Transcript T5 Final Presentation

PHYTOPHTHORA INFESTANS eEF3 FUNCTIONALLY
COMPLEMENTS FOR LOSS OF FUNCTION OF
SACCHAROMYCES CEREVISIAE eEF3
NJGSS 2016: T-5
Tanique Bennett, Kaitlyn Boyle, Emma Carlson, Arjun Gupta, Enoch Jiang,
Ryan Jin, Aishwarya Kalyanaraman, Jeferson Mendoza, Pooja Nahar,
Danielle Pergola, Siri Uppuluri, Sophia Velasquez
➢Fungal pathogens
➢Current combative methods
Overview
➢Targeting eukaryotic
translation
➢Eukaryotic elongation factor 3
➢Materials, methods, procedures
➢Conclusions and implications
(“Potato Blight - Recognising and avoiding”)
Fungal Pathogens
Health & Ecological Threats
● 50% of HIV/ AIDS
patients killed by
fungal infections
annually
● Crop devastation
(Paddock)
Combative Methods
● Diagnosis/treatment of
fungal infections is
insufficient
● Human susceptibility is
increasing
(Knutsem)
Phytophthora infestans (P. infestans)
● Fungus-like species
(oomycete)
● Devastating economic
effects
(Nutter)
○ $2.75 billion lost annually
Antifungal Drugs
(Nail fungus treatment)
● Current drugs are too expensive
and ineffective.
● Many come with various side
effects.
● Antifungal resistance strains
hinder targeting of fungal
diseases
● Drugs are not being specific
enough when targeting.
Amphotericin B is the most common systemic
antifungal drug used to treat life-threatening conditions
(Ocular Pharmacology of antifungal drugs)
Central Dogma
DNA → RNA → Protein
Eukaryotic
Elongation Factor 3
(eEF3)
An Essential
Translation Factor
(Chakraburtty)
eEF3 as a Potential Drug Target
•
•
Highly conserved in single-celled eukarya
•
Ideal candidate for antifungal drug target
Not functionally active in higher
complexity eukaryotes

Critical for survival in lower organisms

Minimal threat of toxicity to host cells
(Dulai)
Preliminary Observations
S. cerevisiae eEF3
•
•
Regions of conservation
between P. infestans and
S. cerevisiae eEF3 genes
P. infestans eEF3
Fission yeast eEF3 can
functionally complement
S. cerevisiae eEF3
(Anderson)
Hypothesis
Recombinant P. infestans eEF3 gene can
functionally complement for the S. cerevisiae
wild-type eEF3 gene, and S. cerevisiae can
survive with P. infestans eEF3.
Plasmid Shuffle as a
Method to Determine
Functional
Complementation
Isolation of Plasmid from Escherichia coli
•
•
E. coli containing empty plasmid was inoculated
Performed MiniPrep to isolate plasmids
Digestion / Gel Electrophoresis
•
Empty plasmid opened using restriction enzymes to
form a modifiable vector
•
Gel electrophoresis run to cross check bands with
standard bands
Gel Purification
● Excision
● Incubation
Gibson Assembly
● P. infestans EF3 gene
● Hybridization
Transformation of vector into E.coli
•
•
Recombinant vector added to competent E.coli cells
Purpose: amplify vector for yeast transfection
Digestion/Gel
Electrophoresis
Purpose: Verify Gibson
Assembly’s success by
digesting & running DNA
•
Several bands matched
•
Indicated success
Transfection of Plasmid into
Saccharomyces cerevisiae
•
The plasmid was
transformed into S.
cerevisiae and plated
onto a petri dish.
Nutritional Selection
& Plasmid Shuffle
Selected for LEU2 marker
•
Surviving colonies
contain both eEF3 genes
Selected against URA4
•
Kills yeast cells with wildtype eEF3
•
Plasmid shuffle: Must
expel wild-type eEF3 to
survive
Plasmid Shuffle In Action
P.I. EF3
ARS
AMP
LEU2
Final Results
Conclusions
•
•
•
P. infestans eEF3 successfully functionally complements the wildtype eEF3 of S. cerevisiae
Great potential for future targeted therapy against fungal
pathogens
Expanded knowledge of functionality and necessity of eEF3
sequence regions
Future Endeavors
•
•
•
Experimental studies of unexplored regions of P.
infestans’ genome
Further research into antifungal drug targets
Specific sequences of eEF3 may be later explored to
elucidate other potential gene functions
Special thanks to...
Overdeck Foundation
State of New Jersey
Mango Concept
Independent College Fund of New Jersey
Johnson & Johnson
Novartis
Allergan
Celgene
NJGSS Alumnae and Parents of Alumnae
Advisor: Dr. Stephen Dunaway
Assistant: Jal Trivedi
Works Cited
Potato Blight - Recognising and avoiding. quickcrop.ie. 2013 Sep 4 [accessed 2016 Jul 26]. https://www.quickcrop.ie/blog/2013/09/potato-blight-recognising-andavoiding/
Paddock. HIV antibody therapy looks promising. Medical News Today. 2016 May 9 [accessed 2016 Jul 28].
http://www.medicalnewstoday.com/articles/310065.php
Knutsen RM. Diagnostic Testing for Fungal Infections. laboratory-manager.advanceweb.com. 2014 Dec 22 [accessed 2016 Jul 25]. http://laboratorymanager.advanceweb.com/features/articles/diagnostic-testing-for-fungal-infections.aspx
Nutter. Phytophthora infestans, continued. [accessed 2016 Jul 28]. http://www.apsnet.org/publications/imageresources/pages/iw00009_2.aspx
Dulai K. Bio 10 Biology Lab . 2016 Feb 16 [accessed 2016 Jul 26]. http://www.csus.edu/indiv/d/dulaik/coursebio10_3cells.htm
hytophthora infestans, continued.
Chakraburtty K. Elongation Factor 3 in Fungal Translation. Encyclopedia of Life Sciences. 2001.
Andersen CBF, et al. Structure of eEF3 and the mechanism of transfer RNA release from the E-site. 2006; 443(7112): 663–668.
Selecting a Nail Fungus Treatment. Global Nail Fungus Organization. [accessed 2016 Jul 28]. http://nationalnailfungus.org/treatment-guide/selecting-a-nail-fungustreatment/