Genetic transformation allows a cell to take up and incorporate foreign genetic material from its environment. This technique is foundational in molecular biology for introducing engineered DNA into host bacteria, such as Escherichia coli. The goal is to force the bacteria to accept a recombinant plasmid, which is DNA created by combining genetic material from two or more sources. This procedure is used to replicate the foreign DNA sequence and often to produce the protein encoded by the new gene. The process requires controlled physical and chemical steps to overcome the cell’s natural resistance to foreign material.
The Essential Components
The two primary ingredients are the bacterial host cell and the recombinant plasmid. E. coli is the most common host because it grows quickly, is easy to manipulate, and its genetics are well understood. The plasmid is a small, circular piece of DNA that replicates independently of the bacterial chromosome.
A recombinant plasmid is engineered to contain a segment of foreign DNA. For the plasmid to function as a genetic carrier, it must possess the Origin of Replication (ori) for copying, the Multiple Cloning Site (MCS) for DNA insertion, and a Selectable Marker. This marker, typically an antibiotic resistance gene, ensures that only transformed bacteria survive in a selective growth environment.
Preparing the Host Cells
Bacteria are naturally resistant to the uptake of large, negatively charged plasmid DNA due to their protective cell wall and membrane. The first step is making the bacterial cells competent, inducing a temporary state receptive to external DNA. This is achieved through chemical competence or electroporation.
Chemical competence involves treating the cells with a cold solution containing divalent cations, such as calcium chloride (\(\text{CaCl}_2\)). The positively charged calcium ions neutralize the negative charges on the DNA and the cell membrane, reducing repulsion. Electroporation uses a brief, intense pulse of electricity to create temporary microscopic pores. Regardless of the method, the final outcome is a batch of fragile host cells ready to receive the recombinant plasmid.
Introducing the Plasmid
Once the bacterial cells are competent, the plasmid DNA is physically introduced. In the chemical competence method, the plasmid DNA is added to the chilled cells and incubated on ice for 15 to 30 minutes to maximize DNA association with the cell surface.
The most characteristic step is the heat shock, involving a rapid, brief temperature shift. The cell-DNA mixture is quickly moved from ice to a warm water bath (typically 42°C) for a short duration, then immediately returned to ice. This sudden fluctuation forces the plasmid DNA through temporary pores in the cell membrane.
Following the heat shock, the transformed cells undergo a recovery period in a nutrient-rich, antibiotic-free liquid medium, usually incubated at 37°C for about an hour. This incubation allows the bacterial membranes to repair themselves and provides time for the antibiotic resistance gene on the plasmid to be expressed. This recovery is necessary for the bacteria to survive the subsequent selective environment.
Identifying Successful Transformants
Transformation has low efficiency, meaning only a small fraction of cells successfully take up the plasmid. The final phase is selection, which separates transformed cells from non-transformed ones. This is accomplished by plating the recovered bacteria onto a selective agar medium containing the antibiotic corresponding to the plasmid’s resistance gene. Only cells that acquired the resistance gene will grow and form colonies; non-transformed bacteria are killed by the antibiotic.
While selection confirms transformation, it does not confirm the plasmid taken up is the desired recombinant one. To verify the plasmid contains the intended foreign DNA insert, screening is necessary. A common technique is blue/white screening, where successful insertion disrupts a gene that produces a blue pigment, causing the colony to appear white. Further verification, such as Polymerase Chain Reaction (PCR) or DNA sequencing, confirms the correct insert is present.