Purification and Analysis of

Mycobacteriophage Alice

Coreen ManleyBiology

College of Arts and Sciences

Dr.Hughes & Dr.BenjaminBiology

College of Arts and Sciences

TimelineTimeline• 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.

Timeline ContinuedTimeline 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.

AliceAlice

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.

Plaque MorphologyPlaque 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

Medium Titer LysateMedium 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

HighHigh TiterTiter LysateLysate

High Titer Lysate Calculation:

2 × 10⁸(2pfu/5µL)×(1000µL/1mL)×10⁸

(0.4 pfu/µL)×(1000µL/1mL)×10⁸(400pfu/mL)×10⁸

4×10¹⁰ pfu/mL

PlaquePlaque MorphologyMorphology

Plate from final Titer Assay

Plaque MorphologyPlaque MorphologyPlate Sequence

PlaquePlaque MorphologyMorphology

The plaques were very small and clear with no apparent abnormalities.

Plaque area 3.1459 mm²

Alice’s capsid is approximately 75-100 nm across and the tail is about 50-75 nm long.

ElectronElectron MicroscopyMicroscopy

PhagePhage DNADNA

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: 29.8µg

RestrictionRestriction DigestsDigests

Tube # 1 2 3 4 5 6

Rxn Buffer 10x 2 µL 2 µL 2 µL 2 µL 2 µL 2 µL

DNA 0.8 µL 0.8 µL 0.8 µL 0.8 µL 0.8 µL 0.8 µL

10 x BSA 2 µL 2 µL 2 µL 2 µL 2 µL 2 µL

Restriction enzyme none Bam HI

0.5 µLCla I2 µL

Eco RI0.5 µL

Hae III1 µL

Hind III0.5 µL

dd H₂O 15.2 µL 14.7 µL 13.2 µL 14.7 µL 14.2 µL 14.7 µL

Total 20 µL 20 µL 20 µL 20 µL 20 µL 20 µL

Fragment #Distance

migrated(cm)

log fragment

length

Fragment length (bp)

1 6.9 3.82 6607

2 10.3 3.51 3236

3 17.2 2.87 741

Bam H1

Fragment #Distance

migrated(cm)

log fragment

length

Fragment length (bp)

1 15.5 3.02 1047

HindIII

Restriction Digest Fragment Measurements

ClaIClaI

Fragment #Distance

migrated(cm)

log fragment

length

Fragment length (bp)

1 5.6 3.95 8913

2 6.1 3.91 8128

3 6.2 3.90 7943

4 6.8 3.85 7079

5 7.1 3.82 6607

6 7.7 3.76 5754

7 8.4 3.70 5012

8 9.1 3.64 4365

9 9.3 3.62 4169

10 10.3 3.53 3388

11 11.6 3.41 2570

12 12.4 3.34 2188

13 14.3 3.16 1445

14 14.8 3.12 1318

15 16.3 2.98 955

16 17.2 2.90 794

17 18.3 2.80 631

Fragment #Distance

migrated(cm)

log fragment

length

Fragment length (bp)

RestrictionRestriction DigestDigest

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.

Structural AnalysisStructural 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.