Isolation and Annotation of Arthrobacteriophage Stu Mair, Chrissy Sessa, Andrea Springman, Natalie Widdows Department of Biology, Baylor University, Waco, Texas and HHMI Science Education Introduction In Vitro Methods In Silico Methods This dot plot demonstrates the similarity of the bacteriophage genomes within their cluster while still conveying the differences between the clusters. The same sequence, containing three genomes for each cluster, was used on both axes, with the significant out of cluster similarities circled. DNA of Nubia, Shrooms, and Caterpillar were sequenced by Illumina sequencing at the Pittsburgh Bacteriophage Institute Genomes were auto-annotated based on algorithms from Glimmer and Genemark The calls from Glimmer and Genemark were verified or altered using after consulting the following programs: NCBI Blast PhagesDB BLAST HHPred Starterator DNA Master ORF’s 82 soil samples were collected, filtered to isolate potential phages, and enriched with host bacteria, arthrobacter sp. A series of plaque assays were performed and 25 plates were identified as positive Single phages were then isolated from positive plates through multiple rounds of plaque assays, spot tests, and serial dilutions These isolated phages were concentrated to a minimum titer of 10^7 and the DNA was extracted Objective: Isolate Arthrobacteriophages from soil and annotate their genomes according to the guidelines of the Science Education Alliance Phage Hunters Advancing and Evolutionary Science program. Background: Arthrobacteriophages are viruses that infect Arthrobacter bacteria, a bacteria commonly found in soil. This project is a part of the SEA-PHAGE program of isolating and analyzing Arthrobacteriophages and their genomes to contribute to a national database and to increase understanding of genomics, evolution, and phage therapy. Discussion The discovery and characterization of these 3 arthrobacteriophages contributes valuable information to a growing body of information regarding phage genomics. Some of this information can be used to answer larger questions and some will be useful for characterizing future, such as the novel characterization of gene 52 as a NPPase, a new contribution to AL phages. Nubia Slippage - YOU GO CHRISSY The other element these phages demonstrate is the vast diversity that is present in phages that infect the same host. Despite all being found within 30 miles of each other, the phamerator map and the dot plot both help to demonstrate the vast differences in each of these bacteriophage genomes. This fact exemplifies why contributions to… In Vitro Results In Silico Results Caterpillar Nubia Shrooms Rounds of Purification 3 Plaques .5mm, lytic plaques 1mm lysogenic plaques .23 mm in diameter, lytic plaques TEM 59.4nm, tail 137nm Head .06nm, tail .220 nm Head 55 nm, tail 120 nm HTL (pfu/ml) 1.4 x 108 7.33 x 1010 1.2 x 109 Genome Characteristics Caterpillar Nubia Shrooms Genome Size 56622 44045 59707 Genes Annotated 88 60 98 Functions Found 19 26 31 %GC Content 51.1% 60.7% 64.5% Phage Cluster AU AK AL Overview Example Annotation: Shrooms Gene 52 Location of soil samples for Caterpillar, Shrooms and Nubia Soil Sample The auto-annotation provided the best possible SD score for available start codons, with Glimmer and Genemark calling the same site. The gene, in its initial form, covered the full coding potential present NCBI gave a hit on a Hypothetical Protein from Laroye, with a Q1:S1 and E-Value of 1e-58. This hit was also seen on PhagesDB. The function of MazG-NPPase was determined from a strong HHPred hit (E=3e-20) confirmed by a relatively weak conserved domain hit. (I want to use a photo for this) Completed Gene Start:28395 Stop:28697 Function: MazG-NPPase Phage Isolation TEM Images DNA Extraction Acknowledgements Caterpillar Shrooms Nubia Genome Sequencing HHMI Science Education Alliance-Phage Hunters Advancing Genomics and Evolutionary Science program Baylor University: Department of Biology Phamerator Map of Caterpillar, Nubia and Shrooms Annotation