1. Cloning of Eukaryotic Elongation Factor 3 from Phytophthora infestans
Team 8
New Jersey Governor’s School in the Sciences 2015

2. Introduction: Fungal Diseases
3 million U.S. cases per year
27% increase in demand for antifungal drugs
Current treatments are ineffective or detrimental
Host toxicity
Emergence of resistance
(Donahue, 2012)
(Tsafrir)

3. Phytophthora infestans as a Re-emerging Agricultural Threat
(Wikimedia, “Phytophthora infestans”)
(Wikimedia)
Irish potato famine. Rapidly reproducing, high mutation rate, resistance (to plant defenses and antifungal).
(W. E. Fry)

4. Looking for a Drug Target: Protein Translation
Ronald
variations on a theme based on species
In looking for ways to treat these diseases, many researchers have targeted specific steps of translation, a process in which proteins are created and are essential for all organisms. In translation, different tRNAs, each with an amino acid, are brought over to the ribosome. By matching their anticodon to the complementary RNA triplet on the mRNA, the tRNAs move into the A site, and bind to the mRNA. As the ribosome translocates, each tRNA shifts over to the next site, and eventually exit at the E, or Exit site. The tRNA’s amino acid is incorporated into the growing polypeptide, which will be released once translation is done.
(Quintanilla)
(Wikimedia)

5. eEF3’s Role in Protein Translation
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’s function in moving tRNA out of E-site
In the process of protein translation, eEF3 is critical in removing deacylated eEF3 from the E-site. After the amino acid from a said tRNA has bound to the growing polypeptide chain, this tRNA must be removed from the ribosome to allow for acylated tRNA to enter the ribosome at the A site. The process of an acylated tRNA moving through the A, P, and E site to ultimately become deacylated is known as translocation and is facilitated by eEF3, which opens the L1 stalk in the ribosome, ejects deacylated tRNA and interacts with eEF1a which brings acylated tRNA to the A site. It is important to note that only lower eukaryotes have this eEF3 protein and higher eukaryotes such as fungi lack even eEF3-like genes.

6. eEF3 as a Drug Target Possible antifungal drug target
Eliminating eEF3 destroys fungi, no effect on host cells
Ronald:
Since eEF3 is only present in lower eukaryotes, it is a novel drug target for fungal related diseases as drugs inhibiting eEF3 would kill fungi, but remain harmless to us. Here is a basic picture of eEF3, showing its major protein domains. Most important is its ABC2 domain which contains a chromodomain important for its translation function in moving the L1 stalk, as mentioned previously. ABC stands for ATP Binding Cassette, which is a family of proteins that are important in ATP binding and hydrolysis to provide energy for other functions.
(Andersen, 2006)
(Andersen, 2006)

7. Research Goal
To clone the eukaryotic elongation factor 3 (eEF3) from the fungus-like oomycete, P. infestans
(Deacon)
The research goal was to insert the eEF3 gene from P. infestans into the plasmid vector, which is a circular piece of DNA that allows foreign DNA to be inserted into it and successfully clone it.

8. Strategy for Molecular Cloning of P. infestans eEF3 by Gibson Assembly
just like do you know what you’ve been doing the last 3 weeks

9. 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)
Our hypotheses were that we would be able to clone the P. infestans eEF3 gene using Gibson Assembly. We also hypothesized that this eEF3 gene would be functionally conserved in S. cerevisiae--that it would continue functioning in protein translation in budding yeast

10. Agarose Gel Electrophoresis
(Wikimedia, “Gel Electrophoresis”)
Objective: separate DNA based on size
Rachael
Agar gel, there are wells, you load your sample
Load onto the negative side
Travels towards the positive side (cathode) as DNA is negative due to the phosphate end group
Attracted towards the opposite charge
Rayleen
Separates the sample, as agar is porous, allowing the DNA fragments to travel through it
smaller pieces of DNA can travel farther and more quickly, following the path of negative to positive of the electric current
In our experiment, we used restriction enzymes to cut our DNA, and separated the separate pieces of DNA to identify the DNA fragments of interest
(Wikimedia “DNA Chemical Structure”)

11. Objective: produce plasmid containing P. infestans eEF3 gene
Gibson Assembly
Goal: to create the recombinant plasmid containing P. infestans eEF3 integrated into previously isolated plasmid
process by which multiple strands of overlapping DNA molecules can be joined together using an isothermal, single-reaction method
pieces that we were combining (Very small microliter amounts)
two gene blocks of eEF3 (too large a gene to do in one piece)
plasmid vector (purified from the gel)
water and master mix
It works due to overlapping ends of the dna
Gibson Assembly is only possible due to the innate ability to combine DNA via overlapping sequences in homologous recombination
The overlaps allow for the integration and mixing of the different fragments
Several enzymes in the mastermix that allow the fragments to combine
After a 1 hour incubation period, the result should be the pictured recombinant plasmid
Objective: produce plasmid containing P. infestans eEF3 gene

