Goals for This Laboratory Course
Unlike most of your labs in college, this lab is very similar to a graduate student rotation project. You have been given the assignment of cloning all five yeast IDH genes using PCR and later express the encoded proteins in bacteria. (Problem #1: how can you express a eukaryotic gene in a prokaryote since the latter cannot splice out introns?) After you have expressed the recombinant protein, determine the molecular weight of the IDH monomer using SDS-PAGE and a western blot. (Problem #2: How will we do a western blot if we do not have an antibody that will bind to the yeast IDH proteins?) In addition, you will want to use the yeast gene as a probe to see if it hybridizes to any orthologs from yeast. Finally, you will want to perform enzyme assays on your recombinant proteins to determine if they still retain their properties when expressed in a prokaryote.
Introduction
The goal of this semester's molecular biology lab is to clone 5 genes from the yeast Saccharomyces cerevisiae. This species of yeast was the first of the big six genome organisms to have its entire genome sequenced. There are many web sites devoted to Saccharomyces cerevisiae but a good place to start looking is <http://genome-www.stanford.edu/Saccharomyces/>. You will want to find all 5 genes and learn more about them. The gene IDP2 is the ortholog of the one you studied in Bio111 Lab.
Weekly Steps in this Project - Each Week Builds on Previous Results
Start at the beginning - Jan. 15/17
1) We will watch a safety video and make stock solutions that will be used throughout the semester. In addition, we will practice pipetting because in molecular biology, you MUST be good at pipetting.
Purify genomic DNA and design PCR primers - Jan. 22/24
1) We will begin by purifying yeast genomic DNA as our template for PCR
2) Next find all 5 genes, restriction sites for each gene, expression plasmid sequence, design PCR primers to produce in-frame fusion protein with His6 epitope and affnity purification tag.
Design PCR Primers, Make Web Pages - Jan. 29/31
1) You will need to finalize the design of your PCR primers and show them to Dr. C.
2) Finish collecting any information about these 5 genes and make your web sites.
Conduct PCR, Finish Web Pages - Feb. 5/7
1) Start PCR to amplify all 5 yeast IDH genes.
2) Final touches on your IDH web pages.
3) We will need some LB +amp plates and this will be a good time to pour them.
Clean PCR, verify on gel, ligate, transform into expression cells - Feb 12/14
1) Clean PCR products.
2) Ligate PCR products into expression vector.
3) Transform ligation into JM109 strain of E. coli.
4) Verify PCR products with gel electrophoresis
Screen for insert and orientation - Feb. 19/21
1) Night before, pick colonies and grow them overnight (O/N)
2) Minprep plasmid DNA
3) Digest plasmids with restriction enzyme
4) Run gel to verify cloning and orientation of PCR product.
5) Save gel for next week.
Southern Blot part 1 - Feb. 26/28
1) Southern blot DNA onto membrane.
Southern Blot part 2 - March 12/14
1) Finish S. blot and see if orthologs bind to one another.
Express IDH Proteins - March 19/21
1) Night before, start O/N culture of cells, forward orientation.
2) Induce protein production.
3) Affinity purify IDH proteins. Freeze in aliquots.
Run Western Blot part 1 - March 26/28
1) Run SDS-PAGE.
2) Transfer to membrane.
Finish Western Blot - April 9/11
1) Bind antibody and detect prtoeins.
2) Determine MW of all 5 IDH proteins.
Run IDH Enzyme Assays - April 16/18
1) Verify NAD+ and NADP+ specificity.
2) Mix and match to optimize reation rates.
Final Wrap Up - April 23/25
1) Finish up any research not completed so far.
Turn in Lab Notebooks and Lab Reports - April 30/May 2
1) Your lab notebooks are due at lab time.
2) Your lab reports are due at lab time.