Quantitative Analysis of Promoter Activity by Green Fluorescent Protein (GFP) Target/Reporter Strategy in a Novel Transgenic alx/fpr-rs2 Null Mouse

2009 ◽  
Vol 58 (S2) ◽  
pp. S252-S255
Author(s):  
Neil Peter Dufton ◽  
R. Hannon ◽  
M. Perretti ◽  
R. Flower
Keyword(s):  
Green Fluorescent Protein ◽  
Quantitative Analysis ◽  
Promoter Activity ◽  
Fluorescent Protein ◽  
Null Mouse ◽  
Green Fluorescent

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.

Differential CTX-M Expression from a Conserved Promoter: Role of Promoter-Associated Spacer Sequences Downstream of the blaCTX-M Regulon

2016 ◽  
Vol 26 (4) ◽  
pp. 284-290 ◽  
Author(s):  
Lin Liu ◽  
Xiangyan Zhang ◽  
Siyuan Yang ◽  
Yao Zhai ◽  
Weijia Liu ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Green Fluorescent Protein ◽  
Promoter Activity ◽  
Expression Vector ◽  
Fluorescent Protein ◽  
Gfp Expression ◽  
Qrt Pcr ◽  
Key Factor ◽  
Green Fluorescent

<b><i>Aims:</i></b> The aim of this project was to explore the different CTX-M expression levels occurring from a single conserved promoter with different spacer sequences, the variation of which is hypothesized to be a key factor in fluctuating levels of CTX-M. <b><i>Methods:</i></b> The <i>bla</i><sub>CTX-M</sub> promoter fragments with five different spacer sequences were amplified, sequenced and cloned into the pUA66 expression vector carrying the green fluorescent protein (GFP) gene. The expression of <i>bla</i><sub>CTX-M</sub> in the transconjugants was analyzed using fluorescence microscopy, flow cytometry and qRT-PCR. <b><i>Results:</i></b> The promoters of all the <i>bla</i><sub>CTX-M</sub> genes were provided by IS<i>Ecp1 </i>and were extremely conserved. The promoter-associated spacer sequences varied from 42 to 127 bp and variations in GFP expression in the five transconjugants were observed. A nucleic acid deletion and point mutation were detected in the spacer sequences by variations in which the expression of <i>bla</i><sub>CTX-M</sub> was influenced. <b><i>Conclusion:</i></b> The different spacer sequences have a significant impact on the activity of the conserved promoter. The shorter spacer sequence between the conserved promoter and the <i>bla</i><sub>CTX-M</sub> gene does not specifically enhance the expression of<i> bla</i><sub>CTX-M</sub>, contrary to previous reports. The expression of <i>bla</i><sub>CTX-M</sub> may be regulated by changes in promoter activity caused by diverse spacer sequences.

scholarly journals Green Fluorescent Protein as a Quantitative Reporter of Relative Promoter Activity in E. coli

BioTechniques ◽  
2000 ◽  
Vol 28 (1) ◽  
pp. 82-89 ◽  
Author(s):  
J.L. Lissemore ◽  
J.T. Jankowski ◽  
C.B. Thomas ◽  
D.P. Mascotti ◽  
P.L. deHaseth
Keyword(s):  
Green Fluorescent Protein ◽  
Promoter Activity ◽  
Fluorescent Protein ◽  
E Coli ◽  
Green Fluorescent

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.

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