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

model. The present study aimed to detect and characterise GFPs expressed in the cytosol of live yeast cells (Saccharomyces cerevisiae) using the Varian Cary Eclipse fluorescence spectrophotometer.

Materials and Methods

(For part numbers see reference 5)

Equipment

Varian Cary Eclipse fluorescence spectrophotometer

Peltier-thermostatted multicell holder (with electromagnetic stirring)

Temperature controller

Temperature probes

Magnetic stirrer bars

Quartz cuvettes (4)

Yeast strains

YRD15 (MATa, his3, ura3, leu2, p+) of the yeast S. cerevisiae was the parental strain used in this study. Genes encoding the GFP derivatives (green, blue, cyan, yellow) and red fluorescent protein (DsRED) were cloned into the yeast expression plasmid pAS1N and transformed into the yeast strain YRD15 as previously described6. Transformants were plated out on yeast minimal medium (0.75% yeast minimal medium w/o amino acids, 2% glucose, 1.5% agar) with selective markers as required and grown at 28C for 35 days.

Protocol

Yeast cells were washed twice in 1ml MilliQ water to remove contaminating media before being resuspended in MilliQ to a final optical density of 0.55 Abs at 650 nm. Cell suspensions (2 ml) were placed in disposable fluorescence cuvettes (Sarstedt) in the multicell holder, which was positioned in the sample compartment of the Cary Eclipse fluorescence spectrophotometer. The temperature within the cuvettes was set to 25C. Using the Scan application, cell suspensions were excited with light of the appropriate wavelength for each specif
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