Mycobacteriophages Isolatedfrom Tropical Soils of Puerto RicoAnaeli ShockeyNicolle RosaMentor: Dr. Rubin
Introduction Mycobacteriophagesare viruses that infectbacteria belonging tothe Mycobacteriumgenus.
Introduction Two cycles: Lysogenic: Infects host, integrates genome andpropagates with the host chromosome. Lytic: Infects, copies DNA, makes new virions, lyses thecell and escapes.
Applications Field of biomedice Elimination of antiabiotic resistant bacteria Phage Therapy
Materials Agar plates Sterilization filters Syringes Phage buffer Microcentrifuge tubes M. smegmatis culture Disposable pipettes 1000µL and 100µL micropipettes Vortexer Centrifuge Shaking Incubator
MethodsIsolatePhagePrepareFiltratePlaquePurificationWeb Pattern(dilutions)High TiterAssaySDS Gel
First Steps: Enrichment Harvesting Plaque Purifications
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.
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.
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.
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.
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.
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.
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.
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.
Electron Micrograph Images Difference in abundance and sizes.Shockage Zombage
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.
Acknowledgement Lab Technician: Giovanni Cruz Christopher Quintanal Dr. Michael Rubin RISE Program