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

PeerJ ◽  
2016 ◽  
Vol 4 ◽  
pp. e2269 ◽  
Author(s):  
Bat-Erdene Jugder ◽  
Jeffrey Welch ◽  
Nady Braidy ◽  
Christopher P. Marquis
Keyword(s):  
Green Fluorescent Protein ◽  
Promoter Activity ◽  
Fluorescent Protein ◽  
Small Scale ◽  
Growth Media ◽  
Reporter System ◽  
Screening Assay ◽  
Fluorescent Reporter ◽  
Fusion Construct ◽  
Green Fluorescent

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.

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

2016 ◽  
Author(s):  
Bat-Erdene Jugder ◽  
Jeffrey Welch ◽  
Nady Braidy ◽  
Christopher P Marquis
Keyword(s):  
Green Fluorescent Protein ◽  
Promoter Activity ◽  
Fluorescent Protein ◽  
Cupriavidus Necator ◽  
Small Scale ◽  
Growth Media ◽  
Reporter System ◽  
Screening Assay ◽  
Fusion Construct ◽  
Green Fluorescent

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.

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

2016 ◽  
Author(s):  
Bat-Erdene Jugder ◽  
Jeffrey Welch ◽  
Nady Braidy ◽  
Christopher P Marquis
Keyword(s):  
Green Fluorescent Protein ◽  
Promoter Activity ◽  
Fluorescent Protein ◽  
Cupriavidus Necator ◽  
Small Scale ◽  
Growth Media ◽  
Reporter System ◽  
Screening Assay ◽  
Fusion Construct ◽  
Green Fluorescent

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.

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

2009 ◽  
Vol 76 (3) ◽  
pp. 978-981 ◽  
Author(s):  
Ana Belén Campelo ◽  
Ana Rodríguez ◽  
Beatriz Martínez
Keyword(s):  
Green Fluorescent Protein ◽  
Lactococcus Lactis ◽  
Fluorescent Protein ◽  
Cell Envelope ◽  
Dot Blot ◽  
Reporter System ◽  
Fast Detection ◽  
Envelope Stress ◽  
Dot Blot Assay ◽  
Green Fluorescent

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.

scholarly journals Genetic Tools To Enhance the Study of Gene Function and Regulation in Staphylococcus aureus

2013 ◽  
Vol 79 (7) ◽  
pp. 2218-2224 ◽  
Author(s):  
Jeffrey L. Bose ◽  
Paul D. Fey ◽  
Kenneth W. Bayles
Keyword(s):  
Staphylococcus Aureus ◽  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Exchange System ◽  
Fluorescent Reporter ◽  
Content Type ◽  
Genetic Tools ◽  
Allelic Exchange ◽  
Green Fluorescent

ABSTRACTThebursa aurealistransposon has been used to create transposon insertion libraries ofBacillus anthracisandStaphylococcus aureus. To provide a set of genetic tools to enhance the utility of these libraries, we generated an allelic-exchange system that allows for the replacement of the transposon with useful genetic markers and fluorescent reporter genes. These tools were tested in the Nebraska Transposon Mutant Library (NTML), containing defined transposon insertions in 1,952 nonessentialS. aureusgenes. First, we generated a plasmid that allows researchers to replace the genes encoding green fluorescent protein (GFP) and erythromycin resistance in the transposon with a noncoding DNA fragment, leaving a markerless mutation within the chromosome. Second, we produced allelic-exchange plasmids to replace the transposon with alternate antibiotic resistance cassettes encoding tetracycline, kanamycin, and spectinomycin resistance, allowing for the simultaneous selection of multiple chromosomal mutations. Third, we generated a series of fluorescent reporter constructs that, after allelic exchange, generate transcriptional reporters encoding codon-optimized enhanced cyan fluorescent protein (ECFP), enhanced yellow fluorescent protein (EYFP), DsRed.T3(DNT), and eqFP650, as well as superfolder green fluorescent protein (sGFP). Overall, combining the NTML with this allelic-exchange system provides an unparalleled resource for the study ofS. aureus.

scholarly journals Analysis of Proteasome-Dependent Proteolysis in Haloferax volcanii Cells, Using Short-Lived Green Fluorescent Proteins

2004 ◽  
Vol 70 (12) ◽  
pp. 7530-7538 ◽  
Author(s):  
Christopher J. Reuter ◽  
Julie A. Maupin-Furlow
Keyword(s):  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Specific Inhibitor ◽  
Haloferax Volcanii ◽  
Reporter System ◽  
Reporter Protein ◽  
Protein Variant ◽  
Fluorescent Reporter ◽  
New Genes ◽  
Green Fluorescent

ABSTRACT Proteasomes are energy-dependent proteases that are central to the quality control and regulated turnover of proteins in eukaryotic cells. Dissection of this proteolytic pathway in archaea, however, has been hampered by the lack of substrates that are easily detected in whole cells. In the present study, we developed a convenient reporter system by functional expression of a green fluorescent protein variant with C-terminal fusions in the haloarchaeon Haloferax volcanii. The levels of this reporter protein correlated with whole-cell fluorescence that was readily detected in culture. Accumulation of the reporter protein was dependent on the sequence of the C-terminal amino acid fusion, as well as the presence of an irreversible, proteasome-specific inhibitor (clasto-lactacystin β-lactone). This inhibitor was highly specific for H. volcanii 20S proteasomes, with a Ki of ∼40 nM. In contrast, phenylmethanesulfonyl fluoride did not influence the levels of fluorescent reporter protein or inhibit 20S proteasomes. Together, these findings provide a powerful tool for the elucidation of protein substrate recognition motifs and the identification of new genes which may be involved in the proteasome pathway of archaea.

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