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Characterization of Fluorescent Reporters for Flow Cytometry-Based Single-Cell Studies in Saccharomyces cerevisiae.
Samuel Evans1,2, Masahiro Tominaga3,4, Zeyu Lu1,2
1ARC Centre of Excellence in Synthetic Biology, Queensland University of Technology, Brisbane, QLD 4000, Australia.
ACS Synthetic Biology
|September 10, 2025
Summary
Fluorescent proteins (FPs) are vital reporters in yeast, but intracellular pH changes can impact their brightness. Researchers recommend using low pKa FPs or adjusting pH for accurate measurements.
Area of Science:
- Biotechnology
- Molecular Biology
- Microbiology
Background:
- Fluorescent proteins (FPs) are essential tools for monitoring cellular states in yeast (Saccharomyces cerevisiae).
- Flow cytometry enables precise single-cell quantification of FP fluorescence.
- Characterizing FPs under dynamic conditions is crucial for reliable reporter applications.
Purpose of the Study:
- To characterize various green, red, and blue FPs for yeast applications.
- To assess the impact of intracellular pH on FP performance.
- To develop and validate biosensors for intracellular pH and cAMP dynamics.
Main Methods:
- Dynamic characterization of fluorophore-containing and fluorogen-activating FPs in batch cultivation.
- Construction and testing of a pH sensor using mNeonGreen and mTagBFP2.
- Development of a cAMP sensor utilizing StayGold and mScarlet-I3 for Förster Resonance Energy Transfer (FRET) analysis.
Main Results:
- FPs with low pKa, including StayGold, E2-Crimson, mTagBFP2, and mScarlet-I3, exhibited stable fluorescence during diauxic growth.
- High acetate concentrations (200 mM) significantly altered intracellular pH, while low concentrations (20 mM) did not.
- The cAMP sensor displayed varying FRET patterns across different growth phases in yeast.
Conclusions:
- Intracellular pH fluctuations substantially influence the brightness of pH-sensitive FPs.
- Selecting FPs with low pKa, maintaining neutral intracellular pH, or implementing pH compensation strategies are critical for accurate FP-based reporting in yeast.
