Post on 24-Feb-2016
description
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.