A molecular biology technique where foreign DNA is introduced into bacterial or yeast cells. This method is analogous to transfection, where the DNA is instead inserted into eukaryotic cells. Both techniques use DNA that typically encodes a gene of interest. This DNA is placed into a plasmid before transformation. Transformation is thought to be first demonstrated in the bacteria Streptococcus pneumoniae by Frederick Griffith in 1928. His famous experiment helped to prove that DNA, not protein, was the unit of hereditary information in the cell.
There are two main reasons to transform cells:
- To produce a large amount of the DNA. The bacteria are used as a host to grow a high number of plasmids. The plasmids can then be isolated from the bacteria to obtain a large amount of purified DNA. The DNA can then be used for sequencing, transfection, or other techniques that require pure DNA.
- To produce a large amount of protein from the DNA. In this case the DNA of interest is a gene that encodes a protein product. If the proper plasmid is used the bacteria can be induced by a compound called IPTG to start expressing the protein. The protein can then be isolated for use in other techniques.
Unlike eukaryotic cells, bacterial cells first require an extra step to encourage them to take up the DNA. This is called making the bacteria competent. The bacteria are placed in a calcium chloride solution and the chloride ions from the solution are able to pass through the bacterial cell membrane. These ions also bring water molecules with them, which causes the cell to swell. This swelling encourages the bacteria to take up foreign DNA, although the exact reason why is unknown. Biotechnology companies sell bacteria cells that are already competent, allowing researchers to skip this step.
Cells can be transformed by using one of two techniques:
- Heat shock
First, plasmid DNA is mixed with the bacteria cells and placed on ice. The cold temperature is thought to help the DNA adhere to the cell membrane. Next, the bacteria are "shocked" by abruptly increasing the temperature. This is done by placing them in a warm (42°C) water bath for a short period of time. The shocked bacteria are more likely to take up DNA. Exact temperature is important to obtain the highest transformation efficiency. If the temperature is too cold the bacteria will not take up the DNA, while if it is too hot the bacteria will die. This method is less efficient than electroporation, meaning fewer bacteria will take up the DNA. However, it is quick, easy, and does not require an electroporator machine.
The bacteria and DNA are mixed together and subjected to a high voltage of electricity. This creates holes in the cell membrane, allowing the DNA to enter the cell. The exact voltage varies rather widely between different strains of bacteria. Electroporation is also used for transfection, although the voltage required is much lower than with transformation. This method is the more efficient than heat shock, but requires a special, expensive device called an electroporator.
After either of these methods are performed it is necessary to let the bacteria recover from the shock. This is done by placing them in a medium such as LB medium and shaking them at a warm temperature for about an hour. In order to increase DNA or protein yields, bacteria that have been transformed are often isolated from untransformed cells by treating the bacteria with an antibiotic. The plasmid DNA often contains a gene whose encoded protein renders the bacteria immune to the antibiotic. Therefore, only bacteria that have been transformed will be able to survive.
For more reading:
- Current Protocols in Molecular Biology, Volumes 1 and 2, 1998