Phage

Ethan Callies

Purpose

• To collect, recreate and analyze phage from the environment

• To sequence DNA from phage

Background• Virus that replicates using bacteria

• Ernest Hankin, 1896– India, something in water cured Cholera

• Frederick Twort, 1915– Discovered small agent that killed bacteria– Work interrupted by WWI

• Félix d'Herelle– September 3, 1917 discovered “an invisible, antagonistic microbe

Phage Therapy

• Phage used as antibacterial agents– Began in 1920’s

• Today is most common for fighting Staphylococcus, Streptococcus, and other infections

My Phage

• Collected from base of tree in River Falls, WI• Moist soil conditions• About 44mm below surface• Lola13

Enrichment of Environmental Samples

• Did this to increase the chance I will get a phage from my sample

Phage Titer/Streak Plate

• Titer– Determine concentration of Plaque forming units

(pfu)

• Streak Plate– Help purify single phage population from mixed

populations

Phage Purification

• Isolate enough pure phage sample to create Lysate or MTL (Medium Titer Lysate)

Phage Lysate

• Used to create large batch of pure phage called HTL (High Titer Lysate)

Web Plates

• Obtain HTL with high enough phage concentration to isolate DNA

Isolate/Purify Genomic DNA

• Want to isolate this in high enough amounts for restriction analysis/sequencing

Gel Electrophoresis

• To compare phage DNA sample to a known DNA sample

Digest Phage DNA

• See how Phage DNA compares to known samples when digested by different enzymes

Electron Microscopy

• Observe individual phage using electron microscopy

Results (After 3 enrichments)

• 3 different morphologies developed• Created streak plate for each– Smaller and bigger morphologies purified very

well– Titrated each out separately with 4 dilutions

Results (after discovering morphologies)

• Smaller morphology to 2nd dilution• Bigger had plaques to 4th dilutionSmall titer:

4.6x10^-4 pfu/mLBigger titer:

5.4x10^-6 pfu/mL• Repeat purification

Results (after repeating purification)

• Smaller morphology disappeared• Bigger morphology to all plates(8 in 10^-2, 2 in 10^-3 and 1 in 10^-4)

Spot test to determine web plate

Countable plate to determine titer

Titer: 2.8x10^-4 pfu/mL

Web Plate

• Plate webbed very well• Small morphology appeared again• Set up 10 web plates

Harvest HTL/Titer/Quanitification

• All 10 plates completely plaqued out• Titer came out to be 2.75x10^-9 pfu/mL• Spectrophotometer read .119 mg/mL

Restrict and analyze phage DNA

Hind III

Hae III

EcoR I

Cla I

Bam HI

DNA

Ladder

Restrict and analyze phage DNA (again)

EcoR I

Nco I

BcII

PstI

Ladder

EM Picture

• Tails are approximately 70 nm

• Heads are approximately 30 nm

Phage most similar to Jawanski

NcoRI

Conclusion

• Many morphologies appeared throughout purifications. While this could be because of contamination, I think it was because of something biological. This could be improved a bit by making things a bit more “sterile” while going through the purification process.

• What caused the different sized plaques to show up? Contamination or biology?