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Cluster classificationmycobacteriophagev6
1. Cluster Classification of
Mycobacteriophages
Isolated From Tropical
Soils of Puerto Rico
Nicole Colón
Alberto Cintrón
Carolina Montañez
Luz Marie Reyes
Dr. Michael Rubin
RISE Program, University of Puerto Rico at Cayey
http://phagesdb.org/
2. Introduction
• W h a t a r e M y c o b a c t e r io p h a g e s ?
- Viruses that infect the Mycobacterial host.
• Wh e re c a n th e y b e fo u n d ?
-They can be found in the environment including soil.
• W h a t d is e a s e s c a n
M y c o b a c t e r ia c a u s e ?
-They cause diseases such as tuberculosis.
• W h a t a r e M y c o b a c t e r io p h a g e s
u s e d fo r?
-They can be used to infect and kill mycobacteria.
(Rubin, M. 2012), (Simmons, M., Snustad, P. 2012).
3. Introduction
• How a re
M y c o b a c t e r io p h a g e s
c h a r a c t e r iz e d ?
-They are characterized based on their
genome.
• C la s s if ic a t io n in C lu s t e r s
-They share characteristics based on
sequence and annotated genomes.
• P r o t e o m ic s
-Study of protein structure and function.
-Can also be used to classify phages.
(Rubin, M. 2012).
5. Lytic Cycle
Is used by virulent phages.
Is characterized by phage
reproduction followed by host
cell lysis.
Stages include:
1. Adsorption
2. Entry
3. Replication
4. Gene Expression
5. Assembly
6. Lysis (Simmons, M., Snustad, P. 2012)
6. Lysogenic cycle
•Is characterized by
three processes:
• I n t e g r a t i o n of
phage DNA into host
genome.
• E x c i s i o n of
prophage.
• E n t r y into the
lytic cycle.
(Simmons, M., Snustad, P. 2012)
7. Objectives
• 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.
8. Problem and Hypothesis
• P r o b l e m : Can we classify
Mycobacteriophages using cluster specific
PCR primers?
• H y p o t h e s i s : Mycobacteriophages
will be classified when cluster specific
primers amplify a PCR product of the
expected size.
9. Mycobacteriophage Clusters Mycobacteriophage Clusters
In Phagesdb With PCR Primers
P ha g e /
C l uA1t e r
s
A2
B1
B2
B3
C1
C2
D
E
F1
H1
H2
I
10. Materials and Methods
Add P la c e P C R P re p a ra ti
R e a g e nts T u b e s in t h e on of Gel
to P C R T h e r m o c y c le r a nd
11. Materials and Methods
A d d L o a d in g Load
P r e p a r a t io D ye to P C R W e lls
n of R e a c t io n s w it h P C R
Ag a ros e R e a c t io n
Gel s
12. Materials and Methods
Run G e l a t 8 0 P ho to g r A n a ly z e
v o lt s aph Gel R e s u lt s
Photo provided
by:ecs.umass.edu
14. Control Gel
M B1 C1 M E M
800 bp
700 bp
400 bp
http://phagesdb.org/
15. Conclusions
• 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.
19. Conclusions
• 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.
21. Conclusions
• 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.
23. Conclusions
• 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.
24. Summary of Conclusions
C o ntro l S iz e in B a s e M yc o b a c te r i C lu s t e r
M y c o b a c t e r io P a ir s opha g e s
pha g e s
Puhltonio (Cluster B1) 700 Phagius_Maximus B2
Colbert (Cluster B1) 700
Suave n/d
Ghost (Cluster C1) 400
Bloo n/d
LRR Hood (Cluster C1) 400
Wilie n/d
Pumpkin (Cluster E) 800
N/d = Not Determined
25. Future 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.
26. 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.
27. Acknowledgments
• Dr. Michael Rubin
• Yadira Ortiz
• RISE Program
-Dra. Eneida Díaz
-Dra. Elena
González
-Dr. Robert Ross
-Melisa Medina
-Valeria Rivera
28.
29. Cluster Classification of
Mycobacteriophages
Isolated From Tropical
Soils of Puerto Rico
Nicole Colón
Alberto Cintrón
Carolina Montañez
Luz Marie Reyes
Dr. Michael Rubin
RISE Program, University of Puerto Rico at Cayey
http://phagesdb.org/
Editor's Notes
Are viruses that infect the micobacterial host Since they are highly diverse, they can be found in the environment including soil. Can cause diseases such as tuberculosis Are used as a model to study biological process such as…
-These are characterized based in their morphology and genome ; using proteomic techniques they can be classified in clusters using genomic sequence comparisons. -Proteome is the complete set of proteins encoded in the genome that is present in all organism and specify their genome. They can be compared to better understand the similarities and differences between phages. -To be classified in clusters they have to share similar characteristics based on their sequence and annotated genomes
Head- DNA Tail- is used to inject the genetic material into the host
Infect the host by attaching to the outer membrane of the bacterial cell. 2) the genetic material is injected into the bacteria Degradation of the host bacterial chromosomal DNA ( eliminate competing instructions from the bacterial genome) Phage replicates 5) phage assembly: the phage genomic DNA is packaged within the mature protein coat 6) Bacterial cell lysis
Used by temperate phage 2) characterized by phage integration into the bacterial chromosome. Steps: 1) the genetic is injected into the bacteria 2) the entering phage genome is confronted with a decision to determine which of two pathways to follow. 3) If the environment conditions are favorable integration occurs. ( the phage inserts its genome into the host chromosome). After this genome is replicated and each dividing cell receive a copy. 4) Unfavorable conditions cause excision ( phage genome exit the host chromosome) followed by the lytic cycle.
Buffer:
Dr. Rubin--- We want to thank Dr. Rubin for his mentoring and for his support during this project Yadira- We want to thank Yadira for being so helpful while doing the project. Rise Program– And also the Rise Program because thanks to opportunities like this we are forming ourselves to become great scientists in the future.
