Cluster Classification of MycobacteriophagesIsolated from Tropical Soils of Puerto RicoPerez Soto, Luis A.1, Ramos Callejas, Khrystall2University of Puerto Rico at CayeyDepartment of Biology1 and Chemistry2RISE ProgramMarch 30,2012Abstract Mycobacteriophages (MBP) are viruses that infect mycobacteria. They have twolife cyles: lytic and lysogenic cycles. Depending on their characteristics, they areclassified into clusters. The purpose of this experiment was to identify the MBPs in thecluster from: A1 to I. After finishing the experiment and analyzing the results we wereable to classify 4 phages in their clusters. The MBPs Bruce, Cemi and Lorenzovegbelong to cluster B2 and Fenixious belonged to cluster E.Introduction MBP are viruses that infect a specific type of bacteria. MBP have two differentlife cycles. They are the lytic and lysogenic cycles; the lytic cycle is characterized byphage reproduction followed by host cell lysis. The lysogenic cycle is characterized byphage integration into the bacterial chromosome. Some of these MBPs have beencharacterized using morphologic, genomic and proteomic techniques. The MBPsisolated from tropical soils of Puerto Rico will be classified into clusters using moleculartechniques. Also, MBPs can be grouped according to how they infect bacterias andreproduce. MBPs are classified in clusters and sub clusters depending on similarities
and differences between their genome. MBPs are classified in a specific clusterbecause they share the same characteristics. Therefore, MBPs are grouped intodifferent groups under a letter. The purpose of this lab procedure is to identify in whatcluster MBP belong to using specific primers in the processes of PCR and gelelectrophoresis.Materials and MethodsPCR Amplification of Mycobacteriophage Genomic DNA The MBP were already isolated in Dr. Rubin’s lab. We transferred 1 mL of MBPHTPL to a clean sterile microtube and centrifuge 10,000 X g for 1 hour at 4ºC toconcentrate the MBP particles. The supernatant was removed with a micropipette(950uL) and disposed of. We used the concentrated MBP on the bottom of themicrotube for the PCR amplification. Next, we did the PCRs for the eight MBPs (Bruce,Carmina, Cemi, Fenixious, Lorenzoveg, NovaAndreas, Phagius_Maximus and Suave)by using the following volumes and reagants:Reagent VolumeNano Pure PCR Grade Water (H2O) 5uLMBP Genomic DNA 5uLCluster Specific Forward Primer 1uLCluster Specific Reverse Primer 1uLPCR Master Mix (Taq Polymerase, Mg++, 12uLBuffer, Nucleotides)Total Volume 24uLThe Cluster specific primer used was for clusters: A1, A2, B1, B2, B3, C1, C2, D, E, F1,H1, H2 and I.Running the thermocyclerAfter preparing the PCR solutions, we place the PCR tubes in the thermocycler andamplified them using the following conditions:
Step Condition Temperature Time 1 Initial Denaturation 95ºC 5 min 2 Denaturation 95ºC 30 sec 3 Annealing 62 ºC 30 sec 4 Extension 72 ºC 2 min 5 Repeat Step 2 to 4 for 25 Cycles 6 Final Extension 72 ºC 7- 10 min 7 Hold 4 ºC for everPreparing the 2% agarose gel At the same time that the thermocycler was working we prepared the agarosegel. To prepare the agarose gel at a 2% concentration, we added 2 grams of agarose,10mL of 10X TAE gel running buffer, and 90mL of distilled water. After mixing theagarose solution, it is heated in a microwave at medium power for approximately 1minute or until completely dissolved. Using gloves we add 4uL of Ethidium bromide tothe agarose solution, and waited until it cooled down to later pour it in a gel mold withthe comb in place. Next, we allowed the gel to solidify for approximately 1 hour.Runnning the agarose electrophoresis We added 2uL of loading dye to the PCR reaction after the thermocyclerstopped. We also loaded the agarose gel (use the same buffer to run the gel). Theelectrophoresis ran at 80 volts charge for 1 hour. When the gel stopped we took apicture with the Gel Doc XR for later analysis. Carmina (lower part). In this gel we were able to classify Bruce in cluster B2Results due to the fact that the primer workedBy using specific primers, we were able and the DNA was replicated. For phageto identify some of the MBP and locate Carmina we didn’t have any resultsthem in their clusters. For Gel 1 we use because when mixing they accidentallythe phage Bruce (upper part) and
use cluster A1 primer for all the so mark as in gel #2 and #1. But we stillreactions. could classify one of the phages. The MBP Lorenzoveg (upper section) wasGel #1: Bruce and Carmina phages classify in cluster B2, but the phage NovaAndreas (lower section) was not classified because the band was almost invisible and we could not be sure that it belonged to that cluster. More work will need to be done with that MBP in order to identify its cluster. Gel #3: Lorenzoveg and NovaAndreasIn Gel #2 the MBP were Cemi (upperpart) and Fenixious (lower part). Wewere able to get some result on the geland classify the MBP. The phage Cemibelongs in cluster B2 and Fenixious incluster E.Gel #2: Cemi and Fenixiuous The agarose gel #4 has the phages Phagius_Maximus (upper part) and Suave (lower part). We didn’t get any band in this gel; it could have been that we didn’t have the primer of their cluster. Gel #4: Phagius_Maximus and SuaveFor the agarose gel #3 the result were alittle cloudy because the bands weren’t
Discussion After preparing the PCR solution with each specific primer for clusters: A1, A2,B1, B2, B3, C1, C2, D, E, F1, H1, H2, and I we prepared the solutions for thethermocycler. In the part of the thermocycler, we had some difficulties because theenergy was outage during the process and the the thermocycler it stopped in cycle 7. Inrespond to this problem we added more Master Mix to the solutions and start runningthe thermocycler again from the beginning. Still with that difficulty we were able tocollect amazing data. We classified the MBPs in the following clusters: Bruce, Cemiand Lorenzoveg belong in cluster B2 and Fenixious to cluster E. The rest of the phageswould be needed to be classified in other experiments with the use of other primers andwithout interruption to the energy source due to a stoppage.AcknowledgementsThe authors thank Dr. Michael Rubin, members of the Howard Hughes and RISEProgram and the teaching assistants Valeria Rivera and Melisa Medina who conductedthe experiment. Also Yadira Ortiz (Laboratory Technician) helped a lot in thedeveloping of the materials. Special thanks to the National Institute of Health forsponsoring the programs.Reference
Rubin Michael, Cluster classification of mycobacteriophages isolated from tropical soilsof Puerto Rico handout, 2012.