1. Reverse Genetic Screen in Toxoplasma gondii
Haley LeFaivre, Stephen L. Denton, and Jason P. Gigley
University of Wyoming
Department of Molecular Biology
INBRE
Introduce topic then myself, Steve, and Gigley as my mentors and advisors in this project.

2. Why Toxoplasma gondii? Parasite effects 1/3 of the human population
Can be re-infected multiple times
Causes toxoplasmosis
No current treatments

3. Invasion of Toxoplasma gondii
The apical end of the parasite attaches to the host cell’s membrane, then special organelles called rhoptries secrete proteolytic enzymes that causes the cell membrane to invaginate the parasite and forms a vacuole that is unsuspected by the host-cell’s defense mechanisms.

4. Membrane Fusion

5. Membrane Fusion Gene Selection
By using the BLAST program, we compared the genes of T. gondii to yeast for susceptible genes that are used in membrane fusion. (steve will you help me explain the Query versus subject of the graph for clarification).

6. Hypothesis
We hypothesized that the genes identified in the homology screen will be required for Toxoplasma gondii secretory organelle biogenesis
T. Gondii as shown in the previous slide on parasite invasion, we suspect that organelles involved in membrane fusion are vital to its fitness and survival. If the parasite cannot synthesize proteins for membrane fusion, then it will effect the parasite fitness making it “sick”

7. Overview of XP53 Possible Calcium ATP-ase Potential Roles
pH Maintenance
Radical Oxygen Species Control
Cell Signaling Pathway’s in Membrane Fusion

8. CRISPR/Cas9 system background
(Sander and Joung, Nature Biotechnology 2014)

9. Our plasmid

10. CRISPR-CAS9 Sequencing Results

11. Experimental Protocol

12. DHFR

13. DHFR Positive Results

14. Summary
To get Adobe Illustrator I would need to pay for it. Are there any websites I can download it for free?

15. Future Directions Testing Toxoplasma gondii Fitness:
Growth and Replication
Motility
Invasion
Egress
Testing Toxoplasma gondii in Mouse Models
Virulence
Protection
Parasitemia

16. Acknowledgments INBRE Stephen L. Denton Daria Ivanova Kevin Fettel
Dr. Jason P. Gigley
University of Wyoming
Unsure if this is the correct format and everyone is accredited