Mycobacteriophages Isolated from Tropical Soils of Puerto Rico

Anaeli Shockey

Nicolle Rosa

Mentor: Dr. Rubin

Introduction

Mycobacteriophages are viruses that infect bacteria belonging to the Mycobacterium genus.

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.

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

Methods

Isolate Phage

Prepare Filtrate

Plaque Purification

Web Pattern (dilutions)

High Titer Assay

SDS 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

follow the same procedure.

Filtration Follow the same four steps of the first harvesting which is

the filtering 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 properly identified plaque.

Incubate.

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.

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.

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.

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°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.

Results: Harvesting and Plaque Purifications

Results: Web Pattern

#1 -3 #2 -4

#3 -4

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.

Electron Micrograph Images Difference in abundance and sizes.

Shockage Zombage

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.

Acknowledgement

Lab Technician: Giovanni Cruz

Christopher Quintanal

Dr. Michael Rubin

RISE Program

Questions?