Yeast protoplast fusion

Make the following solutions ahead of time. They do not need to be sterilized.

Unless specified, all solutions are made in water

CPB (Citrate Phosphate Buffer) solution

Solution A

0.1Molarity (M) Citric Acid (dihydrate)

Solution B

0.2Molarity (M)Na2HPO4

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

3M KCl

0.5 M EDTA (pH = 8.0)

0.5 M CaCl2

Other media

YPD

Autoclaved sterile water

Solutions - made fresh the day of

BMEE solution

3mL 0.5Molarity (M) EDTA

50µL beta-mercaptoethanol

21.95mL MilliQ water

Filter sterilize

0.6 M KCl

20mL 3Molarity (M) KCl

80mL MilliQ water

Filter sterilize

Buffer 1

39mL CPB Buffer

10mL 3Molarity (M) KCl

1mL 0.5Molarity (M) EDTA

Filter sterilize

Buffer 2

A - PEG 3350 50% w/v PEG 3350

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

B - Buffer 2

16.65mL 50% w/v PEG 3350

5mL 3Molarity (M)KCl

2.5mL 0.5Molarity (M) CaCl2

850µL MilliQ water

Filter sterilize

Buffer 3

5mL 3Molarity (M)KCl

2.5mL 0.5Molarity (M) CaCl2

17.5mL MilliQ water

Filter sterilize

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 100mL

93.5mL MilliQ water

4.473g KCl

0.667gYNB with ammonium sulfate OR 0.5g ammonium sulfate + 0.171gYNB 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))

0.14g g of appropriate dropout

Adjust pH to 6.1

3gagar

Run on liquid 30 cycle, leave inside autoclave until later.

2 or 3 days before the experiment, start 3mL pre-cultures in appropriate selection media for the donor ( p1 carrying strain ) and recipient ( background + EP-DNAP ) strains. Grow at 30°Cwith shaking

The day before protoplast fusion, dilute pre-cultures 1:50 into 50mL 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.

The morning of protoplast fusion, make the solutions above and filter-sterilize them.

Take 100µL of culture and add 900µL 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 0.3g of cells of each strain using the formula c1.v1 = c2.v2

Weigh your tubes before adding culture and make a note of it.

Centrifuge at 3000x g, using blank tubes to balance the different culture volumes

Weigh your tubes again, and adjust pellet weight by adding more culture or scooping out pellet.

Aim for a final weight of 0.3g - preferably OVER

Resuspend in 10 mL sterile water and transfer to 15 mL falcon

Centrifuge at 3000x g, and decant supernatant. Pipette out water.

Resuspend in 1.8mL in the same tube BMEE solution TAPPING GENTLY to resuspend

DO NOT VORTEX ANYTHING BEYOND THIS POINT

Incubate at 30°Cfor 0h 30m 0s.

At 15 min, invert a few times to mix gently.

Centrifuge at 3000x g, decant supernatant.

Add3mL 0.6 M KCl and resuspend with serological pipette.

Note: avoid the creation of bubbles

Centrifuge at 3000x g, decant supernatant.

Add 4.8mL Buffer I and resuspend with serological pipette.

Add Zymolyase solution at a total concentration of 6U/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

Incubate at 30°Cstatically for 1h 0m 0s and rotate every 0h 15m 0s 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.

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

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

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

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

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

Resuspend in 3mL Buffer 1

In new 15 mL tubes, mix 1.5mL of donor strain with 1.5mL of recipient strain.

A	B	C
	Donor strain	Donor strain
Recipient strain	Evolution strain	Evolution strain
NA	Control 1	-

Use appropriate EP-DNAPs

The remaining volumes can be carried forward as negative controls.

Mix by inversion gently a few times.

Centrifuge at 700x g,0°C, decant supernatant. Resuspend in 5mL 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.

Incubate at 30°Cfor 0h 30m 0s with occasional inverting.

Centrifuge at 700x g,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 0h 5m 0s

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)

Resuspend in 5mL Buffer 3 with EXTREME GENTLENESS - the cells are protoplasted at this point.

Take media in the autoclave and place in42°Cwater bath, for a minimum of 0h 3m 0s. TIME IT!!!!!!

You should be able to touch the flask with your wrist's bare skin and not feel pain, only warmth.

In a 50 mL falcon tube, add cells

Make 2-3 tubes per fusion:

• One with250µL of cells
• One with 500µL of cells
• One with 1mL 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 240h 0m 0s

On the day after, tape the lid with parafilm.