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  1. 1. Anaeli Shockey Lópeza, Nicolle Rosa MercadobaChemistry Department, University of Puerto Rico-CayeybBiology Department, Univeristy of Puerto Rico-CayeyIsolation and characterization of Mycobacteriophages from tropical soil of Puerto RicoAbstract:Mycobacteriophages are viruses that infect bacteria from the genus Mycobacterium. Theyare ubiquitous and are easily found in different types of soils. Phages are composed of a head,which contains the genetic material, and a tail. Bacteriophages have two possible life cycles:lytic or temperate. Most of them have a temperate life cycle in which they can cause immediatelysis or enter a state of dormancy within the host. The objective of this investigation was toisolate a new phage using soil from Puerto Rico. This is necessary because there are too manyundiscovered bacteriophages that can be of great use to mankind. The methodology for thisinvestigation consisted of isolating a phage using the protocols listed in the SEA-PHAGESresource guide. Two phages were isolated from Gurabo, Puerto Rico and were taken up to thehigh-titter Assay protocol. Future work would include sequencing their DNA.Introduction:Mycobacteriophages are viruses that infectbacteria from the genus Mycobacterium. They areubiquitous and are easily found in different typesof soils. They can be isolated using a simpleprocedure. Although phages are the mostabundant life-form on earth, very few of themhave been identified. Phages insert their geneticmaterial into the bacteria and replicate within itprovoking the lysis of its host. Bacteriophageshave two possible life cycles: lytic or temperate.Most of them have a temperate life cycle in whichthey can cause immediate lysis or enter a state ofdormancy within the host.Hatfull et al. (2008), indicate therecognition of the vast numbers of bacteriophagesin the biosphere has prompted a renewed interestin understanding their morphological and geneticdiversity, and elucidating the evolutionarymechanisms that give rise to them. Thisinvestigation has several important applicationswithin the field of scientific research. An exampleof this within the biomedical field is the possibleelimination of antibiotic resistant bacteria usingphages. Phages can also help us understandcertain aspects of the bacteria that they infect andthe effects that they might have on them. Basedon the immense diversity present in phages wecan also obtain important information on theevolutionary line of these viruses. Some phagesare an example of how viruses can be beneficial tohumans. The objective of this investigation was toisolate a new phage using soil from Puerto Rico.This is necessary because there are too manyundiscovered bacteriophages that can be of greatuse to mankind.Mycobacteriophages can be found allaround the world and are the most numerousbiological entities in the biosphere (Pope et al.,2011). This is a very promising research areasince there is still much to be discoveredconcerning phages. There are yet many importantundiscovered characteristics that may be helpfulin the treatment of bacterial diseases. Theirgenetic diversity provides a promising future inresearch. Phages help us get a betterunderstanding of bacteria as well.Materials and Methods:As instructed by our mentor, for thisexperiment, all of its materials and methods were
  2. 2. recommended by Science Education Alliance(2012).- Sample Collection:The first step of the experiment was thecollection of a soil sample. In this step, as part ofmaintaining everything sterile, you used a pre-packed utensil to recollect the soil sample into asealed and sterile test tube. After collecting thesample, the test tube was sealed and stored atroom temperature. Data such as temperature,climate, soil moisture, GPS site, soil depth, etc.was recorded.- Enrichment:Afterwards, the second step of theexperiment was the enrichment of the soil samplethat was recollected. In this step, you added to asterile 50ml test tube the following: 8ml of sterilewater, 1ml of sterile 10x 7Hq/glycerol broth, 1mlof AD supplement, and 0.1ml of 1000mM CaCl2.To this enrichment solution, you also added 1mlof the bacteria M. smegmatis. In addition, youadded 0.5g of the soil sample to the test tube withthe enrichment solution and the bacteria. Lastly,you incubated the test tube at 37°C at 220rpm for24 hours.- Harvesting:Once 24 hours passed after the enrichmentstep, the test tube was centrifuged for 10 minutes.Then, you poured the supernatant into a newsterile 50ml test tube using sterile filteringtechniques. Once successfully filtered, the testtube was capped and labeled. Next came thesecond part of the harvesting: to plaque. Theplaque process was done on petri dishes and eachplate was divided into three sections. This stepconsisted of using a wooden stick to streak, acrossthe first section of the bottom agar of the petridish, the supernatant that resulted after filtering(This sentence lacks clarity.). Afterwards, anotherwooden stick was used to streak from section oneto section two and then, using a new woodenstick, we steaked from section two to sectionthree. After the streaking was completed, 4.5ml oftop agar with 0.5ml of bacteria were added to theplate. Once the agar solidified, the plates wereincubated at 37°C, for 24 hours, and after positiveresults appeared, the plates were refrigerated.