Transcript Cloning of Eukaryotic Elongation Factor 3 from

Cloning of Eukaryotic
Elongation Factor 3 from
Phytophthora infestans
Team 8
New Jersey Governor’s School in the Sciences 2015
Introduction: Fungal Diseases
● 3 million U.S. cases per year
● 27% increase in demand for
antifungal drugs
● Current treatments are ineffective
or detrimental
o Host toxicity
o Emergence of resistance
(Donahue, 2012)
(Tsafrir)
Phytophthora infestans as a Re-emerging
Agricultural Threat
(Wikimedia, “Phytophthora infestans”)
(Wikimedia)
(W. E. Fry)
Looking for a Drug Target: Protein
Translation
(Quintanilla)
(Wikimedia)
eEF3’s Role in Protein Translation
● Lack of eEF3 in lower
eukaryotes → death
● Ejects tRNA from Esite
● Conserved only in
lower eukaryotes
○ No eEF3-like gene
in higher
eukaryotes
eEF3’s function in moving
tRNA out of E-site
eEF3 as a Drug Target
● Possible
antifungal drug
target
o Eliminating
eEF3
destroys
fungi, no
effect on
host cells
(Andersen, 2006)
(Andersen, 2006)
Research Goal
To clone the eukaryotic elongation
factor 3 (eEF3) from the funguslike oomycete, P. infestans
(Deacon)
Strategy for Molecular Cloning of P.
infestans eEF3 by Gibson Assembly
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)
Agarose Gel Electrophoresis
(Wikimedia, “Gel Electrophoresis”)
Objective: separate DNA
based on size
(Wikimedia “DNA Chemical Structure”)
Gibson Assembly
Objective: produce plasmid containing P. infestans eEF3 gene
E. coli Transformation
Objective: produce many copies
of P. infestans eEF3 plasmid
Access Excellence, “E. coli”
Electrophoresis of DNA Digest
Lane
8
7
6
5
4
3
2
1
● DNA digest:
8 kb
8 kb
Figure
created
by author
o to prepare plasmid
vector for insertion of
P. infestans eEF3
gene
● Verify isolation of
plasmid vector
o Lane 1,5: DNA digest
o Lane 3: Marker
Agarose Gel Electrophoresis of E. coli
Plasmid
Lanes 1 to 8 (right to left)
Expected
Result L8 L7 L6 L5 L4 L3
8 kb
L2 L1
Lanes 1,2,3,6,7,8:
Digested Samples (note
smears)
Lane 5: Undigested
Sample
3.1 kb
Lane 4: Marker
Reasons for Failure: Preliminary
Analysis
● Colonies grew on
ampicillin plate
o All steps up to
transformation
worked
● No DNA was harvested
from MiniPrep isolation
● What went wrong?
(Wikimedia, “E. coli colonies”)
Reasons for Failure: Solution
Reasons for Failed Transformation
Reason for Smearing
Horizontal
integration of
plasmid
(Wikimedia, “Plasmid Replication”)
Degraded
ampicillin (most
likely)
(Brown iGEM, 2008)
Genomic DNA
isolated, not plasmid
(Cooper Pharmaceuticals)
Future Goals for Research:
Transforming P. infestans eEF3 into Model Organism through Plasmid Shuffle
S. cerevisiae
S. cerevisiae
Original
plasmid
OUTWARD
Original
plasmid
URA 3
URA 3
Recombinant
plasmid
Recombinant
plasmid
LEU2
LEU2
Leucine-lacking and 5FOA containing media
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
Supplementary Slide: Gibson Assembly
(Virvliet, “Gibson Assembly”)
Supplementary Slide: Gibson Assembly