Cluster Classification Cluster Classification of Mycobacteriophages of Mycobacteriophages Isolated From Tropical Isolated From Tropical

Soils of Puerto RicoSoils of Puerto RicoNicole Colón

Alberto CintrónCarolina MontañezLuz Marie Reyes

Dr. Michael Rubin

RISE Program, University of Puerto Rico at Cayeyhttp://phagesdb.org/

IntroductionIntroduction• What are Mycobacteriophages?

- Viruses that infect the Mycobacterial host.

• Where can they be found?-They can be found in the environment including soil.

• What diseases can Mycobacteria cause?-They cause diseases such as tuberculosis.

• What are Mycobacteriophages used for?-They can be used to infect and kill mycobacteria.-They are used as a model to study biological processes.

(Rubin, M. 2012), (Simmons, M., Snustad, P. 2012).

• How are Mycobacteriophages characterized?-They are characterized based on their genome.

• Classification in Clusters-They share characteristics based on sequence and annotated genomes.

• Proteomics-Study of protein structure and function.-Can also be used to classify phages.

IntroductionIntroduction

(Rubin, M. 2012).

Bacteriophage StructureBacteriophage Structure

DNAHead

Tail

(Rubin, M. 2012).

Lytic Lytic CycleCycleIs used by virulent phages.

Is characterized by phage reproduction followed by host cell lysis.

Stages include: 1. Adsorption 2. Entry 3. Replication4. Gene Expression5. Assembly6. Lysis (Simmons, M., Snustad, P. 2012)

Lysogenic cycle Lysogenic cycle

•Is characterized by three processes:

• Integration of phage DNA into host genome.• Excision of prophage.• Entry into the lytic cycle.

(Simmons, M., Snustad, P. 2012)

ObjectivesObjectives

• To analyze different unsequenced mycobacteriophages and classify them into their respective clusters using PCR and Gel Electrophoresis.

• To provide genuine research experience for undergraduate students.

Problem and HypothesisProblem and Hypothesis

• Problem: Can we classify Mycobacteriophages using cluster specific PCR primers?

• Hypothesis: Mycobacteriophages will be classified when cluster specific primers amplify a PCR product of the expected size.

Phage/ Cluster

A1

A2

B1

B2

B3

C1

C2

D

E

F1

H1

H2

I

Mycobacteriophage ClustersIn Phagesdb

Mycobacteriophage ClustersWith PCR Primers

Materials and Methods Materials and Methods

Add Reagents to PCR Tubes

Place PCR Tubes in the Thermocycler

Preparation of Gel and

Buffer

Add Loading Dye to PCR Reactions

Load Wells with PCR Reactions

Preparation of Agarose

Gel

Materials and Methods Materials and Methods

Run Gel at 80 volts

Photo provided by:ecs.umass.e

du

Photograph Gel

Analyze Results

Materials and Methods Materials and Methods

Class (Gel #4)

Experimental

Control

ResultsResults

Control GelControl Gel

M B1 C1 M E M

http://phagesdb.org/

700 bp

400 bp

800 bp

ConclusionsConclusions• Amplification of Colbert and

Puhltonio genomic DNAs resulted as belonging to Cluster B1.

• Amplification of Ghost and LRRHood genomic DNAs resulted as belonging to Cluster C1.

• Amplification of Pumpkin genomic DNA resulted as belonging to Cluster E.

Phagius_Maximus GelPhagius_Maximus Gel

ConclusionConclusion

• Amplification of Phagius_Maximus genomic DNA resulted in a PCR product using B2 cluster specific primers.

Suave GelSuave Gel

ConclusionsConclusions• Amplification of Suave genomic DNA

did not result in a PCR product using any cluster specific primers.

• We conclude that Suave does not belong to any of the clusters we tested with our collection of cluster specific primers.

• Further experiments are needed with PCR primers for other clusters.

Bloo GelBloo Gel

ConclusionsConclusions• Amplification of Bloo genomic DNA did

not result in a PCR product using any cluster specific primers.

• We conclude that Bloo does not belong to any of the clusters we tested with our collection of cluster specific primers.

• Further experiments are needed with PCR primers for other clusters.

Wilie GelWilie Gel

ConclusionsConclusions

• Amplification of Wilie genomic DNA did not result in a PCR product using any cluster specific primers.

• We recommend preparing phages from Wilie with greater amount of genomic DNA.

• Further experiments are needed with PCR primers for other clusters.

Summary of ConclusionsSummary of Conclusions

Mycobacteriophages

Cluster

Phagius_Maximus B2

Suave n/d

Bloo n/d

Wilie n/d

N/d = Not Determined

Control Mycobacteriopha

ges

Size in Base Pairs

Puhltonio (Cluster B1) 700

Colbert (Cluster B1) 700

Ghost (Cluster C1) 400

LRR Hood (Cluster C1)

400

Pumpkin (Cluster E) 800

Future DirectionsFuture Directions

• Use cluster specific primers to classify the mycobacteriophages isolated from Puerto Rico.

• Design additional cluster specific primers for clusters J - Q.

• Prepare additional DNA from mycobacteriophage Wilie and repeat the PCR experiments using the cluster specific primers.

• Calculate the expected sizes of all cluster specific amplified PCR products.

References• Hatfull, Graham F., Cresawn, Steven E., Hendrix, Roger, W.

2008. Comparative Genomics of the Mycobacteriophages: Insights into Bacteriophage Evolution. Research in Microbiology Volume 159, Issue 5. P. 332-339.

Ross, Robert. 2012. General Botany Study Guide. Department of Biology UPR Cayey. Puerto Rico pp xxvii, xxviii, xxix.

• Rubin. M, 2012. Experimental Classification of Mycobacteriophages: Theoretical Background on Important Concepts and Techniques.

Simmons, Michael J., Snustad, D. Peter. 2012. Principles of Genetics. John Wiley & Sons, Inc. New Jersey pp. 165, 167, 168.

Acknowledgments

• Dr. Michael Rubin• Yadira Ortiz • RISE Program-Dra. Eneida Díaz-Dra. Elena González-Dr. Robert Ross

-Melisa Medina-Valeria Rivera

Cluster Classification Cluster Classification of of

Mycobacteriophages Mycobacteriophages Isolated From Isolated From

Tropical Soils of Tropical Soils of Puerto RicoPuerto RicoNicole Colón

Alberto CintrónCarolina MontañezLuz Marie Reyes

Dr. Michael Rubin

RISE Program, University of Puerto Rico at Cayeyhttp://phagesdb.org/