- Plaque Purifications:If positive results appeared. the phage(s)that you wanted to purify were to be circled at thebottom of plate so that you could view themclearly. Now to a labeled tube, we added 50µl ofphage buffer. To the circled phage of the plate, weinserted a micropipette tip and then placed it inthe tube with the phage buffer. Afterwards, anew petri dish was labeled and the plaqueprocess described in the second part of theharvesting was repeated. However, instead ofusing the filtering results to plaque, we used thetube with the mixture of the phage buffer and thephage inserted from the plate. Once again, westreaked section one, then from section one tosection two, and section two to section three.Three rounds of plaque purifications wereperformed.- Second Enrichment:The next step was to make anotherharvesting. First of all, a phage was isolated withthe tip of a micropipette and then added into thesame enrichment solution as the first enrichment.Afterwards, it was incubated for 24 hours likein the first enrichment. Next, the test tube wascentrifuged for 10 minutes. Then, we poured thesupernatant into a new sterile 50ml test tube usingsterile filtering techniques. Once successfullyfiltered, the test tube was capped and labeled.- Medium Titer Assay:This step consisted of creating serialdilutions. From the filtration obtained after thesecond filtration, four phage solutions werediluted in four tubes labeled -1 to -4, add 90µl ofphage buffer. Then, we added 10µl of thefiltration to the -1 tube and centrifuge it. Next, weadded 10µl of the -1 tube to the -2 tube andcentrifuge. This process was repeated for the 4tubes. Afterwards, we added 10µl of each tube(filtration and -1 to -4 tubes) to a sample of 0.5mlof bacteria. Once the solution sat for 15-30minutes we added 4.5ml of top agar to thebacteria solution and spread the solution on aproperly identified plaque. The plates wee
  3. 3. incubated after solidifying and checked after 24hours. Once the plate that was successful wasidentified (the one with the “web” pattern), weadd 6ml of phage buffer to it and placed it in therefrigerator for 24 hours. Next, we extracted thephage buffer ___the plaque, filtered, andrefrigerated it.- High Titer Assay:In this step, 10 plates were infected withthe bacteriophage. First of all, we labeled 10plates and labeled a sterile 50ml test tube. To thetest tube, we added 5ml of bacteria culture andthen infected it with 10µl of the dilution thatcompletely lysed the bacteria. The bacteria wasincubate and shaken at 37°C for 30 minutes.Afterwards, 45ml of top agar was addedto the testtube. Five ml of the mixture was distributed ontoeach plate and incubated at 37°C for 24 hours.After the time has passed and the web pattern wasobtained, 6ml of phage buffer was added to eachof the 10 plates, and using sterilized utensils theagar was broken and mixed with the buffer. Next,place the plates were placed in the incubator at37°C for four hours. After the time has passed,the phage buffer was extracted from all of theplates and placed in a sterile 50ml test tube. Asthe last step, the tube was centrifuged andfiltered.- Rapid Isolation, Separation, and Visualizationof Mycobacteriophages Capsid Proteins:This step is performed with the extractionof phage buffer from the plate with the “web”pattern from the Medium Titer Assay step (Notclear!). One ml of Mycobacteriophage High TiterPhage Lysate (HTPL) was transferred to a cleansterile microtube and centrifuged at 10,000xg forone hour at 4°C. Afterwards, 950µl of thesupernatant was aspirated. Next, a sample bufferwas prepared by adding 25µl of Beta-mercaptoethanol (BME) to 475µl of LaemmliSample Buffer (LSB) and vortexed completely.Later, 20µl of the LSB plus BME solution wasadded to the Mycobacteriophage virion coatprotein pellet. After that was done, (from here oncontinue to use passive voice with past participleverbs for the rest of the report. You are reportingon the methodology that you used. It has to bepast tense and to make it objective the voice mustbe passive.). boil the sample for two minutes, coolthe protein sample for two minutes and centrifugeit briefly. Now you prepare the gel. This is donepreparing 1x of running buffer by adding 100mlof 10x Tris Glycine SDS buffer to 900ml ofdistilled water. Next, remove the gel from thepackaging, remove the tape from the bottom ofthe gel, carefully remove the comb using evenpressure, and rinse the wells using distilled water.Assemble the gel in the apparatus and addappropriate amounts of 1x running buffer.Afterwards, load