In molecular biology, transformation is the genetic alteration of a cell resulting from the direct uptake and incorporation of exogenous DNA (originating outside the cell) from its surroundings through the cell membrane. In yeast, there are numerous ways to perform transformation. One method that is used is Electroporation. Here is a good summary of the different transformation methods, with their pros and cons: Genetic Transformation of Yeast
It’s a technique in which an electrical field is applied to cells in order to increase the permeability of the cell membrane, allowing DNA to be introduced into the cell.
- Electroporation efficiency = number of transformants per microgram of DNA
- Electroporation frequency= transformation efficiency per viable cells
“Freezing of intact cells in sorbitol with calcium at -80C results in higher transformation efficiency by electroporation, giving more than 10^6 transformants/μg of plasmid DNA after thawing.” Reference: High-efficiency electroporation by freezing intact yeast cells with addition of calcium, by Suga and Hatakeyama.
“Pretreatment of yeast cells with lithium acetate (LiAc) and dithiothreitol (DTT) enhances the frequency of transformation by electroporation. The method shows improvements of 6–67-fold in wild-type strains derived from commonly used Saccharomyces cerevisiae genetic backgrounds. In addition, 15–300-fold improvement in transformation frequency was achieved with several mutant strains of S. cerevisiae that transformed poorly by conventional procedures.” Reference: An improved protocol for the preparation of yeast cells for transformation by electroporation, by Thompson et al.
Preparation (using Suga & Hatakeyama ‘s protocol)
Grow S. Cerevisiae cells in YPD medium to a density of approximately 1x10^7 cells/ml at 30°C.
Microorganism: S. Cerevisiae cells Chemicals: YPD Equipment: Plates (for plate culture) or microfuge tubes (for liquid culture); spectrophotometer od600, thermocycler (used for PCR- to control temprature changes at a specific incremental rates) Temperature req.: 30°C
• Place the cultures on ice for 15 min just before harvesting. Collect the cells by centrifugation and wash the resulting pellet three times with ice-cold sterilized water.
• Suspend this pellet in ice-cold freezing buffer containing 0.6-2.5M sorbitol, 5-10 mM CaCl2, and 10mM of 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES, pH=7.5) to give a density of approximately 5x 10^8 cells/ml
Microorganism: S. Cerevisiae cells Chemicals: Ice, Sterilized water, Buffer, Sorbitol, CaCl2, HEPES with pH 7.5 Equipment: Centrifuge, pellet, spectrophotometer od600
Dispense samples (0.1ml) of the cell suspension in 1.5ml microcentrifuge tubes, slowly freeze them and store by placing them directly in a -80C freezer. Cooling Rate: 10C/min
Microorganism: cell suspension Chemicals: Equipment: Microcentrifuge tubes Temperature Req: -80C
• For each electroporation, quickly thaw the frozen competent cells in a water bath at 30C Warming rate: 200C/min
• Wash once at with 1ml of ice-cold 1.0 M sorbitol by centrifugation.
• Re-suspend the final pellet in 1.0M sorbitol to give a density of 1-2x10^9 cells/ml.
Microorganism: Frozen cells (at -80C) Chemicals: Sorbitol, Equipment: Centrifuge, spectrophotometer od600
• Mix the cell suspension with 0.5-10ng of purified plasmid DNA and then transfer to a chilled cuvette with a 0.2 cm electrode gap.
• Apply a high electric pulse to the cell suspension, by using the Bio Rad Gene Pulsar II with Pulse Control Plus (or equivalent)
Microorganism: Cell suspension Chemicals: Equipment: Cuvette (chilled), Electroporation system w/ electrodes Other: plasmid
Immediately dilute the electroporated cells in 1ml of ice-cold 1.0 M sorbitol and spread samples (0.1-0.2 ml) on minimal selection plates. Note: The sorbitol acts as an osmotic stabilizer
Microorganism: Electroporated Cells Chemicals: Sorbitol Equipment: Dilution equipment, , Selection Plates
Summary of Reagents & Equipments Need to Complete the above protocol
YPD, Ice, Sterilized water, Buffer, Sorbitol, CaCl2, HEPES with pH 7.5, Plasmid
Plates, Spectrophotometer od600, Centrifuge, Pellet, Microcentrifuge tubes, Cuvette (chilled), Electroporation system w/ electrodes, Selection Plates, Dilution equipment (tubes, pipette, etc.…), PCR
Electric field strength: 9.5-11KV/cm
In the work done by Suga & Hatakeyama, a constant electric pulse of 11kV/cm with 25microF and 200 Ohms was used on a cell concentration of 2x10^9cells/mL