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DmpstrDiver: It’s Just a Phage Introducing DmpsterDiver: DmpstrDiver is a bacteriophage, or phage, a virus that infects bacteria. Viruses are non-living agents that must infect living cells including plants, animals, and microbes, to replicate, by hijacking the infected cells’ machinery. Some phages are lysogenic, meaning they can replicate for longer because they do not kill the “replication factory” immediately. Other phages are lytic, meaning they kill the bacteria cell quickly. Viruses have a capsid made of proteins that surrounds their genetic material. They also have tails that are used to connect the phage to the host cell. Mycobacteriophages are viruses that infect mycobacteria such as Mycobacterium smegmatis. DmpstrDiver was collected (arrested) from a sample of dirt inside a dumpster outside of a residence hall on Miami University's campus. The phage was then isolated and the genome was sequenced and annotated. RESULTS Geneva Mommsen, Ciara Lawson, Jana Cable, Martha Carter, Elsa Clenny, Justin Crookes, Rachana Gollapudi, Sarah Hiner, Pierce Kurek, Svitlana Podolyan, Leah Stetzel, Julie Donna, Ryann Brzoska, Gabrielle Lopez, David Ream, Iddo Friedberg, Gary Janssen, Mitchell Balish Methods How to Read a Genome: Criteria to find a word in a text; anything in between two spaces Criteria to find a gene in a genome; we use the LOCH Principle: L- Length… there is a minimum length for a gene O- Overlap… real estate is expensive in a phage’s genome, so overlapping of start and stop codons from different genes is common. Overlaps of 1, 4, and 7 nucleotides are strong considerations. C-Coding Potential… Statistical makeup of bases in the gene. H- Homology… the genes in one organism tend to be similar to genes in other organisms, so we look for similar sequences to verify that are gene is “real”. Using Artemis Stop codons Suggested genes Possib le ORFs Base Pair assigned number Nucleotid es Suggested Amino Acids Collect soil/garbage sample from environment. Isolate phage using an enrichment method. Created a stock solution of phage. DmpstrDiver’s titer: 5.36 x 10 10 PFUs/mL Purify the phage. Plate with Mycobacterium Smegmatis to test for plaques. Streak these plaques several times to further purify the phage. Use electron microscopy to observe the morphology of the phage. Extract phage DNA. Students annotate genome using programs such as Genemark, Artemis, BLAST. DNA sent to the Pittsburgh Bacteriophage Institute. Analyzed and organized into a fasta file. Functions of the phage’s proteins are predicted using InterProScan and other software. Electron Microscopy: magnification: 100,000x hook protein at tail end Siphoviridae morphology • The total number of phage particles in the biosphere is 10 30 , more than stars in the observable universe •There are more phage particles than all other biological entities added together, including bacteria and archea •There are approximately 10 24 viral infections per second 1 • If all viruses on earth were laid end-to-end they would stretch on for 250 million light years •“From one cubic meter of sea water to the next, there was more genetic diversity in viruses than found in any other known group of organisms” 2 Acknowledgments: Miami University Center for Advanced Microscopy and Imaging, Howard Hughes Medical Institute, Pittsburgh Bacteriophage Institute References: 1 Hatfull, G. Bacteriophages: Nature’s Most Successful Experiment (2008) Microbiology Today Nov: 189. 2 Hamilton, G. Virology: The Gene Weavers (2006) Nature 441: 683-85. DmpstrDiver’s Plaques: *plaques are bacterial mats in which phages are grown lytic Pinprick plaque low plaque density Results: Genome length: 112,285 GC content: 60.6% 230 predicted genes Arresting Officer Clenny and the prosecution team at the arrest site Woah! They really can figure out what I do!
