Editing Pichia EXpression (section)

== <br>Making Yeast Competent Cells and Yeast Cell Transformation<br>  ==

Disposables and Reagents 

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1. Bacto-Yeast Extract (Fisher) 

2. Bacto-Peptone (Fisher) 

3. Glucose (Dextrose) (Fisher) 

4. Bacto-agar (Fisher) 

5. Deionized H2O 

6. Glycerol (Sigma) 

7. DMSO (Dimethyl sulfoxide) (Sigma) 

8. 1.5 ml centrifuge tubes (appendorf) 

9. PEG 3350 (Sigma) 

10. LiAc (Lithium acetate dihydrate) (Sigma) 

11. Salmon sperm DNA (Invitrogen) 

12. YNB (Yeast Nitrogen Base) (Sigma) 

13. MES (2-(N-morpholino)ethanesulfonic acid) 

14. ADE (Sigma) 

15. Glucose 

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'''Equipments''' 

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1. Water bath (VWR) 

2. Centrifuge (Eppendorf) 

3. 30 °C shaker and incubator (VWR) 

4. Standard petri dishes (VWR) 

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'''Procedure''' 

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1. Make yeast competent cells (Modified from Gietz &amp; Schiestl, 2007) 

1) Get the yeast strains and streak on YPAD plates. Let cells grow 2 d before inoculation. 

2) 1st Inoculation: inoculate one colony into 25 ml YPAD liquid medium. Grow cells overnight at 30 °C with shaking speed around 200 rpm. 

3) 2nd inoculation. Transfer the 25 ml cell culture into 75 ml YPAD medium. Grow cells at 30 oC for 4 h. 

4) Harvest cells by centrifugation at 3000 g for 5 minutes, wash cells with 0.5 volumes of sterile H2O. 

5) Centrifuge again with 3000 g for 5 minutes. 

6) Re-suspend cells in 0.01 volumes of sterile H2O, transfer to a suitable centrifuge tube and pellet at 3000 g for 5 min at 20 oC. 

7) Re-suspend cell pellet in 0.01 volumes of filter sterilized frozen competent cell solution ('''5&nbsp;% v/v glycerol, 10% v/v DMSO'''). 

8) Dispense 50 ul cells into 1.5 ml appendorf tubes. 

9) Place the tubes into box with Styrofoam or cardboard. (Slowly freezing is essential for good survival rates). 

10) Store the box into -80 oC freezer. (Cells can be kept at this condition for up to one year). 

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2. yeast cell transformation 

11) If use competent cells stored at -80 oC, thaw cells at 37 oC water bath for 15-30 S. if use freshly made competent cells, go to step 2). 

12) Centrifuge at 13,000 g for 2 minutes to remove the supernatant. 

13) Make the transformation solution for the planned number of transformation plus one extra (negative control) (see the recipes). 

14) Add the solution to the cell pellet, votex to re-suspend the cells. 

15) Incubate in a 42 oC water bath for 40 minutes. 

16) Centrifuge at 13,000 g for 30 seconds and remove the supernatant. 

17) Pipette 1 ml of sterile H2O into the transformation tube to re-suspend the pellet. 

18) Plate 200 ul of the cell suspension onto the YNB+MA plate growth plate with your selection marker. 

19) Incubate plates at 30 oC for 2~4 d. 

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'''Recipes''' 

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<u>1. Make YPAD plate &amp; liquid medium:</u> 

1% Bacto-Yeast Extract – 10 g/L 

2% Bacto-Peptone – 20 g/L 

2% Glucose (Dextrose) – 20 g/L 

If making YPD plates, add 20 g Bacto-agar. 

Fill up to 1 liter* with deionized HOH. 

NO pH adjustments 

(Note: autoclave agar &amp; glucose separately, to avoid caramelization) 

<u>2. Transformation solution:</u> 

PEG 3350 (50&nbsp;% (w/v)): 260 μl 

LiAc 1 M: 36 μl 

Salmon sperm DNA (10mg/ml): 10 μl 

Plasmid: 3 μl 

Sterile H2O: 51 μl 

Total: 360 μl 

<u>3. YNB+ MA plate (100 ml):</u> 

YNB: 0.67 g 

MES (20 mM): 0.39 g 

ADE: 0.01 g 

Agar: 2 g 

Glucose: 2 g 

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'''References''' 

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1. Gietz R.D., Schiestl R.H. (2007). Frozen competent yeast cells that can be transformed with high efficiency using the LiAc/SS carrier DNA/PEG method. Nat Protoc 2(1): 1-4. 

2. Lu Y., Chanroj S., Zulkifli L., Johnson M.A., Uozumi N., Cheung A., Sze H. (2011). Pollen tubes lacking a pair of K+ transporters fail to target ovules in Arabidopsis. Plant Cell 23(1): 81-93. 

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