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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