Construction and use of a Cupriavidus necator H16 soluble hydrogenase promoter (PSH) fusion to gfp (green fluorescent protein)

Construction and use of a Cupriavidus necator H16 soluble hydrogenase promoter (PSH) fusion to gfp (green fluorescent protein)

Hydrogenases are metalloenzymes that reversibly catalyse the oxidation or production of molecular hydrogen (H2). Amongst a number of promising candidates for application in the oxidation of H2 is a soluble [Ni–Fe] uptake hydrogenase (SH) produced by Cupriavidus necator H16. In the present study, mol...

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Main Authors: Jugder, Bat-Erdene, Welch, Jeffrey, Braidy, Nady, Marquis, Christopher P.
Format: Online
Language:English
Published: PeerJ Inc. 2016
Online Access:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4974937/
id pubmed-4974937
recordtype oai_dc
spelling pubmed-49749372016-08-19 Construction and use of a Cupriavidus necator H16 soluble hydrogenase promoter (PSH) fusion to gfp (green fluorescent protein) Jugder, Bat-Erdene Welch, Jeffrey Braidy, Nady Marquis, Christopher P. Biotechnology Hydrogenases are metalloenzymes that reversibly catalyse the oxidation or production of molecular hydrogen (H2). Amongst a number of promising candidates for application in the oxidation of H2 is a soluble [Ni–Fe] uptake hydrogenase (SH) produced by Cupriavidus necator H16. In the present study, molecular characterisation of the SH operon, responsible for functional SH synthesis, was investigated by developing a green fluorescent protein (GFP) reporter system to characterise PSH promoter activity using several gene cloning approaches. A PSH promoter-gfp fusion was successfully constructed and inducible GFP expression driven by the PSH promoter under de-repressing conditions in heterotrophic growth media was demonstrated in the recombinant C. necator H16 cells. Here we report the first successful fluorescent reporter system to study PSH promoter activity in C. necator H16. The fusion construct allowed for the design of a simple screening assay to evaluate PSH activity. Furthermore, the constructed reporter system can serve as a model to develop a rapid fluorescent based reporter for subsequent small-scale process optimisation experiments for SH expression. PeerJ Inc. 2016-07-26 /pmc/articles/PMC4974937/ /pubmed/27547572 http://dx.doi.org/10.7717/peerj.2269 Text en ©2016 Jugder et al. http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, reproduction and adaptation in any medium and for any purpose provided that it is properly attributed. For attribution, the original author(s), title, publication source (PeerJ) and either DOI or URL of the article must be cited.
repository_type Open Access Journal
institution_category Foreign Institution
institution US National Center for Biotechnology Information
building NCBI PubMed
collection Online Access
language English
format Online
author Jugder, Bat-Erdene
Welch, Jeffrey
Braidy, Nady
Marquis, Christopher P.
spellingShingle Jugder, Bat-Erdene
Welch, Jeffrey
Braidy, Nady
Marquis, Christopher P.
Construction and use of a Cupriavidus necator H16 soluble hydrogenase promoter (PSH) fusion to gfp (green fluorescent protein)
author_facet Jugder, Bat-Erdene
Welch, Jeffrey
Braidy, Nady
Marquis, Christopher P.
author_sort Jugder, Bat-Erdene
title Construction and use of a Cupriavidus necator H16 soluble hydrogenase promoter (PSH) fusion to gfp (green fluorescent protein)
title_short Construction and use of a Cupriavidus necator H16 soluble hydrogenase promoter (PSH) fusion to gfp (green fluorescent protein)
title_full Construction and use of a Cupriavidus necator H16 soluble hydrogenase promoter (PSH) fusion to gfp (green fluorescent protein)
title_fullStr Construction and use of a Cupriavidus necator H16 soluble hydrogenase promoter (PSH) fusion to gfp (green fluorescent protein)
title_full_unstemmed Construction and use of a Cupriavidus necator H16 soluble hydrogenase promoter (PSH) fusion to gfp (green fluorescent protein)
title_sort construction and use of a cupriavidus necator h16 soluble hydrogenase promoter (psh) fusion to gfp (green fluorescent protein)
description Hydrogenases are metalloenzymes that reversibly catalyse the oxidation or production of molecular hydrogen (H2). Amongst a number of promising candidates for application in the oxidation of H2 is a soluble [Ni–Fe] uptake hydrogenase (SH) produced by Cupriavidus necator H16. In the present study, molecular characterisation of the SH operon, responsible for functional SH synthesis, was investigated by developing a green fluorescent protein (GFP) reporter system to characterise PSH promoter activity using several gene cloning approaches. A PSH promoter-gfp fusion was successfully constructed and inducible GFP expression driven by the PSH promoter under de-repressing conditions in heterotrophic growth media was demonstrated in the recombinant C. necator H16 cells. Here we report the first successful fluorescent reporter system to study PSH promoter activity in C. necator H16. The fusion construct allowed for the design of a simple screening assay to evaluate PSH activity. Furthermore, the constructed reporter system can serve as a model to develop a rapid fluorescent based reporter for subsequent small-scale process optimisation experiments for SH expression.
publisher PeerJ Inc.
publishDate 2016
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4974937/
_version_ 1613622199209426944

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