Purification and Analysis of Mycobacteriophage Alice

1. Purification and Analysis of Mycobacteriophage Alice

2. Coreen ManleyBiologyCollege of Arts and Sciences • Dr.Hughes & Dr.Benjamin • Biology • College of Arts and Sciences

3. Timeline • 9/2/09: Collected first soil sample, Mushroom • 9/9/09: Collected second soil sample, Garden; Enrichment of Mushroom • 9/14/09: No plaques from Mushroom’s enrichment, sample thrown out. Direct plating of Garden • 9/16/09: Plaques from direct plating. Spot test on Garden’s direct plating plaques. • 9/21/09: Spot test confirms the presence of phage. Proceeded to first phage-titer assay to purify the phage from sample Garden. • 9/23/09: All plates from first titer have plaques. Continued to second titer. • 9/28/09: Second titer produced two plaque sizes. When the third titer was preformed, both size plaques were picked separately. • 9/30/09: Both of the third titer plates sequences showed signs of two different sized plaques. Concluded that the plaques are caused by the same phage. Flooded plates and began lysate preparation.

4. Timeline Continued • 10/5/09: Titer calculation, made a phage stock from a ten plate lysate, and calculated the dilution for an optimal web pattern. • 10/7/09: Continue from 10/5, Harvested the phage lysate, sterilized, and filtered the lysate; Dilution of lysate to the 10¹¹. • 10/12/09: DNA preparation • 10/19/09: Analyze phage using electron microscopy • 10/21/09: Concentration,volume, and yield calculations; Restriction enzyme digest calculations • 10/26/09: Preparation of restriction digests • 10/28/09: Electrophorese of the sample and look at EM photos. • 2/18/10: Began structural analysis on Alice.

5. Alice • Gathered the soil sample at 31°41′17.50″N, 97°38′52.77″W • The sample was collected from 6 month old compost pile. • The soil was warm and damp since the sample was gathered in the afternoon. The sample contained quite a bit of lose, decaying organic matter.

6. Plaque Morphology • Three phage-titer assays were preformed to purify and isolate the phage. • On the second titer there were two discernibly different sized plaques. • After picking both plaques and plating them, it became apparent that the Alice produces two different sized plaques

7. Medium Titer Lysate • Medium Titer Lysate was prepared and collected from the 10^-4 plate and used in a spot test to determine the titer of the final lysate • The final lysate (High Titer Lysate) was gathered on 10/07/09

8. HighTiterLysate • HighTiterLysateCalculation: 2 × 10⁸ (2pfu/5µL)×(1000µL/1mL)×10⁸ (0.4 pfu/µL)×(1000µL/1mL)×10⁸ (400pfu/mL)×10⁸ 4×10¹⁰ pfu/mL

9. PlaqueMorphology PlatefromfinalTiterAssay

10. Plaque Morphology Plate Sequence

11. PlaqueMorphology • Theplaqueswereverysmallandclearwithnoapparentabnormalities. • Plaquearea 3.1459 mm² • Alice’scapsidisapproximately 75-100 nmacrossandthetailisabout 50-75 nmlong.

12. ElectronMicroscopy

15. PhageDNA • Concentration: 628.4 ng/µL • Totalvolume: 53.00 µL • Total DNA yield: 33.3µg • Used 3.5µg of DNA for digests • Totalµg of DNAafterdigests: 29.8µg

16. RestrictionDigests

18. Restriction Digest Fragment Measurements Bam H1 HindIII

19. ClaI

20. RestrictionDigest • IncomparisonwiththerestrictiondigestsfromtheSEAWiki, therearenodiscerniblematcheswithAlice’sdigest. Howeverit is a very large phage and most likely has a lot of DNA. It would fall into a category with large heads and very short tails.

21. Structural Analysis • Alice has over 120 putative genes to this point and 24 tRNAs. • The most similar phage in genomic structure to Alice found thus far is ETO8.