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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).
- 4. Bacteriophage Structure DNA Head Tail (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
- 13. Results Class (Gel Experiment Control #4) al
- 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.
- 17. Conclusion • Amplification of Phagius_Maximus genomic DNA resulted in a PCR product using B2 cluster specific primers.
- 18. Suave Gel
- 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.
- 20. Bloo Gel
- 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.
- 22. Wilie Gel
- 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
- 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.