3.7 Transforming Cells with Plasmids
As you might imagine, it is not easy to force large DNA molecules into bacterial cells. E. coli is protected by two membranes, the outer membrane and an inner plasma membrane, and large, charged molecules do not ordinarily pass through phospholipid membranes. The organism also has protective enzymes; the restriction endonucleases we already have learned about and other nucleases that are ready to attack foreign DNA as it entered the cell. In 1974, Lederberg and Cohen developed a method to partially overcome these natural barriers.
Intact DNA does not ordinarily enter bacterial cells. First, treat E coli cells at 0o with a dilute solution of CaCl2 to weaken the cell wall. Next, added DNA forms hydroxy-calcium-phosphate bonds and the complex adsorbs to the cells. The calcium-phosphate complex not only adsorbs to cells, but the DNA is protected inside the complex.
Next, Raise the temperature to 37-40o to "heat shock" the cells and force DNA inside. During the heat shock, a few of the cells take up complete molecules of DNA. Usually about 1 in 1000 DNA molecules yield a transformant. This mean that 105-107 transformants may be obtained for each microgram of pBR322 DNA. Other methods may be used. For example, DNA may be driven into cells by an electric pulse which creates a temporary pore in the membranes, a technique known as electroporation.
Special host strains of E. coli are used that lack the nucleases that would otherwise attack and degrade the inserted plasmid DNA (see host strain section)