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.

Why Toxoplasma gondii? Parasite effects 1/3 of the human population Can be re-infected multiple times Causes toxoplasmosis No current treatments http://media-cache-ec0.pinimg.com/736x/cf/26/4a/cf264a68248ff3d0df1bf2f2a89641c1.jpg

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.

Membrane Fusion

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). http://slideplayer.com/25/7616149/big_thumb.jpg

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”

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

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

Our plasmid

CRISPR-CAS9 Sequencing Results

Experimental Protocol http://en.academic.ru/pictures/enwiki/51/300px-Splicing_by_Overlap_Extension_PCR.svg.png

DHFR

DHFR Positive Results

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

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

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