Mutagenesis

Today will be an educational blog post. This past week I started the beginning of my own experiment. Now, the big picture of the experiment is to learn more about the binding site of anti-depressant drugs. Not a lot is known about HOW these drugs work on a molecular level. We know they bind to the transporter in the cell membrane (see earlier post), and plug the gate, so that neurotransmitters cannot get back into the cell. We are not quite sure where the drug actually binds though. These experiments will test different areas on the transporter protein to see if they are involved in the binding. Before we go any further, let's work our way down to the molecular level.

• The neuron cell is enclosed by a cell wall
• The cell wall has transporters embedded in it. (rainbow colored thing in picture)
• The transporter is a large coiled protein.
• Protein is made of a long string of amino acids. (The numbers in the darker image are used to identify specific amino acids. So the 320th amino acid on the protein chain is F for phenylalanine. There are 20 different amino acids)
• Amino acids are coded from the DNA sequence.

Now let's work back up:

• If we change letters in the DNA, a different amino acid will be coded.
• If we have a different amino acid, the shape, and or chemical properties will change in that section of the protein.
• That change in shape and chemical properties may or may not effect the ability for the transporter to do it's job.
• If the mutation causes more neurotransmitters to get through the transporter, than we know that the mutation effected the binding area of the inhibitory drug, and is there for an important section of the protein. 

By making many different mutations, we can begin to see what area the drug is actually binding to, and what areas it could be forced to bind to. Mutagenesis basically means that a mutation is created at a defined site in the DNA molecule. I have found 15 locations on the transporter that I would like mutate. I figured out which amino acids are in those locations. I decided what amino acid I wanted to change it to. I then found the triplet on the DNA that codes for that specific amino acid. For example, on the DNA strand TGT would code for a cystine amino acid. If I want to mutate that into tyrosine, I would change it to TAT. 

It's as simple as that. After hours of preparation and research to figure out what to mutate, where to mutate, and what to mutate it to, we just delete a triplet and add a new one. Bam.