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Analysis of Unknown Sample 15. Emilia Czyszczon IT 490 April 18, 2013. Sample Unknown 15. Lytic Phage Plaque Characteristics of Unknown 15 Obvious, large, clearly defined circles Characteristics of Cluster A3 Plaques Obvious, large, clear circles.
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Analysis of Unknown Sample 15 Emilia Czyszczon IT 490 April 18, 2013
Sample Unknown 15 • Lytic Phage • Plaque Characteristics of Unknown 15 • Obvious, large, clearly defined circles • Characteristics of Cluster A3 Plaques • Obvious, large, clear circles
Phage proteins were isolated and analyzed • A 10-plate infection from (10^0 – 10^-10) was conducted to obtain the correct lysis plate concentration • The phage was then plated and flooded with phage buffer to obtain a high titer concentration from a 10-plate infection • Proteins were collected from the flooded plates and run through Pulse-Tube-Cryogenics • Proteins were run through Mass Spec and data was used to determine phage cluster.
Mass Spec Data • The Mass Spec Data returned protein compisition of the unknown phage • Unknown Phage 15 was run against Unknown Phage 1, positive control, and negative control • The positive control was phage Czyszczon1 from Cluster E • We utilized the Purdue Proteiomics Pipeling and Spectrum Mill analysis for our data.
Spectrum Mill Data Unknown 15 vs. Unknown 1 • Acadian_52, Helicase Type III Restriction Subunit, 564 ( 1 read) – cluster B5 • Baka_gp15, function unknown, 275 (1 read) – cluster J • Bxz2_11, function unknown, 514 (4 reads) – cluster A3 • Bxz2_17, function unknown, 330 (12 reads) – cluster A3 • Bxz2_23, function unknown, 198 (11 reads) – cluster A3 • Bxz2_24, function unknown, 132 (9 reads) – cluster A3 • Butters_42, function unknown, 131 (1 read) – cluster N • Che8_77, function unknown, 72 (1 read) – cluster F • EpicPhail-Draft_55, function unknown, 672 (4 reads) – cluster A3 • Evanesce-Draft_22, function unknown, 364 (1 read) – cluster Q • Evanesce-Draft_40, function unknown, 41 (1 read) – cluster Q • PG1_56, function unknown, 226 (2 reads) – cluster B1 • Rockstar_15, major capsid protein, 332 (8 reads) – cluster A3 • Rockstar_49, ThyX, 244 (1 read) – cluster A3 • Rockstar_51, ribonucleotide reductase, 673 (7 Reads) – cluster A3 • Shauna1_57, function unknown, 166 (1 read) • Sheen-Draft_14, function unknown, 566 (1 read) – cluster A7 • Stinger_25, Minor Tail, 358 (6 reads) – cluster B4 • Wildcat_52, Putative peptidoglycan binding domain (1 read) - SINGLETON
Unknown 15 vs. Negative Control • Adephagia_30, Lysin A, 551 (1 read) – cluster K • Alice_235, function unknown, 251 (1 read) – cluster C • Archie-DRAFT_18, function unknown, 577 (1 read) – cluster L • Bxz2_11, function unknown, 154 (6 reads) – cluster A3 • Bxz2_17, function unknown, 330 (2 reads) – cluster A3 • Bxz2_23, function unknown, 198 (4 reads) – cluster A3 • Che9c_57, function unknown, 56 (1 read) – cluster I2 • Cheetobro_Draft_5, function unknown, 120 (4 reads) – cluster K • Cheetobro_Draft_9, function unknown, 445 (3 reads) – cluster K • Cooper_72, function unknown, 72 (2 reads) – cluster B4 • Dori_51, phage antirepressor, 257 (8 reads) - SINGLETON • Evanesce_Draft_19, function unknown, 80 (1 read) – cluster Q • Eureka_20, tail assembly chaperone, 153 (1 read) – cluster E • MacnCheese_62, function unknown, 299 (1 read) – cluster K • Ovechkin_Draft_98, function unknown, 110 (1 read) – cluster F1 • Trixie_7, function unknown, 316 (5 reads) – cluster A2 • Turbido_17, Scaffolding, 175 (2 reads) – cluster A2
Unknown 15 vs. Positive Control • Alice_129, putative minor tail protein, structural, 948 (1 read) – cluster C • BAKA_gp15, function unknown, 275 (1 read) – cluster J • Bxz2_3, function unknown, 106 (1 read) – cluster A3 • Bxz2_11, function unknown, 514 (1 read) – cluster A3 • Bxz2_17, function unknown, 330 (7 reads) – cluster A3 • Bxz2_23, function unknown, 198 (5 reads) – cluster A3 • Bxz2_25, function unknown, 148 (1 read) – cluster A3 • Cheetobro-Draft_9, function unknown, 445 (1 read) – cluster K • Evanesce-Draft_10, function unknown, 318 (1 read) – cluster Q • Ibhubesi_2, Terminase Large Subunit, 545 (5 reads) – cluster F • Liefie_3, portal, 504 (1 read) – cluster G • MacnCheese_62, function unknown, 299 (1 read) – cluster K • Norbert-Draft_42, function unknown, 63 (1 read) – cluster A3 • Rockstar_49, ThyX, 244 (2 reads) – cluster A3 • Stinger_25, Minor Tail, 358 (6 reads) – cluster B4
Spectrum Mill Data and Tuberculosis • In the unknown sample 15 run on the Spectrum Mill data it appears that the phage can also infect Mycobacterium tuberculosis H37Rv because many of the proteins expressed in the sample encompass a total of 83 protein reads. • From previous papers that I have read, cluster A is known to infect tuberculosis bacteria as well.
Pham of Cluster A • Pham 1839 • 144 phages in Pham • Bxz2 with gene Bxz2_15(no function) • EpicPhail with gene EpicPhail-Draft_16 (no function) • Rockstar with gene Rockstar_13 (capsid assembly protease) • None of these particular genes present in the Spectrum Mill or Pipeline Data • Genes that DO show up in the data • Bxz2: 11,17, 23, 24, 25 • EpicPhail: 55 • Rockstar: 15, 49, 51
Analysis • Data from both the Spectrum Mill and the Proteomics Pipeline indicated for the Unknown Phage 15 to be from Cluster A3 • Cluster Identification • Phages in Cluster A3 showing up in Pipeline during analysis of Unknown 15 • Bzx2 • EpicPhail • Rockstar
