Producing molecular biology reagents without purification

• Published in: PloS one 2021-06, Vol.16 (6), p.e0252507-e0252507
• Main Authors: Bhadra, Sanchita, Nguyen, Vylan, Torres, Jose-Angel, Kar, Shaunak, Fadanka, Stéphane, Gandini, Chiara, Akligoh, Harry, Paik, Inyup, Maranhao, Andre C, Molloy, Jenny, Ellington, Andrew D
• Format: Article
• Language: English
• Subjects: Antibiotics; Bacteria; Bacterial proteins; Biological products; Biology; Biology and Life Sciences; Cameroon - epidemiology; Cellular manufacture; Chemical engineering; Chemical properties; Chemical tests and reagents; Cloning; Cold storage; COVID-19; COVID-19 - diagnosis; COVID-19 - epidemiology; COVID-19 - virology; COVID-19 Testing - methods; COVID-19 Testing - standards; Diagnostic Tests, Routine - standards; DNA polymerase; Dry ice; E coli; Engineering and Technology; Enzymes; Escherichia coli - genetics; Escherichia coli - metabolism; Freeze drying; Gene Expression; Geobacillus stearothermophilus - genetics; Geobacillus stearothermophilus - metabolism; Ghana - epidemiology; Humans; Indicators and Reagents - chemistry; Indicators and Reagents - metabolism; Indicators and Reagents - standards; Indicators and Reagents - supply & distribution; Infrastructure; Liquid nitrogen; Medicine and health sciences; Methods; Molecular biology; Molecular Diagnostic Techniques; Plasmids; Plasmids - chemistry; Plasmids - metabolism; Production processes; Proteins; Reagents; Real-Time Polymerase Chain Reaction - methods; Real-Time Polymerase Chain Reaction - standards; Recombinant Proteins - biosynthesis; Recombinant Proteins - genetics; Research and Analysis Methods; SARS-CoV-2 - genetics; Severe acute respiratory syndrome coronavirus 2; Synthetic biology; Synthetic Biology - methods; Transformation, Bacterial; United Kingdom - epidemiology
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0252507

We recently developed 'cellular' reagents-lyophilized bacteria overexpressing proteins of interest-that can replace commercial pure enzymes in typical diagnostic and molecular biology reactions. To make cellular reagent technology widely accessible and amenable to local production with minimal instrumentation, we now report a significantly simplified method for preparing cellular reagents that requires only a common bacterial incubator to grow and subsequently dry enzyme-expressing bacteria at 37°C with the aid of inexpensive chemical desiccants. We demonstrate application of such dried cellular reagents in common molecular and synthetic biology processes, such as PCR, qPCR, reverse transcription, isothermal amplification, and Golden Gate DNA assembly, in building easy-to-use testing kits, and in rapid reagent production for meeting extraordinary diagnostic demands such as those being faced in the ongoing SARS-CoV-2 pandemic. Furthermore, we demonstrate feasibility of local production by successfully implementing this minimized procedure and preparing cellular reagents in several countries, including the United Kingdom, Cameroon, and Ghana. Our results demonstrate possibilities for readily scalable local and distributed reagent production, and further instantiate the opportunities available via synthetic biology in general.