Transformation (Biotechnology technique)

Transformation is an important technique used to insert exogenous DNA into a host cell. One of the many techniques to transform cell is electroporation. It's a mechanical transformation method that utilizes electrical pulse to create temporary pores in the cell membrane. The pores allow the DNA molecule to pass the cell membrane and enter the cell. The high voltage of electricity causes hydrophobic pores to form at cell membrane with approximately 2 nm.

Parameter of electroporation

• Voltage

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

• Cell

Electro-competent cells are used to increase the electroporation efficiency. Competence means the ability of a cell to take up extracellular ("naked") DNA from its environment. Yeast cells are made electrocompetent by washing it in non-ionic solution to remove all salts , ensuring 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.

At 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 with increasing DNA concentration in the electroporation buffer. But please note that 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 act as 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 by osmotic stabilizer such as sorbitol. Thus, sorbitol act as a cryoprotectant and osmoregulator.

Spread

Spread refers to a method of distributing bacteria evenly over the surface of an agar plate media. A small volume of microbial culture is spread evenly over the agar surface using a glass spreader. Spread procedure:

• A glass spreader should be sterilized using heat of the Bunsen burner flame. First, dip the glass spreader in alcohol (70% ethanol), shake off the excess alcohol, and ignite the residue. Then, allow the spreader to cool.
• Spread the microbial culture evenly on the surface of media plate. Pressure is applied evenly as the glass spread along the media surface.
• Allow the microbial culture to be absorbed by the media plate.
• Incubate the plate up-side down to avoid water droplets (condensation) on the plate.

It is important to pick a single colony to isolate pure genetic clones. A colony is a group of cells that originated from the same source; therefore, all the cells in a single colony contain identical genetic information.

Antibiotic selection

The media used in molecular cloning is a rich media that promotes microbial growth. Selective media can be used to avoid confusion in selecting between transformed colonies and non-transformed colonies. A selective media is prepared by adding antibiotics such as ampicillin, hygromycin, zeocin-bleomycin derivative, and kanamycin. The antibiotic used must match with antibiotic resistance gene encoded in a plasmid vector, i.e., if a plasmid vector contains β-lactamase gene (ampicillin resistance gene), then ampicillin must be used as the selective agent.

Microbes that have been transformed will contain the particular antibiotic resistance gene encoded in the plasmid vector. Antibiotic resistance gene allows the transformed colonies to be selected out of non-transformed colonies. Because transformed colonies express certain proteins needed to resist the antibiotic effect, they can survive in the media containing antibiotic where the non-transformed colonies cannot.

The effectiveness of a particular antibiotic resistance system is effected by:

• The selective agent (antibiotic).

It should completely inhibit the nontransformed growth.

• The resistance gene.

It should express the protein needed to fully resist the selective agent effect. The resistant gene expression level can be controlled by transcriptional and translational control signals that are used to it.

• The material to be selected.

The material to be selected in this case is microbe cells. The microbe cells should be sensitive to the antibiotic effect.

Ampicillin is commonly used for bacterial selection. However yeast is not susceptible to ampicillin; hence, it cannot be used as a selective agent for yeast transformation screening. One of many antibiotics that can be used in yeast transformation screening is zeocin. Zeocin is part of bleomycin/phleomycin-type antibiotics isolated from Streptomyces. Zeocin causes cell death by binding and cleaving DNA inside the cell. Sh ble gene (Streptoalloteichus encode Zeocin resistance protein hindustanus bleomycin gene). This protein will bind to zeocin in a stoichiometric manner causing inhibition of the zeocin DNA strand cleavage activity.