ISOLATION OF NOVEL MYCOBACTERIOPHAGES FROM TROPICAL SOILS OF PUERTO RICO

Aida Vargas de JesusDanilo Trinidad Pérez Rivera

Lizbeth Perez CastroJavier M. Zavala Ayala

http://corvusart.com/

RISE ProgramDepartment of BiologyProf. Eneida Díaz PérezProf. Michael Rubin

Objectives

Present the bacteriophages of interest.

Establish adequate hunting strategies.

Explain the processes carried out for preparing the phages after localization.

Demonstrate growth acquired from the project so far.

What is a “phage”?

Bacteriophages, informally known as “phages”, are viruses capable of infecting a bacterium and replicating inside of it. (Snustad and Simmons, 2011)

These are typically composed of proteins which encapsulate their DNA or RNA genome.

www.theepochtimes.comwww.shapeways.com

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. (Brüssow at al. 2004)

Virulent-Lytic:

Straightforward cycle by lysing all bacteria they infect.

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

Why should we search for phages?

Due to their specific nature, many phages can shed light not only on viruses, but also on their host. (Silva et al. 2001)

Moreover, identification of “wild species” can reveal how the given type of phage is “evolving”.

Understanding these two things is transcendental to medical applications. (Clark and March 2006)

Mycobacteria & Bacillus

http://www.genomeindia.org/mycobacterium/http://www.fallingpixel.com/bacillus-bacteria-3d-model/142

M. smegmatis B. cereus

Experimental Design

Questions Can new phages be found in the environment

in Puerto Rico? What environments should be inspected to

obtain a phage? Hypotheses

New phages are widespread throught most ecosystems, therefore, there should be phages in Puerto Rico.

The probability of finding a phage in a given environment depends on if it is adecuate for the phages host.

Procedure

httpwww.teachthechildrenwell.comscience.

html

http://www.exportersindia.com/swastikaelectric/products.htm

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

Soil

EnrichmentFiltration

Purification

Dilutions Ten Plate Preparation

(Rubin and Vázquez 2012)

ResultsData for the soil samples recollected by D.Perez

Sample Coordinates

Environmental description Location Date

DTP - #1

18° 04’46.08”N; 66° 21’47.29”W

Dry; Superficial (1 cm); 20.6°C Coamo, PR

Feb/06/145:45 AM

DTP - #2

18° 02’11.57”N; 66° 22’22.25”W

Moist; Superficial (1 cm); 21.7°C

Los Baños de Coamo

Feb/19/146:36 AM

DTP - #3

18° 07’12.19”N; 66° 09’26.60”W

Mud-like; Superficial (3 cm); 21.7°C Cayey, PR

Mar/06/149:52 AM

Results

Sample Coordinates Description Location Date

DTP - #4

18° 07’12.75”N; 66° 09’26.89”W

Moist soil; Superficial (1 cm); 17.8°C Cayey, PR

Mar/18/149:13 AM

DTP - #5

18° 07'5.430" N;66° 09'42.58" W

Moist soil; Superficial (1 cm); 17.8°C Cayey, PR

Mar/18/149:24 AM

DTP - #6

18° 07’12.19”N; 66° 09’26.60”W

Mud-like consistency; Superficial (3 cm); 18.3°C Cayey, PR

Mar/18/149:43 AM

ResultsData for the soil samples recollected by A.Vargas

Sample Coordinates Description Location Date:Time

AV - #1 18°4’27” N; 66.11’56” W

Moist; Superficial sample (1cm); 24°C

Cayey, PR

Feb/2/143:56PM

AV - #2 18°28’4” N; 66°19’52” W

Very moist, sample of dirt underneath clover leaves (1cm); 30°C

Dorado, PR

Mar/2/143:05 PM

AV - #3 18°7’30” N; 66°15’48” W

Normal moisture (underneath grass), 1cm;23°C

Aibonito, PR

Mar/9/1411:07 AM

AV - #4 18°7’14”N;66°16’22”W

Dry (underneath dried leaves; chicken nesting area), 1cm; 23°C

Aibonito, PR

Mar/9/1411:36 AM

ResultsSample Coordinates Description Location Date:Time

AV - #5 18°4’19”N;66°12’20”W

Some moisture, 1cm; 27°C

Cayey, PR Mar/16/144:19PM

AV - #6 18°4’29”N;66°11’56”W

Moist (underneath grass and dried feces), (1cm) 27°C

Cayey, PR Mar/16/144:45PM

AV - #7 18° 4’31”N;66°11’51”W

Wet (mud) Superficial (1cm); 27°C

Cayey, PR Mar/16/146:24PM

Cacimar

Wilsonn

Results

ResultsData for the soil samples recollected by L. Perez

Sample Coordinates Description Location Date:Time

LP - #1 18º 16’ 16’ N, 65º 58’

