*This page is part of undergraduate assignment for Molecular Biology at Davidson College
BIO 306: Molecular Biology Course Webpage
By Elizabeth Shafer
Molecular Biology is a 300 level biology course taught at Davidson College, by the illustrious Dr. A. Macolm Campbell. This course focuses on techniques used in Molecular Biology in conjunction with providing information on cellular processes. This webpage was designed to outline lab work I completed during this course on cloning isocitrate dehydrogenase and to link to a paper I wrote reviewing an article in nature. The following links will take you to either project.
As part of my Molecular Biology course this spring, my class is attempting to clone and express the genes that encode the 5 isoforms of the Saccharomyces’ gene isocitrate dehydrogenase: IDH1, IDH2, IDP1, IDP2, and IDP3.
Isocitrate dehydrogenases are enzymes that oxidize isocitrate and are involved in glutamate biosynthesis, isocitrate biosynthesis, and the tricarboxylic acid cycle.
Cloning and Sequence Information about Isocitrate Dehydrogenase
The 5 isoforms we are interested in differ in DNA sequence, amino acid sequence, restriction fragment map, the primers they require, the fusion protein they produce with the plasmid, and the coenzyme that they require to catalyze reactions.
Each isoform is described in the following links:
In our experiment we will isolate each gene, amplify the gene with PCR, and clone it in to a plasmid pQE –30UA for expression. To insure that each insert is in the correct orientation within the plasmid, we will digest the plasmid with a restriction enzyme that is known to cut at a certain location with in the insert. If the insert is in the correct orientation then a Southern blot will reveal the correct size band, if not the Southern blot will yield a band of a different size. Because each gene varies in sequence and the restriction enzymes that cut them, we will use a specific enzyme for each gene will expect different size bands to result for each gene insert. Digestion Experiment Design.
This phenomena could be attributed to a number of variables in the process of isolating, amplifing, and cloning the gene as well as to the biological function of the gene itself.
Plasmids which have the insert in the correct position will express the protein encoded by the insert. The structure of the isocitrate dehyrdogenase proteins provide insight into its biochemical role within the cell. Orthologs are genes with a similar sequence in differ species. Information on orthologs reveal how conserved a protein is across species, thereby suggesting the protein’s universal importance in the cell’s functioning.
Check out this links to see more about the protein structure and orthologs of isocitrate dehydrogenase.
Created by: Elizabeth Shafer. Email questions to firstname.lastname@example.org