Jul 11, 2024

Public workspaceYeast protoplast fusion

DOI
https://dx.doi.org/10.17504/protocols.io.5qpvokrxdl4o/v1
  • is Sparrow1,
  • Ulschan Bathe2,
  • Kristen Van Gelder2
  • 1UIUC;
  • 2UF
  • Yeast Protocols, Tools, and Tips
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 11, 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
Concentration0.1 Molarity (M) Citric Acid (dihydrate)

Solution B
Concentration0.2 Molarity (M) Na2HPO4

Combine Amount34.8 mL A and Amount65.2 mL B and bring up final volume to Amount200 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

Amount3 mL Concentration0.5 Molarity (M) EDTA
Amount50 µL beta-mercaptoethanol
Amount21.95 mL MilliQ water

Filter sterilize
0.6 M KCl

Amount20 mL Concentration3 Molarity (M) KCl
Amount80 mL MilliQ water

Filter sterilize
Buffer 1

Amount39 mL CPB Buffer
Amount10 mL Concentration3 Molarity (M) KCl
Amount1 mL Concentration0.5 Molarity (M) EDTA

Filter sterilize
Buffer 2

A - Concentration50 % w/v PEG 3350

Add Amount10 g PEG + Amount8 mL water, stir with high heat and adjust volume to 25 mL when done

B - Buffer 2

Amount16.65 mL Concentration50 % w/v PEG 3350
Amount5 mL Concentration3 Molarity (M) KCl
Amount2.5 mL Concentration0.5 Molarity (M) CaCl2
Amount850 µL MilliQ water

Filter sterilize
Buffer 3

Amount5 mL Concentration3 Molarity (M) KCl
Amount2.5 mL Concentration0.5 Molarity (M) CaCl2
Amount17.5 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 Amount100 mL
Amount93.5 mL MilliQ water
Amount4.473 g KCl
Amount0.667 g YNB with ammonium sulfate OR Amount0.5 g ammonium sulfate + Amount0.171 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))
Amount0.14 g g of appropriate dropout
Adjust pH to 6.1
Amount3 g agar
Run on liquid 30 cycle, leave inside autoclave until later.
Yeast culture
2 or 3 days before the experiment, start Amount3 mL pre-cultures in appropriate selection media for the donor (p1 carrying strain) and recipient (background + EP-DNAP) strains. Grow at Temperature30 °C with shaking

2d
The day before protoplast fusion, dilute pre-cultures 1:50 into Amount50 mL YPD culture and incubate shaking at Temperature30 °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 Amount100 µL of culture and add Amount900 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 Amount0.3 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 Centrifigation3000 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 Amount0.3 g - preferably OVER

Resuspend in 10 mL sterile water and transfer to 15 mL falcon
Centrifuge at Centrifigation3000 x g, 00:05:00 , and decant supernatant. Pipette out water.
5m
Resuspend in Amount1.8 mL in the same tube BMEE solution TAPPING GENTLY to resuspend


DO NOT VORTEX ANYTHING BEYOND THIS POINT
Incubate at Temperature30 °C for Duration00:30:00 .

At 15 min, invert a few times to mix gently.
30m
Centrifuge at Centrifigation3000 x g, 00:05:00 , decant supernatant.
5m
AddAmount3 mL 0.6 M KCl and resuspend with serological pipette.

Note: avoid the creation of bubbles
Centrifuge at Centrifigation3000 x g, 00:05:00 , decant supernatant.
5m
Add Amount4.8 mL Buffer I and resuspend with serological pipette.

Add Zymolyase solution at a total concentration of Concentration6 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 Temperature30 °C statically for Duration01:00:00 and rotate every Duration00:15:00 by hand.

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

Additionally, set up your Temperature42 °C water bath next to the fume hood. Use a thermometer to make sure the temperature is right.

1h 15m
Centrifuge at Centrifigation700 x g, 0°C, 00:10:00 , decant supernatant.

Add Amount3 mL 0.6 M KCl. Resuspend gently by adding the buffer along the side of the tube.

Centrifuge at Centrifigation700 x g, 0°C, 00:10:00 , decant supernatant.

Add Amount3 mL 0.6 M KCl. Resuspend gently by adding the buffer along the side of the tube.

Centrifuge at Centrifigation700 x g, 0°C, 00:10:00 , decant supernatant.
30m
Resuspend in Amount3 mL Buffer 1

In new 15 mL tubes, mix Amount1.5 mL of donor strain with Amount1.5 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 Centrifigation700 x g, 0°C, 00:10:00 , decant supernatant. Resuspend in Amount5 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 Temperature30 °C for Duration00:30:00 with occasional inverting.

30m
Centrifuge at Centrifigation700 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 Duration00:05:00

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 Amount5 mL Buffer 3 with EXTREME GENTLENESS - the cells are protoplasted at this point.

Take media in the autoclave and place inTemperature42 °C water bath, for a minimum of Duration00:03:00 . 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 withAmount250 µL of cells
  • One with Amount500 µL of cells
  • One with Amount1 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 Temperature30 °C for up to Duration240:00:00


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