Real-time flow cytometric quantification of GFP expression and Gfp-fluorescence generation in Saccharomyces cerevisiae

A genetic and analytical methodology was developed based on a green fluores cent mutant protein (Gfp(S65T)) that allows the real-time quantification of gene expression in Saccharomyces cerevisiae. Using the UAS(GAL1-10)/CYC1 p romoter and plasmids that an maintained in different copy numbers per cell, wild-type GFP and mutant GFP(S65T) Were expressed in low to high concentra tion. Flow cytometric analysis was then applied to directly quantify Gfp((S 65T)) (both Wild type and mutant protein expression at the single-cell leve l, and to indirectly measure the concentrations of non-fluorescent apoGfp(( S65T)) and fluorescent fluorescent Gfp((S65T)), which is autocatalytically formed from the apoprotein. Kinetics of apoGfp((S65T))/Gfp((S65T)) conversi on during aerobic growth showed that the time required for complete apoGfp( (S65T)) conversion is limited only by the amount of apoprotein that is expr essed. When GFP(S65T) was expressed in single copy, the apoprotein did not accumulate and was instantly converted into its fluorescent form. The data indicate that an instant quantification of gene expression in S. cerevisiae is achievable based on Gfp(S65T), even if the gene is transcribed from a v ery strong promoter. (C) 2000 Elsevier Science B.V. All rights reserved.