This document describes the isolation of mycobacteriophages from soil samples in Puerto Rico. Two students isolated three unique phages and characterized them through plaque assays, enrichment techniques, and SDS-PAGE gel electrophoresis. Two distinct phages, named Shockage and Zombage, were identified based on their protein band patterns and plaque morphology. Further sequencing of the phage genomes was proposed to better characterize the newly discovered viruses.
3. Introduction
Two cycles:
Lyseogenic: Infects host, integrates genome and
propagates with the host chromosome.
Lytic: Infects, copies DNA, makes new virions, lyses the
cell and escapes.
4. Applications
Field of biomedice
Elimination of antiabiotic resistant bacteria
Phage Therapy
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 follow
the same procedure.
Filtration
Follow the same four steps of the first harvesting which is the
filtering 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 properly
identified plaque.
Incubate.
10. Medium Titer Assay
Result: Web pattern (arrangement of plaques in which
almost 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 the
bacteria.
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 agar
and 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 and
Visualization 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 two
minutes. Centrifuge.
Prepare the gel.
Prepare 1x running buffer.
Carefully handle the gel and assemble it. Add the running
buffer.
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°14'48.53"N 66° 0'6.55"W
Sunny/clear morning. 25.6°C. Next to trees and
compost.
Dry soil and taken 5.74 inches deep.
17. SDS Gel
AS1 is named Shockage
and we can see its protein
bands.
AS2 and 3 are the same
phage based on their
protein band similarity and
it is named Zombage.
19. Conclusion
Mycobacteriophages are viruses that infect bacteria and
have applications in the field of biomedicine.
Two different phages were isolated: Shockage and
Zombage.
These phages were taken up to the High Titer Assay
Protocol and the SDS gel.
The next step would include to sequence their DNA.
