Quantitative analysis of gene expression with an improved green fluorescent protein. p6.
The fast and easy in vivo detection predestines the green fluorescent protein (GFP) for its use as a reporter to quantify promoter activities. We have increased the sensitivity of GFP detection 320-fold compared to the wild-type by constructing gfp+, which contains mutations improving the folding efficiency and the fluorescence yield of GFP+. Twelve expression levels were measured using fusions of the gfp+ and lacZ genes with the tetA promoter in Escherichia coli. The agreement of GFP+ fluorescence with beta-galactosidase activities was excellent, demonstrating that the gfp+ gene can be used to accurately quantify gene expression in vivo. However, expression of the gfp+ gene from the stronger hsp60 promoter revealed that high cellular concentrations of GFP+ caused an inner filter effect reducing the fluorescence by 50%, thus underestimating promoter activity. This effect is probably due to the higher absorbance of cells containing GFP+. Thus promoters with activities differing by about two orders of magnitude can be correctly quantified using the gfp+ gene. Possibilities of using GFP variants beyond this range are discussed.
Scholz
O
O
Lehrstuhl für Mikrobiologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany.
Thiel
A
A
Hillen
W
W
Niederweis
M
M
D017920:Antiporters / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics; D018834:Chaperonin 60 / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005470:Fluorometry; D015870:Gene Expression; D017930:Genes, Reporter; D005813:Genes, Synthetic; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins; D007763:Lac Operon; D008164:Luminescent Proteins / Q000032:analysis* / Q000737:chemistry / Q000235:genetics; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D017920:Antiporters; D001426:Bacterial Proteins; D018834:Chaperonin 60; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; C079051:tetA protein, Bacteria; D049452:Green Fluorescent Proteins