written report bacteriophages

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  • 1.1 Isolation, Purification and Characterization of Isinaayusferti, Bacteriophage Isolated from Tropical Soil Leyda Villagrasa, John L. Rivera Melndez Department of Biology, RISE Program, UPR Cayey Abstract: Mycobacteriophages are currently being studied for their potential to treat different diseases. The purpose of this investigation is to find bacteriophages in tropical soils of Puerto Rico, isolate and characterize them so they can later be studied. In total of five samples were taken from Cidra, Caguas, and Cayey, P.R Enrichment, filtration, and streak protocol were performed to isolate and characterize the phage. A mycobacteriophage was found in the third sample at Caguas P.R. and named Isinaayusferti. After obtaining the positive result, purification, purification of enrichment, filtration, dilutions, spot testing, medium titer phage lisate, ten plates and high titer phage lisate were done. Purification is to isolate and have the phage in its purest form, enrichment is to create conditions that favor the replication of phages and filtration is to separate the supernatant from the pellet of particle matter. In addition, dilutions are to reduce the quantity of phages, spot testing is to determine whether spots on a bacterial lawn are actually plaques, ten plates are to increase and concentrate the amount of phages and the titer phage lysates are to determine the number of phages that form plaques in the solution.Gel electrophoresis was performed to separate the physical properties of the phages proteins and thereby determine the phages amino acid sequence. Quantification of the number of plaque forming units in the solution, analyzation, and the characterization of the phage are future plans for this study. Clonation and mutation of DNA genome can be done for future works. Also, in therapeutic uses, antibiotics resistant to bacteria can be created. Introduction: Viruses that infect bacteria are known as bacteriophages. The host bacteria can be harmless such as Mycobacterium smegmatis or Bacillus cereus. It can also be infectious such as Mycobacterium tuberculosis or Mycobacterium leprae (Todar 2012). Bacteriophages are viruses that infect bacteria and that depend on the bacteria to replicate. Their structure is composed of a protein capsid which holds its genetic material and a tail used to inject the genetic material into the host bacteria (Hatfull et al. 2006). There are two types of bacteriophages in this study. One is mycobacteriophage known to infect members of the genus mycobacteria such as Mycobacterium smegmatis, Mycobacterium tuberculosis and Mycobacterium leprae. The other type of bacteriophage is Bacillusphage. It infects bacteria from the genus Bacillus such as Bacillus cereus, Bacillus pumilus, and Bacillus licheniformis (SEA 2012). As part of the hypothesis, Mycobacteriophages and Bacillusphages will be found on the tropical soils of Puerto Rico. The bacteria that were used for the semester investigation are Mycobacterium smegmatis and Bacillus cereus. Bacteriophages can have two types of lifecycles that are determined by their genome. A virulent phage is one constantly seeking for host bacteria to reproduce by injecting its genetic material into it, replicating until the bacteria cannot hold anymore and the bacteria undergoes lysis. This is called the lytic cycle. The temperate phages integrate their genetic material with the bacterial chromosome. Temperate phages remain passive for a long time and have no immediate visible effect on their hosts.This is called the lysogenic cycle. Temperate phages are the only ones with the capability to also go through the lytic cycle. This is possible only if environmental conditions change and are unfavourable for the lysogenic cycle. In the lytic cycle, the bacteriophage infects its host cell by attaching to the membrane then injecting the genetic material through the tail. This degrades the host bacterial chromosome to prevent any competition thus replicating the DNA. The DNA then expresses the genes for the proteins composing the phages outer viron coat or capsid. The last step is when lysis occurs in the cell

2. 2 membrane. In the lysogenic cycle, infection of the genetic material occurs. Then depending on the environmental conditions, the phage decides which of the two life cycles it will undergo. When it chooses the lysogenic pathway, it inserts its genome into the host chromosome by integration. This is known as the prophage. Now, the phage genome is replicated along with the bacterial DNA, and each dividing cell receives a copy of the prophage. If conditions are unfavourable for cell growth, the phage changes to the lytic pathway and its genome exits the host by excision. This produces cell lysis and the liberation of new bacteriophages (Pope et al. 2011). Bacteriophages are important because they can be used as treatments and creation of antibiotics for diseases such as Leprosy and