Jul 07, 2023

Public workspaceHigh-Efficiency Yeast Electroporation V.3

DOI
dx.doi.org/10.17504/protocols.io.5qpvorr69v4o/v3
  • 1University of Arkansas
Jeffrey A Lewis
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DOI: dx.doi.org/10.17504/protocols.io.5qpvorr69v4o/v3
Protocol Citation: Stephanie Hood, Jeffrey A Lewis 2023. High-Efficiency Yeast Electroporation. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvorr69v4o/v3Version created by Jeffrey A Lewis
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: Working
We use this protocol and it's working
Created: November 16, 2022
Last Modified: July 07, 2023
Protocol Integer ID: 72872
Funders Acknowledgement:
NSF
Grant ID: MCB-1941824
Abstract
For when extremely high transformation efficiencies are needed (e.g., for transforming plasmid DNA libraries). Adapted from An improved yeast transformation method for the generation of very large human antibody libraries.

Overview:
Using strain BY4742, transformation efficiencies were optimal using 400 µl of concentrated cells and 1 µg of plasmid DNA, yielding ~3.5 million transformants per electroporation. It is possible that other strains may have different optima. The protocol is designed to allow for 2 electroporations per culture (yields ~1ml of concentrated cells) and can be scaled up for producing libraries.

Guidelines
The OD600 values are based on those obtained from a Unico 1100RS Spectrophotometer.
Materials
Growth Media:
YPD (1% yeast extract, 2% peptone, 2% dextrose)
2x YPD (2% yeast extract, 4% peptone, 4% dextrose)

Reagents:
  • 2M Lithium Acetate (LiOAc)
  • 1 mM Dithiothreitol (DTT)
  • 1M Sorbitol
  • 2M Sorbitol
  • 100 mM CaCl2
  • Sterile ddH2O

Electroporation Materials:
2 mm gap electroporation cuvettes (VWR Cat# 89047-208) Bio-Rad Gene Pulser Electroporation System


Recipes:

Electroporation Buffer (500 ml):
  • 250 ml 2M Sorbitol
  • 5 ml 100 mM CaCl2
  • 245 ml ddH2O

Conditioning Buffer (100 ml) :
  • 50 ml sterile ddH2O
  • 5 ml 2M LiOAc
  • 1 ml 1M DTT
  • 44 mL sterile ddH2O
Note: Add in order, as precipitation will occur if undiluted LiOAc and DTT are added together

Outgrowth Media (100 ml):
  • 50 ml 2x YPD
  • 50 ml 1M Sorbitol


Safety warnings
Lithium acetate is harmful if ingested and irritating to the eyes and skin. Wear appropriate personal protective equipment, including eye protection, lab coat, long pants, closed toed shoes, and nitrile gloves. Be sure to follow your Institution's guidelines for proper disposal.
2 days before starting experiment
2 days before starting experiment
2d
2d
Streak out yeast strains to be transformed onto YPD agar and grow at 30°C for 2 days.
2d
Day before experiment
Day before experiment
16h
16h
For each transformation, inoculate cells into 4 separate tubes containing 5 ml of YPD, and grow to saturation overnight at 30°C with 270 rpm orbital shaking.

16h
Day of experiment: preparation of electrocompetent cells
Day of experiment: preparation of electrocompetent cells
4h 15m
4h 15m
Pool the individual 5-ml overnight cultures into a sterile 50-ml conical tube and vortex to mix.
5m
Measure the OD600 of the saturated cells.
  • Generally need to make a 1:20 or 1:50 dilution to be within the linear range of the spectrophotometer.
5m
In a 500-ml flask, dilute cultures to be transformed to a starting OD600 of 2.0 in 100 ml of 2x YPD.

Example: If the overnight culture has an OD600 of 10, use the solution dilution equation (C1V1 = C2V2) to solve for the amount of cells to add:
  • C1 = 2 OD600 units (target OD600), V1 = 100 ml (target volume), C2 = 10 OD600 units (saturated OD600), V2 = ? (what we are solving for)
  • Rearrange the equation: V2 = C1V1 / C2
  • V2 = (2 OD600 units x 100 ml) / 10 OD600 units = 20-ml saturated cells
  • For 100 ml total, add 20-ml saturated cells to 80-ml 2x YPD.
5m
Grow the 100-ml inoculated cultures at 30°C with 270 rpm orbital shaking for 4 hours.
4h
While cultures are incubating: prepare media
While cultures are incubating: prepare media
15m
15m
Place sterile water on ice and store at 4°C until ready to use.
  • 100 ml of sterile water per transformation
5m
Prepare electroporation buffer, place on ice, and store at 4°C until ready to use.
  • 100 ml of electroporation buffer per transformation.

