How to add foreign DNA to bacteria
Using modern laboratory techniques, it is relatively easy to add pieces of foreign DNA to bacteria. To do this, scientists first package their DNA of interest within a circular DNA molecule (a vector). They then use various techniques to induce bacteria to take up the vector.
Why add DNA to bacteria?
Scientists add foreign DNA sequences to bacteria for two reasons:
- To make it easier to work with the DNA sequence. Once inside bacteria, a stretch of DNA can readily be copied and its sequence determined.
- To make a foreign protein within bacteria. If the introduced DNA is a gene that encodes a protein, scientists can study the gene’s protein product by expressing it in bacteria.
Packaging foreign DNA for bacteria
If you tried to put your gene of interest into bacteria without any extra DNA surrounding it, you’d fail! The foreign gene alone has no instructions to tell the bacteria to make copies of it. For this reason, it would be overlooked by bacteria (or even chopped up by bacterial enzymes), so subsequent generations of bacteria would not contain ‘your’ sequence of DNA.
To overcome these issues, scientists package their DNA of interest into vectors – circular DNA molecules that look very similar to pieces of bacterial DNA. The process of putting a gene into a vector is called molecular cloning or gene cloning.
Most vectors are based on plasmids, which are small circular sequences of DNA that occur naturally within bacteria. Plasmid vectors can accept a few genes’ worth of DNA. Others, called bacterial artificial chromosomes or BACs, can contain much longer DNA sequences. A vector has an ‘origin of replication’ – a stretch of DNA that ensures it gets replicated (copied) by the host bacterium. Often, it also contains a promoter sequence so that the introduced gene can be expressed (and a protein produced).
Get information sheet: Bacterial DNA – the role of plasmids
Cloning DNA into a vector, step by step
To introduce foreign DNA into a circular vector, scientists carry out a three-step process:
- Cutting out the gene. Scientists first remove their gene of interest from the DNA sequences on either side of it. They can use restriction enzymes to do the cutting. These enzymes, which came originally from bacteria, cut DNA at specific sites in the sequence. If there’s not enough DNA for successful cutting or no suitable restriction enzyme recognition sites around the gene, scientists first use polymerase chain reaction (PCR) to make many more copies. By designing their PCR primers carefully, they can introduce new restriction sites on either side of the copied DNA sequence.
- Opening up the vector. Next, scientists make a cut in the circular DNA sequence of the vector. They use the same restriction enzymes as they used to cut out the gene in step 1. This turns the vector into a linear molecule and makes it ready to accept the new piece of DNA.
- Sticking the vector and the gene together. The final step in cloning is to incorporate the DNA of interest into the vector. Scientists mix the gene and the opened vector together with a bacterial enzyme called DNA ligase. The ligase sticks DNA ends together to form a single circular molecule that includes both the vector and the gene.
Getting the vector into bacteria
Once a vector that contains foreign DNA has been constructed in the lab, it is introduced into bacterial cells. Scientists do this by creating tiny holes (pores) within the bacterial cell membrane. It’s fairly easy to make the pores – you can do it by suddenly heating the bacterial culture by several degrees or by passing an electric shock through the culture. The vector enters the bacteria while the pores are open. The pores close again quickly – otherwise, the bacteria would die!
Once bacteria have recovered from the process of introducing DNA (called transformation), they can be cultured in the lab. Because the vector has an origin of replication, it is copied and passed to daughter cells in the same way as the bacterium’s own DNA.
Get information sheet: Bacterial transformation
Adding DNA makes GM bacteria
Bacteria that contain foreign DNA are considered to be new, genetically modified organisms (GMOs). For this reason, the conditions of their use are strictly controlled. Most GM bacteria are produced to be lab tools – for making copies of DNA, producing proteins and so on – and never leave the lab.
Get theme: New Zealand views on biotech
- 13 March 2014