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Analysis of
Unknown Sample
15
Emilia Czyszczon
IT 490
April 18, 2013
Sample Unknown 15
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Lytic Phage
Plaque
Characteristics of
Unknown 15
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Obvious, large,
clearly defined
circles
Characteristics of
Cluster A3 Plaques
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Obvious, large, clear
circles
Phage proteins were isolated
and analyzed
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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
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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
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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
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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.
Spectrum Mill Unknown 15
Pham of Cluster A
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Pham 1839
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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)
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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
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Analysis
 Data
from both the Spectrum Mill and the
Proteomics Pipeline indicated for the
Unknown Phage 15 to be from Cluster A3
 Cluster Identification
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Phages in Cluster A3 showing up in Pipeline
during analysis of Unknown 15
 Bzx2
 EpicPhail
 Rockstar