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2. Active diagnostic ingredients (ADIs) for PCR: A mini‐bioreactor producing dNTPs with silica immobilized R5‐kinases.

Author

Bird, Anna R. and Hall, Elizabeth A. H.

Abstract

Low availability of routine nucleic acid amplification testing (NAAT) during infection outbreaks, especially in less resourced environments, was highlighted by the Covid pandemic. One of the barriers lies with the supply chain and cost of the active diagnostic ingredients (ADIs) that are the reagents for NAATs. This work explores a novel synthesis method to produce a key NAAT reagent, namely the 2′‐deoxynucleoside 5′‐triphosphate (dNTPs), via a reusable enzyme bioreactor, that can be integrated into a NAAT workflow. A self‐immobilizing R5‐silaffin kinase fusion enzyme was designed for immobilization on silica, converting dNMPs to their respective dNTP ADIs for PCR in a R5‐kinase mini‐bioreactor, designed to be implemented in a reusable device, stable over 2 months, when stored at 4°C. The performance is demonstrated for PCR reactions of the lambda genome and showed successful amplification up to 7.5 kb. In comparison with commercial dNTPs, in Plasmodium malariae NAATs, a high linear correlation was shown between the Ct value and the log(Copy Number), with lower incidence of false positives than with the commercial dNTPs. Overall a pathway to generate deoxynucleotides from monophosphate precursors was demonstrated, and an immobilized enzyme mini‐bioreactor investigated as a proof‐of‐principle for work‐flow integration with NAAT in low‐resource research and diagnostics labs. [ABSTRACT FROM AUTHOR]

Published
2024
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4. Applications of Paper-Based Diagnostics

Author

Akram, Muhammad Safwan, Daly, Ronan, da Cruz Vasconcellos, Fernando, Yetisen, Ali Kemal, Hutchings, Ian, Hall, Elizabeth A. H., Castillo-León, Jaime, editor, and Svendsen, Winnie E., editor

Published
2015
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24. Applications of Paper-Based Diagnostics

Author

Akram, Muhammad Safwan, primary, Daly, Ronan, additional, da Cruz Vasconcellos, Fernando, additional, Yetisen, Ali Kemal, additional, Hutchings, Ian, additional, and Hall, Elizabeth A. H., additional

Published
2014
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38. Plasmid-encoded genes influence exosporium assembly and morphology in Bacillus megaterium QM B1551 spores

Author

Manetsberger, Julia, Hall, Elizabeth A. H., Christie, Graham, Hall, Lisa [0000-0001-9572-9854], Christie, Graham [0000-0001-7177-9646], and Apollo - University of Cambridge Repository

Subjects
Spores, Bacterial, Membrane Glycoproteins, spores, fungi, exosporium, Computational Biology, Bacillus, hairy nap, Bacillus cereus, Bacterial Proteins, Microscopy, Electron, Transmission, Microscopy, Fluorescence, Cell Wall, Genes, Bacterial, indigenous plasmids, Mutation, Research Letter, Environmental Microbiology, Bacillus megaterium, bacteria, Plasmids
Abstract

Spores of Bacillus megaterium QM B1551 are encased in a morphologically distinctive exosporium. We demonstrate here that genes encoded on the indigenous pBM500 and pBM600 plasmids are required for exosporium assembly and or stability in spores of this strain. Bioinformatic analyses identified genes encoding orthologues of the B. cereus-family exosporium nap and basal layer proteins within the B. megaterium genome. Transcriptional analyses, supported by electron and fluorescent microscopy, indicate that the pole-localized nap, identified here for the first time in B. megaterium QM B1551 spores, is comprised of the BclA1 protein. The role of the BxpB protein, which forms the basal layer of the exosporium in B. cereus spores, is less clear since spores of a null mutant strain display an apparently normal morphology. Retention of the localized nap in bxpB null spores suggests that B. megaterium employs an alternative mechanism to that used by B. cereus spores in anchoring the nap to the spore surface., Essential components of the outermost shell that envelopes Bacillus megaterium spores are encoded on plasmid-located genes., Graphical Abstract Figure. Essential components of the outermost shell that envelopes Bacillus megaterium spores are encoded on plasmid-located genes.

Published
2015

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