cloning of eukaryotic elongation factor 3 from phytophthora infestans team 8 new jersey governor’s...
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Cloning of Eukaryotic Elongation Factor 3 from Phytophthora infestans
Team 8New Jersey Governor’s School in the Sciences 2015
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Introduction: Fungal Diseases
● 3 million U.S. cases per year● 27% increase in demand for
antifungal drugs● Current treatments are ineffective
or detrimentalo Host toxicityo Emergence of resistance
(Tsafrir)
(Donahue, 2012)
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Phytophthora infestans as a Re-emerging Agricultural Threat
(W. E. Fry)
(Wikimedia, “Phytophthora infestans”) (Wikimedia)
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Looking for a Drug Target: Protein Translation
(Quintanilla) (Wikimedia)
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eEF3’s Role in Protein Translation
eEF3’s function in moving tRNA out of E-site
● Lack of eEF3 in lower eukaryotes → death
● Ejects tRNA from E-site
● Conserved only in lower eukaryotes○ No eEF3-like gene
in higher eukaryotes
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eEF3 as a Drug Target● Possible
antifungal drug targeto Eliminating
eEF3 destroys fungi, no effect on host cells
(Andersen, 2006)
(Andersen, 2006)
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Research Goal
To clone the eukaryotic elongation factor 3 (eEF3) from the fungus-like oomycete, P. infestans
(Deacon)
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Strategy for Molecular Cloning of P. infestans eEF3 by Gibson Assembly
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Hypotheses
● The eEF3 gene from P. infestans can be cloned using the Gibson Assembly method
● The P. infestans eEF3 gene is functionally conserved in S. cerevisiae (baker’s yeast)
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Agarose Gel Electrophoresis
(Wikimedia “DNA Chemical Structure”)
(Wikimedia, “Gel Electrophoresis”)
Objective: separate DNA based on size
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Gibson Assembly
Objective: produce plasmid containing P. infestans eEF3 gene
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E. coli Transformation
Access Excellence, “E. coli”
Objective: produce many copies of P. infestans eEF3 plasmid
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Electrophoresis of DNA Digest Lane 8 7 6 5 4 3 2 1
● DNA digest: o to prepare plasmid
vector for insertion of P. infestans eEF3 gene
● Verify isolation of plasmid vector o Lane 1,5: DNA digesto Lane 3: MarkerFigure
created by author
8 kb
8 kb
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Agarose Gel Electrophoresis of E. coli Plasmid
Lanes 1 to 8 (right to left)
Lanes 1,2,3,6,7,8: Digested Samples (note smears)
Lane 5: Undigested Sample
Lane 4: Marker
L7 L5 L4L6 L3 L2 L1L8
8 kb
3.1 kb
ExpectedResult
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Reasons for Failure: Preliminary Analysis
● Colonies grew on ampicillin plateo All steps up to
transformation worked
● No DNA was harvested from MiniPrep isolation
● What went wrong?
(Wikimedia, “E. coli colonies”)
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Reasons for Failure: Solution
Reason for Smearing
Genomic DNA isolated, not plasmid
(Brown iGEM, 2008)
Reasons for Failed Transformation
Horizontal integration of
plasmid
Degraded ampicillin (most
likely)
(Wikimedia, “Plasmid Replication”)
(Cooper Pharmaceuticals)
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LEU2
Future Goals for Research:
Leucine-lacking and 5-FOA containing media
S. cerevisiae
Recombinant plasmid
URA 3
Transforming P. infestans eEF3 into Model Organism through Plasmid Shuffle
Original plasmid
S. cerevisiae
URA 3
Original plasmid
LEU2
Recombinant plasmid
OUTWARD
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Acknowledgements
We extend our gratitude to the following individuals and groups:
● Dr. Stephen Dunaway ● Mitchell Dittus● Astré Bouchier ● Justyna Pupek● Drew University ● AT&T ● Bayer Healthcare
● Independent College Fund of New Jersey/Johnson & Johnson
● The Overdeck Family Foundation● NJGSS Alumnae, Parents, and Corporate Matching
Funds● The State of New Jersey● Board of Overseers of New Jersey Governor’s School in
the Sciences● New Jersey Governor’s School in the Sciences
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Supplementary Slide: Gibson Assembly
Supplementary Slide: Gibson Assembly
(Virvliet, “Gibson Assembly”)