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.As said by Hatfull, et al. (2008), therecognition of the vast numbers of bacteriophagesin the biosphere has prompted a renewal ofinterest in understanding their morphological andgenetic diversity, 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, as said byPope, et al. (2011).This is a very promisingresearch due to the fact that there is still much tobe discovered concerning phages. There are yetmany important undiscovered characteristics thatmay be helpful in the treatment of bacterialdiseases. Their genetic diversity provides apromising future in research. Phages help us get abetter understanding of bacteria as well.Materials and Methods:As instructed by our mentor, for thisexperiment, all of its materials and methods werebased on Science Education Alliance (2012).
- Sample Collection:The first step of the experiment is thecollection of a soil sample. In this step, as part ofmaintaining everything sterile, you must use apre-packed utensil to recollect the soil sample intoa sealed and sterile test tube. After collecting thesample, the test tube must be sealed and stored atroom temperature. Data such as temperature,climate, soil moisture, GPS site, soil depth, etc.should be recorded.- Enrichment:Afterwards, the second step of theexperiment is the enrichment of the soil samplethat was recollected. In this step, you add to asterile 50ml test tube 8ml of sterile water, 1ml ofsterile 10x 7Hq/glycerol broth, 1ml of ADsupplement, and 0.1ml of 1000mM CaCl2. To thisenrichment solution, you add 1ml of the bacteriaM. smegmatis. In addition, you add 0.5g of thesoil sample to the test tube with the enrichmentsolution and the bacteria. Lastly, you incubate thetest tube at 37°C at 220rpm for 24 hours.- Harvesting:Once 24 hours pass after the enrichment,the test tube is centrifuged for 10 minutes. Then,you pour the supernatant into a new sterile 50mltest tube using sterile filtering techniques. Oncesuccessfully filtered, the test tube is capped andlabeled. Afterwards comes the second part of theharvesting: to plaque. The plaque process is doneon petri dishes and each plate should be divided inthree sections. This step consists of using awooden stick to streak, across the first section ofthe bottom agar of the petri dish, the supernatantthat resulted after filtering. Afterwards, anotherwooden stick is used to streak from section one tosection two and then, using a new wooden stick, itused to streak from section two to section three.After the streaking is complete, 4.5ml of top agarwith 0.5ml of bacteria are added to the plate. Afterthe agar solidifies, the plates must be incubated at37°C, and, after 24 hours, if positive resultsappear, the plates must be refrigerated.- Plaque Purifications:If positive results appear, the phage(s) thatyou want to purify should be circled at the bottomof plate so that you can view it clearly. To alabeled tube, add 50µl of phage buffer. To thecircled phage of the plate, insert a micropipette tipand then place it in the tube with the phage buffer.Afterwards, label a new petri dish and repeat theplaque process described in the second part of theharvesting. However, instead of using the filteringresults to plaque, you use the tube with themixture of the phage buffer and the phage youinserted from the plate. And with that, once again,you streak section one, then from section one tosection two, and section two to section three.Three rounds of plaque purifications areperformed.- Second Enrichment:The next step is to make anotherharvesting. First of all, a phage is isolated with thetip of a micropipette and then added into the sameenrichment solution as the first enrichment.Afterwards, follow the same incubationinstructions as the first enrichment. Once 24 hourspass after the enrichment, the test tube iscentrifuged for 10 minutes. Then, you pour thesupernatant into a new sterile 50ml test tube usingsterile filtering techniques. Once successfullyfiltered, the test tube is capped and labeled.-Medium Titer Assay:This step consisted of creating serialdilutions. From the filtration obtained from thesecond filtration, four phage solutions will bediluted. In four tubes labeled -1 to -4, add 90µl ofphage buffer. Then, add 10µl of the filtration tothe -1 tube and centrifuge it. Next, add 10µl of the-1 tube to the -2 tube and centrifuge. Repeat thisprocess until -4 tube. Afterwards, add 10µl ofeach tube (filtration and -1 to -4 tubes) to asample of 0.5ml of bacteria. Let the solution sitfor 15-30 minutes. After the time has passed, add4.5ml of top agar to the bacteria solution andspread the solution on a properly identifiedplaque. Incubate the plates after solidifying andcheck after 24 hours. Once the plate that wassuccessful is identified (the one with the “web”pattern), add 6ml of phage buffer to it and place inthe refrigerator for 24 hours. After the time has
