TITLE: Yeast protoplast fusion
AUTHORS: is Sparrow, Ulschan Bathe, Kristen Van Gelder

[DESCRIPTION]
This protocol was based on the original protocol by Ulschan Bathe and Kristen Van Gelder from the Hanson Lab at UF in 2023.

It details how to fuse 2 yeast protoplasts. It is focused on obtaining an evolution strain with the system OrthoRep, but should work for the fusion of any 2 yeast strains provided the selection scheme is functional.

[STEPS]
SECTION: Media and buffers
1. Make the following solutions ahead of time. They do not need to be sterilized.

Unless specified, all solutions are made in water
SECTION: Media and buffers
1.1. CPB (Citrate Phosphate Buffer) solution

Solution A
0.1 Molarity (M) Citric Acid (dihydrate)

Solution B
0.2 Molarity (M)Na2HPO4 

Combine 34.8 mL A and 65.2 mL B and bring up final volume to 200 mL with MilliQ water

SECTION: Media and buffers
1.2. 3M KCl
SECTION: Media and buffers
1.3. 0.5 M EDTA (pH = 8.0)
SECTION: Media and buffers
1.4. 0.5 M CaCl2
SECTION: Media and buffers
1.5. Other media

YPD

Autoclaved sterile water
SECTION: Media and buffers
2. Solutions - made fresh the day of
SECTION: Media and buffers
2.1. BMEE solution

3 mL 0.5 Molarity (M)  EDTA
50 µL beta-mercaptoethanol
21.95 mL MilliQ water

Filter sterilize
SECTION: Media and buffers
2.2. 0.6 M KCl

20 mL 3 Molarity (M) KCl
80 mL MilliQ water

Filter sterilize
SECTION: Media and buffers
2.3. Buffer 1

39 mL CPB Buffer
10 mL 3 Molarity (M) KCl
1 mL 0.5 Molarity (M) EDTA

Filter sterilize
SECTION: Media and buffers
2.4. Buffer 2

A - 50 % w/v PEG 3350

Add 10 g PEG + 8 mL water, stir with high heat and adjust volume to 25 mL when done

B - Buffer 2

16.65 mL 50 % w/v PEG 3350
5 mL 3 Molarity (M)KCl
2.5 mL 0.5 Molarity (M) CaCl2
850 µL MilliQ water

Filter sterilize
SECTION: Media and buffers
2.5. Buffer 3

5 mL 3 Molarity (M)KCl
2.5 mL 0.5 Molarity (M) CaCl2
17.5 mL MilliQ water

Filter sterilize
SECTION: Plating media
3. Plating media (i.e., selective media) must be made in 100-150 mL batches. Each 25 mL supports a single plating, thus 100 mL supports 1 fusion with 2 platings at different concentrations

For 100 mL
93.5 mL MilliQ water
4.473 g KCl
0.667 gYNB with ammonium sulfate OR 0.5 g ammonium sulfate + 0.171 gYNB without ammonium sulfate (monosodium glutamate can also be used as an alternative nitrogen source if ammonium sulfate cannot be used due to interfering with antibiotics (G418 or Nat))
0.14 g g of appropriate dropout
Adjust pH to 6.1
3 gagar
Run on liquid 30 cycle, leave inside autoclave until later.
SECTION: Yeast culture
4. 2 or 3 days before the experiment, start 3 mL pre-cultures in appropriate selection media for the donor (p1 carrying strain) and recipient (background + EP-DNAP) strains. Grow at 30 °Cwith shaking

SECTION: Yeast culture
5. The day before protoplast fusion, dilute pre-cultures 1:50 into 50 mL YPD culture and incubate shaking at 30 °C

Be very mindful of the rate of growth of the cells - OD after outgrowth should not exceed 7 but should be above 2. Depending on your strain, you will need to figure out optimal time for starting the culture. 
SECTION: Protoplast fusion
6. The morning of protoplast fusion, make the solutions above and filter-sterilize them. 
SECTION: Protoplast fusion
7. Take 100 µL of culture and add 900 µL YPD to it, then measure the OD600. 

Multiply the OD by the dilution factor (x10)

Using the approximation that, at OD600 = 4.5, for 20 mL of culture, ~0.45 g of cells are yielded, calculate the volume of culture necessary to obtain 0.3 g of cells of each strain using the formula c1.v1 = c2.v2

SECTION: Protoplast fusion
8. Weigh your tubes before adding culture and make a note of it.
SECTION: Protoplast fusion
9. Centrifuge at 3000 x g, 5 min, using blank tubes to balance the different culture volumes

SECTION: Protoplast fusion
10. Weigh your tubes again, and adjust pellet weight by adding more culture or scooping out pellet. 

