A. pullulans chemical transformation

A. pullulans Transformation Protocol (Published in DOI: 10.1083/jcb.202402114)

NOTE: This protocol yields enough for 4-5 transformations. If you intend to do more, scale up and grow more cells. If you need less, extra comp cells can also be frozen at -80°C for several months and still work well with little to no loss in competency. 

Media and Solutions

YPD Liquid for growing cells

For 1L dissolve the following in 900 ml diH2O:

10 g Yeast extract

20 g peptone

Separately, make 40% glucose

            Add 400 g glucose to a final volume of 1L using diH2O, divide between 4-10 bottles

Autoclave each separately. After media cools, add 100 ml 40% glucose to the 900 ml YEP. The final concentration should be 4% glucose. If you autoclave the glucose in the YEP, the media darkens, and cells do not grow as well. 

YPD selection plates

For 1L dissolve the following in 900 ml diH2O:

10g Yeast extract

20g peptone

20g BD Bacto^TM agar (214050, VWR)

Separately, make 40% glucose (see above)

Autoclave the YEP agar, add 50 ml 40% glucose (2% final concentration) and allow to cool to ~60°C. After the media is cooled, add the desired antibiotics at the following final concentrations and immediately pour the plates:

• 174 µl Hygromycin B stock (HYG). Final conc.: 70.4 µg/ml (174 µl/L) (400051-1MJ, Millipore)
• 500 µl G418 stock (G418, 300 mg/ml). Final conc.:150 µg/ml G418 (G64000-5.0, Research Products International)
• 500 µl Nourseothricin stock (NAT, 100 mg/ml). Final conc.: 50 µg/ml (N1200-1.0, Research Products International)

Drop out uracil (doURA) plates

For 1L dissolve the following in 1L diH2O

6.71 BD Difco^TM Yeast Nitrogen Base without Amino Acids (BD291940, FisherScientific)

0.77 g Complete Supplement Mixture minus uracil (1004-100, Sunrise Science Products)

20 g BD Bacto^TM agar (214050, VWR)

Autoclave, add 50 ml 40% glucose (see above), and pour plates.

SORB (Good for 6 months)

1 ml 10x TRIS EDTA pH 8 (100 mM Tris, 10 Mm EDTA) (filter sterilized or autoclaved)

1 ml 1M LiOAc (filter sterilized or autoclaved)

5 ml 2M Sorbitol (filter sterilized)

Fill to 10 ml with sterile dH2O in a 15 ml conical

40% PEG (Make fresh, use within a few days)

4 ml 50% (w/v) PEG 3350 stock solution (filter sterilized or autoclaved)

500 ml 10x TE (sterile)

500 ml 1M LiOAc (sterile)

