Preparation and Use of Cellular Reagents: A Low‐resource Molecular Biology Reagent Platform

Abstract

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

Open Educational Resources for distributed hands-on teaching in molecular biology

“…These reductions have enabled new hands-on modules in three other courses as well as training workshops for high school teachers. Further reductions can be achieved, for instance with the use of cellular reagents [10,69] to replace enzyme purification protocols.…”

Vivid COVID-19 LAMP is an ultrasensitive, quadruplexed test using LNA-modified primers and a zinc ion and 5-Br-PAPS colorimetric detection system

“…Future research should address some minor limitations: 1) since our ZBP master mix is based on proprietary commercial enzymes, adapting this assay to use open source enzymes 41 , 66 , 93 , 118 – 120 (e.g., Bst large fragment derivatives, MashUp-RT, RTX, etc.) can facilitate scalability by reducing costs, broadening access, and mitigating reagent supply constraints; 2) developing a lyophilized version of our ZBP master mix to eliminate the necessity for cold chain storage and transport 66 , 119 , 120 ; 3) while we implemented a machine-guided mobile application to help classify color change, it depends on a stable environment provided by a light box. In future work an automated analysis without a stable environment could be implemented that can make system decisions closer to a human operator.…”

Specific Nucleic AcId Ligation for the detection of Schistosomes: SNAILS

“…Whilst the optimisation of biosensor probes has been an important aspect of this study, it is conceivable that other aspects of the 'SNAILS' assay could be refined in future studies. For example, the volumes or concentrations of assay reagents could be further optimised and open access enzymes (e.g., T7 polymerase) [53,54] could also be tested to reduce costs even further. Likewise, additional fluorescent molecule-binding aptamers could be utilised to enable compatibility with additional laboratory or in-field equipment [54,55] or enable assay multiplexing (detection of several DNA sequences within the same assay well).…”

“…For example, the volumes or concentrations of assay reagents could be further optimised and open access enzymes (e.g., T7 polymerase) [53,54] could also be tested to reduce costs even further. Likewise, additional fluorescent molecule-binding aptamers could be utilised to enable compatibility with additional laboratory or in-field equipment [54,55] or enable assay multiplexing (detection of several DNA sequences within the same assay well). The global availability of PCR equipment has also rapidly expanded during the last several years and could be exploited to improve the accessibility of 'SNAILS' biosensor assays in different geographical locations.…”