Transcript Presentation - Harlem Children Society

Genomic Screening
Princess Kara Parker-Smith
Marlyn Gonzalez and Dr. Peter Lipke
Hunter College, CUNY
Harlem Children Society
Genomic Screening
Introduction 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 GPIproteins 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 Screening
Introduction Part II
Why 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.
Genomic Screening
Visual Preparation
Cell wall (inner
layer)
Cell
membrane
Cell wall (outer
layer)
mRNA
protein
Gene encoded for Reporter
GPI tagged with a green
florescent protein (Alphaagglutinin gene) inserted into
the plasmid
Genomic Screening
Procedure
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
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
B) Processing protein to remove
carbohydrates
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.
Genomic Screening
Procedure
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
Molecular
Weight
Standard
250 KD
150
100
75
50
37
25
20
15
10
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 Screening
Conclusion
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 Screening
References
1.
http://images.google.com/images?q=Western+Blotting&hl=en
2.
Instructions for Binding Proteins to Beads
3.
http://www.bio.davidson.edu/courses/genomics/method/Westernblot.html
4.
http://images.google.com/imgres?imgurl=http://www.piercenet.com/media
/PDetectFig
24.gif&imgrefurl=http://www.piercenet.com/Proteomics/browse.cfm%3Ffl
dID%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
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Acknowledgements
Marlyn Gonzalez
Naomi Thomas
Dr. Peter Lipke
Hunter College, CUNY
Dr. Sat Bhattacharya
Harlem Children Society
Ms. Redway
Thank you!