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Unknown 13

This work discusses the research focusing on the taxonomy of the bacteriophage Unknown 13 prior to genomic sequencing. Various methods such as protein production and isolation, Orbitrap GC/MS, and comparisons against databases like PhagesDB and NCBInr were employed. The findings suggest that Unknown 13 likely belongs to Cluster J, with distinct protein profiles. Morphology observations indicate the presence of small, temperate plaques. Challenges in genomic overlap and fixed taxonomical assignments highlight the need for genomic sequencing to confirm these relationships.

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Unknown 13

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  1. Unknown 13 A Phage Mystery IT 227 – Purdue University Robert Agee 04/18/2013

  2. Getting Started (Intro/Methods) • Objective: Decide phage taxonomy prior to genomic sequencing • Procedure: • Protein production and isolation • Orbitrap GC/MS • SpectrumMilldatabase comparisons • PhagesDB, NCBInr • Omics Discovery Pipeline comparison • Vs. Unknown 14 • Vs. Positive Control (Czyszczon1) • Vs. Negative Control

  3. Rounding up Suspects (Results) • PhagesDB, all species

  4. NCBInr, Mycobacterium species

  5. Top 20 Highest Scoring Proteins • Unknown 13 (PhagesDB)

  6. Top 20 Highest Scoring Proteins • Unknown 13 (NCBInr)

  7. Top 20 Highest Scoring Proteins • Unknown 13 vs. 14

  8. Top 20 Highest Scoring Proteins • Unknown 13 vs. Positive Control (Czyszczon1)

  9. Top 20 Highest Scoring Proteins • Positive Control (Czyszczon1)

  10. Top 20 Highest Scoring Proteins • Unknown 13 vs. Negative Control

  11. Exhibit A (Protein Comparison) • Expectations: • Smegmatis genes in Negative Control • Similar protein profile across comparisions • Surprises: • Most functions unknown • Phage proteins in Negative Control • Genomic overlap in database • Tuberculosis and Smegmatis • Diverse phams in phage samples

  12. Exhibit A (Protein Comparison) Pt2 • Unknown 13 vs. other phages: • Similar A cluster proteins and phams (genome ‘backbone’) • Bxz2 proteins • 271, 1842, 4284 • Different and specific proteins and phams in Uk13 • EpicPhail, BAKA proteins, BAKA_gp28 (major tail subunit) • 748, 2872

  13. Exhibit A (Protein Comparison) Pt3 • To summarize, Unknown 13: • Comparison against databases strongly suggests Cluster J. • Comparison against other samples strongly suggests Cluster J, particularly phams748 and 2872. • To summarize, Positive Control (Czyszczon1): • Comparison and individual protein profile are inconclusive.

  14. The Line-up (Morphology) • Cluster A suspects Bxz2 Unknown 13 web plate EpicPhail

  15. The Line-up (Morphology) Pt 2 • Cluster J suspects Ariel Unknown 13 web plate Omega

  16. Exhibit B (Morphology) • To summarize: • Unknown 13 - many small, temperate plaques 1-5mm in diameter. • Bxz2 – fewer, larger temperate plaques • EpicPhail – many small, temperate plaques • Ariel – small, temperate plaques • Omega – small, temperate plaques • Evidence is inconclusive, but suggests Cluster A or J are correct suspicions.

  17. Deliberation (Discussion/Thoughts) • Based on the protein profiles and morphology notes, Unknown 13 is mostly likely a member of Cluster J. • As an aside, the positive control remains unclustered. • Genomic sequencing is necessary to confirm both.

  18. Project Thoughts • Known/Discovered • Protein profile and morphology • Unknowns • Genomic sequence • Challenges • Genomic overlap in databases • Cluster vsSubcluster proteins • Fixed taxonomical assignments (highly fluid genome)

  19. References • http://www.phagesdb.org/ • http://www.omicsdp.org/ • http://chem.agilent.com/

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