12. Objective: produce many copies of P. infestans eEF3 plasmid
E. coli Transformation
Objective: produce many copies of P. infestans eEF3 plasmid
We transformed in our newly created plasmid from the Gibson Assembly into the E. coli.
Tried to select for E.coli which accepted the plasmid, as well as force the E. coli to accept it
Plasmid contained an ampicillin resistant gene
Placed E. coli on ampicillin petri dish, so the E. coli would only survive if it accepted the plasmid
Ensure only E. coli with plasmid of interest would grow and reproduce
Reason for this: a method to copy the plasmid multiple times, very quickly
Similar to PCR, and allows the creation of large amounts of plasmid, which would be used later on in the experiment
Access Excellence, “E. coli”

13. Electrophoresis of DNA Digest
Lane
DNA digest:
to prepare plasmid vector for insertion of P. infestans eEF3 gene
Verify isolation of plasmid vector
Lane 1,5: DNA digest
Lane 3: Marker
8 kb
8 kb
dna digest-> prepare vector plasmid to uptake the eEF3 gene
this gel verified that we were able to open up the vector
in wells, loaded dna digest let it run
lane 3 control lanes 1,5 had our dna digest samples
an image could not be taken due to prolonged exposure, would cause mutations in plasmid vector
these fragments were quickly cut out and stored in eppendorfs for the gibson assembly and transformation
Figure created by author

14. Agarose Gel Electrophoresis of E. coli Plasmid
Expected
Result
Lanes 1 to 8 (right to left) L7 L5 L4 L6 L3 L2 L1 L8
Lanes 1,2,3,6,7,8: Digested Samples (note smears)
Lane 5: Undigested Sample
Lane 4: Marker
8 kb
3.1 kb
2nd electrophoresis to assess the state/presence of DNA in the samples
lane 4- marker for comparison
lane 5- undigested run to ensure DNA was actually present
Other lanes - Samples digested by same restriction enzymes (BAMHI, Xho1) these samples were digested and run through electrophoresis to isolate the P. Infestans eEF3 gene and ensure its presence within plasmids (to make sure the Gibson Assembly worked)
All lanes except for 4 show cloudy, white columns instead of clear,delineated bands
The lane all the way to the left is an example of what the electrophoresis results would’ve looked like if nothing had gone wrong

15. Reasons for Failure: Preliminary Analysis
Colonies grew on ampicillin plate
All steps up to transformation worked
No DNA was harvested from MiniPrep isolation
What went wrong?
(Wikimedia, “E. coli colonies”)

16. Reasons for Failure: Solution
Reasons for Failed Transformation
Reason for Smearing
Horizontal integration of plasmid
(Wikimedia, “Plasmid Replication”)
Degraded ampicillin (most likely)
Transformation of bacteria failed:
One possibility = the integration of the plasmid containing the eEF3 gene and the ampicillin resistance gene into the circular genomic DNA
The more likely explanation is the degradation of the ampicillin used on the plates the bacteria was growing on
Less active ampicillin concentrated on the plate, no selective pressure for bacteria with the eEF3 gene to grow, so other bacteria grew
Right off the bat, ruled out defective or old reagents.
Since we could not transform the plasmid successfully, the enzymes cut the genomic DNA (no plasmid) which results in numerous amounts of fragments with various lengths for digested DNA in lanes 1,2,3,6,7,8 (rather than single, delineated bands)
A possibility for the smear in lane 5 is DNA shearing →larger strands of DNA, in this case undigested DNA, are more susceptible to shearing due to micropipetting
(Brown iGEM, 2008)
Genomic DNA isolated, not plasmid
(Cooper Pharmaceuticals)

17. Future Goals for Research:
Transforming P. infestans eEF3 into Model Organism through Plasmid Shuffle
S. cerevisiae
S. cerevisiae
URA 3
URA 3
Original plasmid
Original plasmid
OUTWARD
LEU2
Recombinant plasmid
LEU2
Recombinant plasmid
S. cerevisiae
AKA baker’s yeast
Eukaryotic model organism used in many genetic studies
Can be cultured on media
Auxotrophic markers
Integrated into recombinant plasmid
LEU2
URA3
Leucine-lacking and 5-FOA containing media

18. 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

19. Supplementary Slide: Gibson Assembly
(Virvliet, “Gibson Assembly”)
Supplementary Slide: Gibson Assembly