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  • 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?