Phage Status Report Emily Lehmann and Ellie Kriese
Phages 101 • Phage is short for Bacteriophage • The name Bacteriophage means, “eater of the bacteria” • Phages are viruses that infect bacteria • Benefits from discovery: • Super Virus • Medical Therapy – use as a treatment for infectious diseases • Genetic Diversity – learn more about genetic information • All information came from Science News online magazine article, • All the World’s a Phage written by John Travis.
BarretLemon Emily: Lemon: In high school my friends would always call me Lemon from my last name Lehmann. Ellie: Barrett: Named after my 7th grade biology teacher who inspired me to pursue a degree in biology.
Sample Source - Collected on September 22, 2013 at 3:30pm near Chippewa Falls Wisconsin -GPS Coordinates: 41 degrees 54’ 35’ N 91 degrees 22’ 45” W -Garden Soil that was located near tomato plants and under grass clippings, as well as compost which included food scraps and horse feces -Soil was very loose and moist
Plaque Morphology Emily picked 1 & 2 Ellie picked 3 & 4 • Plaque Size: Varies from • 1mm to 3mm • Clear without halo • When we first started this experiment Ellie picked and went forth with the larger plaques and Emily picked and went forth with the smaller plaques. First Soil Sample Titer 10^-4 plate
Titer of HTL 43 Plaques on the 10^-6 plate 43plaques/10mL x 100mL/1mL x 10^6 = 4,300,000,000 PFU/mL 4.3X10^9 PFU/mL
Concentration: .135mg/mL Yield: 13.5mg A260/280 Ratio: 1.686 Which is slightly below the optimal amount of 1.8, meaning that there may be small amounts of proteins and other debris in the sample. DNA Isolation
Restriction Digest Digest 1: 1 KB Ladder Undigested BamHI ClaI EcoRI HaeIII HindIII Results: Unfortunately, none of the enzymes appeared to cut the DNA except minor cuts in HindIII, which is not enough to distinguish whether or not this is a unique phage.
Restriction Digest Digest 2: NcoI EcoRV SalI PstI 1 KB Ladder Results: Again these enzymes did not cut the DNA again making it difficult to distinguish whether or not this is a unique phage.
Similarities to the Database After looking at our Gel results: - We felt the phage, Laryoe, was the most similar to our phage’s digest results - For the enzymes, EcoR I, BamH I, and Cala I, both our phage and the data base phage showed little or no digestion with these enzymes.
Electron Microscope Phage 1: Head diameter: 1cm= 62.5 nm Tail Length: 2.3cm= 143.75 nm Phage 2: Head diameter: 0.9cm= 56.25nm Tail Length: 2.3cm= 143.75nm Emily’s Electron Microscope Image 2 1
Electron Microscope Phage 1: Head Diameter: 1.1cm= 68.78nm Tail Length: 2.3 cm= 143.75nm Ellie’s Electron Microscope Image 1 1
Conclusions When analyzing the electron microscope images we were able to conclude that we have the same phage due to the length and appearance of the tail. The head diameters were also fairly similar. The structure of our phage appears to be siphoviridae structure with a very wide tail, making it unique from any other phage found in the class and database.
Why Choose our Phage -This phage was found in Chippewa Falls WI which differentiates it from the other phages many of which were found by Dr. Klyzack. -There is enough DNA that was collected to send it for sequencing -This phage is unique because while there was a variation in size of plaques, the appearance of the plaque did remain uniform. -Also this phage continuously plagued out plates all the way through 10^-3, it produced many plaques when it was not diluted.