passed, extract the phage buffer the plaque andfilter it. Afterwards, place it in the refrigerator.- High Titer Assay:In this step, 10 plates will be infected withthe bacteriophage. First of all, you label 10 platesand label a sterile 50ml test tube. To the test tube,add 5ml of bacteria culture and then infect with10µl of the dilution that completely lysed bacteria.Incubate and shake at 37°C for 30 minutes.Afterwards, pour 45ml of top agar to the test tube.Distribute 5ml of the mixture onto each plate andincubate at 37°C for 24 hours. After the time haspassed and the web pattern is successful, add 6mlof phage buffer to each of the 10 plates, break theagar using sterilized utensils and mix with thebuffer. Next, place the plates in the shakingincubator at 37°C for four hours. After the timehas passed, extract the phage buffer from all ofthe plates and place it in a sterile 50ml test tube.As the last step, centrifuge and filter.- Rapid Isolation, Separation, and Visualization ofMycobacteriophages Capsid Proteins:This step is performed with the extractionof phage buffer from the plate with the “web”pattern from the Medium Titer Assaystep.Transfer 1ml of Mycobacteriophage HighTiter Phage Lysate (HTPL) to a clean sterilemicrotube and centrifuge at 10,000xg for one hourat 4°C. Afterwards, aspirate 950µl of thesupernatant. Next, prepare a sample buffer byadding 25µl of Beta-mercaptoethanol (BME) to475µl of Laemmli Sample Buffer (LSB) andvortex completely. Later, add 20µl of the LSBplus BME solution to theMycobacteriophagevirion coat protein pellet.After that is done, boil the sample for twominutes, cool the protein sample for two minutesand centrifuge it briefly. Now you prepare the gel.This is done preparing 1x of running buffer byadding 100ml of 10x Tris Glycine SDS buffer to900ml of distilled water. Next, remove the gelfrom the packaging, remove the tape from thebottom of the gel, carefully remove the combusing even pressure, and rinse the wells usingdistilled water. Assemble the gel in the apparatusand add appropriate 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-RadBiosafeCoomassie 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:Following all of the previous discussedmaterials and methods the following data wasrecorded and the following results were found inthe experiment. 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.Thesoilsamplewastakenfrom an urban sitenexttotreesandcompost. Inaddition,thesoilwasdryandthedepthfromwhere itwastakenwas 5.74 inches.After the first enrichment and harvesting,positive phage results were found when the soilsample was used. From the plate with positiveresults, 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. For
phage #1, the pattern was on the plate with thedilution -3, for phage #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:It is suspected that the reason why positiveresults for phages after the enrichment and firstplating were present because of the sample depthand location (next to compost). With the positivephage results, three phages were identifiedbecause they had different sizes. The difference insizes means that each phage is morphologicallydifferent from the other. Moreover, this wouldmean that each phage that is a different size wouldbe 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. After 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.After creating a medium based on the“web” pattern, a protein gel was run. 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 can isolateMycobacteriophages, which are viruses that infectbacteria. From a single soil sample, taken fromGurabo, Puerto Rico, two different phages havebeen isolated. Phage #1 is named Shockage andphage #2 is named Zombage. The next step foreach of these phages would be to sequence theirDNA in order to finish the phage characterizationand in order to determine if the isolated phage is,in fact, unique. Furthermore, the isolation ofphages has applications in the field ofbiomedicine. As mentioned before, an example ofthis is the possible elimination of antibioticresistant bacteria using phages. In addition,phages can also be helpful in order to understandcertain 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:http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0016329