ISOLATION OF A NOVEL MYCOBACTERIOPHAGE

Lizbeth Pérez Castro

Javier Zavala Ayala

Mónica Del Moral

Félix Vallés

WHAT IS A BACTERIOPHAGE?

• A bacteriophage is any virus that infects any bacterium.

• Mycobacteriophages are viruses that infect mycobacteria.

• Found in different bacterial colonies.

• Mostly found in the soil and sea water.

• Resistant to antibiotics.

http://news.bbcimg.co.uk/media/images/66413000/jpg/_66413229_m0900092-bacteriophage_virus-

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BACTERIAS FOR HOSTING

1. M. smegmatis (Smeg)

• fast growing rate and non-pathogenic behaviour

• Aerobic organisms

• Found in water, soil and plants

2. Bacillus

• Obligate aerobes or facultative anaerobes

• Well understood- used as a model for differentiation, gene regulation, and cell cycle events in bacteria.

• Common in soil

Both are:

Gram positive

Acid fast bacteria

Morfology- rodshape

BACTERIOPHAGE STRUCTURE

• Phages consist of 3 basic components:

• Capsid-contains the genetic material

• Genetic material- DNA

• Tail-serves to attach to bacteria and DNA passes through tail and into bacterium

http://en.wikipedia.org/wiki/Bacteriophage

PHAGE LIFE CYCLES

• Temperate-Lysogenic:

• Replicate and lyse or dormant state- incorporate its genetic material into DNA of host.

• Virulent-Lytic:

• Straightforward cycle by lysing all bacteria they infect.

http://biomedfrontiers.org/infection-2014-3-28/

INTRODUCTION

• Problem: Can novel bacteriophages be isolated from tropical soil of Puerto Rico?

• Hypothesis: Unique bacteriophages will be isolated and characterized from a collected soil sample of Puerto Rico.

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13229_m0900092-bacteriophage_virus-spl.jpg

PROCEDURE

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http://quizlet.com/6760545/lab-3-streak-plate-

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Soil

EnrichmentFiltration

Purification

HTPLTen Plate Preparation

MY BACTERIOPHAGE

• Name: Nitidusvenutus

• Located in: Carretera 943 Sector Los Chinos, Gurabo

• Coordinates: 18º 16’ 7’ N, 65º 58’ 32’ W

• Collected: February 24, 2014

• It was found in a rural area next to cow feces on a template day.

LOCATION

MY BACTERIOPHAGE

RESULTS

http://faculty.plattsburgh.edu/donald.slish/lambdatitre.html

MY BACTERIOPHAGE: MUSAMODEL

• Name- Musamodel

• Located in: 1-9 calle Barcelona, Urb.Terralinda, Caguas P.R

• Coordinates: 88˚14’6”N , 66˚2’39”W

• Collected: March 16, 2014

• Time: 7:51pm

• Temperature: 79˚

• About 3 inches deep and 1 foot closeto concrete

• Found next to a plantain tree

• Soil was moist and loose

LOCATION

SAMPLES COLLECTED1st 2nd 3rd 4th 5th 6th 7th

Date 2/6/14 2/18/14 2/24/14 3/3/14 3/10/14 3/16/14 3/16/14

Time 7:53 am 7:06 am 9:55 pm 9:38 am 6:26 am 7:45 pm 7:51 pm

Coordinates 18˚14’6”N,

66˚2’40”W

18˚14’13”N,

66˚2’35”W

18˚14’7”N,

66˚2’39”W

18˚14’18”N,

66˚2’42”W

18˚14’7”N,

66˚2’40”W

18˚14’6”N,

66˚2’39”W

18˚14’17”N,

66˚2’42”W

Temperature 75˚ 75˚ 79˚ 81˚ 73˚ 79˚ 79˚

Soil

description

Moist, 3

inches deep

Moist, 1 inch

deep

Moist, 1

inch deep

Moist, with

moss, 1 inch

deep

Moist, 1

inch

Moist,

loose, 5

inches

deep

Moist, loose,

3 inches

deep

Site

description

Urban, 5 feet

close to

concrete

Urban, 1 foot

from

concrete

Urban, 2

feet from

concrete

Urban, ½

foot from

concrete

Urban, 3

feet away

from

concrete

Urban, 1

foot close

to

concrete

Next to a

plantain

plant, 1 foot

close to

concrete

RESULTS

CONCLUSION

• Two mycobacteriophages were found:

1. Nitidusvenutusa. Located in Carretera 943 Sector Los Chinos, Gurabo.

b. Based on the clear plaques, we can assume it has a lytic life cycle.

2. Musamodela. Located in 1-9 calle Barcelona, Urb. Terralinda, Caguas P.R.

b. Up to now, we believe it has a lytic life cycle because of clear plaques.

c. Three purifications were completed.

FUTURE PLANS

• Nitidusvenutus;• Isolation of the bacteriophage’s DNA• Bioinformatics analyses and sequencing.

• Musamodel;• 3rd purification- to have more isolated plaques and measure diameter• Spot test• Make phage stocks-

• Empirical test

• 10 plate preparation

• Analyze • Bioinformatics and sequencing

ACKNOWLEDGEMENT

• Dr. Michael Rubin

• Juan Apiz

• Eduardo Correa

• Gustavo Martínez

• Giovanni Cruz

• Edwin Alvarado