• Save
Phages presentation  final final final
Upcoming SlideShare
Loading in...5

Like this? Share it with your network

  • Full Name Full Name Comment goes here.
    Are you sure you want to
    Your message goes here
    Be the first to comment
    Be the first to like this
No Downloads


Total Views
On Slideshare
From Embeds
Number of Embeds



Embeds 2

http://portafolionicollerosamercado.wordpress.com 2

Report content

Flagged as inappropriate Flag as inappropriate
Flag as inappropriate

Select your reason for flagging this presentation as inappropriate.

    No notes for slide


  • 1. Mycobacteriophages Isolatedfrom Tropical Soils of Puerto RicoAnaeli ShockeyNicolle RosaMentor: Dr. Rubin
  • 2. Introduction Mycobacteriophagesare viruses that infectbacteria belonging tothe Mycobacteriumgenus.
  • 3. Introduction Two cycles: Lysogenic: Infects host, integrates genome andpropagates with the host chromosome. Lytic: Infects, copies DNA, makes new virions, lyses thecell and escapes.
  • 4. Applications Field of biomedice Elimination of antiabiotic resistant bacteria Phage Therapy
  • 5. Materials Agar plates Sterilization filters Syringes Phage buffer Microcentrifuge tubes M. smegmatis culture Disposable pipettes 1000µL and 100µL micropipettes Vortexer Centrifuge Shaking Incubator
  • 6. MethodsIsolatePhagePrepareFiltratePlaquePurificationWeb Pattern(dilutions)High TiterAssaySDS Gel
  • 7. First Steps: Enrichment Harvesting Plaque Purifications
  • 8. Second Enrichment and Filtration Enrichment Isolate a phage with the tip of a micropipette. Add in the same solution as the first enrichment and followthe same procedure. Filtration Follow the same four steps of the first harvesting which is thefiltering process.
  • 9. Medium Titer Assay: Dilutions From the filtration, dilute four phage solutions. In four tubes labeled from -1 to -4, add 90uL of phage buffer. To the -1 tube, add 10uL of the filtration and centrifuge. To the -2 tube, add 10uL of the -1 tube and centrifuge. Repeat this process up to the -4 tube. Add 10uL of each tube (including the filtration) to a sample of bacteria. Let sit for 15 – 30 minutes. Add top agar to the bacteria and spread the solution on a properlyidentified plaque. Incubate.
  • 10. Medium Titer Assay Result: Web pattern (arrangement of plaques in whichalmost all of the bacteria was lysed) Add 6mL of phage buffer to plaques #1 -3, #2 -4, and #3-4 Place in the refrigerator. Extract the phage buffer from each plaque. Filter each and, once again, place it in the refrigerator.
  • 11. High Titer Assay Determine which was the dilution the completely lysed thebacteria. Add 10µl of dilution to solution of agar and bacteria. Distribute the mixture on 10 plates and incubate. Add phage buffer to all of the plates. Break apart the agarand mix with the buffer. Place the plates in the incubator, shaking for 4 hours. Extract the phage buffer, centrifuge, and filter.
  • 12. Rapid Isolation, Separation andVisualization of Caspid Proteins Medium: phage buffer extracted from web pattern in MTA 1ml of HTPL to a microtube and centrifuge for an hour. Aspirate the supernatant. Prepare sample buffer. Boil the samples for two minutes and cool them down for twominutes. Centrifuge. Prepare the gel. Prepare 1x running buffer. Carefully handle the gel and assemble it. Add the runningbuffer.
  • 13. Cont. SDS Gel Load samples and molecular weight markers. Run gel for 30 minutes. Strain the gel in a plastic tray. Wash the gel three times. Stain the gel for one hour with gentle shaking. Rinse the gel for 30 minutes. Photograph on white light box. Store in water in a zip lock bag.
  • 14. Results: Location Gurabo, PR. 18°1448.53"N 66° 06.55"W Sunny/clear morning. 25.6°C. Next to trees andcompost. Dry soil and taken 5.74 inches deep.
  • 15. Results: Harvesting and PlaquePurifications
  • 16. Results: Web Pattern#1 -3 #2 -4#3 -4
  • 17. SDS Gel AS1 is named Shockageand we can see its proteinbands. AS2 and 3 are the samephage based on theirprotein band similarity andit is named Zombage.
  • 18. Electron Micrograph Images Difference in abundance and sizes.Shockage Zombage
  • 19. Conclusion Mycobacteriophages are viruses that infect bacteria andhave applications in the field of biomedicine. Two different phages were isolated: Shockage andZombage. These phages were taken up to the High Titer AssayProtocol and the SDS gel. The next step would include to sequence their DNA.
  • 20. Acknowledgement Lab Technician: Giovanni Cruz Christopher Quintanal Dr. Michael Rubin RISE Program
  • 21. Questions?