Cluster classification of mycobacteriophages isolated from tropical soils of puerto rico
Cluster Classification of Mycobacteriophages Isolated from Tropical Soils of Puerto Rico Nicole Colón¹, Alberto Cintron¹, Carolina Montañez¹, Luzmari Reyes¹ University of Puerto Rico, Cayey campus¹Abstract: Mycobacteriophages have been studied through time for a number of reasons. They have beenused as model systems for the study of biological processes, such as phage infection and the dogmacentral. This study aims to analyze different unsequenced mycobacteriophages and classify them intotheir respective clusters using PCR and Gel Electrophoresis. Each group of researchers received a specificMycobacteriophage. The bacteriophages’ DNA were amplified using the PCR method, and were lateranalyzed by running an electrophoresis gel.Introduction: For many decades bacteriophages have beenknow as viruses that infect bacteria. Each “Mycobacteriophages are viruses that infectBacteriophage is composed of a head and a tail bacteria belonging to the mycobacteria(figure 1.1). The head, stores the genetic genus”(Rubin 2012). Mycobacteriophages canmaterial, while the tail is used to inject the be found in a variety of soils. They can begenetic material into the host bacterial cell. classified as harmless bacteria or disease causingPhages can be classified according to their agents, such as tuberculosis. The size of a phagemorphology and nucleic acids. The International depends on the average number of phageComittee on Taxonomy on Viruses has study particles liberated when an infected bacterium isand classified over 2,475 species in 2011. lysed.
Bacteriophages can be isolated by enriching soil For many years, Mycobacteriophagessamples in a nutrient media containing the have been studied and analyzed in order tobacterial host. Doing this will let the phage understand biological processes. Around 2,400reproduce, increase in number, and form Mycobacteriophages have been analyzed andplaques. The plaques represent the cycles of characterized. “Over 70 universities and collagesinfection and cell lysis which identify the phage. around the United States, have isolated, purified,After identification, the plaques are purified for and characterized Mycobacteriophages from soilfurther characterization. samples” (Rubin 2012). This experiment analyzes and examines specific phages in orderThe Mycobacteriophages are classified into to assign them into clusters.clustesr using specific primers. Table 1 showsthe Mycobacteriophage Clusters in Phages database. For this experiment we only used fromcluster A to cluster I because the rest of the Headcluster didn’t have design primers. Tail Figure 1.1 Shows the structure of a bacteriophage.
specific forward and reverse primers were also incorporated. In addition, 12µl of the PCR Master Mix, which contained Taq Polymerase, Buffer, Nucleotides, Mg2+, were added. The tubes were placed in a thermocycler for amplification. Once amplified, the electrophoresis method was performed. The agarose gel was prepared using 200mL of TAE buffer and 4g of agarose. After that, 2µl of loading dye were added to the reagents. TheTable 1 Shows the Mycobacteriophage ClustersIn Phagesdb. wells of the agarose gel were loaded and theyMaterials and Methods: were left to run for one hour at 80 volts. This whole procedure was repeated with five gels ofDuring this experiment, four specific different Mycobacteriophages, including theMycobacteriophages genomic DNA were control gel.assigned. Different Genomic DNA weredesignated from Mycobacteriophages classifiedas Phagus_Maximus, Suave, Bloo and Wilie. Results:The preparation of the phage DNA’s and theforward and reverse primers were previously The gel was analyzed and photographs weremade. Test tubes were labeled from 1 to 15. To taken using a gel documentation system. Theeach tube a certain amount of each reagent was bands of some of the Mycobacteriophagesadded. First, 5µl of Nano Pure PCR Grade indicated that they were part of a specific cluster. The thicker upper bands are bright,Water (H2O) was added. Later, 5µl of each because they contain the genome, and the onesMycobacteriophage genomic DNA was added to that ran towards the bottom of the wells areeach tube. Following this step, 1µl of the
the primer replications of a specific region of thegenome. Figure 1.2 shows the controls on anagarose gel. In the control gel amplification ofColbert and Puhltonio genomic DNAs resultedin PCR products using B1 cluster specificprimers. Thus these phages are verified asbelonging to Cluster B1 and their size pair baseis 700. As well, Ghost and LRRHood resulted inPCR products as belonging to Cluster C1. Andtheir size is 400 base pair. Also for Pumpkin,which DNA resulted, as being part of Cluster Eand its size was 800 base pair. Figure 1.3 the Figure 1.2 Shows the Control Gel.middle contains the results of the experimentalagarose gels. It contains theMycobacteriophages known as Bloo and Suave.In the experimental gel neither Wilie nor Blooshowed an amplified PCR product. The final gelwas named as class gel containing twoMycobactheriophage. Figure 1.3 left bottomcontains the Mycobactheriophage Suave, whichdid not show any amplified PCR product. Whilein Figure 1.3, top left, contains the Figure 1.3 Shows the three gels made during this Research Experience. From left to right areMycobactheriophage Phagus_Maximus, whose the Class Gel #4, Experimental Gel and Control Gel.amplification of its genomic DNA resulted in a Discussion:PCR product about 500 base pair using B2 This study provides information aboutclusters specific primers. the classification of each Mycobacteriophage.
The conclusion during this experiment was that • Mycobacteriophage Database.only Phagus_Maximus could be classified as [unknown]. http://phagesdb.org/belonging to cluster B2. The results of the other • Ross, Robert. 2012. GeneralMycobactheriophages were not clear; therefore Botany Study Guide. Department of Biology UPR Cayey. Puertowe cannot arrive to any conclusions. The Rico pp xxvii, xxviii, xxix.Mycobacteriophage named Wilie had • Rubin. M, 2012. Experimentalambiguous results, because it did not show any Classification ofbands. Wilie had a low amount of DNA, that’s Mycobacteriophages: Theoretical Background on Importantthe reason why there weren’t any amplify Concepts and Techniques.products on the gel. In order to classify thisMycobacteriophage it will be necessary to • Simmons, Michael J., Snustad, D. Peter. 2012. Principles ofprepare the phage with a greater amount of DNA Genetics. John Wiley & Sons, Inc.and if is necessary test the phage with a new set New Jersey pp. 165, 167, 168.of designed cluster primers. TheMycobacteriophages classified as Wilie, Suaveand Bloo did not showed any amplified PCRproduct. In order for them to be classified isnecessary to design new PCR cluster primersfrom J trough Q to test on them.Acknowledgements:Special thanks to: our mentor Dr.Rubin, theTA’s, Melisa Medina and Valeria Rivera,Yadira Ortiz and RISE. Also to Dr. EneidaDíaz, and Dr. Elena González.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.