Working with plasmids.

Working with plasmids.

Plasmids are relatively small, replicate very quickly and are thus easy to study and to manipulate. It is easy to determine the sequence of its DNA, that is, to find out the sequence of the letters (A, C, G and 1) and number them. Certain letter combinations -such as CAATTG - are easy to cut with the help of specific enzymes (see proteins). Ilese cutting enzymes, called restriction enzymes, are part of the Genetic Engineering "tool-kit" of biochemists. So if I want to splice a gene from fish into a plasmid, I have to take the following steps: I place a large number of a known plasmid in a little test tube and add a specific enzyme that will cut the plasmid at only one site. After an hour I stop the digest, purify the cut plasmid DNA and mix it with copies of the fish gene; after some time the fish gene places itself into the cut ring of the plasmid. I quickly add some "glue" from my "tool-kit" - an enzyme called ligase - and place the mended plasmids back into bacteria, leaving them to grow and multiply. But how do I know which bacteria will have my precious plasmid? For this reason I need MARKER GENES, such as antibiotic resistance genes. So I make sure my plasmid has a marker gene before I splice my fish gene into it. If thA I plasmid is marked with a gene antibiotic resistance I can now add specific antibiotic to the food supply of the bacteria. All those which do not have the plasmid will die, and all those that do have the plasmid will multiply.