Yeast Transformation

What’s Transformation?

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 he different transformation methods, with their pros and cons: Genetic Transformation of Yeast

What's Electroporation?

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.

Important distinctions:

• Electroporation efficiency = number of transformants per microgram of DNA
• Electroporation frequency= transformation efficiency per viable cells

Findings:

“Freezing of intact cells in sorbitol with calcium at -80C results in higher transformation efficiency by electroporation, giving more than 106 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. [1]

Preparation (using Suga & Hatakeyama ‘s protocol)

Step 1:

Grow S. Cerevisiae cells in YPD medium to a density of approximately 1x107 cells/ml at 30°C.

Microorganism: S. Cerevisiae cells Chemicals: SD-URA, Dextrose/Glucose, 5-FOA, Leucine, Agar Equipment: Plates (for plate culture) or microfuge tubes (for liquid culture); spectrophotometer od600 Temperature req.: 30°C

Step 2:

• 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 108 cells/ml

Microorganism: S. Cerevisiae cells Chemicals: Ice, Sterilized water, Buffer, Sorbitol, CaCl2, HEPES with pH 7.5 Equipment: Centrifuge, pellet, spectrophotometer od600

Step 3:

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

Microorganism: cell suspension Chemicals: Equipment: Microcentrifuge tubes Temperature Req: -80C

Electroporation

Step 4:

• For each electroporation, quickly thaw the frozen competent cells in a water bath at 30C and 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 1-2x109 cells/ml.

Microorganism: Frozen cells (at -80C) Chemicals: Sorbitol, Equipment: Centrifuge, spectrophotometer od600

Step 5:

• Mix the cell suspension with 0.5-10mg 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

Step 6:

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

Reagents:

SD-URA, Dextrose/Glucose, 5-FOA, Leucine, Agar, Ice, Sterilized water, Buffer, Sorbitol, CaCl2, HEPES with pH 7.5, Plasmid

Equipment:

Plates, Spectrophotometer od600, Centrifuge, Pellet, Microcentrifuge tubes, Cuvette (chilled), Electroporation system w/ electrodes, Selection Plates, Dilution equipment (beaker, pipette, etc.…)