scholarly journals Using Green Fluorescent Protein to Correlate Temperature and Fluorescence Intensity into Bacterial Systems

2016 ◽  
Vol 4 (1) ◽  
pp. 49-57
Author(s):  
K. Beltran ◽  
◽  
J.M. De Jesus-Miranda ◽  
J. A. Castro ◽  
L. A. Mandujano-rosas ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescence Intensity ◽  
Fluorescent Protein ◽  
Green Fluorescent ◽  
Bacterial Systems

scholarly journals Use of Green Fluorescent Protein To Assess Urease Gene Expression by Uropathogenic Proteus mirabilis during Experimental Ascending Urinary Tract Infection

1998 ◽  
Vol 66 (1) ◽  
pp. 330-335 ◽  
Author(s):  
Hui Zhao ◽  
Richard B. Thompson ◽  
Virginia Lockatell ◽  
David E. Johnson ◽  
Harry L. T. Mobley
Keyword(s):  
Gene Expression ◽  
Urinary Tract Infection ◽  
Green Fluorescent Protein ◽  
Urinary Tract ◽  
Tract Infection ◽  
Fluorescence Intensity ◽  
Proteus Mirabilis ◽  
Fluorescent Protein ◽  
Green Fluorescent

ABSTRACT Proteus mirabilis, a cause of complicated urinary tract infection, expresses urease when exposed to urea. While it is recognized that the positive transcriptional activator UreR induces gene expression, the levels of expression of the enzyme during experimental infection are not known. To investigate in vivo expression of P. mirabilis urease, the gene encoding green fluorescent protein (GFP) was used to construct reporter fusions. Translational fusions of urease accessory gene ureD, which is preceded by a urea-inducible promoter, were made withgfp (modified to express S65T/V68L/S72A [B. P. Cormack et al. Gene 173:33–38, 1996]). Constructs were confirmed by sequencing of the fusion junctions. UreD-GFP fusion protein was induced by urea in both Escherichia coli DH5α and P. mirabilis HI4320. By using Western blotting with antiserum raised against GFP, expression level was shown to correlate with urea concentration (tested from 0 to 500 mM), with highest induction at 200 to 500 mM urea. Fluorescent E. coli and P. mirabilis bacteria were observed by fluorescence microscopy following urea induction, and the fluorescence intensity of GFP in cell lysates was measured by spectrophotofluorimetry. P. mirabilis HI4320 carrying the UreD-GFP fusion plasmid was transurethrally inoculated into the bladders of CBA mice. One week postchallenge, fluorescent bacteria were detected in thin sections of both bladder and kidney samples; the fluorescence intensity of bacteria in bladder tissue was higher than that in the kidney. Kidneys were primarily infected with single-cell-form fluorescent bacteria, while aggregated bacterial clusters were observed in the bladder. Elongated swarmer cells were only rarely observed. These observations demonstrate that urease is expressed in vivo and that using GFP as a reporter protein is a viable approach to investigate in vivo expression ofP. mirabilis virulence genes in experimental urinary tract infection.

scholarly journals Using Fluorescence Intensity of Enhanced Green Fluorescent Protein to Quantify Pseudomonas aeruginosa

Chemosensors ◽  
2018 ◽  
Vol 6 (2) ◽  
pp. 21 ◽  
Author(s):  
Erin Wilson ◽  
Macduff Okuom ◽  
Nathan Kyes ◽  
Dylan Mayfield ◽  
Christina Wilson ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Green Fluorescent Protein ◽  
Fluorescence Intensity ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Green Fluorescent

scholarly journals The Mechanism of Chlorine Damage Using Enhanced Green Fluorescent Protein-Expressing Escherichia coli

Water ◽  
2019 ◽  
Vol 11 (10) ◽  
pp. 2156
Author(s):  
Mizozoe ◽  
Otaki ◽  
Aikawa
Keyword(s):  
Escherichia Coli ◽  
Cell Wall ◽  
Green Fluorescent Protein ◽  
Cell Membrane ◽  
Fluorescence Intensity ◽  
Enhanced Green Fluorescent Protein ◽  
Hypochlorous Acid ◽  
Fluorescent Protein ◽  
E Coli ◽  
Green Fluorescent

