Yeast strain construction and transformation

The protocol below is an adaptation of the protocol described by the commercial Frozen-EZ Yeast Transformation II kit (Zymo Research)

For yeast strain integration, i.e., genetic modification with CRISPR/Cas9-based technique:

1. Prepare yeast competent cells by inoculating parent strain colony into yeast peptone with 2% dextrose (YPD) media and grow overnight at 30°C and 220 rpm in a shaker incubator.
2. Dilute the saturated overnight culture by 50-fold, i.e., 100 µl culture in 5 ml fresh YPD media, and incubate for 4 to 6 hours until OD600 reaches 0.8 to 1.0. Note that the yeast competent cells are usually prepared in a batch and can be frozen at -80°C for future use; therefore, a volume of at around 40 ml of culture is typically prepared.
3. For yeast strain integration, i.e., genomic modifications, 2.5 to 5 ml cells of OD600 0.8 to 1.0 were used per integration reaction, depending on the integration efficiency. For convenience, 5 ml of culture will be used in below.
4. Pellet the cells at 3500 x g for 4 min and discard the supernatant.
5. Wash the cells twice using 125 µl Frozen-EZ solution 1 per 5 ml of culture in YPD media (or 1 ml solution 1 per 40 ml of culture); pellet the cells at 3500 x g for 4 min and discard the supernatant. Keep the cells on ice!
6. Add 25 µl Frozen-EZ solution 2 per 5 ml of culture to resuspend the pellet. Note to take into account the volume of the cells so typically a little less than calculated volume of solution 2 will be added. Keep the cells on ice!
7. At this stage, can choose to either continue with integration or store the competent cells at – 80°C; no need to flash freeze, typically we just transfer the cells from ice to freezer directly and thaw on ice the next time you take the cells out from freezer.
8. To continue with integration, add 25 µl of competent cells to 250 µl of Frozen-EZ solution 3 in an eppendorf tube, and keep the tube on ice.
9. Add 100 ng of the Cas9 plasmid (encodes Cas9 enzyme, the gRNA and selected by G418 resistance) and 500 ng of the linear DNA repair template to the eppendorf tube with the mixture. Note that the linear DNA repair template is designed to harbor a 35 to 45 bp homology region with the adjacent genome sequences, at 5’ and 3’ ends of the cutting site. The linear DNA template is usually PCR-ed off from a plasmid if multiple expression cassettes are included, and in author’s experience can range from 70 to 10k bp.
10. Place the eppendorf tube into 30°C incubator, ideally on a rotator, and incubate for 45 to 60 min.
11. Centrifuge the mixture at 300 x g for 2 min and discard the supernatant.
12. Resuspend the cell pellet with 1 ml of fresh YPD media, followed by a 2 to 3 hour recovery period at 30°C, ideally on a rotator.
13. Plate the cells onto YPD media plates supplemented with G418 (250 to 400 mg/liter) to select for colonies with successfully integrated DNA linear template. Note that these plates are commercially available (Teknova).
14. Incubate the plates for 2 to 3 days and hopefully you will see the colonies!

For plasmid transformations, switch steps 9, 12, 13 as following:

9. Add 50 ng of plasmid. Note that the yeast selection marker encoded by the plasmid should match the dropout media plate of choice!

12. Resuspend the cells by 150 µl of sterile water.

13. Plate the cells onto dropout synthetic media plates. Again these plates are commercially available (Teknova).