ERIN HARVEYFRESHMAN NGRI RESEARCH LAB

DR. HUGHES, DR. BENJAMIN

HHMI Mycobacteriophage Project

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

Enrichment Plating

Sample A – enrichment plating

Three distinct plaque morphologies

Sample B was direct plated – no results

Purification 1

Incubated 48 hoursMix 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

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)

Purifications 3 and 4

Purification 310-3:

1 plaque (cloudy, difficult to see bull’s eye)

5 mm in diameter M. smeg very clumpy

on this and other platesLabeled 1 plaque (C)

to move forward with 4th 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

MTL, Empirical Assay and Titer Calculation

MTL spot test produced strange double-spot

Empirical Assay repeated 1st time: odd progression from

x1/5 to x5; mixed morphologies

2nd time: odd progression from x1/5 to x5; mixed morphologies Prompted “just in case”

purificationThird Empirical Assay

allowed to soak for 8.5 hours to increase titer

Calculated titer: 3.1x109 pfu/mL

Electron Microscopy

Note there are no tails attached to round “heads”

Estimated about 275 nm in length

Hexagonal heads

DNA Extraction, Analysis and Gel Electrophoresis

EM grids prepared before DNA extraction

DNA extraction performed three times 1st time: lost most of my

sample trying to put plunger back in

2nd time: class yields low, everyone repeated

3rd time: yield similar to concentration of 2nd trial; 360.5 ng/μL; peak at 260 nm

154 μL of DNA solution; 55.5 μg of DNA

Gel electrophoresis performed twice 1st: no cuts made;

assumed human error 2nd time: cuts on Hae

III and Pst I Estimated 53.05 kbp

in length

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

Sequencing

• A4 phage• 53.49 kbp• Approximately 80 Glimmer predictions

Example Gene Annotation

When annotating we look at: Computer predictions Start codon Shine-Delgarno score Coding potential BlastX homology to other phage

gp25993-27456

Glimmer prediction (pink)GeneMark prediction (orange)GeneMark TB prediction (blue)Chose longest TB prediction

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

Coding Potential

Coding potential matches in the 1st ORFNo match in 2nd and 3rd

OK because this gene is in the 1st ORF

BLASTx results

Basic Local Alignment Search Tool

X compares proteins

Homology with gp32 of phage Bxz2; score 86.3 Cover : 29% Q1:S1

QUESTIONS?

Thank You