Cluster classification of the mycobacteriophage Bruce                    Lysander Borrero-Romero and Alberto Citrón-Colón ...
it is ready, and the host bacteria is lysed.Next, the progeny is released, and it isable to infect other bacteria. Thechar...
primer (1ul), cluster specific reverse       do not have any bands, therefore they areprimer (1ul), PCR master mix (which ...
References                       Rico. Howard Hughes and RISE                                 Programs. Puerto Rico pp. 4,...
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Cluster classification of the mycobacteriophage bruce


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Cluster classification of the mycobacteriophage bruce

  1. 1. Cluster classification of the mycobacteriophage Bruce Lysander Borrero-Romero and Alberto Citrón-Colón MBRS-RISE Program University of Puerto Rico at CayeyAbstract (The abstract has too many run-on sentences. It is an indication of notenough revision of the text by the group.) Mycobacteriophages are viruses that infect a certain type of bacteria calledmycobacteria. The objective of this experiment was to classify and identify the cluster ofmycobacteriophages. These viruses can have either one of two cycles. One of the cyclesis called the lytic cycle which occurs when the virus injects its DNA into the bacteria andmore viruses form resulting in the destruction of the host. The other cycle is called thelysogenic cycle . It can have two phases. One occurs when the virus injects its DNA intothe bacteria host, but this time the DNA is incorporated into the bacteria’s chromosomeand divides along with the bacteria without destroying it immediately. The other phaseof the lysogenic cycle is that it can transform into a lytic cycle. Furthermore, we useddifferent techniques like PCR and electrophoresis for this experiment. We added differentprimers containing the clusters to our mycobacteriophage so we could determine, if theyappeared, to what clusters they belonged . In the electrophoresis technique we saw aband which meant that our mycobacteriophage, called Bruce, had regions complementaryto those of the cluster. As a result, Bruce was complementary to only one of the clusters.We compared our results with the mycobacteriophages of our other classmates, and itseemed that Bruce, along with other mycobacteriophages, were from the same cluster. Introduction The viruses called bacteriophages areresponsible for infecting other kinds ofbacteria such as E. Coli (Simmons andSnustad, 2012). Mycobacteriophages,just like bacteriophages, are viruses that (a) (b)affect a certain type of bacteria (Rubin, Figure 1.1 (a) Phage tail, (b) Phage capsid2012). Some examples ofmycobacteriophages are Mycobacterium Mycobacteriophages, just likeleprae (known to cause leprosy), bacteriophages, haves two life cycles.Mycobacterium tuberculosis (known to One is called the lytic cycle and thecause tuberculosis), and Mycobacterium other is called the lysogenic cycle. In thegastri (known to be a resident in the lytic cycle, the phage, attach to thehuman stomachs). The structure of a host bacteria, and inject the hereditarymycobacteriophage consists in a head, material (DNA). After the introductionwhich contains the genetic material of the hereditary material, the DNA from(DNA), and a tube-like protein structure the phage replicates, and the DNA fromin the form of a tail, which is tube-like the host bacteria is degraded. The phageprotein structure. The job of the tail is to assembles its body. After the phagesattach to the host and inject its DNA. assembles,
  2. 2. it is ready, and the host bacteria is lysed.Next, the progeny is released, and it isable to infect other bacteria. Thecharacteristic step in this life cycle is thelysis, and that is why it is called lyticcycle. Figure 1.3 Lysogenic Cycle The purpose of studying mycobacteriophages is to learn and understand the life cycle. They can also Figure 1.2 Lytic Cycle be used in the area of medicine to treat diseases against mycobacterias. In contrast, the lysogenic cycleinvolves both the lytic and the lysogenic Materials and methodscycle. In the lysogenic life cycle, the In order to do the clusterphage gets attached to the bacteria and classification, first theinjects its genome. The phage’s genome mycobacteriophage was prepared. Theis integrated into the host bacteria mycobacteriophage was prepared bychromosome, and it replicates along with transferring 1ml of mycobacteriophageit