Yeast GFP Clone Collection

Overview
The Yeast GFP (Green Fluorescent Protein) Clone Collection of Saccharomyces cerevisiae tagged open reading frames (ORF) were generated by Dr. Erin O’Shea and Dr. Jonathan Weissman at University of California-San Francisco (Huh et al., 2003).The strategy for constructing the Yeast GFP Clone Collection is described below.

The Yeast GFP Clone Collection is a S. cerevisiae yeast strain collection expressing full-length ORFs containing an Aequorea victoria GFP (S65T) tag (Tsien, 1998) at the C-terminus end. The GFP fusion proteins are integrated into the yeast chromosome through homologous recombination and are expressed using endogenous promoters. The Yeast GFP Clone Collection consists of 4,159 GFP tagged ORFs comprising 75% of the yeast proteome. The GFP fusion proteins were localized to 22 distinct subcellular locations using fluorescence microscopy.

Applications
The Yeast GFP Clone Collection can be used for the following applications:

• Analyze response to specific external stimuli or growth conditions
• Assay the effects of mutation on a protein of interest and subsequent subcellular localization
• Understand complex regulatory networks for protein targeting
• Study organellar proteomics
• Evaluate protein-protein interactions (specially useful for proteins for which minimal functional data exists)

Strategy
The strategy used to generate the Yeast GFP Clone Collection is described below.

Figure 1

Advantages 
Using the Yeast GFP Clones for localization studies offers the following advantages:

• Low false positive rate
• Expression of wild-type levels of proteins from endogenous promoters avoids common problems associated with protein overexpression
• Non-invasive methods for analyzing GFP fluorescence without the need for any external cofactors
  

Replacements 
As a distributor of Yeast GFP Clone Collection, Thermo Fisher Scientific cannot be held responsible for errors in genotype phenotype, and localization. Replacements are issued at the discretion of Thermo Fisher Scientific.

References

• Huh W, Falvo JV, Gerke LC, Carroll AS, Howson RW, Weissman JS, and O'Shea EK (2003) Global Analysis of Protein Localization in Budding Yeast Nature 425:686-691.
• Tsien RY (1998) The Green Fluorescent Protein Annu Rev Biochem 67:509-544.

Introduction 
Instructions for preparing glycerol stocks and handling plates upon receipt of the Yeast GFP Clones are described below. Protocols for using the Yeast GFP Clones in specific applications are not included in this manual.

Preparing glycerol stocks
Prepare a glycerol stock culture within 2 weeks of receiving the clone stab.

1. Use a sterile loop to inoculate a 50 mL tube containing 5 mL YPD media with the clone stab. You can also use SD-His medium.
   
2. Incubate the cells at 30ºC with shaking overnight or until the culture is turbid.
   
3. Add 0.9 mL sterile 80% glycerol and mix thoroughly.
   
4. Dispense the stock into ultra-low temperature vials and freeze at –80ºC.
   
5. Revive the yeast by transferring a small portion of the frozen sample onto a YPD agar plate.

Inoculation from plates
Allow the plate to thaw completely and resuspend the cells before replication as yeast has a tendency to settle to the bottom (flocculate) of the well during growth. Avoid freeze-thaw as it decreases cell viability.

Avoiding cross contamination

• Wash the lids with ethanol before returning them to their 96-well plates.
• When re-sealing 96-well plates, freeze plates, remove a few plates at a time, and seal them before returning them to the freezer.
• Remove the foil seal only when the plate is frozen and allow the plates to thaw with the lids removed under a hood.

YPD medium 
Yeast Extract Peptone Dextrose Medium (1 liter)
1% yeast extract
2% peptone
2% dextrose (D-glucose)

1. Dissolve the following in 1000 mL of water:
   
   • 10 g yeast extract
     
   • 20 g peptone
     
   • 20 g dextrose (see note below if making plates)
   Optional: Add 20 g agar, if making plates.
   


2. Autoclave for 20 minutes on liquid cycle.
   
   
3. Store medium at room temperature or cool the medium and pour plates. The shelf life is approximately 1–2 months.
   

Note: If making plates, omit dextrose from Step 1. Autoclaving agar and dextrose together will cause the
dextrose to caramelize. Prepare a separate stock solution of 20% dextrose and autoclave or filter-sterilize. After the YPD broth (900 mL volume) has been autoclaved, add 100 mL of 20% dextrose to the medium.