SEA-PHAGES
Fatma Sahin
April 4th, 2018
Introduction: Bacteriophage is a phage where it plays in the role of being a virus which infects bacteria host and replicates within bacteria. Phages are not able to replicate on their own and due to this reason, they demand a bacterial host in order to reproduce. These viruses are only specific for their own host. The purpose of this lab is to be successful at amplifying phages presented in the environmental soil sample. The first soil sample was collected from Pine Oak Forest, since the first round of the experiment not successful, a second soil sample was collected from Kennidy Rauchbach’s front lawn. The second soil sample has brought many successful plaque assays and it was able to be to kill the host bacteria, which was, Anthrobacter sp. Anthrobacter sp. is another name from bacterium, this bacterium usually lives in soil, which is why we collected a soil sample. The goal of this lab is to understand bacteriophages and to isolate the phage from the environmental soil sample.
Methods:
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Then add growth media until the sample is submerged beneath the 2-3 ml of liquid, cap the tube and invert several times to mix thoroughly, incubate the tube while shaking vigorously in a shaking incubator at 250 rpm for a couple hours, then allow the sample to sit.
• Enrichment: o In enrichment, the soil gets filled up to 15mL in a 50mL tube with 1x Arthobacter sp. phage buffer to the soil and vortex, then add 10mL of Lennox LB media, adding on 2.5mL of Anthrobacter sp. culture. Then the sample gets incubated for five days with shaking at
2. Drop a gummy bear into each of your prepared beaker or cup and place the beaker or cup
The green fluorescent protein (GFP) gene is a naturally occurring gene from a bioluminescent jellyfish. The gene allows for objects and animals to glow in the dark when activated by the presence of the sugar arabinose in the pGLO plasmid. The GFP gene is often used as a marker for gene expression and genetic transformation. The pGLO plasmid is a genetically engineered plasmid used as a vector in biotechnology to generate genetically modified organisms(GMO). M. Chalfie et. al. (1994) explain that a complementary DNA for GFP produces a fluorescent product when expressed in E. coli cells as the expression of GFP can be used to monitor gene expression and protein localization in living things.
When those steps are finished, the temperature is held at 20°C. In step one, the hot start is initiated by incubating the tubes for five minutes at 95°C and adding the water. 0.4 ul Taq DNA polymerase to each tube while disallowing the tubes to cool and without taking. time to mix the reaction solution after adding the Taq polymerase.
After the addition of the media, we insert an aeration tube inside and cover the lid with a cotton plug and start giving them aeration. This preparation has to be put on for 3 days under proper sunlight and 25-30 degree Celsius to observe if the culture is healthy/ potent or not depending on the color each culture portrays (The nanochloropsis culture should have a grass-green color to be seen as potent and the isochrysis culture should have a dark brown color to be seen as potent), if the colors seem dull and light, then that might mean that the culture is impotent.
Diagnosis is important not only for prescribing effective drugs but for preventing the evolution of resistant microorganisms (Mori and Notomi, 2009). Traditionally, the microbiology laboratory identified etiologic agents of infectious disease by the direct examination and culture of clinical specimens. Methods of identifying and differentiating microorganisms responsible for microbial infection mainly relied on microbial morphology, staining properties of the organism and its growth variables. However, a major restriction is that >99% of the microorganisms observed through a microscope are not cultivable by these direct techniques (Rastogi and Sani, 2011).
...nvironmental Microbiology. New York: A John Wiley & Sons, Inc; 1992. pp. 125?156. Accessed December 2, 2013.
Sharp, Richard. "Bacteriophages: Biology and History." Journal of Chemical Technology and Biotechnology. John Wiley & Sons, Ltd., 19 June 2001. Web. 11 Apr.
3.) Divide your 30g of white substance into the 4 test tubes evenly. You should put 7.5g into each test tube along with the water.
Prepare sufficient electrophoresis buffer (usually 1x TAE or 0.5x TBE) to fill the electrophoresis tank and to cast the gel.
One primary weakness of phage therapy is its failure to function properly in digestive tracks (Ly-Chatain 2014). When human subjects orally consumed phage particles, fecal E. coli counts failed to decrease (Bruttin and Brüssow 2005). This research demonstrates phage particles’ inability to function properly in areas with a low pH, and their lack of capability in treating diarrheal diseases. Moreover, phage therapy is also not suitable for presumptive treatment. Bacteriophages’ narrow host range, will place limitations on presumptive treatment, which are treatment courses that begin prior to the identification of the pathogen (Hyman and Abedon 2010). Furthermore, a negative stigma around phages is omnipresent due to their unfamiliarity and their characterization as viruses, making them unpopular in society (Loc-Carrillo and Abedon 2011). Phages are able to actively replicate and evolve during manufacturing and use like any living organisms (Loc-Carrillo and Abedon 2011); thus, faith in phage therapy is low. By exhausting the possible disadvantages of phage therapy, full knowledge of the topic of phage therapy can be
Bacteriophages are viruses that attack bacteria. Bacteriophages are obligate intracellular parasites. Bacteriophages are infections, which are a hereditary matter pressed inside a protein layer. A few infections contain a little lipid (fat) or hints of different substances. Infections are not cells. They are little particles that increase just inside living cells. Phages can't reproduce or engender outside their host cell, phages are not helpless to anti-infection agents, phages are omnipresent, phages are the most plenteous life-structure on earth, phages can get by in pretty much any environment, and they can be discovered both inside and outside bacterial cells.
2. In the large beaker, put water and boil it completely. After that, remove the beaker from heat. 3. Sample tubes (A-D) should be labeled and capped tightly.
Leboffe, M. J., & Pierce, B. E. (2010). Microbiology: Laboratory Theory and Application, Third Edition 3rd Edition (3rd Ed.). Morton Publishing
Begin collecting samples with the pure hexane. Keep adding hexane so that the silica gel column does not run dry. Collect one 20 ml sample. Repeat with 90:10 hexane and collect 4 20-mL bottles. Repeat with 80:20 hexane and collect 2 20-mL samples.
Phages are found wherever bacteria exist like in the soil, water , intestines of animals etc. (Stephen Mc Grath & Sinderen, 2007). Phages are utilized worldwide as a substitute of antibiotics for more than 90 years and can possibly be used as a cure for multi- drug-resistant strains of many bacteria in former Soviet Union, France, United States of America, Italy, etc. (Keen, 2012). It has been observed that seasonal epidemics of cholera is negatively correlated with the prevalence of environmental cholera phages (Shah M. Faruque et al., 2004). So, there’s an inverse relationship between the bacteria and the phages. That means if the number of bacteria increases then the amount of phages decreases and vice versa. By understanding their mechanism, bacteriophages can be used as a therapeutic agent to kill those antibiotic resistant