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.

The addition of foreign DNA makes the host bacterium a new, genetically modified organism

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.

Find out more in these articles:

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).

Read the article, Bacterial DNA – the role of plasmids, for further information.

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.

Find out more in these articles:

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.

Find out more about bacterial transformation.

A vector that delivers genes to plants

The Ti (tumour inducing) plasmid from the bacterium Agrobacterium tumefaciens is a special example of a plasmid that is used as a vector in biotechnology. Agrobacterium lives close to the roots of plants. Unusually, it ‘pushes’ its Ti plasmid into plant root cells. In the wild, this causes cancer in the plant.

In the laboratory, scientists have used the ‘plasmid-pushing’ ability of Agrobacterium to help make transgenic plants. The scientists clone their gene of interest into a Ti plasmid, along with DNA sequences that make sure the gene can be expressed in plants. They then reintroduce the vector to Agrobacterium cells and infect plants with the modified Agrobacterium, which delivers the vector to plant cells. The result is a transgenic plant.

Find out more about transgenic plants in this interactive.

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. Find out more about what we think about biotechnology in New Zealand in this article.

Useful links

This animation from the Association of the British Pharmaceutical Industry shows the steps involved in cloning a gene and introducing the recombinant vector to bacteria

 

Published 13 March 2014