Electroporation Buffer (500 ml):
  • 250 ml 2M Sorbitol
  • 5 ml 100 mM CaCl2
  • 245 ml ddH2O
10m
Prepare conditioning buffer, and store at room temperature until ready to use.
  • 20 ml of conditioning buffer per transformation.


Conditioning Buffer (100 ml) :
  • 50 ml sterile ddH2O
  • 5 ml 2M LiOAc
  • 1 ml 1M DTT
  • 44 mL sterile ddH2O
Note: Add in order, as precipitation will occur if undiluted LiOAc and DTT are added together.
After 4 hour incubation
After 4 hour incubation
51m
51m
Split yeast culture into two 50-ml conical tubes, collect cells by gentle centrifugation at 1500 rcf for 3 minutes, and decant the media.

3m
Resuspend (wash) each cell pellet by gently pipetting with 25-ml ice-cold sterile water, centrifuge at 1500 rcf for 3 minutes, and decant the media.

3m
Wash the cell pellets again with 25-ml ice-cold sterile water, centrifuge at 1500 rcf for 3 minutes, and decant the media.
3m
Wash the cell pellets with 25-ml of ice-cold electroporation buffer, centrifuge at 1500 rcf for 3 minutes, and decant the media.
3m
Resuspend the cell pellets in 10-ml conditioning buffer and pool into a single 125-ml flask. Incubate at 30°C with 270 rpm orbital shaking for 30 minutes.
30m
Pour cells into a 50-ml conical tube, centrifuge at 1500 rcf for 3 minutes, and decant the media.

Note: follow institutional waste disposal procedures for unused or ‘spent’ conditioning buffer.
3m
Wash the cells with 50-ml ice-cold electroporation buffer, centrifuge at 1500 rcf for 3 minutes, and decant the media.
3m
Resuspend the cell pellet to a final volume of 1 ml by adding 100-200 µl electroporation buffer.
  • If you need to add more or less electroporation buffer than this, you likely need to optimize your initial inoculation OD600.
3m
Keep cells on ice until electroporation.
Electroporation, outgrowth, and plating
Electroporation, outgrowth, and plating
1h 8m
1h 8m
Pre-chill electroporation cuvette(s) on ice.
  • 1 cuvette per transformation.
Prepare Outgrowth Media.
  • 8 ml per sample, aliquoted into 50-ml conical tubes.

Outgrowth Media:
  • 50 ml 2x YPD
  • 50 ml 1M Sorbitol
For each electroporation, transfer 1 µg of DNA and 400 µl of concentrated cells to a microcentrifuge tube and gently mix by pipetting.
Pipet concentrated cells plus DNA into a pre-chilled Gently electroporation cuvette and keep on ice for 5 minutes.
5m
Electroporate the cells with the following instrument settings:
  • Voltage: 2.5 kV
  • Capacitance: 25 µF
  • Resistance: 200 Ohms

  • Typical time constant ranges from 3.0 to 4.5 milliseconds.
Using a sterile Pasteur pipette, transfer electroporated cells into the 50-ml conical tube containing 8 ml of outgrowth media.
  • To reduce the amount of cells left in cuvette, ~500 µl of the outgrowth media can be pipetted into the cuvette and then transferred back into the 50-ml conical with the rest of the outgrowth media.
Incubate the 50-ml conical tube containing the cells plus 8-ml conditioning media at 30°C without shaking for 1 hour, mixing every 15 minutes by gentle inversion.
  • Orbital shaking reduces transformation efficiency, possibly due to increased fragility of electroporated cells.
1h
Following outgrowth, collect cells by gentle centrifugation at 1500 rcf for 3 minutes, and decant the media.
3m
For auxotrophic marker selection, resuspend cells in the appropriate yeast dropout media (e.g., for uracil selection, resuspend cells in SC -Ura). For drug marker selection, resuspend cells in YPD.
For auxotrophic marker selection, plate directly on selective media. For drug marker selection, can either plate directly to selective media, or can plate to YPD and replica print to selective media the next day.
  • Replica printing often increases the number of transformants recovered when using drug markers.
  • Alternatively, can perform selection in liquid or semi-solid (low-percentage agarose) media and then pellet the transformants.
  • To determine the total number of transformants (e.g., if plating to confluency or performing a liquid or semi-solid selection), plate 100 µl of 10-fold serial dilutions down to a 10-5 dilution.