So far in the genetic engineering journey we have learnt how to isolate or produce a DNA fragment, and amplify it in vitro using PCR. Bacterial transformation is a method to amplify a DNA fragment in vivo (in a living organism), and to engineer bacteria to produce a protein. In this article we will look at the steps involved in this process.
Inserting a DNA fragment into a vector
A vector is something used to transfer DNA into another cell. Often a vector is a plasmid (a small circle of DNA) which is what we will use as an example in this article. Another type of vector is a bacteriophage – a virus which can infect bacteria and therefore inject the DNA fragment.
To insert a DNA fragment into a plasmid, we need our old friends the restriction enzymes. The vector is cut open using the same restriction enzyme that was used to isolate the DNA fragment, so the sticky ends will be complementary to each other. The DNA fragment and plasmid will join with complementary base pairing, then the enzyme DNA ligase is added to join the sugar-phosphate backbones with phosphodiester bonds. Recombinant DNA has been formed, because the DNA is now a combination of the DNA fragment and the plasmid vector.
Transforming bacterial cells
The plasmid vector must now be taken up by bacterial cells. However, bacteria will not take up the plasmid unless the permeability of the cell-surface membrane is increased. There are two methods to do this:
- Electroporation: an electromagnetic field is passed through a solution containing the bacteria and plasmid.
- Heat-shock: a solution containing the bacteria and plasmid are placed on ice then transferred to 42°C for about a minute.
The plasmid will now be inside some of the bacterial cells, but not all of them. So the next step is to identify which bacteria contain the plasmid. To enable this, the plasmid is designed to contain a marker gene. This marker gene often codes for antibiotic resistance, but could also code for fluorescence.
The bacteria are spread onto an agar plate containing an antibiotic. Each individual bacteria will divide by binary fission and produce a little colony. However, only bacteria which contain the antibiotic resistance marker gene will be able to survive and divide, so any bacteria which did not take up the plasmid will be destroyed. A colony of transformed cells can now be cultured further until there are a huge number of bacteria containing the plasmid.
Producing a protein
Often when doing genetic engineering you want the transformed cells to transcribe and translate the DNA fragment to produce the protein. In order for this to work, the plasmid must contain a promoter region before the DNA fragment and a terminator region after the fragment. The promoter region acts as the “start here” flag to tell RNA polymerase that it must start transcribing at that point. The terminator region acts as the stop signal (similar to the stop codon). Sometimes the promoter can also be used as the “on switch”, and the fragment will only be transcribed if something specific is present e.g. a certain antibiotic. This enables transcription to be turned on and off by the scientist.
- A DNA fragment is inserted into a vector using a restriction enzyme and DNA ligase.
- Electroporation or heat-shock enables the bacteria to become transformed and take up a plasmid vector.
- A marker gene allows for selection of successfully transformed bacterial colonies.
- Promoter and terminator regions in the vector allow transcription of the DNA fragment.