1. ERIN HARVEY FRESHMAN NGRI RESEARCH LAB DR. HUGHES, DR. BENJAMIN HHMI Mycobacteriophage Project

2. Basics of Soil Sample A Finder's Name: Erin Harvey Year Found: 2010 Sample found in: Denton, Texas; USA GPS Latitude: 33°11’29.46” N GPS Longitude: 97°05’48.44”W Discovery Notes:  Dry soil (local soils tend to be alkaline – NOT tested)  95° outside temp  Collected with a spoon and Ziploc bag

3. Enrichment Plating Sample A – enrichment plating Three distinct plaque morphologies Sample B was direct plated – no results

4. Purification 1 Incubated 48 hours Mix of small and large bull’s eye plaques  5 mm in diameter  2-3 mm in diameter Labeled two plaques for next purification  A: 5 mm in diameter  B: 2 mm in diameter

5. Purification 2 Bull’s Eye B --Initial plaque was 2 mm in diameter 10 -3 plate: ~21 bull’s eye plaques about 3 mm in diameter ~4 bull’s eye plaques about 1 mm in diameter Labeled two plaques to use for 3rd round A: 4 mm C: 1 mm Difference in plaque sizes due to incubation times (48 vs 24 hours)

6. Purifications 3 and 4 Purification 3 10 -3 :  1 plaque (cloudy, difficult to see bull’s eye)  5 mm in diameter  M. smeg very clumpy on this and other plates Labeled 1 plaque (C) to move forward with 4 th round of purification Purification 4 -1: web pattern of small plaques -2: ~200 small plaques -3: 4 plaques (2 small; 2 large bull’s eye) -4: no plaques

7. MTL, Empirical Assay and Titer Calculation MTL spot test produced strange double-spot Empirical Assay repeated  1 st time: odd progression from x1/5 to x5; mixed morphologies  2 nd time: odd progression from x1/5 to x5; mixed morphologies  Prompted “just in case” purification Third Empirical Assay allowed to soak for 8.5 hours to increase titer Calculated titer: 3.1x10 9 pfu/mL

8. Electron Microscopy Note there are no tails attached to round “heads” Estimated about 275 nm in length Hexagonal heads

9. DNA Extraction, Analysis and Gel Electrophoresis EM grids prepared before DNA extraction DNA extraction performed three times  1 st time: lost most of my sample trying to put plunger back in  2 nd time: class yields low, everyone repeated  3 rd time: yield similar to concentration of 2 nd trial; 360.5 ng/ μL; peak at 260 nm  154 μL of DNA solution; 55.5 μg of DNA Gel electrophoresis performed twice  1 st : no cuts made; assumed human error  2 nd time: cuts on Hae III and Pst I  Estimated 53.05 kbp in length

10. Try 1 Try 2 Gel Electrophoresis Lanes from left to right: Ladder, Uncut, Bam HI, Cla I, Eco RI, Hae III, and HindI III; on right side only: last lane is Pst I

11. Sequencing A4 phage 53.49 kbp Approximately 80 Glimmer predictions

12. Example Gene Annotation When annotating we look at:  Computer predictions  Start codon  Shine-Delgarno score  Coding potential  BlastX homology to other phage

13. gp25993-27456 Glimmer prediction (pink) GeneMark prediction (orange) GeneMark TB prediction (blue) Chose longest TB prediction

14. The Start Codon and SD score Left option  SD score 400  Shorter gap with next gene Right option  SD score 130  Longer gap (~50 bp) with next gene

15. Coding Potential Coding potential matches in the 1 st ORF No match in 2 nd and 3 rd OK because this gene is in the 1 st ORF

16. BLASTx results Basic Local Alignment Search Tool X compares proteins Homology with gp32 of phage Bxz2; score 86.3  Cover : 29%  Q1:S1

17. QUESTIONS? Thank You