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Production of plasmid-encoded chlamydial proteins in host cells

Production of plasmid-encoded chlamydial proteins in host cells. Jeffrey Burnett Dr. Dan Rockey Lab. Chlamydia trachomatis. most common STI 2.8 million cases annually causes: Pelvic Inflammatory Disease Ectopic pregnancy Infertility Preventable blindness asymptomatic.

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Production of plasmid-encoded chlamydial proteins in host cells

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  1. Production of plasmid-encoded chlamydial proteins in host cells Jeffrey Burnett Dr. Dan Rockey Lab

  2. Chlamydia trachomatis • most common STI • 2.8 million cases annually • causes: • Pelvic Inflammatory Disease • Ectopic pregnancy • Infertility • Preventable blindness • asymptomatic

  3. Gram (-) bacterium Obligate intracellular aerobe Cannot make its own ATP Inclusion Biphasic lifecycle Non-infectious metabolically active form Infectious “spore” form C. trachomatis

  4. Background knowledge • Horoschak and Moulder • Decrease in amount of cells • Bose and Liebhaber • No decrease in DNA synthesis • Greene and Zhong • Interactions between chlamydia and host cell cycle

  5. Background knowledge McCoy cells – blue (nuclei), green (cytoskeleton), red (chlamydial protein transfection - CT223) McCoy cells – blue (nuclei), green (cytoskeleton)

  6. Background information • Rockey et al • Inclusion membrane proteins (inc) • CT119 - incA • Identification of CT 223 as inc gene

  7. My Project • Two projects: • Find active domain of CT 223 • Double transfection with genes in inc operon

  8. Project #1

  9. Project #1 - conclusion • Active site of CT223 is c-terminus 56 AA sequence • To do: • What is the relevance? • Note: Chlamydia blocks cytokinesis • Find binding partners

  10. Project #2 - part one • Make expression vectors • Transformation • Send for sequencing

  11. Project #2 - part one

  12. Project #2 - part one

  13. Project #2 - part two • Goal of part two: • Attain double transfection of same host mammalian cell • Conclude whether or not there is an effect on polynuclear cells

  14. Project #2 - part two

  15. Project #2 - part two

  16. Results • No effect on polynuclear morphology between single transfection CT223 and double transfections • To do: • Transfections with CT225, and CT226 to see if same conclusion can be drawn

  17. Summary • Project #1 • Found active domain of CT223 - last 56 aa • To Do: relevance / active partner in host • Project #2 • Found no increase in rate of polynuclear cells in double-transfected cells • To Do: look at other genes (CT225, CT226)

  18. Acknowledgements • Howard Hughes Medical Institute • Dr. Kevin Ahern • Dr. Dan Rockey and his Lab • Damir Alzhanov • Department of Biomedical Sciences in the college of Veterinary Medicine

  19. Thank you for your time. Questions?

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