Aim for a final weight of 0.3 g - preferably OVER

SECTION: Protoplast fusion
11. Resuspend in 10 mL sterile water and transfer to 15 mL falcon 
SECTION: Protoplast fusion
12. Centrifuge at 3000 x g, 5 min, and decant supernatant. Pipette out water.
SECTION: Protoplast fusion
13. Resuspend in 1.8 mL in the same tube BMEE solution TAPPING GENTLY to resuspend


DO NOT VORTEX ANYTHING BEYOND THIS POINT
SECTION: Protoplast fusion
14. Incubate at 30 °Cfor 30 min. 

At 15 min, invert a few times to mix gently.
SECTION: Protoplast fusion
15. Centrifuge at 3000 x g, 5 min, decant supernatant.
SECTION: Protoplast fusion
16. Add3 mL 0.6 M KCl and resuspend with serological pipette. 

Note: avoid the creation of bubbles
SECTION: Protoplast fusion
17. Centrifuge at 3000 x g, 5 min, decant supernatant.
SECTION: Protoplast fusion
18. Add 4.8 mL Buffer I and resuspend with serological pipette.

SECTION: Protoplast fusion
19. Add Zymolyase solution at a total concentration of 6 U/mL

Note: Hanson Lab storage buffer for Zymolyase is as follows. If using this recipe make at a concentration of 5 U/uL, then add 5.8 uL :
                                                   
i.     100 mg (2000 U) of zymoylase from amsbio (Cat# = 120491-1)
                                                  
ii.     0.1 M Na2PO4 (pH 7.5, adjusted with NaOH)
                                                 
iii.     50% glycerol
                                                 
iv.     3 mM β-ME

To make 400 uL of solution
1. 100 mg zymolyase
2. 200 uL glycerol
3. 192 uL 0.1 M Na2PO4 (pH 7.5, adjusted with NaOH)
4. 8 uL of a 1:100 beta-mercaptoethanol dilution in 0.1 M Na2PO4

SECTION: Protoplast fusion
20. Incubate at 30 °Cstatically for 60 min and rotate every 15 min by hand.

Use these incubations to make the plating media and autoclave it.

Additionally, set up your 42 °Cwater bath next to the fume hood. Use a thermometer to make sure the temperature is right.

SECTION: Protoplast fusion
21. Centrifuge at 700 x g, 10 min, 0 °C, decant supernatant. 

Add 3 mL 0.6 M KCl. Resuspend gently by adding the buffer along the side of the tube.

Centrifuge at 700 x g, 10 min, 0 °C, decant supernatant. 

Add 3 mL 0.6 M KCl. Resuspend gently by adding the buffer along the side of the tube.

Centrifuge at 700 x g, 10 min, 0 °C, decant supernatant. 
SECTION: Protoplast fusion
22. Resuspend in 3 mL Buffer 1

SECTION: Protoplast fusion
23. In new 15 mL tubes, mix 1.5 mL of donor strain with 1.5 mL of recipient strain. 


 


The remaining volumes can be carried forward as negative controls. 

Mix by inversion gently a few times.

SECTION: Protoplast fusion
24. Centrifuge at 700 x g, 10 min, 0 °C, decant supernatant. Resuspend in 5 mL of Buffer 2.

The PEG will make resuspension difficult, so try your best with the serological pipette. Tiny, visible clumps of cells are OK, but should be avoided as they can make identification of emerging colonies difficult later on - avoid using the small pipettes.
SECTION: Protoplast fusion
26. Centrifuge at 700 x g, 10 min, 0 °C, decant supernatant. 

The media should be done autoclaving at this point. Take out, add glucose and other required auxotrophic additives, and return back to the autoclave to maintain liquidity for at least 5 min

BE VERY CAREFUL NOT TO ADD ADDITIVES WHICH LOSE YOUR SELECTION SCHEME!!!! - PAY ATTENTION TO SELECTION MARKER IN P1 (LEU2) AND SELECTION MARKER IN EP-DNAP CONSTRUCT (In our case, this is URA3, but other plasmids have HIS3)
SECTION: Protoplast fusion
27. Resuspend in 5 mL Buffer 3 with EXTREME GENTLENESS - the cells are protoplasted at this point. 

SECTION: Protoplast fusion
25. Incubate at 30 °Cfor 30 min with occasional inverting.

SECTION: Protoplast fusion
28. Take media in the autoclave and place in42 °Cwater bath, for a minimum of 3 min. TIME IT!!!!!!
You should be able to touch the flask with your wrist's bare skin and not feel pain, only warmth.

SECTION: Protoplast fusion
29. In a 50 mL falcon tube, add cells

Make 2-3 tubes per fusion:
One with250 µL of cells
One with 500 µL of cells
One with 1 mL of cells

Gently, add 25 mL of plating media, mix gently, and then pour in labelled plates. Allow plates to solidify and incubate at 30 °C for up to 14400 min


SECTION: Protoplast fusion
30. On the day after, tape the lid with parafilm.