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Unknown Phage 11

Unknown Phage 11. Leah Liston. Cluster Identification. Unknown 11 is identified with Cluster Q Other Phage in Cluster Q Long time singleton: Giles Evanesce HH92 OBUPride. Plaque Observations. Small and round Slightly turbid Cluster Q Lightly turbid plaques Small. Phamerator - Phams.

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Unknown Phage 11

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  1. Unknown Phage 11 Leah Liston

  2. Cluster Identification • Unknown 11 is identified with Cluster Q • Other Phage in Cluster Q • Long time singleton: Giles • Evanesce • HH92 • OBUPride

  3. Plaque Observations • Small and round • Slightly turbid • Cluster Q • Lightly turbid plaques • Small

  4. Phamerator- Phams • Pham 1278 is unique to Cluster Q • Evanesce Gene 11 (441bp) • Giles Gene 10 (441bp) • HH92 Gene 10 (441bp) • OBUPride Gene 10 (441bp) • The amino acid sequence: • (K)AAGAIADASTDEAAAAK(A) • At beginning of the genome located after the terminases, protease, portal, and capsid genes

  5. Pham 1278 • In most phages, between the capsid protein and the tail assembly chaperones proteins there is usually major tail subunit proteins • No gene in Giles can be predicted • Pham 1278 protein associated with the unpredicted major tail subunit gene • Unique gene because of this • Unknown 11 from Mass Spec had 97% alignment • Biggest Cluster Q indicator

  6. Spectrum Mill • Compared against phage database top scores • Evanesce gene 11 – Pham 1278 – Cluster Q specific • Evanesce gene 10 – Pham 1326 – Cluster Q specific • Evanesce gene 25 – Pham 3145 – Cluster Q specific • Evanesce gene 16 – Pham 1283 – Cluster Q specific • Evanesce gene 40 – Pham 1292 – Cluster Q specific • Bxz2 gene 23 – Pham 1842 – Not in Cluster Q • Evanesce gene 9 – Pham 1325 – Cluster Q specific • Evanesce gene 7 – Pham 1311 – Cluster P & Q only • DUF935 – Unknown function but consists of several bacterial protein including Bacteriophage Mu gp29 • Bxz2 gene 17 – Pham 1841 – Not in Cluster Q

  7. Spectrum Mill • Compared against Mycobacterium Database • Evanesce gene 11 – Pham 1278 Cluster Q specific • Evanesce gene 10 - Pham 1326 – Cluster Q specific • Elongation factor Tu – M. trivale • RNA polymerase beta – M. chitae • EndopeptidaseClp 1 – Mycle • Glyceraldehyde-2-phosphate dehydrogenase – M. tuberculosis H37Rv • Evanesce gene 25 – Pham 3145 – Cluster Q specific • ATP-dependent Clp protease – M. tuberculosis CDC1551 • Alcohol dehydrogenase – M. smegmatis

  8. Spectrum Mill Results • Phage Database • High alignments with Evanesce genes • High alignment with Pham 1278 Cluster Q specific • High alignment with Cluster Q specific genes • Mycobacterium Database • Alignment with elongation factor • Critical function: in translation energy source for binding of tRNA to the A site in ribosomes • EndopeptidaseClp 1 • Major role in degradation of misfolded proteins

  9. Pipeline – Comparison with Positive Control Czyszczon1 • Both Contain • Elongation Factor Tu • ChaperoninGroEL • Glyceraldehyde-3-phosphate dehydrogenase • Fatty acid synthetase I • Glutamine synthetase GLNA1 • Evanesce Gene 22 – Pham 3143 • Only in Cluster Q on Phamerator – possible mutations • Only Unknown 11 • Glycerol kinase • RNA polymerase beta • Evanesce Genes: 17, 43, 11, 10, 21

  10. Chromatography Graphs Unknown 11 Positive Control – Czyszcon1

  11. Future Direction • Phamerator • Look at cluster Q genes that are shared in other phages • Gene Function?

  12. References • Hatfull, G. “The Secret Lives of Mycobacteriophages.” (2012). Advances in Virus Research • Hatfull, G. “Comparative genomic analysis of sixty mycobacteriophage genomes: genome clustering, gene acquisition and gene size. (2010). J Mol Biol, 397(1), 119-143 • Spectrum Mill • Phamerator • NCBI – Blastp alignments

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