Final Bacteriophages

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  • 1.Isolation, Purification, and Characterization of Gemeos, a novel phage isolated from tropical soils. Amanda Icazatti Burtell, Fernando Pacheco Ocasio, and RISE Program University of Puerto Rico- Cayey Campus Abstract Viruses are the most abundant organisms on the Earth and are found in a great number of environments. Bacteriophages are viruses that infect bacteria. The purpose of this research is to isolate a bacteriophage (also abbreviated as phage) in tropical soils using soil as the environmental sample and doing enrichment to see if any phage can be obtained directly from soil. Since phages are viruses and viruses need a living host in order to reproduce, two host bacteria are being used in this experiment: Mycobacterium smegmatis and Baccilus cerous. These bacteria do not represent a threat to health since they are nonpathogenic and safe to use in a learning laboratory. The soil sample were enriched with bacteria and placed at 37C for 24 hours to stimulate ideal conditions for phage growth. The culture would then be centrifuged and filtrated. The filtration will be used to do Petri dishes and check if any phages were present. If phages do appear, a single plaque would be isolated and purified using streaking techniques with the purpose of isolating a single phage colony. After the fourth purification, a plaque would be isolated that has already been purified to perform dilutions. The dilutions are used to show which concentration of phages is the best for obtaining a web pattern, which is the phase where the optimums concentration of the phage is found. Different soil samples were analyzed from tropical soils of Puerto Rico and a false positive was obtained. An phage adoption was made. The phage was obtained from soil samples from Sao Pablo, Brazil. This phage was named Gmeos and its host is Baccilus cereus. From initial observations the page appears to be a lysogenic phage. Further experimentations, like genome sequencing, will give more details about the type of phage that has been isolated. Key words: Virus, bacteriophages, Mycobacterium smegmatis, Baccilus cereus, lytic and lysogenic cycle, and electrophoresis. Introduction Viruses are the most abundant infectious agents on Earth. They straddle between the living and nonliving. Therefore, viruses need to infect a living host in order to replicate and reproduce themselves. Bacteriophages are viruses that infect bacteria (Snustad and Simmons 2012). These agents infect a variety of bacteria, which can be either harmful or helpful to human beings. Bacteriophages have many useful characteristics. They can be used to manipulate the genetic constitution of bacteria and even to create antibiotics resistant to bacteria. Bacteriophages are identified according the type of life cycle they have. There are two ways that a virus can replicate itself in a bacteria and that is through the lytic cycle or through the lysogenic cycle. In the lytic cycle the bacteriophage infects the host by attaching itself to the membrane of the bacteria cell. The viruss DNA is then injected into the bacteria and the bacterias DNA will be degraded. The bacteriophage then replicates its genetic information inside the bacteria until, the phage ruptures or lysis the bacteria, liberating mature bacteriophages that can infect more bacteria (Pendleton 2013). In the lysogenic cycle, the phages DNA is integrated into the bacteria chromosome. The phage infects the bacterial host in the same way as the lytic cycle. Once the phages DNA is injected into the bacteria, the replication of its genetic material can take one of

