Cloning of Eukaryotic Elongation Factor 3 from Phytophthora infestans

Team 8New 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 detrimentalo Host toxicityo Emergence of resistance

(Tsafrir)

(Donahue, 2012)

Phytophthora infestans as a Re-emerging Agricultural Threat

(W. E. Fry)

(Wikimedia, “Phytophthora infestans”) (Wikimedia)

Looking for a Drug Target: Protein Translation

(Quintanilla) (Wikimedia)

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

eEF3 as a Drug Target● Possible

antifungal drug targeto 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 fungus-like 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 “DNA Chemical Structure”)

(Wikimedia, “Gel Electrophoresis”)

Objective: separate DNA based on size

Gibson Assembly

Objective: produce plasmid containing P. infestans eEF3 gene

E. coli Transformation

Access Excellence, “E. coli”

Objective: produce many copies of P. infestans eEF3 plasmid

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

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

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”)

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)

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

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

Supplementary Slide: Gibson Assembly

(Virvliet, “Gibson Assembly”)