Team:BYU Provo/Large Phage

From 2013.igem.org

(Difference between revisions)

Latest revision as of 19:26, 6 September 2013

Overview

- still needs work

The goal of the Large Phage Project is to make a mutant phage that has a capsid much larger than the normal ~200nm T4 capsid. Our intent is to create a protocol where large phage with stable phenotypes will form making it possible to "pick a size" when using phage as a delivery system. The impact of these large phage could mean the difference in making a drug treatment effective and efficient.

Through the use of our mutagen 5'bromodeoxyuridine, a good mutagenesis protocol and the use of a sensitive cesium chloride gradient. We were able to mutagenize and isolate giant mutant t4 phage.

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+**still needs work
-ADD large Phage project overview
+:The goal of the Large Phage Project is to make a mutant phage that has a capsid much larger than the normal ~200nm T4 capsid. Our intent is to create a protocol where large phage with stable phenotypes will form making it possible to "pick a size" when using phage as a delivery system. The impact of these large phage could mean the difference in making a drug treatment effective and efficient.
+:Through the use of our mutagen 5'bromodeoxyuridine, a good mutagenesis protocol and the use of a sensitive cesium chloride gradient. We were able to mutagenize and isolate giant mutant t4 phage.
+[[File:EMpic-1.jpg|400px]]
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-==March==
-===3/15/13===
-===3/18/13===
-===3/20/13===
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-===3/29/13===
-==April==
-===4/1/13===
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-==May==
-===5/1/13===
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-===5/15/13===
-===5/17/13===
-===5/20/13===
-Today we need to run a dilution series to test the titer of our mutated phage stock.
-We also need to start selecting for small plaques and learning how to pick them and titer them out.
-We also should run a UV test on the mutated phage stock compared to the normal stock.
-We picked one plaque off of the 180 sec UV plate sample. We suspended it in 1 mL of broth, and then UV-ed 20 uL samples at 45 second intervals. The number of plaques decreased the longer the samples sat under UV light.  The samples were irradiated from 0 sec to 4 min 30 sec.
-As a control, we diluted the T4Do stock to 10^-6 and tested 20 uL at 45 sec intervals (up to 6 min) under UV light.
-Also, we diluted the T4 mutagenized stock to 10^-6 and tested 20 uL at 45 sec intervals (up to 4 min 30 sec) under UV light.
-UV tests were done by placing 20 uL spots on parafilm and placed in a BSL-2 hood with the UV light turned on.
-===5/22/13===
-Results from 5/20/13 -
-We left the plates in the incubator for 48 hours, which caused contamination on many plates to grow.
-When checked at 24 hours, the T4 mutagenized stock had a web plate at 10^-3 and less than 5 plaques at 10^-6. This experiment will need to be redone from 10^0 down through 10^-6.  The whole mutagenesis may need to be redone if this only represents a dilution of our titer when we were trying to grow it in liquid culture.
-(We infected with ___ mL of phage at ___ titer in ____ vol of resuspended bacteria.)
-Under UV light, the T4Do stock (diluted to 10^-6) has 19 plaques after being irradiated for six minutes (down from almost cleared at 0 min).
-Under UV light, the 180 sec UV plate spot (diluted in 1 mL) has a few hundred plaques on it, but the amount dropped significantly at 4 min 30 sec from when it was UV-ed for 0 min.
-We will re-titer the T4-Mut stock so we can learn whether it was diluted or whether an infection worked.
-We will also re-run the UV test comparing the T4-Mut (10^-3) with the T4-Do stock (at 10^-6) for survivability. The mutagenized phage should survive better.
-Since the loss of plaques seemed to level off for the T4Do stock at 5:15 (23 plaques) and 6 min (19 plaques), we will test a 7 min and 8 min timepoint to see if it stays level, suggesting these phage have multiple genomes and are severely mutated.
-[[File:Apple.jpeg|600px|thumb|center|T4 mutant 0 and 1.5 minutes under UV light.]]
-[[File:Blackberry1.jpeg|600px|thumb|center|T4 mutant 3 and 4.5 minutes under UV light.]]
-[[File:Cranberry.jpeg|600px|thumb|center|T4 mutant 6 minutes under UV light.]]
-[[File:Dingleberry.jpeg|600px|thumb|center|T4 mutant 7.5 minutes under UV light.]]
-[[File:Eggplant.jpeg|600px|thumb|center|T4 mutant 9 minutes under UV light.]]
-[[File:Eggplant.jpeg|600px|thumb|center|T4 mutant 0 and 1.5 minutes under UV light.]]
-===5/24/13===
-===5/27/13===
-===5/29/13===
-Today we wanted to see if we can transfer our current experiment from using E. coli W3110 as the host to E. coli B as the host. We set up multiple experiments.
-. We spot tested the following phage samples on W3110 and B: T1, T2, T3, T4Do stock, T4 mutated stock, T4-UV one-plaque plate harvest, T4-UV-mutated one-plaque plate harvest.
-. We did a dilution series on the T1 stock to see how concentrated it is. We will need to know the PFUs in order to grow a successful liquid culture and mass produce it for our large phage amplification procedure.
-. We also infected two samples of E. coli B, each with 10 uL of 10^-6 T4Do to observe plaque formation.
-===5/31/13===
-==June==
-===6/3/13===
-===6/5/13===
-===6/7/13===
-===6/10/13===
-===6/12/13===
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-===6/28/13===
-==July==
-===7/1/13===
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-===7/31/13===
-==August==
-===8/2/13===
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-===8/23/13===
-===8/26/13===
-===8/28/13===
-===8/30/13===
 ==September==

Team:BYU Provo/Large Phage

From 2013.igem.org

Latest revision as of 19:26, 6 September 2013

Contents

September

9/2/13

9/4/13

9/6/13

9/9/13

9/11/13

9/13/13

9/16/13

9/18/13

9/20/13

9/23/13

9/25/13

9/27/13

9/30/13