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- 1. Abstract Methods Direct Plating Spot Plating Plaque Purification via Serial Dilution High Titer Lysate DNA Extraction EM Grid DNA Sequencing Further Analysis/ Annotate DNA Enrichment Isolate Soil Sample Figure 1: Virus infecting bacterial host. 1.Webbed Plate 2.Flood 3.Harvest 4.Titer Results Figure 2b Plaques produced by mycobacteriophage Lilliputian. The plaques are turbid, and extremely small with a diameter of approximately one millimeter. The plates were incubated at 30 degrees Celsius for 48 hours. Figure 2: Figure 3b Electron micrograph of mycobacteriophage Lilliputian. The average tail length is approx. 208 nm. The head is positively stained and isometric with an average diameter of approx. 72.5 nm. Figure 4b Restriction digest of the DNA of mycobacteriophage Lilliputian. We used an agarose gel and incubated the digests first at 37C for 30 minutes, then 4C overnight. The sole enzyme that digested the DNA for Lilliputian was HaeIII. Conclusion and Future Direction References By Katie Milliken and Peri Shohnen Figure 3: Figure 4: Based upon comparison of the evidence listed below with information on phagesdb.org, mycobacteriophage Lilliiputian appears to be a member of subcluster A4. 1) The EM showed a positively stained capsid- similar to members of A4 2) The only enzyme to digest was HaeIII - extremely similar to members of A4 For Lilliputian, the next steps would be to: redo the electron micrograph sequence the DNA compare to other sequenced DNA and confirm the cluster/ subcluster Additionally through comparison, Pearbear appears to be a member of subcluster B1. 1) The EM showed phages with long flexible tails and an isometric head- like that of subcluster 2) The enzymes with digest Pearbear were HaeIII and ClaI For Pearbear, the next steps would be to: redo the electron micrograph sequence the DNA compare to other sequenced DNA and confirm the cluster/ subcluster http://oceanworld.tamu.edu/resources /oceanography-book/microbialweb.htm http://www.phagesdb.org http://www.hypertextbookshop.com/bi ofilmbook/v005/r002/Contents/02_Lab _Exercises/02_Protocols_and_Method s/02_Student_Version/03_Drop_Plate _Method.html SEA-PAHGES Lab Manuel and Resource Information Bacteriophages are highly diverse and adundant organisms. Because of this, we utilize bacteriophages for evolutionary research and molecular biology. Our goal was to indentify and analyze mycobacteriophages and compare them to existing samples. Lilliputian and Pearbear were discovered through infection of mycobacteria M. Smegmatis. Through analysis it was determined that phage Lilliputian is likely to be a member of subcluster A4, and phage Pearbear is likely to be a member of B1. Through DNA sequencing and further analysis, these suggestions can be confirmed. A Voyage into Mycobacteriophage Isolation and Analysis Introduction Bacteriophages are viruses that infect bacterium. In particular, we are isolating mycobacteriophages that infect the host Mycobacterium Smegmatis. The purpose for isolating bacteriophages is three-fold. 1.They are a rich resource for understanding molecular biology because they are abundant and highly genetically diverse. 2.They contribute to bacterial pathogenesis. 3.They are useful for mapping evolutionary pathways. Mycobacteriophages specifically have the potential to serve as therapeutic agents for drug resistant bacteria, such as tuberculosis. The goal of this project is to define the diversity of the bacteriophage population and to understand the evolutionary mechanisms that shape it. The immediate goal is to collect and analyze specific mycobacteriophage data and compare them. Figure 5: Serial Dilution. Figure 3a: Electron micrograph of phage Pearbear. Pearbear has an isometric head and a flexible tail. The average head diameter is approx. 60nm but due to positive staining, an accurate measurement could not be made. The average tail length is approx. 267nm. Enzymes: DNA Ladder BamHI ClaI Hind III HaeIII EcoRI Figure 2a Plaques formed by mycobacteriophage Pearbear. Pearbear produces plaques that are clear and approximately 1 mm in diameter. Pearbear infects M. smegmatis. This plate was incubated at 30C for 48 hours. Figure 4a: Restriction digest of Pearbear. The digests were run in a .7% agarose gel alongside a DNA ladder at 30C for 30 minutes. The gel shows that enzymes ClaI and HeaIII cut Pearbears DNA, suggesting that Pearbear Belongs to subcluster B1. Ladder BamHI ClaI HindIII HeaIII EcoRI