when the cell divides. The phage stays HTPL into a sterile microtube. Then, itin the lysogenic cycle as long as the is centrifuged 10,000x for 1 hour, andconditions are favorable, if not they go this way the particles from thethrough the lytic life cycle, and it starts mycobacteriophage get to be moreall over again. When the phage genome concentrated. A micropipette is used tois going to leave the host chromosome remove 950 micro liters of thethe process is called excision, and it is supernatant, which is the materialconsidered a recombination mechanism. floating on the surface. The preparationThe lysogenic life cycle is better, of the primers is done by re-suspendingbecause you have more options. You primers in PCR grade water to acan either stay in the chromosome or concentration of 10 micrograms (ug) /undergo lysis When the genome is micro liters (ul).attached to the chromosome, it is better The Polymerase Chain Reactionsprotected. If the phage stays with the are going to be set up for the assignedchromosome of the host and keeps mycobacteriophage genome using eachdividing when it enters the lytic cycle, it of the cluster specific primerwill have a lot of progeny. combination by adding the indicated volume for each reagent. ???. The reagent, in accordance with their respective volumes, are: nano pure PCR, grade water (5ul), assigned mycobacteriophage genomic DNA from step #3 (5ul), cluster specific forward
  3. 3. primer (1ul), cluster specific reverse do not have any bands, therefore they areprimer (1ul), PCR master mix (which is not complementary.composed of Taq polymerase, buffer,nucleotides, Mg+) (12ul) ?? I don’tunderstand the use of so manyparentheses.. The total volume is goingto be 24 micro liters (ul). After addingthe PCR reaction components, the PCRtubes are added in the thermocycler, andare amplified using the cycling conditionmethods. The 2% agarose gel is preparedby adding 2 grams of agarose, 10ml of Figure 1.4 Bruce Cluster10x TAE gel running buffer, and 90 ml Electrophoresisof distilled water. Afterwards, youmicrowave the gel on medium power Discussionand let it gently boil for about 1 minute. According with the electrophoresisUse gloves to add 4 ul of Ethidium technique, it suggests that Bruce hasBromidium (EtBr). After adding EtBr, regions complementary to only onelet it cool and pour it into a clean gel particular primer. The primer was theapparatus. Next, mold it with the cluster B2 primer and we can see thiscomb??? in order to make the wells. Let result because it is the only band in thethe gel solidify for around 1 hour. Later, gel. This means that Bruce may belong2 micro liters of loading dye is added to to that specific cluster. Determining the10 micro liters of the PCR reaction after cluster in which Bruce belongs to, is athermocycling, and load the 2% great advantage, because we can have anagarose gel. After loading the agarose idea of the genome of the phage and thegel, the gel will be run at 80 volts for 1 target DNA of the mycobacteriophage.hour and 45 minutes. Finally, the gel We can also assume that Bruce, Cemi,will be photographed using a gel Lorenzoveg, and NovaAndreas are in thedocumentation system. Every group same cluster because they all have bandshad different mycobacteriophages to test on the well that had the cluster B2with different clusters in the experiment. primer.Our group did electrophoresis toseparate molecules based on their massand charge. We added the cluster Acknowledgementsspecific primers so we could see a band Dr. Michael Rubin, for providingin the gel which meant that the us with the essential informationmycobacteriophage contained regions and materials to do the both thecomplementary to the designed primers. paper and the lab. Results Dr. Eneida Díaz and Dr. Elena Our result with the mycobacteriophage Gonzalez, for allowing Dr. Rubincalled Bruce was that apparently it had be our profesor for the day.regions which were complementary tothe cluster B2 primer as Figure 1.4below demonstrates. The other regions
  4. 4. References Rico. Howard Hughes and RISE Programs. Puerto Rico pp. 4, 5,Ross, Robert. 2012. General 6, 13, 14, 15, 17.Botany Study Guide. Department Simmons, Michael J., Snustad,of Biology UPR Cayey. Puerto D. Peter. 2012. Principles ofRico pp xxvii, xxviii, xxix. Genetics. John Wiley & Sons,Rubin, Michael. 2012. Cluster Inc. New Jersey pp. 165, 167,Classification of 168.Mycobacteriophages Isolatedfrom Tropical Soils of Puerto