酵母单杂交

Two-hybrid assay of yeast transformation Qi's protocol

(This method can be used for both laboratory and industrial yeast)

Yeast strain we used for two-hybrid is S. cerevisiae HF7c (MATa ura3-52 his3-200 ade2-101 lys2-801 trp1-901 leu2-3,112 gal4-542 gal80-538 LYS2::GAL1UAS-GAL1TATA-HIS3 URA3::GAL4 17mers(x3)-CyC1TATA-LacZ), which contains the two reporter genes LacZ and HIS3, was used in two-hybrid analysis. Yeast can be co-transformation with plasmids carrying the different GAL4 DNA binding domain-target protein fusions (TRP1marker) and plasmid carrying the GAL4 activation domain (LEU2marker) to perform the protein-protein interaction study or two-hybrid screening. The transformation method is modified from Schiestl and Gietz (1989)

GAL4 DNA binding domain vector (pGBT8 or pGBT9, difference only in polylinker, relatively low expression, TRP1 marker)

GAL4 activation domain vector (pGAD424, low expression; pGADGH, high expression, LEU2 marker )

Transformation protocol for unique plasmid or for two known plasmids

1. From the stock (-70ªC) the yeast were inoculated on YPAD plate for 2 days at 28-32ªC

2. Inoculate 2-3 independent yeast colonies to 10 ml YPAD medium for the preinoculation, shaking at 28-32ªC for 10 to 12 hours, check OD600.

3. Make an inoculation of the volume necessary for the transformation. (Usually 10 ml of OD600 0.3 cells or 5 ml of OD600 0.8 is used for each sample transformation.

You can use the formula: OD

--------

2n

------------- x Vinoc. = Vpreinoc.

ODpre

OD is OD600 of cells you will use for transformation, 0.3-0.8 of OD600 is good for LiOAc transformation. ODpre is OD600 of the preinoculation. n is the generation time of yeast, different yeast strain has different generation time. The generation time for HF7C is 1.5 hours in rich medium and 2 hours in minimal medium. Vinoc. The volume you need for the transformation. Vpreinoc. is the volume you need take from preinoculation.

Shaking at 28-32ªC and an overnight inoculation is recommended

4. Check the OD of yeast cells. If OD is in the desired range, Cent. cells at 4000 rpm for 5 min. at room temperature.

5. Wash cells with half a volume of sterile water and cent. as step 4.

6. Wash cells with 0.2 volumes of 0.1M LiOAc/TE solution and cent. as step 4.

7. Resuspend cells in 1/100 volume of 0.1M LiOAc/TE solution if OD is 0.3, 2/100 for OD 0.8. Shake briefly at 30 ªC for 30 min. (At this step, competent cell can be stored at -70ªC in 7% DMSO)

8. During step 7 is going on, prepare DNA for transformation. Add 2 µl of 10 mg/ml denatured salmon DNA and plasmid (plasmids) to the bottom of an eppendorf tube. Total volume salmon DNA and plasmid mix should be less than 10 µl. After 30 min. incubation, add 100 µl of competent cells to each tube and mix by pipetting several times and than leave at 30ªC for 30 min.

9. Add 600 µl of 40%PEG (40%PEG/0.1 M LiOAc/TE), mix well then place tubes at 30ªC with gently shaking (optional) for 45 min.

10. 70 µl DMSO is added to each tube to give10% final concentration and the contents are mixed by inversion, follow by a heat shocked at 42ªC for 5 min.

11. Cells are spun in a microcentrofigue for (just enough to pellet) (3-5 seconds), washed with H20 or TE and resuspended in 500 µl H20 or TE. An aliquot of 10 to 100 µl transformed cells are plated to SD medium with different combination of supplements. After 10 min at room temperature, the plates are incubated at 30ªC for 3-6 days for transformants.

Solutions: All solution used in transformation should be prepared freshly from the stocks.

Stock solutions: 10xTE (100 mM Tris, 10 mM EDTA, pH7.5)

10x LiOAc (1 M 10x LiOAc, pH7.5), filter

50% PEG 4000 filter or autoclave

Selection plates for the transformation of: For all plates Adenine and lysine should be added to SD medium (0.7% yeast w/o base, 20 g Difco agar, and 2% glucose added after the autoclave). The supplements can be added before or after (sterile stock) autoclaving to 40 mg/l. 3-AT should be added when medium is below 60 ªC and the plate should be kept at dark and no more than one week. It is better if all plates are prepared one day before the transformation.

Combinations: * only necessary for selection of pGAD vector

1 2 * 3 4 5 6

His+leu His+Trp* His 3-AT (0, 5, 10 mM)