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Interactives -- DNA

Genetic Engineering : Engineering a Plasmid
 

After the lux gene is isolated, it will be inserted into a small, circular piece of bacterial DNA, called a plasmid. Bacteria can naturally pick up plasmids from their environment or from other bacteria. Plasmids usually contain a few genes, including genes that make their host bacterium resistant to antibiotics.

Molecular biologists can use many different types of plasmids with special characteristics that are helpful for different purposes. Plasmids usually have known restriction enzyme sites, allowing scientists to cut them apart with a certain type of restriction enzyme and then add in a gene.

The bacteria used in genetic engineering experiments are often a special variety of the common gut bacteria Escherchia coli, or E. coli, that can survive in the lab only when given special nutrients. The plasmid we will use here has a gene that allows its bacterial host to survive on growth medium that lacks one of those nutrients; bacteria without the plasmid die if grown on the modified growth medium.

Once the plasmid and lux gene have been cut with restriction enzymes, their DNA will have sticky ends. Since sticky ends attach together randomly, the plasmid sometimes closes up on itself, and the ends of the gene sometimes get stuck together. Occasionally, however, the sticky ends of the plasmid and the gene adhere, and the lux gene is inserted into the plasmid.

Another enzyme called ligase is now added. It seals up the bond between the lux gene and the plasmid DNA.

Now that we have plasmids with our lux gene, we need to get them into bacteria. To do this, we mix the bacteria with the plasmids and then spread them onto a growth medium in a Petri dish. Many bacteria will naturally pick up plasmids, and some of those plasmids will have the lux gene. How do we know which bacteria have the gene and which don't? Remember that our plasmid has a gene which lets bacteria grow on a modified growth medium that is missing a special nutrient. Only bacteria containing that plasmid can survive; the rest will die.

Next, to find bacteria containing plasmids with the lux gene, we simply dim the lights; the lux gene causes bacteria to glow in the dark.



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