Parameters for electroporation

Voltage

The electrical condition for electroporation for each microorganism varies. For example, yeast, such as Saccharomyces cerevisiae, has an optimal electrical condition of 40μL cell, 1.5 kV for approximately 5ms.

Cells

Electro-competent cells are used to increase the electroporation efficiency. Yeast cells are made electrocompetent by washing, with the use of a non-ionic solution in order to remove all salts. This process ensures that the charge is not conducted through the medium. The optimal electroporation for yeast is achieved when using yeast from mid or late-logarithmic phase. The yeast logarithmic phase can be divided into three parts:

• Early-log phase: the period when cell densities are about 10⁷ cells/mL.
• Mid-log phase: the cultures have densities between 1 and 5 × 10⁷ cells/mL.
• Late-log phase: occurs when cell densities are between 5 × 10⁷ and 2 × 10⁸ cells/mL.

In the logarithmic phase, the growth rate is rapid, and the rate of cell division exceeds the rate of cell death. Cells are metabolically active and carry sufficient genome replication to conduct efficient DNA repair. Thus, cell at this phase can tolerate harsh treatment such as electroporation.

DNA

The frequency of transformation is increased by increasing DNA concentration in the electroporation buffer. However, too much DNA concentration can also lower the transformation efficiency. The exact DNA concentration must be determined for each experiment.

Electroporation Media

To avoid sample arcing during electroporation, it is important to wash the electrocompetent cell thoroughly to remove the growth media from the cell. Washing can be performed with water or with non-ionic solution such as glucose, glycerol, sucrose, sorbitol, or polyethylene glycol. The presence of ionic material may cause arcing during electroporation.

Sorbitol is preferably used because it acts as a non-permeating cryoprotectant that significantly reduces the cell membrane damage during thawing. Damage in the membrane significantly reduces the transformation efficiency.

During the electroporation procedure, the temperature increases and the cells experience membrane injuries caused by rapid water influx and efflux due to extracellular and intracellular large osmotic non-equilibrium. This type of injury can be prevented with an osmotic stabilizer such as sorbitol. Thus, sorbitol acts as a cryoprotectant and osmoregulator.