Cytosolic expression of Green Fluorescent Protein (GFP) and its derivatives in the yeast Saccharomyces cerevisiae: Detection in vivo using the Agilent Cary Eclipse

Importance of the Topic

The remarkable ability of green fluorescent protein (GFP) and its derivatives to mature autonomously without exogenous cofactors has transformed in vivo imaging of cellular processes. GFP enables noninvasive, real-time monitoring of gene expression, protein localization, and physiological dynamics in living cells, reducing damage associated with conventional fluorescence staining.

Study Objectives and Overview

This application note demonstrates the detection and characterization of cytosolically expressed GFP variants in Saccharomyces cerevisiae using an Agilent Cary Eclipse fluorescence spectrophotometer. The study aims to establish reliable protocols for recording excitation and emission spectra under controlled temperature and stirring conditions.

Methodology and Instrumentation

Yeast strain YRD15 was transformed with plasmids encoding green, blue, cyan, yellow GFP derivatives and red fluorescent protein (DsRed). Transformants were grown on selective minimal medium, washed, and adjusted to an optical density of 0.55 at 650 nm.

Instrumentation Used:

• Agilent Cary Eclipse fluorescence spectrophotometer
• Peltier-thermostatted multicell holder with magnetic stirring
• Temperature controller and probes
• Quartz cuvettes

Cell suspensions (2 mL) were placed in the multicell holder at 25 °C. Emission scans were performed following excitation at each protein’s peak wavelength, based on published maxima.

Main Results and Discussion

Recorded emission spectra matched expected maxima for GFP (509 nm), BFP, CFP, YFP, and DsRed (583 nm). Internal filters on the Cary Eclipse minimized cellular autofluorescence and scatter, which is especially critical for UV-excited proteins like BFP. Low background noise enabled clear discrimination of multiple fluorescent markers and reliable spectral analysis in live cells.

Benefits and Practical Applications

This approach provides a rapid, reproducible platform for multiwavelength fluorescence measurements in living yeast. It supports studies of gene expression, subcellular localization, and protein–protein interactions via FRET without cell disruption or extrinsic dyes.

Future Trends and Potential Applications

Expanding the spectrum of fluorescent proteins and integrating automated, high-throughput multiwavelength scanning will enhance live-cell assays in drug discovery, synthetic biology, and dynamic interaction mapping. Development of red-shifted and near-infrared fluorophores promises deeper tissue imaging and increased multiplex capacity.

Conclusion

The Agilent Cary Eclipse system, equipped with temperature control and multicell stirring accessories, offers a versatile and sensitive solution for characterizing cytosolic fluorescent proteins in live yeast. Its low intrinsic background and flexible scanning capabilities facilitate advanced in vivo fluorescence assays.

References

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