Producing molecular biology reagents without purification

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.

doi:10.1371/journal.pone.0252507

Cited by 10 publications

(16 citation statements)

References 45 publications

(53 reference statements)

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“…Although cellular reagents can be used for a variety of molecular biology applications, their use in nucleic acid amplification assays is especially noteworthy because, in most cases, their performance has been shown to be on par with that of pure proteins (Bhadra, Nguyen, et al., 2021; Bhadra, Pothukuchy, et al., 2018). When performing nucleic acid amplification reactions such as PCR or LAMP, it is imperative to minimize sources of cross‐contamination with templates.…”

Section: Commentarymentioning

confidence: 99%

“…Cellular reagents prepared using Basic Protocol 1 usually contain some viable bacteria (Bhadra, Nguyen, et al, 2021). Although over time, and during storage, bacterial viability declines, it may be necessary in some cases, such as for off-site distribution, to ensure the absence of live bacteria in preparations of cellular reagents.…”

Section: Preparation Of Lyophilized Cellular Reagentsmentioning

confidence: 99%

“…Both thermostable and mesophilic proteins, such as MLV RT, have been used in the cellular reagent format without pre‐processing. In most analytical applications, such as qPCR, reverse transcription qPCR, endpoint PCR analyzed by agarose gel electrophoresis, and LAMP read using fluorogenic OSD probes (Jiang et al., 2015), the performance of cellular reagents is comparable to that of reactions carried out with purified counterparts, as measured by amplification efficiency, detection limit, and time to result (Bhadra, Nguyen, et al., 2021; Bhadra, Pothukuchy, et al., 2018). Similarly, cellular reagents can also be used as synthetic biology tools for building new DNA constructs via multi‐enzymatic assembly methods, such as Golden Gate and Gibson assembly procedures (although current efficiencies may be suboptimal for the construction of large libraries; Bhadra, Nguyen, et al., 2021; Bhadra, Pothukuchy, et al., 2018).…”

Section: Commentarymentioning

confidence: 99%

“…The accumulating PCR amplicons may be analyzed in real time using DNA-binding dyes or nucleic acid probes, for applications in diagnostics or detection. PCR products may also be visualized at the endpoint, for instance, by using agarose gel electrophoresis, and may then be used in downstream reactions, such as DNA assembly (Bhadra, Nguyen, et al, 2021;Bhadra, Pothukuchy, et al, 2018). In this protocol, Taq DNA polymerase cellular reagents produced by following Basic Protocol 1 are rehydrated and used instead of pure Taq DNA polymerase for performing a real-time PCR assay designed to amplify a region of the Chlamydia trachomatis 16S rRNA gene (see Table 2).…”

Section: Using Taq Dna Polymerase Cellular Reagents For Pcrmentioning

confidence: 99%

“…These cellular reagents, which can be used directly to carry out numerous molecular biology reactions (see below), do not require protein purification or a cold chain. As such, they are considerably simpler and cheaper to produce than purified or commercial enzymes (Bhadra, Nguyen, et al., 2021).…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2022

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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

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Section: Commentarymentioning

confidence: 99%

Section: Preparation Of Lyophilized Cellular Reagentsmentioning

confidence: 99%

Section: Commentarymentioning

confidence: 99%

Section: Using Taq Dna Polymerase Cellular Reagents For Pcrmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

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Biocatalytic synthesis of 2′‐deoxynucleotide 5′‐triphosphates from bacterial genomic DNA: Proof of principle

Bird

Molloy

Hall

2023

Biotech & Bioengineering

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2′-deoxynucleoside 5′-triphosphates (dNTPs) are the building blocks of DNA and are key reagents which are incorporated by polymerase enzymes during nucleic acid amplification techniques, such as polymerase chain reaction (PCR).These techniques are of high importance, not only in molecular biology research, but also in molecular diagnostics. dNTPs are generally produced by a bottom-up technique which relies on synthesis or isolation of purified small molecules like deoxynucleosides. However, the disproportionately high cost of dNTPs in lowand middle-income countries (LMICs) and the requirement for cold chain storage during international shipping makes an adequate supply of these molecules challenging. To reduce supply chain dependency and promote domestic manufacturing in LMICs, a unique top-down biocatalytic synthesis method is described to produce dNTPs. Readily available bacterial genomic DNA provides a crude source material to generate dNTPs and is extracted directly from Escherichia coli (step 1). Nuclease enzymes are then used to digest the genomic DNA creating monophosphorylated deoxynucleotides (dNMPs) (step 2). Design and recombinant production and characterization of E. coli nucleotide kinases is presented to further phosphorylate the monophosphorylated products to generate dNTPs (step 3). Direct use of the in-house produced dNTPs in nucleic acid amplification is shown (step 4) and their successful use as reagents in the application of PCR, thereby providing proof of principle for the future development of recombinant nucleases and design of a recombinant solidstate bioreactor for on-demand dNTP production.

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