Tuberculosis. They can also be used to further study DNA mutation in genetics. Materials and Methods: For the semester investigation of the PHAGES program, soil samples were taken from distinct areas of Puerto Rico to see if mycobacteriophages or bacillophages could be found and isolated in tropical soils. A series of procedures were done to find, isolate and characterize bacteriophages. For each sample, the location was recorded (by GPS o Google Earth), date and time of the collection, the temperature, the depth at which the sample was obtained, soil description, and site description. The first five soil samples were taken. Two were taken from Cidra, P.R., two from Caguas P.R. and one from Cayey, P.R. These samples were taken with plastic spoons and then placed in sealed ziplock bags. Each sample was divided into two subsamples: one to localize phages of M. smegmatis and the other to localize phages of B. cereus. Each subsample weighted .5 grams. After performing aseptic technique, on a one 50 mL conical tube, it was labelled with initials, the date, the location where the sample was collected, the sample number, and the name of the corresponding bacteria hosts. Next, .5 grams of soil was weighed and mixed with 10 mL of master mix, and 1 mL of the bacteria M. smegmatis were added. The tubes were added to the shaking incubator at 112 rpm and 37C for 24 hours. The same process was done for the samples of B. cereus with the exception that Tryptic Soy Broth (TSB) was used instead of master mix. The next day the samples were put on the centrifuge at 3,000 rotations per minute (rpm) for 15 minutes. This formed a pellet of particle matter, containing most of the bacterial cells at the bottom of the tube, and at the top a liquid called supernatant. Using a 5 mL pipette connected to a 0.22-m filter, 5 mL of the supernatant was transferred. The same was done for B. cereus. After doing the filtrate process, the streak protocol was done. Here two different plates were used, one for M. smegmatis and one for B. cereus. They were labeled and divided into 3 regions. After aseptic technique was done, streak protocol was made in the plates using the filtrates. Luria base top agar (LB) was used for M. smegmatis and tryptic soy agar (TSA) was used for B. cereus. Sterile swabs were inserted into the filtrate and then the streak process was done by streaking from quadrant 1 to quadrant 3 to separate the sample from a bigger concentration to a smaller one. For the two different samples 4.5 mL of top agar with .5 mL of host bacteria were mixed and added to the plates from gradient 3 to gradient 1. The plates were left for 30 minutes to solidify and then placed in the incubator for 24 hours at 37C. If the plates showed negative results, all the previous processes were to be repeated until a page was found. If they showed positive for phages then the next step was to do plaque purification. In this process a micropipette was used to pick a single putative plaque and put it into 50 L of phage buffer, and the streak protocol was repeated on a new plate. This purification was repeated two more times, by taking a single putative plaque from the one before. Once three purifications are obtained, a second enrichment was done from the third purification. Secondly, 10 mL of master mix or TSB, depending on the phage, was mixed with 1mL of host bacteria, and a plaque from the purified phage. Lastly, the tube was placed in the shaking incubator at 112rpm for 24 hours at 37C. Centrifugation at 3000 rpm and filtration were once again performed. The next step was to do dilutions. This was done by first taking eight microtubes and labelling them with the initials and the corresponding dilution number. Then 90 L of the phage buffer (PB) was added to each microtube. From the third purification 3. 3 filtrate, 10L were taken and added to the first microtube, it was then vortexed and centrifuged. The next seven dilutions were done by taking 10L from the one before it, then vortexing and centrifuging. From these eight dilutions, the spot test was performed. For the spot test, one plate is used for the eight dilutions to see which one will provide the highest concentration of phages. The plate is divided into eight gradients, each gradient is labelled from 1 to 8 and, also identified with the date, initials and name of the process. Then one 10 L drop from each dilution is put on its corresponding gradient. After waiting 10 minutes for the drops of dilutions to settle, the plate is put in the incubator for 24 hours at 37C. The plate was observed the next day to see which dilutions showed potential. For this research, only the two dilutions with the most potential were used to find out which would most likely show a web pattern. After preparing the work area aseptically, five culture tubes, each with .5 mL of host bacteria were infected with 10 L of the optimum phage dilutions. Three were for the dilution that showed the best potential, and the other two were done with the second best diluti