A simple and immediate method for simultaneously evaluating expression level and plasmid maintenance in yeast.

Area of Science:

• Molecular Biology
• Biotechnology
• Cell Biology

Background:

• Yeast expression systems are crucial for producing recombinant proteins.
• Accurate assessment of expression levels and plasmid stability is essential for optimizing these systems.
• Current methods for evaluating plasmid maintenance can be time-consuming.

Purpose of the Study:

• To develop a simple, rapid, and simultaneous method for assessing yeast expression levels and plasmid maintenance.
• To utilize green fluorescent protein (GFP) and flow cytometry (FCM) for this dual analysis.
• To provide a more efficient alternative to existing assessment techniques.

Main Methods:

• Employing green fluorescent protein (GFP) as a reporter.
• Utilizing flow cytometry (FCM) for quantitative analysis.
• Analyzing both the average GFP fluorescence intensity and the population of GFP-expressing cells.

Main Results:

• FCM analysis of GFP fluorescence intensity provides rapid quantification of expression levels.
• Single-cell FCM analysis of the GFP-expressing population accurately reflects plasmid retention rates.
• This method eliminates the need for lengthy incubation periods (2-3 days) required for colony counting.

Conclusions:

• FCM analysis with a GFP reporter is a suitable and efficient method for evaluating yeast expression systems.
• This technique aids in selecting optimal expression vectors and host strains.
• It facilitates the establishment of robust yeast expression systems with desired characteristics.