1 / 25

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

teresa
Télécharger la présentation

Phage

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  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?

More Related