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Genomic Screening Princess Kara Parker-Smith Marlyn Gonzalez and Dr. Peter Lipke Hunter College, CUNY Harlem Children Society
Genomic ScreeningIntroduction Part I Problem: Do the kre1 and cwp1 mutants of Saccharomyces cerevisiae play a role in the cleavage of the GPI- anchor of the GPI glycoproteins? Goal of Project: Identify the genes that play a role that allows the GPI- proteins to be incorporated into the cell wall. My Goal: Perform a western blot to compare the weight and size of the reporter GPI proteins yielded from the kre1 and cwp1 mutants and Wild Type of Saccharomyces cerevisiae.
Genomic ScreeningIntroduction Part IIWhy are we doing this? • We are trying to locate the genes responsible for the formation of the GPI proteins during cell wall development so that a drug can be made targeting every gene responsible in the creation of the cell wall, killing the fungi, Candida albicans. • However, Candida albicans is unsafe to work with because it is a pathogenic fungi. Thus, we worked with Saccharomyces cerevisiae. • Alpha- agglutinin, a cell adhesion protein, is produced in Saccharomyces cerevisiae and is homologous to the Als protein, which binds the infectious fungus, Candida albicans, to the mucus membranes of human beings. • The alpha- agglutinin was tagged with a green florescent light protein, making it a reporter GPI protein.
Gene encoded for Reporter GPI tagged with a green florescent protein (Alpha-agglutinin gene) inserted into the plasmid Cell wall (inner layer) mRNA Cell membrane protein Cell wall (outer layer) Genomic ScreeningVisual Preparation
Genomic ScreeningProcedure Step 1: Grow out mutant and wild type Saccharomyces cerevisiae in 3 separate flasks in media Step 2: Spin cells down and collect supernatant Step 3: See Diagram A) Protein detection and purification B) Processing protein to remove carbohydrates 1) Immobilize GFP antibodies onto the beads (in blue) 2) Run supernatant through the beads so the GFP proteins can stick to the GFP antibodies 3) Toss the proteins that go through the frit. 4) Run a wash buffer (3X) through the beads to release any loosely binded proteins that have similar properties to GFP 5) Run an elusion buffer through the beads to collect purified GFP binded to alpha- agglutinin proteins 6) Collect purified GFP binded to alpha agglutinin in a microcentifuge tube 1) Immobilize EndoH enzyme onto the beads (in blue) 2) Run the purified GFP binded to the alpha agglutinin through the beads so that the EndoH can deglycosylate the protein 3) Collect the protein in a microcentifuge tube.
Molecular Weight Standard 250 KD 150 100 75 50 37 25 20 15 10 Genomic ScreeningProcedure Step 4: Perform BCA Assay( used to determine concentration of proteins in sample. The concentration is used to determine the quantity of protein we need to place in our SDS polyacrylamide gel to see enough expression of protein.) Step 5: Perform western blot, encompassing SDS polyacrylamide gel Step 6: Incubate the nitrocellulose membrane in a series of wash buffers and antibodies Step 7: Wrap up nitrocellulose membrane and head to dark room for inspection
Genomic ScreeningConclusion Anticipated Results We do not know exactly what to expect, however we do know that if the reporter GPI proteins secreted out of the Wild Type Saccharomyces cerevisiae are lighter in weight and smaller than the reporter GPI proteins secreted from the kre1 and cwp1 mutants of Saccharomyces cerevisiae, then the gene sequence deleted in the mutants are needed for the healthy development of the cell wall of Saccharomyces cerevisiae. This would be a favorable outcome, however any result will produce helpful information.
Genomic ScreeningReferences • http://images.google.com/images?q=Western+Blotting&hl=en • Instructions for Binding Proteins to Beads • http://www.bio.davidson.edu/courses/genomics/method/Westernblot.html • http://images.google.com/imgres?imgurl=http://www.piercenet.com/media/PDetectFig 24.gif&imgrefurl=http://www.piercenet.com/Proteomics/browse.cfm%3FfldID%3D8259A7B6-7DA6-41CF-9D55 AA6C14F31193&h=307&w=375&sz=16&tbnid=8Kqu10CT QlkJ:&tbnh=96&tbn w=118&hl=en&start=9&prev=/images%3Fq%3DWest ern%2BB lotting%26sv num%3D10%26hl%3Den%26lr%3D
Acknowledgements Marlyn Gonzalez Naomi Thomas Dr. Peter Lipke Hunter College, CUNY Dr. Sat Bhattacharya Harlem Children Society Ms. Redway Thank you!