Status Report #2

By: Ashley BueFigure 1: This is a close up image of my plaques from 10/15/2013. In this picture there are a few of my strange bacterial growths in the plaques.

Phage Fun Facts• Phages’ play a essential role in the environment and our human lives.• Currently, there are a few selectively studies working to develop

current medical conditions.• One of these studies has developed a biodegradable film that is

covered with phage. This film has been successful used to cover wounds and prevent bacterial infections. • Also, there is treats using phages being developed for burn victims.

Reference:Kirby, Breeann, and Jeremy J. Barr. "Going Viral." The Scientist. www.the-scientist.com, 1 Sept. 2013. Web. 04 Dec. 2013.

• Named after my High School Biology Teacher, Rochelle Gathje. • She influenced me to pursue a career in biology and through her classes I found my passion for biology.• She even graduated from UWRF!

Phage Name: Gathje

Figure 2: University of Wisconsin-River Falls mascot.

Source of Sample• On 9/8/13 I collected my sample from

under a pine tree in a flower bed with cedar mulch. The top soil appeared dry and it contained numerous roots and pieces of mulch. • The weather was about 70⁰ F with an

overcast sky but not raining.• GPS Coordinates: 43⁰ 49’ 31 N 91⁰

58’ 21” W• 10 Minutes outside of Lanesboro, MN

Figure 3: Pictured is my hometown of Lanesboro, MN

Development of Plaques

Figure 4: These are my first major plaque growths with the bacterial spots. Plaques from 9-26-13

Figure 8: Plaques from 10/12/13 show growth again after redoing previous procedure because of contamination.

Figure 7: 10/15/13 plaques which contain no bacterial growth.

Figure 9: Plaques from 10/22/13, one of my final plates.

Figure 5: Plaque from 10/3/12 that contains a bacterial growth.

Figure 6: Spot test from 10/8/13 all plaques contain the bacterial growth.

Plaque Morphology

• Titer of HTL: 5.4x10^9• Size: 8 to 10 mm• Appearance: Clear, Round• Had Strange Appearance to Start

Figure 9: This is my 10^ -5 HTL Titration plate created on 10/22/2013.

DNA Isolation• Concentration: 0.1775 μg/μl

• Yield: 17.75 μg• A260/280: 1.699

• The ratio of A260/280 should be around 1.8 so mine is a little low but the number is still good.

• I have enough DNA to preform all procedures.

Restriction Enzyme Results #1

1 Kb LadderUndigested DNABamHIClaIEcoRIHaeIIIHindIII

Figure 10: My first gel results from 10/31/13.

Gel Electrophoresis #1

Fragment Lengths for Restriction Enzymes #1

Enzyme Fragment Number # of Base Pairs

BAMHI 3 9544.43

2 8605.97

1 7368.43

CLAI 1 8806.43

ECORI 1 8806.43

HAEIII 5 485.51

4 320.923 260.92

2 212.13

1 92.68

HINDIII 5 8806.43

4 6130.20

3 1864.45

2 734.51

1 260.91

Figure 11: This chart depicts my fragment length from my first gel.

Analysis • When looking at this gel, I can

determine that my phage is possible related to three other phages in the class, SJD, JD, and TT.

• With only these results it is hard to determine which ones are related or the same phage.

• By studying the phages further I should be able to determine which ones are related.

Restriction Enzyme Results #2

PST ISAL IECO RVNCO IKB Ladder

Figure 12: This is my second gel from 11/12/13.

Gel Electrophoresis #2

Enzyme Fragment Number Fragment Length Enzyme Fragment Number Fragment Length

PST I 12 11053.76 ECO RV 1 12114.9

11 10085.56 NCO I 4 5558.21

10 8396.15 3 3063.16

9 6989.72 2 2326.7

8 5818.89 1 2027.8

7 4844.18 SAL I 5 11053.75

6 3679.51 4 6377.5

5 2794.86 3 5309.21

4 2326.7 2 3679.51

3 1936.96 1 2794.86

2 1612.5

1 1117.53

Fragment Length for Restriction Enzymes #2

Figure 13:

Analysis of Second Set of Restriction Enzymes • From the second gel, I am able to conclude that it is not related to SJD

and JD.• My gel is completely different from both of these gels, both are

picture below.

Figure 14: JD gel had different cuts than mine and SJD.

Figure 15: SJD gel had different cuts than mine and JD.

Electron Microscope Results

3

5

4

1

2• 1, 2 are normal siphoviridae phages.

• 3 is missing the DNA inside its head.

• 4 has a broken open head which has allowed the dye to enter.

• 5 is a normal siphoviridae phage with a curled tail.

Figure 16: This is my image from the electron microscope.

Phage 1: Tail-96.88nm Head-43.75 nm

Phage 2:Tail-100nm Head-40.63nm

Phage 3:Tail-100 nm Head-43.75 nm

Final Conclusions • For all of this information I can conclude that my phage is for sure not

related to JD or SJD because of the second gel.• A third gel was ran to determine if mine and TT were related. This gel

showed very poor results so I can not determine if the two are related. Also there is not a good electron microscope image of TT so I can’t clearly determine if the two images look similar.• My phage shows no exact similarities to any sequenced phage in the

database.

Interesting!

• My phage started to develop strange bacterial growths in the center of my plaques.• Slowly these growths started to

disappear as a purified the phage.

Figure 17: This is a streak plate created from the bacterial growths in the center of my plaqes.

WHY MY PHAGE!• Unique bacteria growth!• I have enough DNA.

• I have take accurate notes on all procedures.• My phages is not taken from on of Karen’s samples.