2. two pathways. The phages can choose to stay attached to the bacteria chromosome and it can replicate itself along with the bacterias genetic material. In this stage of integration, the combined DNA is called a prophage. If the environmental conditions become unfavorable for bacterial cell growth, the phage genome exists the host chromosome by a recombination process called excision. Once separated from the host chromosomes the phage genome begins to arm itself like in the lytic process, resulting in cell lysis and the production of new mycobacteriophages. In this study, bacteriophages where isolated from soil samples. An enrichment technique was used with two host bacteria with the purpose of expanding the possibilities of finding bacteriophages and to study the similarities between bacteriophages. The host bacteria used were Mycobacterium smegmatis and Baccilus cereus. A mycobacteriophage infects specifically the bacteria genre Mycobacterium and a bacilophage infects strictly the bacteria genre Baccilus. In addition to the enrichment technique, purifications and dilutions of a phage colony were carried out to identify and characterize a novel phage. Materials and Methods Mycobacteriophage and bacilophage were used as models to study how a virus infects bacteria. These bacteriophages have the capacity to infect specific bacteria such as Mycobacterium smegmatis and Bacillus cerous, respectively. Both bacteria are nonpathogenic, therefore, they are safe to use for a laboratory experiment. The use of techniques in microbiology is fundamental in the process of studying and characterizing isolated bacteriophages. Also, it is important to understand the importance of the aseptic technique, which helps to minimize the chance of contamination (SEA PHAGE Part 1). Procedures Since viruses are the most abundant agents on Earth, they can be found in any kind of environment. The finding of bacteriophages was carried out through the collection of 1.000 g of soil samples from a tropical environment. Specific information such as coordinates, temperature, hour, and address from the place where the soil sample was collected is needed. After the collection of the soil samples, approximately 0.5000 g were used for the enrichment culture technique. This was performed to create conditions that favor replication of the specific bacterial phages. By seeding the sample with host bacteria (0.5-mL) and adding a medium broth to the mixture (5.0-mL) conditions would be optimized for bacterial growth and the phages that are specific to that bacterial species infect the bacterial cells and replicate in higher concentrations (SEA PHAGES, 2012). When the enrichment culture technique was done, incubation of the soil samples at 37C for 24 hours was required. The incubated enrichment cultures were ready for a plaque assay. A requirement for performing this process was to centrifuge to obtain a supernatant (~ 5.0- mL) with the bacteria and phages. In addition, a filtration had to be performed to isolate the bacteria from the phages (if any present) by the removal of liquid 3. from the supernatant that was centrifuged. Afterwards, the filtrate obtained was diluted in petri dishes by the streaking process, which consisted in making rifling through the petri dish three times to obtain different concentration of phages. Petri dishes were incubated at 37C for 24 hours. After the incubation period, plaques may be found in the petri dishes. If different morphologies can be identified, it indicates that a variety of phages were found. In order to isolate a single phage population from samples with potentially mixed a purification process had to be done. In this process a single plaque was isolated from the petri dish and a dilution process through the streaking technique was performed again. This process was performed four times, to obtain accurate results. When the purification process was completed, another enrichment culture technique was performed to amplify the quantity of the phages that were purified. The third purification stock filtrate, obtained from the second enrichment culture technique, was used to perform a polyacrylamide gel. The purpose of performing such a process was to separate the phages capsid proteins in order to understand its composition with a chemical denaturant, SDS. The protein sample was obtained from the supernatant of a centrifugation performed for the third purification stock filtrate. A series of eight dilutions were performed with the purpose of obtaining the purified phage in different concentrations. After the dilutions, a Spot test was performed (Image 3). Results Sample Coordinates Details Depth Temperature Date Place Sample #1 AMIB Lat: 18.215651 Lon: -66.044790 Humid and Dry soil 3 25C 02/02/14 Caguas, PR Sample #2 AMIB Lat: 18.234378 Lon: -66.061491 Dry soil 2 25C 02/17/14 Caguas, PR Sample #3 AMIB Lat: 18.234604 Lon: -66.06138 Humid and dry soil 1 27C 02/24/14 Caguas, PR Sample #4 AMIB Lat: 18.213103 Lon: -66.041709 Humid and damp Soil 3 26C 04/03/14 Caguas, PR Sample #5 AMIB Lat: 18.215715 Lon: -66.044879 Dry soil 1 25C 11/03/14 Caguas, PR Sample #6 AMIB Lat: 18.234378 Lon: -66.061491 Dry soil 1 26C 25/03/2014 Caguas, PR Sample #1 FPO Lat: 18.11 15 Lon: 65.57 54 Dry soil 3 24C 02/04/14 San Lorenzo, PR Sample #2 FPO Lat: 18.11 15 Lon: 65.57 53 Dry soil 4 26C 02/18/14 San Lorenzo, PR Sample #3 FPO Lat: 18.7 6 Lon: 66.9 42 Humid soil 3 28C 02/18/14 Cayey, PR Sample #4 FPO Lat: 18.11 15 Lon: 65.57 53 Humid soil 4 22C 02/26/14 San Lorenzo, PR Sample #5 FPO Lat: 18.2 26 Lon: 66.35 35 Humid soil 6 30C 02/03/14 Ponce, PR Table 1. Soils samples collected in Tropical soils Eleven soil samples were analyzed for the phages hunting process (five samples one person and six samples one person). A phage was found in one partners 5th soil sample after 3 days of incubation. The enrichment culture technique 4. process had to be repeated twice for the soil sample to retrieve the obtained result. No positive results were obtained after the process repeated. A soil sample collected from Sao Paolo, Brazil gave positive results for five phage plaques. The phage was found on April 2nd , 2024 in a populated area near a river at a 19C temperature. The phage was named Gemeos. Process was performed until the Third Purification Stock Filtrate (Image 1). A polyacrylamide gel was p


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