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

Promoter Activity ◽

Fluorescent Protein ◽

Cupriavidus Necator ◽

Small Scale ◽

Growth Media ◽

Reporter System ◽

Screening Assay ◽

Fusion Construct ◽

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.

Construction and use of aCupriavidus necatorH16 soluble hydrogenase promoter (PSH) fusion togfp(green fluorescent protein)

PeerJ ◽

10.7717/peerj.2269 ◽

2016 ◽

Vol 4 ◽

pp. e2269 ◽

Cited By ~ 4

Author(s):

Bat-Erdene Jugder ◽

Jeffrey Welch ◽

Nady Braidy ◽

Christopher P. Marquis

Keyword(s):

Promoter Activity ◽

Fluorescent Protein ◽

Small Scale ◽

Growth Media ◽

Reporter System ◽

Screening Assay ◽

Fluorescent Reporter ◽

Fusion Construct ◽

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 H2is a soluble [Ni–Fe] uptake hydrogenase (SH) produced byCupriavidus necatorH16. 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 PSHpromoter activity using several gene cloning approaches. A PSHpromoter-gfp fusion was successfully constructed and inducible GFP expression driven by the PSHpromoter under de-repressing conditions in heterotrophic growth media was demonstrated in the recombinantC. necatorH16 cells. Here we report the first successful fluorescent reporter system to study PSHpromoter activity inC. necatorH16. The fusion construct allowed for the design of a simple screening assay to evaluate PSHactivity. 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.

Identification of mycoplasmal promoters in Escherichia coli using a promoter probe vector with Green Fluorescent Protein as reporter system

Gene ◽

10.1016/s0378-1119(98)00260-1 ◽

1998 ◽

Vol 215 (1) ◽

pp. 213-222 ◽

Cited By ~ 12

Author(s):

S. Dhandayuthapani ◽

W.G. Rasmussen ◽

J.B. Baseman

Keyword(s):

Escherichia Coli ◽

Fluorescent Protein ◽

Reporter System ◽

Promoter Probe ◽

Promoter Probe Vector ◽

Novel reverse genetics of genotype I and III Japanese encephalitis viruses assembled through transformation associated recombination in yeast: The reporter viruses expressing a green fluorescent protein for the antiviral screening assay

Antiviral Research ◽

10.1016/j.antiviral.2021.105233 ◽

2021 ◽

pp. 105233

Author(s):

Chenxi Li ◽

Xuan Chen ◽

Yanyang Zhou ◽

Jingbo Hu ◽

Xinjie Wang ◽

...

Keyword(s):

Japanese Encephalitis ◽

Reverse Genetics ◽

Fluorescent Protein ◽

Screening Assay ◽

Genotype I ◽

Use of Green Fluorescent Protein To Monitor Cell Envelope Stress in Lactococcus lactis

Applied and Environmental Microbiology ◽

10.1128/aem.02177-09 ◽

2009 ◽

Vol 76 (3) ◽

pp. 978-981 ◽

Cited By ~ 3

Author(s):

Ana Belén Campelo ◽

Ana Rodríguez ◽

Beatriz Martínez

Keyword(s):

Lactococcus Lactis ◽

Fluorescent Protein ◽

Cell Envelope ◽

Dot Blot ◽

Reporter System ◽

Fast Detection ◽

Envelope Stress ◽

Dot Blot Assay ◽

ABSTRACT A Lactococcus lactis reporter system suitable to detect cell envelope stress in high-throughput settings was developed by fusing the CesR-regulated promoter of llmg0169 to the gfpuv gene. A dot blot assay allowed fast detection of green fluorescent protein (GFP) fluorescence even at low production levels. Unexpectedly, this promoter was also induced by mitomycin C via CesR.