Date post: | 20-Dec-2014 |
Category: |
Technology |
Upload: | tmiranda1 |
View: | 183 times |
Download: | 0 times |
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/