Characterization of Fluorescent Reporters for Flow Cytometry-Based Single-Cell Studies in Saccharomyces cerevisiae

Abstract

Fluorescent proteins (FPs) are commonly used as reporters to examine intracellular genetic, molecular, and biochemical status. Flow cytometry is a powerful technique for accurate quantification of single-cell fluorescent levels. Here, we characterize green, red, and blue FPs for use in yeast Saccharomyces cerevisiae. Fluorophore-containing FPs and fluorogen-activating FPs (YFAST and FrFAST) were characterized dynamically in batch cultivation. FPs with a low pK_a, StayGold, E2-Crimson, mTagBFP2, and mScarlet-I3, showed relatively stable fluorescence in diauxic growth when they were expressed under the control of the TEF1 promoter. A pH sensor, based on the fusion of mNeonGreen and mTagBFP2, was used to investigate the dynamic change of intracellular pH in batch cultivation. High-concentration acetate (200 mM) interfered with intracellular pH dramatically, whereas low-concentration acetate (20 mM) could not. Using StayGold and mScarlet-I3, an Epac-based cAMP sensor was constructed, showing varied Förster resonance energy transfer (FRET) patterns in different growth phases in S. cerevisiae CEN.PK113–7D and EBY100 backgrounds. In summary, the change in intracellular pH can significantly affect the brightness of pH-sensitive FPs. It is important to use FPs with a low pK_a, neutralize intracellular pH, or compensate pH impacts when FPs are used as reporters.