*NOTE: Mix well before use

Protocol 

1. Inoculate 5 ml of YPD (with 2x glucose, 4%) and grow over night (18-24 hours) at 24°C (or room temp) with agitation (200 rpm or on a roller drum) 
   1. NOTE: Fresh plates are best (2-3 days old) but plates up to a week old can be used.
   2. NOTE: If you are only growing for 18 hours, you may need to pick 4-5 colonies into a single culture tube especially if the colonies are small (2 days of growth)
   3. NOTE: To ensure one of your culture tubes is at the correct density the following day, you can inoculate multiple tubes and do a 2-fold serial dilution between each.
2. Inoculate 50 ml YPD (4% glucose) in a 250 ml flask by adding media from the overnight culture. The final density after back diluting should be ~4-6x10⁶ cells/ml.
   1. NOTE: Typically, the overnight culture should be at a density of ~4-6x10⁷ cells/ml and thus you add all 5 ml to the 50 ml culture in the flask to achieve the desired final concentration. If the cultures are overgrown (~1x10⁸ cells/ml or more) they do not grow well after back diluting. If the cultures are undergrown, you may not have enough cells to inoculate all 50 ml. Starting at a density of ~3x10⁶ cells/ml after back diluting will cause cells to stay in lag phase for a few hours and take much longer to grow. This is especially true of the ura3 auxotrophic strain. 
3. Grow cells for ~2.5 hours at 24°C at 200 rpm. In this time the cell number should double. 
   1. NOTE: The final cell density should be between 1.0 and 1.5 x 10⁷ cells/ml. Much higher than this and there is a dip in transformation efficiency. Cells harvested at a lower density (~0.5x10⁷ cells/ml) also work provided that these cells grew after back diluting. If the cells do not grow well (do not double in 4 hours), they do not transform well. Do not overgrow.
4. Transfer cells to a 50 ml conical tube and centrifuge at ~3000 rpm (~2200 xg) for 8 min
5. Dump supernatant and resuspend cells in 1 ml sterile dH2O and transfer to a 1.5 ml epi tube.
6. Centrifuge in tabletop centrifuge 10 sec, 10,000 rpm
7. Remove supernatant and resuspend cells in 500 µl SORB 
8. Repeat spin and remove supernatant
9. Resuspend cells in 250 µl SORB. 
10. Aliquot these into 5 epi tubes (~60-65 µl in each). 
    1. NOTE: Each tube should have a total of 1-1.5 x 10⁸ cells, if the cells were grown to a density of 1-1.5 x 10⁷ before harvesting. If cells were harvested at a lower density, resuspend in a smaller volume of SORB and make fewer aliquots to keep the number of cells per tube at around 1x10^8.
11. OPTIONAL: If freezing cells, then place these aliquots directly in the -80°C freezer. They can be stored for several months (even more than a year). To use, simply thaw at room temperature for ~1-2 min.
12. Add 2.5 µl 40% glucose to each tube.
13. Add the following in order and mix by flicking the tube after each addition:
    1. 10 µl Carrier DNA (previously denatured 8 min at 95 degrees and stored on ice)
       1. Fish Sperm DNA, 10 mg/ml 0 (11467140001, Roche)
    2. 5-20 µl transformation DNA (aim for at least 1000 ng/µl of cleaned PCR product or uncleaned digest. I typically use ~10 µg total DNA. Usually, more DNA is better. However, I have not tested more than ~30 µg total. If doing 3-part PCR, add each PCR product at equal molarity.)
    3. 600 µl 40% PEG solution
14. After everything is added, gently pipette up and down or invert several times to mix. Make sure there are no more clumps of cells. 
15. Place on the wheel (or on a rotating tube inverter) at 24°C for 1 hour in the 1.5 ml epi tubes. 
16. While cells are incubating, warm plates to 24°C and add beads for spreading.
17. After 1 hour, add 30 µl 40% glucose and invert to mix.
18. Heat shock the cells in a 37°C for 15 min.
    1. NOTE: Do not heat shock for longer or at a higher temperature as this kills most of the cells. 
19. Pellet yeast 10 sec 10,000 rpm
20. Carefully remove ALL of the PEG solution with a pipette. 
21. Select transformants:
    1. Selecting on dropout media:
       1. Resuspend the cells in 250 µl sterile water (or 1M sorbitol, both work). Plate 50 µl of the cells on one plate and the rest on a second plate. Grow at 24°C. You should see colonies in 3 days.
    2. Selecting with antibiotics 
       1. Resuspend the cells in 1 ml 1M sorbitol.
       2. Add this to 1 ml YPD in a glass culture tube.
       3. Allow cells to recover 4 hours at 24°C on the wheel, then remove 1 ml (half the culture), spin down, and plate on selection media (see top of protocol). 
       4. Allow the remaining 1 ml culture to continue recovering at 18°C overnight. Then, spin down the rest of the culture and plate on a second selection plate. 
          1. NOTE: A 4-hour recovery appears best. Cells grow little during this time, but have more transformants than cultures plated earlier, suggesting that new integration events happen during this time. I usually split the transformation and also recover overnight just in case some integration events take longer. If desired, you can plate all cells after only 4 hours, but these should still be split between two plates.  

       5. You should see colonies in 2-3 days. Typically, there are ~100 colonies on each plate.