Transformation

Bacterial Transformation and Selection of Plasmid-Carrying Bacteria in DNA Cloning

Plasmids and other DNA molecules can be introduced into bacteria through a process known as transformation. This typically involves non-pathogenic strains of E. coli commonly used in laboratories. During transformation, specially prepared bacterial cells undergo thermal shock (e.g., exposure to elevated temperatures), which encourages the uptake of foreign DNA.

Transformation of Recombinant Plasmids into Bacteria Using Thermal Shock

The underlying mechanisms of this process remain somewhat enigmatic, and no definitive explanation has been provided. However, it appears that thermal shock alters membrane fluidity and/or induces the formation of pores, thereby facilitating the passage of DNA into the cells.

In gene cloning, plasmids usually contain an antibiotic resistance gene, which enables the survival of bacteria in the presence of a specific antibiotic. Consequently, bacteria that have successfully taken up the plasmid can be selected on plates containing the antibiotic. Bacteria lacking the plasmid will die, while those carrying it can survive and proliferate. Each surviving bacterium forms a small, colony-like cluster composed of genetically identical cells, all harboring the same plasmid.

After transforming the bacteria, they should be cultured on antibiotic-containing media for colony selection.

Not all colonies will necessarily contain the desired plasmid. This is due to the fact that, during the ligation of DNA ends, the fragments may not always adhere in the intended manner. Therefore, it is necessary to isolate DNA from several colonies to determine which ones possess the appropriate plasmid. Techniques such as restriction enzyme digestion and PCR are commonly employed to assess the plasmids. This approach allows for the selection of cloned bacteria and confirms the successful cloning of DNA.