Green fluorescent protein in Saccharomyces cerevisiae: real-time studies of the GAL1 promoter.
Green fluorescent protein (GFP) was used to study the regulation of the galactose-inducible GAL1 promoter in yeast Saccharomyces cerevisiae strains. GFP was cloned into the pGAL110 vector and transformed into the yeast strains. Time course studies comparing culture fluorescence intensity and GFP concentration were conducted along with on-line monitoring of GFP expression. Our results demonstrated that GFP fluorescence could be used as a quantifiable on-line reporter gene in yeast strains. The effect of an integrated GAL10p-GAL4 transcription cassette was investigated. Induction time studies showed that there was no significant difference in GFP expression level by adding galactose at different culture times. A wide range of galactose concentrations was used to study the initial galactose concentration effect on GFP expression kinetics. A minimum of 0.05 g/L galactose doubled the GFP fluorescence signal as compared to the control, whereas 0.1 g/L gave the highest specific GFP yield. A simple analytical model was proposed to describe GFP expression kinetics based on the experimental results. In addition, this GFP-based approach was shown to have potential use for high-throughput studies. The use of GFP as a generic tool provided important insights to the GAL expression system and has great potential for further process optimization applications.
Li
J
J
Medical Biotechnology Center, University of Maryland Biotechnology Institute, Baltimore, Maryland 21201, USA.
Wang
S
S
VanDusen
W J
WJ
Schultz
L D
LD
George
H A
HA
Herber
W K
WK
Chae
H J
HJ
Bentley
W E
WE
Rao
G
G
D015153:Blotting, Western; D004268:DNA-Binding Proteins; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D000431:Ethanol / Q000378:metabolism; D005656:Fungal Proteins / Q000378:metabolism; D005690:Galactose / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism; D049452:Green Fluorescent Proteins; D007700:Kinetics; D008164:Luminescent Proteins / Q000235:genetics* / Q000378:metabolism*; D008954:Models, Biological; D008962:Models, Theoretical; D011401:Promoter Regions, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D013053:Spectrophotometry; D013997:Time Factors; D014157:Transcription Factors / Q000378:metabolism; D014170:Transformation, Genetic
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C052130:GAL4 protein, S cerevisiae; D008164:Luminescent Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D049452:Green Fluorescent Proteins; D000431:Ethanol; D005947:Glucose; D005690:Galactose