Open AccessQuantitative analysis of gene expression with an improved green fluorescent protein
Author(s) -
Scholz Oliver,
Thiel Anja,
Hillen Wolfgang,
Niederweis Michael
Publication year - 2000
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1046/j.1432-1327.2000.01170.x
Subject(s) - green fluorescent protein , promoter , reporter gene , microbiology and biotechnology , fluorescence , gene , gene expression , lac operon , chemistry , biology , escherichia coli , biochemistry , physics , quantum mechanics
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 β‐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.