This study investigated how chlorine inactivates and damages Escherichia coli cells. E. coli that had transformed to express enhanced green fluorescent protein (EGFP) at the cytoplasm was treated with chlorine. Damage to the cell membrane and cell wall was analyzed by measuring the fluorescence intensity of the leaked EGFP, then accounting for the fluorescence deterioration. At pH 7, E. coli was lethally damaged after treatment with chlorine, but significant leakage of EGFP was not observed. In contrast, significant leakage of EGFP was observed at pH 9, even though E. coli was not as inactivated as it was at pH 7. Flow cytometry was used to confirm the fluorescence intensity of the remaining EGFP inside the cells. No significant fluorescence loss was observed in the cells at pH 7. However, at pH 9, the fluorescence intensity in the cells decreased, indicating leakage of EGFP. These results suggest that hypochlorous acid inactivates E. coli without damaging its cell membrane and cell wall, whereas the hypochlorite ion inactivates E. coli by damaging its cell membrane and cell wall. It was possible to confirm the chlorine damage mechanism on E. coli by measuring the fluorescence intensity of the leaked EGFP.

Reversible biomechano-responsive surface based on green fluorescent protein genetically modified with unnatural amino acids

2015 ◽  
Vol 51 (1) ◽  
pp. 232-235 ◽  
Author(s):  
Johan Longo ◽  
Chunyan Yao ◽  
César Rios ◽  
Nguyet Trang Thanh Chau ◽  
Fouzia Boulmedais ◽  
...  
Keyword(s):  
Amino Acids ◽  
Green Fluorescent Protein ◽  
Fluorescence Intensity ◽  
Unnatural Amino Acids ◽  
Fluorescent Protein ◽  
Genetically Modified ◽  
Uniaxial Stretching ◽  
Green Fluorescent

Uniaxial stretching of modified GFP “clicked” onto an elastomer leads to a repeatable and reversible decrease of its fluorescence intensity.

scholarly journals Use of a Green Fluorescent Protein-Based Reporter Fusion for Detection of Nitric Oxide Produced by Denitrifiers

2003 ◽  
Vol 69 (7) ◽  
pp. 3938-3944 ◽  
Author(s):  
Shixue Yin ◽  
Mayuree Fuangthong ◽  
William P. Laratta ◽  
James P. Shapleigh
Keyword(s):  
Nitric Oxide ◽  
Green Fluorescent Protein ◽  
Fluorescence Intensity ◽  
Nitrite Reductase ◽  
Fluorescent Protein ◽  
Reductase Activity ◽  
Rrna Gene ◽  
Fluorescent Intensity ◽  
Fusion Analysis ◽  
Green Fluorescent

ABSTRACT To determine if green fluorescent protein could be used as a reporter for detecting nitric oxide production, gfp was fused to nnrS from Rhodobacter sphaeroides 2.4.3. nnrS was chosen because its expression requires nitric oxide. The presence of the fusion in R. sphaeroides 2.4.3 resulted in a significant increase in fluorescent intensity of the cells, but only when nitrite reductase was active. Cells lacking nitrite reductase activity and consequently the ability to generate nitric oxide were only weakly fluorescent when grown under denitrification-inducing conditions. One of the R. sphaeroides strains unable to generate nitric oxide endogenously was used as a reporter to detect exogenously produced nitric oxide. Incubation of this strain with sodium nitroprusside, a nitric oxide generator, significantly increased its fluorescence intensity. Mixing of known denitrifiers with the reporter strain also led to significant increases in fluorescence intensity, although the level varied depending on the denitrifier used. The reporter was tested on unknown isolates capable of growing anaerobically in the presence of nitrate, and one of these was able to induce expression of the fusion. Analysis of the 16S rRNA gene sequence of this isolate placed it within the Thauera aromatica subgroup, which is known to contain denitrifiers. These experiments demonstrate that this green fluorescent protein-based assay provides a useful method for assessing the ability of bacteria to produce nitric oxide.

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