1. Purification and Analysis of Mycobacteriophage Alice

2. Coreen Manley Biology College 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. High Titer Lysate High Titer Lysate Calculation : 2 × 10 ⁸ (2pfu/5µL)×(1000µL/1mL)×10 ⁸ (0.4 pfu/µL)×(1000µL/1mL)×10 ⁸ (400pfu/mL)×10 ⁸ 4×10¹ ⁰ pfu/mL

9. Plaque Morphology Plate from final Titer Assay

10. Plaque Morphology Plate Sequence

11. Plaque Morphology The plaques were very small and clear with no apparent abnormalities. Plaque area 3.1459 mm ² Alice ’ s capsid is approximately 75-100 nm across and the tail is about 50-75 nm long.

12. Electron Microscopy

15. Phage DNA Concentration : 628.4 ng / µL Total volume : 53.00 µL Total DNA yield: 33.3µg Used 3.5µg of DNA for digests Total µg of DNA after digests : 29.8 µg

16. Restriction Digests Tube # 123456 Rxn Buffer 10 x 2 µL DNA 0.8 µL 10 x BSA 2 µL Restriction enzyme none Bam HI 0.5 µL Cla I 2 µL Eco RI 0.5 µL Hae III 1 µL Hind III 0.5 µL dd H ₂ O 15.2 µL 14.7 µL 13.2 µL 14.7 µL 14.2 µL 14.7 µL Total 20 µL

18. Fragment # Distance migrated(c m) log fragment length Fragment length (bp) 16.93.826607 210.33.513236 317.22.87741 Bam H1 Fragment # Distance migrated(c m) log fragment length Fragment length (bp) 115.53.021047 HindIII Restriction Digest Fragment Measurements

19. ClaI Fragment # Distance migrated(c m) log fragment length Fragment length (bp) 15.63.958913 26.13.918128 36.23.907943 46.83.857079 57.13.826607 67.73.765754 78.43.705012 89.13.644365 99.33.624169 1010.33.533388 1111.63.412570 1212.43.342188 1314.33.161445 1414.83.121318 1516.32.98955 1617.22.90794 1718.32.80631 Fragment # Distance migrated(c m) log fragment length Fragment length (bp)

20. Restriction Digest In comparison with the restriction digests from the SEA Wiki, there are no discernible matches with Alice’s digest. However it 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.