Question

Describe the mechanism of transformation.

Short answer

The key steps in the process of transformation include the preparation of competent cells, the uptake of plasmid DNA, the recovery period, the selection and verification of transformants, and the incorporation of foreign DNA into the host genome.

Step-by-step solution

Preparation of Competent Cells

In the transformation process, bacterial cells must be made competent, or capable of taking up foreign DNA molecules. This is accomplished by treating the cells with divalent cations (such as calcium chloride) and chilling them on ice, creating small reversible pores in the cell membrane. This treatment increases cell permeability to DNA molecules.

DNA Uptake

Next, the competent cells are mixed with foreign DNA, typically a circular DNA molecule called a plasmid. The plasmid often carries a gene of interest that the researcher wants to introduce into the cells, along with resistance genes, allowing for easy selection of successfully transformed cells. Heat shock treatment (a rapid shift from ice-cold temperature to a warmer one) is used to facilitate the uptake of plasmid DNA but only a small percentage of cells will take up the plasmid.

Recovery Period

After heat shock, the transformed cells are incubated in a rich growth medium, such as Lysogeny broth (LB), for a short period, allowing the cells to recover and express any new plasmid-encoded genes, like antibiotic resistance genes.

Selection and Verification of Transformants

The transformed cells are then spread onto selective agar plates containing an antibiotic (e.g., ampicillin) that kills non-transformed cells (those without the resistance gene from the plasmid). After incubation, only those cells that have successfully taken up and expressed the plasmid-encoded antibiotic resistance genes will survive and form colonies on the plate. These colonies can be picked and analyzed to verify the presence of the gene of interest by various molecular techniques, such as polymerase chain reaction (PCR) or DNA sequencing.

Incorporation of Foreign DNA into the Host Genome

In some cases, the foreign DNA may be incorporated into the host bacterial genome either by homologous recombination or, in some bacterial species, through specialized mechanisms (e.g., site-specific recombination systems). Once the DNA has been integrated into the host genome, it becomes a stable part of the bacterium's genomic DNA, passing to its daughter cells upon cell division.