Jul 10, 2024

Yeast protoplast fusion

DOI
https://dx.doi.org/10.17504/protocols.io.5qpvokrxdl4o/v1
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is Sparrow
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DOI: https://dx.doi.org/10.17504/protocols.io.5qpvokrxdl4o/v1
Protocol Citation: is Sparrow, Ulschan Bathe, Kristen Van Gelder 2024. Yeast protoplast fusion. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvokrxdl4o/v1
License: This is an open access  protocol  distributed under the terms of the  Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol
Created: May 13, 2024
Last Modified: July 10, 2024
Protocol  Integer ID: 99664
Keywords: yeast protoplast fusion this protocol, yeast protoplast fusion, yeast protoplast, yeast strain, yeast, fusion, hanson lab, original protocol by ulschan bathe, original protocol
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Abstract
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.
Media and buffers
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
0.1 Mass Percent Citric Acid (dihydrate)

Solution B
0.2 Mass Percent Na2HPO4

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

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

Filter sterilize
0.6 M KCl

20 mL 3 Mass Percent KCl
80 mL MilliQ water

Filter sterilize
Buffer 1

39 mL CPB Buffer
10 mL 3 Mass Percent KCl
1 mL 0.5 Mass Percent EDTA

Filter sterilize
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 Mass Percent KCl
2.5 mL 0.5 Mass Percent CaCl2
850 µL MilliQ water

Filter sterilize
Buffer 3

5 mL 3 Mass Percent KCl
2.5 mL 0.5 Mass Percent CaCl2
17.5 mL MilliQ water

Filter sterilize
Plating media
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 g YNB with ammonium sulfate OR 0.5 g ammonium sulfate + 0.171 g YNB 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 g agar
Run on liquid 30 cycle, leave inside autoclave until later.
Yeast culture
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 °C with shaking

2d
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.
16h
Protoplast fusion
1w 3d 3h 33m
The morning of protoplast fusion, make the solutions above and filter-sterilize them.
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

Weigh your tubes before adding culture and make a note of it.
Centrifuge at 3000 x g, 00:05:00 , using blank tubes to balance the different culture volumes

5m
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

Resuspend in 10 mL sterile water and transfer to 15 mL falcon
Centrifuge at 3000 x g, 00:05:00 , and decant supernatant. Pipette out water.
5m
Resuspend in 1.8 mL in the same tube BMEE solution TAPPING GENTLY to resuspend


DO NOT VORTEX ANYTHING BEYOND THIS POINT
Incubate at 30 °C for 00:30:00 .

At 15 min, invert a few times to mix gently.
30m
Centrifuge at 3000 x g, 00:05:00 , decant supernatant.
5m
Add3 mL 0.6 M KCl and resuspend with serological pipette.

Note: avoid the creation of bubbles
Centrifuge at 3000 x g, 00:05:00 , decant supernatant.
5m
Add 4.8 mL Buffer I and resuspend with serological pipette.

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

Incubate at 30 °C statically for 01:00:00 and rotate every 00:15:00 by hand.

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

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

1h 15m
Centrifuge at 700 x g, 0°C, 00:10:00 , 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, 0°C, 00:10:00 , 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, 0°C, 00:10:00 , decant supernatant.
30m
Resuspend in 3 mL Buffer 1

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


ABC
Donor strainDonor strain
Recipient strainEvolution strainEvolution strain
NAControl 1-
Use appropriate EP-DNAPs


The remaining volumes can be carried forward as negative controls.

Mix by inversion gently a few times.

Centrifuge at 700 x g, 0°C, 00:10:00 , 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.
10m
Incubate at 30 °C for 00:30:00 with occasional inverting.

30m
Centrifuge at 700 x g, 0°C, 00:10:00 , 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 00:05:00

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)
15m
Resuspend in 5 mL Buffer 3 with EXTREME GENTLENESS - the cells are protoplasted at this point.

Take media in the autoclave and place in42 °C water bath, for a minimum of 00:03:00 . TIME IT!!!!!!
You should be able to touch the flask with your wrist's bare skin and not feel pain, only warmth.

3m
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 240:00:00


1w 3d
On the day after, tape the lid with parafilm.