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Yeast toxicity assays
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High-efficiency transformation of yeast and yeast toxicity assay
1. Insert gene of interest (here we use ceg3 gene as example) into pYES2NTA (Invitrogen), which offers the URA3 gene for selection in yeast.
2. Prepare at least 1 ug pYES2NTA-Ceg3 plasmids by using Zyppy plasmid miniprep kit (Zymo research).
3. Inoculate wild-type W303 yeast strain into 5 mL of liquid YPD medium and incubate with shaking overnight at 30°C. This culture will give sufficient cells for 5 transformations.
4. Harvest the culture in a 1.5 mL sterile tube at 3000 rpm for 1 min.
5. Pour off the medium, resuspend in 1 ml of sterile water and spin again, twice.
6. Pour off the water, resuspend in 1 mL of 100 mM lithium acetate (LiAc).
7. Pellet the cells at 3000 rpm for 1 min and remove the LiAc.
8. Resuspend the cells to a final volume of 250 uL with 100 mM LiAc.
9. Boil a 1 mL sample of single-stranded carrier DNA (Sigma) for 5 mins and quickly chill in ice water.
10. Vortex the cell suspension and pipette 50 ul samples into 1.5mL labeled tube. Pellet the cells and remove the LiAc buffer.
11.The basis “transformation mix” consists of the following ingredients:
Carefully add them in the order listed:
-240ul of PEG (50% w/v, MW=3350Da)
-36ul of 1M LiAc
-25ul of single-stranded carrier DNA (2.0 mg/mL)
-50ul of water and 1 ug Plasmid DNA (pYES2NTA-Ceg3 here)
12. Vortex tubes vigorously until the cell pellet has been completely mixed. This usually takes about 1 min.
13. Incubate for 30 mins at 30°C.
14. Heat shock for 30 mins in a water bath at 42°C.
15. Spin at 5000 rpm for 15 sec and remove the transformation mix.
16. Pipette 50 ul sterile water into these tubes and resuspend the pellet by pipetting it up and down gently.
17. Plate the heat-shocked cells onto SD-ura plates containing 2% glucose.
18. Incubate these plates at 30°C incubator for three days.
19. Inoculate single yeast colony from plates into 2 mL SD-Ura selective medium containing glucose and incubate with shaking overnight at 30°C.
20. The overnight cultures were serially diluted 5-fold with sterile water.
21. 5 ul of each dilution was spotted on SD-ura plates containing glucose or galactose.
22. Plates were incubated at 30°C for 48 h before images were acquired.
YPD medium or plates:
1% yeast extract
2% peptone
2% D-glucose
2% agar (for plates only)
SD-Ura minimal medium or plates (1 L):
6.7 g Yeast nitrogen base w/o amino acids
20 g Agar (for plates only)
100 mL AA-Mix w/o Uracil (100x concentrate)
2% D-glucose or galactose
AA-Mix w/o Uracil (100x concentrate):
L-Arginine (HCl) 200 mg/100ml
Adenine (add NaOH) 400 mg/50 ml
L-Aspartic acid 1000 mg/100ml
L-Histidine 200 mg/50 ml
L-Glutamic acid (sodium salt) 1000 mg/100ml
L-Leucine 600 mg/50 ml
L-Lysine (HCl) 300 mg/100 ml
L-Methionine 200 mg/50 ml
L-Phenylalanine 500 mg/100 ml
L-Tryptophane 400 mg/50 ml
L-Serine 4000 mg/100 ml
L-Threonine 2000 mg/100 ml
L-Tyrosine 300 mg/100 ml
L-Valine 1500 mg/100 ml
- Luo, Z and Fu, J(2022). Yeast toxicity assays. Bio-protocol Preprint. bio-protocol.org/prep1910.
- Fu, J., Zhou, M., Gritsenko, M. A., Nakayasu, E. S., Song, L. and Luo, Z.(2022). Legionella pneumophila modulates host energy metabolism by ADP-ribosylation of ADP/ATP translocases. eLife. DOI: 10.7554/eLife.73611
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