Week+of+2-31-10

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Looking at the overall format and such of my wiki, there is room for improvement. I will start by making things more clear to the "lay" reader, but there are some reorganization and revamping of pages that could be done, particularly because not much work remains before getting into the lab.

This week I reviewed old material, and continued preparing to go into the lab to conduct mutagenesis, starting with choosing and ordering a primer. i also made some modifications to my Mutagenesis summary page, linking some confusing vocab terms to the Terms and Concepts page, as well as adding a table of contents to that page.

The primer that we have decided on is a modification of face 1. We will be changing one of several Lysine amino acids (symbol K). We chose this particular amino acid because it has a highly polar side chain (a positive one, to be specific). Changing a highly polar amino acid on a region that is evolutionarily very well conserved is likely to have an effect on the performance of the NPC2 protein after the primer is inserted to the primary sequence, amplified, and introduced to a culture of cells. By observing the effect on the protein, we hope to glean some information on the function of face 1 (currently believed to be a mediator in a reaction with one of NPC2's partners). We will begin by changing the Lysine to a nonpolar, hydrophobic amino acid, followed by a negative, polar, hydrophilic amino acid. The Lysine positions we are considering are shown by the arrows on this image of the primary amino acid sequence: The position on the 3D model can be seen here:

As stated on my Mutagenesis page, a primer should be 25-40 bases long so as to minimize the chances of secondary structure formation and maximize ease of annealing. An amino acid is coded for by 3 bases, so four amino acids on either side of the target lysine should be sufficient. For the leftmost Lysine indicated in the above picture, this would yield a primary amino acid sequence primer of: KLPV(new amino acid)SEYP.