2. Unknown Phage 11 Leah Liston

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

4. Plaque Observations Small and round Slightly turbid Cluster Q – Lightly turbid plaques – Small

5. 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

6. 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

7. 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

8. 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 – Endopeptidase Clp 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

9. 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 – Endopeptidase Clp 1 Major role in degradation of misfolded proteins

10. Pipeline – Comparison with Positive Control Czyszczon1 Both Contain – Elongation Factor Tu – Chaperonin GroEL – 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

11. Chromatography Graphs Unknown 11 Positive Control – Czyszcon1

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

13. 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