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

Green Fluorescent Protein (GFP) and its variants enable non‐invasive, real‐time tracking of cellular processes in living cells. Their autocatalytic maturation without external cofactors and bright fluorescence have revolutionized studies of gene expression, protein localization, and interaction dynamics in intact biological systems.

Study Objectives and Overview

This application note details the detection and characterization of GFP, BFP, CFP, YFP, and DsRed expressed in the cytosol of Saccharomyces cerevisiae using the Agilent Cary Eclipse fluorescence spectrophotometer. The primary goal was to establish in vivo measurement protocols for multiple fluorescent proteins and to demonstrate robust multiwavelength detection capabilities.

Methodology and Instrumentation

Yeast strain YRD15 was transformed with plasmids encoding various fluorescent proteins. Cultures were grown, washed, and standardized to an optical density of 0.55 at 650 nm. Two‐milliliter cell suspensions were placed in quartz cuvettes within a Peltier‐thermostatted multicell holder at 25 °C. Fluorescence measurements used the Agilent Cary Eclipse spectrophotometer equipped with internal excitation/emission filters. Excitation and emission settings were chosen based on each probe’s spectral maxima. Instrumentation included:

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

Main Results and Discussion

Distinct emission spectra matching known maxima were obtained for all fluorescent proteins. Cellular autofluorescence and scatter were minimized by internal monochromator filters, crucial for UV‐excited or low‐intensity probes. The system reliably differentiated signals from GFP variants and DsRed within live yeast cells, demonstrating high sensitivity and negligible background interference.

Benefits and Practical Applications

This workflow enables simultaneous monitoring of multiple fluorescent tags in vivo, supporting studies of protein trafficking, gene expression reporting, and FRET‐based interaction assays. Rapid, reproducible measurements without cell damage make it valuable for molecular biology research, QA/QC, and industrial analytics.

Future Trends and Potential Applications

Future developments may include automated multiwavelength scanning for high‐throughput live‐cell assays, design of novel fluorescent probes with enhanced photostability, and real‐time tracking of dynamic interactions in more complex model organisms.

Conclusion

The Agilent Cary Eclipse platform with temperature‐controlled multicell sampling provides a versatile and reliable solution for characterizing fluorescent proteins in living cells. Its capacity for simultaneous multi‐probe detection advances the study of cellular mechanisms in real time.

References

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