10’ W

Loose, and moist Gurabo,PR

Feb/3/147:34 AM

LP - #2 18º 6’ 58’ N, 66º 9’ 19’ W

Moist and Chunky Cayey, PR

Feb/17/2014

8:20 AM

LP - #3 18º 16’ 7’ N, 65º 58’ 32’

W

Loose, and moist next to cow feces

Gurabo, PR

Feb/24/2014

8:05 AM

Results

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

SDS – PAGE Results1 2 3 4 5 6 7 8 9 10

1 2 3 4 5 6 7 8 9 10

1 – Marker 2 – Cacimar 3 – Incognito 4 – Isinaayusferti 5 – Nitidusvenutus 6 – Serotinus 7 – Wilsonn 8 – Figaro 9 – Gêmeos

Puerto Rico has phages

Where should you hunt?

X

Summary

Understanding and characterizing bacteriophages provides plenty of useful information.

Puerto Rico has an ample amount of uncharacterized phages in its soil.

One should search for phages where their host could possibly be found.

Future Work

Describe the structure of these phages utilizing Electron Microscopy.

Sequence and annotate the genetic material of these phages.

Lessons learned

Losing is winning.

Practice makes perfect.

Friends make it a lot easier.

Patience is a virtue.

Quitters never win.

Special Thanks

The RISE program for opening the door for us to such a great opportunity.

Dr. Rubin for volunteering to give us such a full and enriched experience.

Nicolle Rosa and Juan Apiz for their help as our assigned TA.

Gustavo and Giovanni for always being there to help.

The cafeteria for the free “sample collectors”.

Our moms for the Ziplock bags.

Referrences

Asai DJ, Bailey C, Barker LP, Bradley KW, Khaja R, Lewis MF. 2012. Sea-Phages: Resource Guide. Chevy Chase, Maryland: Howards Hughes Medical Institute; 168 p.

  Brüssow H, Canchaya C, Hardt W. 2004. Phages and the Evolution

of Bacterial Pathogens: from Genomic Rearrangements to Lysogenic Conversion. Microbiol. Mol. Biol. Rev. 68(3): 560-602 p.

Cantón R, Horcajada JP, Oliver A, Garbajosa PR, Vila J. 2013. Inappropriate use of antibiotics in hospitals: The complex relationship between antibiotic use and antimicrobial resistance. Enferm. Infecc. Microbiol. Clin; 31(4): 3-11.

  Clark JR, March JB. 2006. Bacteriophages and biotechnology:

vaccines, gene therapy and antibacterials. Trends Biotechnol; 24(1): 212-218.

Delbück, M. 1945. The Burst Size Distribution in the Growth of Bacterial Viruses (Bacteriophages). J Bacteriol; 50(2): 131-135.

  Leung PCS, Shu S, Kenny TP, Wu P, Tao M. 2010 March.

Development and validation of gene therapies in autoimmune diseases: Epidemiology to animal models. Autoimmun Rev; 9(5): A400-A405.

Rubin M, Vázquez E. 2012. Microbacteriophage Proteomics: From Genotype to Phenotype (There and Back Again)!.Cayey, PR. Howard Hughes Program, Department of Biology; 20 p.

Silva AJ, Elgersma Y, Costa RM. 2001. From genes to therapies: the role of animal models. Clin Neurosci Res; 1(3): 187-193.

Snustad P, Simmons MJ. 2011. Principles of Genetic, 6th Edition. Hoboken, N.J.: J Wiley; 784 p.

ISOLATION OF NOVEL MYCOBACTERIOPHAGES FROM TROPICAL SOILS OF PUERTO RICO

Aida Vargas de JesusDanilo Trinidad Pérez Rivera

Lizbeth Perez CastroJavier M. Zavala Ayala

http://corvusart.com/

RISE ProgramDepartment of BiologyProf. Eneida Díaz PérezProf. Michael Rubin