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Phage

Phage. Ethan Callies. Purpose. To collect, recreate and analyze phage from the environment To sequence DNA from phage. Background. Virus that replicates using bacteria Ernest Hankin , 1896 India, something in water cured Cholera Frederick Twort , 1915

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Phage

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  1. Phage Ethan Callies

  2. Purpose • To collect, recreate and analyze phage from the environment • To sequence DNA from phage

  3. Background • Virus that replicates using bacteria • Ernest Hankin, 1896 • India, something in water cured Cholera • Frederick Twort, 1915 • Discovered small agent that killed bacteria • Work interrupted by WWI • Félix d'Herelle • September 3, 1917 discovered “an invisible, antagonistic microbe

  4. Phage Therapy • Phage used as antibacterial agents • Began in 1920’s • Today is most common for fighting Staphylococcus, Streptococcus, and other infections

  5. My Phage • Collected from base of tree in River Falls, WI • Moist soil conditions • About 44mm below surface • Lola13

  6. Enrichment of Environmental Samples • Did this to increase the chance I will get a phage from my sample

  7. Phage Titer/Streak Plate • Titer • Determine concentration of Plaque forming units (pfu) • Streak Plate • Help purify single phage population from mixed populations

  8. Phage Purification • Isolate enough pure phage sample to create Lysate or MTL (Medium Titer Lysate)

  9. Phage Lysate • Used to create large batch of pure phage called HTL (High Titer Lysate)

  10. Web Plates • Obtain HTL with high enough phage concentration to isolate DNA

  11. Isolate/Purify Genomic DNA • Want to isolate this in high enough amounts for restriction analysis/sequencing

  12. Gel Electrophoresis • To compare phage DNA sample to a known DNA sample

  13. Digest Phage DNA • See how Phage DNA compares to known samples when digested by different enzymes

  14. Electron Microscopy • Observe individual phage using electron microscopy

  15. Results (After 3 enrichments) • 3 different morphologies developed • Created streak plate for each • Smaller and bigger morphologies purified very well • Titrated each out separately with 4 dilutions

  16. Results (after discovering morphologies) • Smaller morphology to 2nd dilution • Bigger had plaques to 4th dilution Small titer: 4.6x10^-4 pfu/mL Bigger titer: 5.4x10^-6 pfu/mL • Repeat purification

  17. Results (after repeating purification) • Smaller morphology disappeared • Bigger morphology to all plates (8 in 10^-2, 2 in 10^-3 and 1 in 10^-4)

  18. Spot test to determine web plate Countable plate to determine titer Titer: 2.8x10^-4 pfu/mL

  19. Web Plate • Plate webbed very well • Small morphology appeared again • Set up 10 web plates

  20. Harvest HTL/Titer/Quanitification • All 10 plates completely plaqued out • Titer came out to be 2.75x10^-9 pfu/mL • Spectrophotometer read .119 mg/mL

  21. Restrict and analyze phage DNA

  22. Restrict and analyze phage DNA (again)

  23. EM Picture • Tails are approximately 70 nm • Heads are approximately 30 nm

  24. Phage most similar to Jawanski NcoRI

  25. Conclusion • Many morphologies appeared throughout purifications. While this could be because of contamination, I think it was because of something biological. This could be improved a bit by making things a bit more “sterile” while going through the purification process. • What caused the different sized plaques to show up? Contamination or biology?

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