Bacteria are commonly used as host cells for making copies of DNA in the lab because they are easy to grow in large numbers. Their cellular machinery naturally carries out DNA replication and protein synthesis.
Amazing bacteria
Bacteria are incredibly versatile organisms that have the unique ability to take in foreign DNA and replicate (or copy) it. This gives them an evolutionary advantage and helps them survive changes in their environment. For example, bacteria can acquire DNA that makes them resistant to antibiotics.
The bacterial genome is contained on a single, circular chromosome. This genetic material floats freely in the cell, unlike eukaryotic organisms where the genetic material is enclosed within a nuclear membrane.
Bacteria may sometimes contain smaller circles of DNA, called plasmids, which have a much smaller number of genes. Plasmids can be swapped between bacteria in a process called conjugation.
Using plasmids in the lab
Plasmids can be used as vectors to carry foreign DNA into a cell. Once inside the cell, the plasmid is copied by the host cell’s own DNA replication machinery.
In the lab, plasmids are specifically designed so that the DNA they contain will be copied by bacteria.
Plasmid essentials
Laboratory-designed plasmids contain a small number of genes that help transformation. These include:
- An origin of replication. This is the specific sequence of nucleotides where DNA replication begins.
- A multiple cloning site. This site contains recognition sites for specific restriction enzymes. These restriction enzymes can be used to ‘cut’ the plasmid so foreign DNA can be ‘pasted’ in by ligation.
- A resistance gene. This gene codes for a protein the bacteria need in order to survive in a particular growth medium, for example, when a specific antibiotic is present.
Inserting genes into plasmids
The piece of DNA or gene of interest is cut from its original DNA source using a restriction enzyme and then pasted into the plasmid by ligation.
The plasmid containing the foreign DNA is now ready to be inserted into bacteria. This process is called transformation.
Bacterial transformation
Before transformation, bacteria are treated with a chemical called calcium chloride, which causes water to enter into the cells and makes them swell. These swollen bacteria are then known as competent bacteria.
Next, plasmid DNA (containing the foreign DNA) is mixed with the competent bacteria and the solution is heated. The plasmid DNA enter the bacteria through small pores created in the cell membranes. Once in the host cell, the plasmid DNA is copied many times by the bacteria’s own DNA replicating machinery.
How do you know if it worked?
After transformation, bacteria are grown on a nutrient rich food called agar. Only bacteria containing a plasmid with antibiotic resistance will grow in the presence of antibiotic.
For example, if the bacteria are grown on agar containing the antibiotic ampicillin, only the bacteria that have been transformed with a plasmid containing the resistance gene for ampicillin will survive.
Transformed bacteria can then be grown in large amounts. The DNA of interest, or the protein coded for by the DNA, can then be isolated and purified.
When is transformation used?
Bacterial transformation is used:
- To make multiple copies of DNA, called DNA cloning.
- To make large amounts of specific human proteins, for example, human insulin, which can be used to treat people with Type I diabetes.
- To genetically modify a bacterium or other cell.
