ERIN HARVEY FRESHMAN NGRI RESEARCH LAB DR. HUGHES, DR. BENJAMIN HHMI Mycobacteriophage Project.
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Transcript of ERIN HARVEY FRESHMAN NGRI RESEARCH LAB DR. HUGHES, DR. BENJAMIN HHMI Mycobacteriophage Project.
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ERIN HARVEYFRESHMAN NGRI RESEARCH LAB
DR. HUGHES, DR. BENJAMIN
HHMI Mycobacteriophage Project
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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
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Enrichment Plating
Sample A – enrichment plating
Three distinct plaque morphologies
Sample B was direct plated – no results
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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
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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)
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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
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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
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Electron Microscopy
Note there are no tails attached to round “heads”
Estimated about 275 nm in length
Hexagonal heads
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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
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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
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Sequencing
• A4 phage• 53.49 kbp• Approximately 80 Glimmer predictions
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Example Gene Annotation
When annotating we look at: Computer predictions Start codon Shine-Delgarno score Coding potential BlastX homology to other phage
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gp25993-27456
Glimmer prediction (pink)GeneMark prediction (orange)GeneMark TB prediction (blue)Chose longest TB prediction
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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
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Coding Potential
Coding potential matches in the 1st ORFNo match in 2nd and 3rd
OK because this gene is in the 1st ORF
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BLASTx results
Basic Local Alignment Search Tool
X compares proteins
Homology with gp32 of phage Bxz2; score 86.3 Cover : 29% Q1:S1
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QUESTIONS?
Thank You