins are acquired from commercial sources, reagent expense is often high, and accessibility may be hampered by shipping delays, customs barriers, geopolitical constraints, and the need for a constant cold chain. Such limitations to the widespread availability of protein reagents, in turn, limit the expansion and adoption of molecular biology methods in research, education, and technology development and application. Here, we describe protocols for producing a low-resource and locally sustainable reagent delivery system, termed "cellular reagents," in which bacteria engineered to overexpress proteins of interest are dried and can then be used directly as reagent packets in numerous molecular biology reactions, without the need for protein purification or a constant cold chain. As an example of their application, we describe the execution of polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) using cellular reagents, detailing how to replace pure protein reagents with optimal amounts of rehydrated cellular reagents. We additionally describe a do-it-yourself fluorescence visualization device for using these cellular reagents in common molecular biology applications. The methods presented in this article can be used for low-cost, on-site production of commonly used molecular biology reagents (including DNA and RNA polymerases, reverse transcriptases, and ligases) with minimal instrumentation and expertise, and without the need for protein purification. Consequently, these methods should generally make molecular biology reagents more affordable and accessible. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Preparation of cellular reagents Alternate Protocol 1: Preparation of lyophilized cellular reagents Alternate Protocol 2: Evaluation of bacterial culture growth via comparison to McFarland turbidity standards Support Protocol 1: SDS-PAGE for protein expression analysis of cellular reagents Basic Protocol 2: Using Taq DNA polymerase cellular reagents for PCR Basic Protocol 3: Using Br512 DNA polymerase cellular reagents for loop-mediated isothermal amplification (LAMP) Support Protocol 2: Building a fluorescence visualization device.

Cell-free systems are a rapidly expanding platform technology with an important role in the engineering of biological systems. The key advantages that drive their broad adoption are increased efficiency, versatility, and low cost compared to in vivo systems. Traditionally, in vivo platforms have been used to synthesize novel and industrially relevant proteins and serve as a testbed for prototyping numerous biotechnologies such as genetic circuits and biosensors. Although in vivo platforms currently have many applications within biotechnology, they are hindered by time-constraining growth cycles, homeostatic considerations, and limited adaptability in production. Conversely, cell-free platforms are not hindered by constraints for supporting life and are therefore highly adaptable to a broad range of production and testing schemes. The advantages of cell-free platforms are being leveraged more commonly by the biotechnology community, and cell-free applications are expected to grow exponentially in the next decade. In this study, new and emerging applications of cell-free platforms, with a specific focus on cell-free protein synthesis (CFPS), will be examined. The current and near-future role of CFPS within metabolic engineering, prototyping, and biomanufacturing will be investigated as well as how the integration of machine learning is beneficial to these applications.