the sample and the molecularweight markers. Now you run the gel at 200 voltsfor 30 minutes until the dye reaches,approximately, 1cm from the bottom of the gel.Next, strain the gel in a plastic tray using Bio-RadBiosafe Coomassie Blue G-250 strain. Wash thegel in distilled water for 5 minutes and remove thewater (this step is repeated three times). Next, add50ml of Coomassie Blue G-250 stain to the geland stain for one hour with gentle shaking. Afterthe staining is complete, rinse the gel with waterfor 30 minutes. Now you’re ready to photographthe gel on a white light box. The gel can be storedin water (in a zip lock plastic bag) or dried and thebands can be carefully excised using washedgloves and clean unused razor blades and placedin sterilized microtubes for subsequent proteinidentification by mass spectroscopy.Results:The following results were found in theexperiment. First of all we have the soilrecollection data. The temperature at 8:00am onFebruary 19, 2013 was 25.6°C and the day wassunny and clear. The sample was taken in Gurabo,Puerto Rico at these coordinates:18°1448.53"N 66° 06.55"W. The soil samplewas taken from an urban site next to trees andcompost. In addition, the soil was dry and thedepth from where it was taken was 5.74 inches.After the first enrichment and harvesting,positive phage results were found when the soilsample was used. From the plate with positive
  4. 4. results, three phages were identified because oftheir difference in sizes. Since they were treatedas three different phages, each of them requiredthree plaque purifications. The purification of thefirst phage resulted in morphologically smallphages. The purification of the second and thethird phage resulted in morphologically mediumsized phages, both suspected to be the same size.After the second enrichment, filtration and -1 to -4dilutions, each phage yielded a “web” pattern. Forphage #1, the pattern was on the plate with thedilution -3. For phages #2 and #3, the pattern wason the plate with the dilution -4.After extracting the phage buffer fromeach of the “web” pattern plates, a medium wascreated and analyzed with the SDS gel. The gelwas loaded with a marker, other phages, and thethree suspected phages. After the whole procedurewas complete, the protein bands of all threephages could be seen and the bands of phages #2and #3 were extremely similar.Discussion:Positive results for phages obtained afterthe enrichment and first plating was mostprobably due to the sample depth and location(next to compost). With the positive phage results,three phages were identified because they haddifferent sizes. The difference in sizes means thateach phage is morphologically different from theother. Moreover, this would mean that each phagethat is a different size would be a different phage.After the plaque purifications werecompleted, phages #2 and #3 were suspected to bethe same phage because their sizes were relativelythe same. However, they continued to be treatedas different phages until the protein gel step wascompleted to determine if they were the samephage or not. Once the dilutions were completed,the “web” pattern of each phage was chosen basedon the arrangement of plaques in which almost allof the bacteria was lysed. In addition, in the“web” pattern, all of the plaques must be incontact with each other.A protein gel was run once a medium wascreated based on the “web” pattern. The proteinbands of phage #1, now named Shockage, weredifferent from the rest of the phages. The proteinbands of phages #2 and #3 were practically thesame; therefore, it is assumed that both phages arethe same. Phage #2 is now named Zombage.Conclusion:Through this experiment, we were able toisolate Mycobacteriophages, which are virusesthat infect bacteria. From a single soil sample,taken from Gurabo, Puerto Rico, two differentphages have been isolated. Phage #1 is namedShockage and phage #2 is named Zombage. Thenext step for each of these phages would be tosequence their DNA in order to finish the phagecharacterization and determine if the isolatedphage is, in fact, unique. Furthermore, theisolation of phages has applications in the field ofbiomedicine. As mentioned earlier, an example ofthis is the possible elimination of antibioticresistant bacteria using phages. In addition,phages can also be helpful in understandingcertain aspects of the bacteria that they infect andthe effects that they might have on them.References:Science Education Alliance. 2012. SEA-PHAGESResource Guide. Howard Hughes MedicalInstitute; Chevy Chase, MA.Hatfull G, Cresawn S, Hendrix R. 2008.Comparative genomics of themycobacteriophages: insights into bacteriophageevolution. Research in Microbiology. 159(5): 332-339.Pope WH, Jacobs-Sera D, Russell DA, PeeblesCL, Al-Atrache Z, et al. 2011. Expanding theDiversity of Mycobacteriophages: Insights intoGenome Architecture and Evolution. [Internet][Cited 2013 May 14] PLoS ONE 6(1)doi:10.1371/journal.pone.0016329 Availablefrom: