Team:BYU Provo/Notebook/LargePhage/Summerexp/Period1/Dailylog

From 2013.igem.org

(Difference between revisions)

Latest revision as of 17:53, 27 September 2013

Large Phage July - August Notebook: September 1 - September 15 Daily Log

Large Phage

9/2/13

Results from 8/28/13: Fractions 20 and 21 produced clearage on 10^0, and many small plaques at 10^-2. #4 had one plaque on 10^-2, and #1 had one plaque on 10^0.

Today we infected 0.5 mL of E. coli with: 100 uL of #1 10^0, 100 uL of #4 10^0, 10 uL of #20 10^-2, 10 uL of #21 10^-2, and for controls we did 10 uL of T4 wild type 10^-6 and 1 uL of T4 10^-7. We also plated a lawn of E. coli as a control.

BDM KW

9/6/13 Results from 9/2:

In general, the plaques for the mutagenized phage seem to be smaller than the pre-mutagenized phage. From this it would appear that we have large phage! But, it's important to make sure that we have a stable phage, so from here we will be picking the small plaques from the mutant plate and plating them out so we can observe if there is a steady result of small plaques. BDM,KW

9/9/13

Today we picked plaques from phages found in the 20 and 21 fractions of the Cesium Chloride gradient that had the smallest plaques. We plated them out with .075 agarose in hopes that the plaques would be bigger on the whole, so differences would be more easily seen.

The results were somewhat disappointing, it appeared that each plaques was smeared either because we allowed the phage to infect e.coli too long before we plated them, because the concentration was not high enough for the agarose to solidify, or a combination of the two.

9/11/13

9/13/13

We picked plaques and put them in eppendorf tubes numbered:

1: T4 control phage
2: 2 on 20-7
3: 3 on 20-7
4: 4 on 20-7

We grew liquid cultures of #1 and #2 (10 uL in .5 mL of E. coli B) for 3 hours. We then added 100 uL of chloroform and pelleted the bacteria.

We prepared seven EM grids (300 mesh). Each grid was prepared by adding 10 uL of sample to the grid for 1 minute, wicking off the droplet with filter paper, adding 10 uL of 2% phosphotungstic acid for 1 minute, wicking off the droplet, and placing back in the grid box. Grid label Sample description

These next two samples were prepared by inserting a 23.5 gauge needle in a plaque on a plate and rinsing it in 50 uL of LB broth. B7 - T4 fraction 21 plaque 7-1 B8 - T4 fraction 21 plaque 7-2 These three samples were prepared by incubating E. coli and T4 together to try to get pictures of both. B6 - T4 + E. coli incubated together for 2 minutes B9?- T4 incubated with E. coli for 10 mins C7 - T4 incubated with E. coli for 40 mins These two samples were prepared by trying to propagate 10 uL of the samples used in B7 and B8 using a liquid culture technique as described above. B10 - T4 fraction 21 plaque 7-1 liquid culture C6 - T4 fraction 21 plaque 7-2 liquid culture

We imaged all grids except B10. Also, B9 may be mislabeled as some of the grids fell out of the box as the technician was loading the grid into the machine.

KW BDM

@@ Line 1: / Line 1: @@
 {{TeamBYUProvo}}
+<br>
-==July==
+{| width="100%"
-===7/1/13===
+| colspan="3" | <font color="#333399" size="5" font face="Calibri">
-===7/3/13===
+: '''Large Phage July - August Notebook: September 1 - September 15 Daily Log'''</font>
-===7/5/13===
+<br>
-Bryan did extra research into the best protocol to get the results that we are hoping for. He found a paper entitled, "Genetic Control of Capsid Length in Bacteriophage T4" By:A. H. DOERMANN, F. A. EISERLING, AND LINDE BOEHNER
+<br>
-===7/8/13===T7
+|- valign="top"
-Today we need to address some problems we ran into and make some plans for the future.
+| style="width: 22%; background-color: transparent;"|
-. Plaque sizes aren't consistent, even though we are using the same agarose each time.
+<font color="#333399" size="3" font face="Calibri">
-<br>2. We haven't been able to observe plaque phenotypes staying the same through multiple generations
-<br>3. We have tried multiple liquid cultures and rounds of mutagenesis but we haven't been able to observe clearage in a flask yet.
+<font size = "4">
-Good things to model--
+: <u> '''Large Phage''' </u> </font>
-<br>- Change in plaque size vs. agarose concentration
-<br>- Mutant vs wild type phage under UV - graph survival rates
-===7/10/13===
+: [[Team:BYU Provo/Notebook/LargePhage/Winterexp|March-April]]
-Today we are trying out a different but similar protocol. We are hoping to see clearage in our overnight cultures which we haven't seen so far. This new protocol describes that the phage will super infect the bacteria this would explain why our phage wasn't clearing the flask. The paper states,
-:"After one round of infection and lysis,all remaining cells become infected. Some of these
-:lyse on schedule, liberating phage which superinfect the majority of cells which are still unlysed. This
-:superinfection causes inhibition of lysis (9). Starting at 150 min, samples (5 ml) are removed periodically
-:and shaken with chloroform to test for the degree of lysis which can be induced. When that test shows that
-:most of the cells can be induced to lyse so that clearing is nearly complete (usually at about 180 min
-:after initial infection),the culture is chilled in an ice bath and the pregnant bacteria are pelleted by
-:centrifugation at about 5,000 rpm for 10 min in a cold centrifuge."
-We inoculated a 250 uL flask with 25 mL of LB broth, 100 uL (20 ug/mL) of adenine, and a loop of E. coli.  We inoculated 25 mL of M9 with the same ingredients. We let these flasks incubate for ~2 hours until moderately turbid.
+: [[Team:BYU Provo/Notebook/LargePhage/Springexp|May-June]]
-After the bacteria is moderately turbid, we added another 100 uL of adenine and 200ul (20 ug/mL) uracil to the 25ml flasks of E. coli B in LB and in M9 along with 500 ul (200 ug/mL) mutagen 5-bromo 2-deoxyuridine and 500 ul T4 phage (1x10^9 pfu/mL) and put that in the shaker to determine if the flask will clear in about three hours.
+: [[Team:BYU Provo/Notebook/LargePhage/Summerexp|July-August]]
-After 150 minutes, we removed 5 mL of bacterial culture and placed it in a 15 mL tube with 1 mL of chloroform.  The LB culture cleared very quickly, while the M9 culture did not clear very well.  At 180 minutes, the LB cleared even better and the M9 cleared slightly better. Due to time constraints, we decided to harvest the pregnant bacteria at 180 minutes.  We pelleted the bacteria in a centrifuge at 3500 rpm for 10 minutes and resuspended each pellet in 2 mL of broth.  We transferred it to a 15 mL tube and added 10 uL of nuclease mix, 5 uL of 1 M MgCl2, and 2 mL of chloroform. We mixed it gently and let it incubate for 30 minutes.  Then, we centrifuged it at 2700 rpm for 10 minutes.  The supernatant was removed and transferred to a new 15 mL tube.
+: [[Team:BYU Provo/Notebook/LargePhage/Fallexp|September-October]]
-===7/12/13===
+</font>
-Today we did a titer on:
-LB and M9+ Supernatant
-LB and M9+ Lysis inhibited pellet
-Re-titer of Mutagenesis 3 and 4
-===7/15/13===
-[[File:July15.jpg|400px|]]
-[[File:July15-1.jpg|400px]]
-[[File:July15-2.jpg|400px]]
-These results gave us some very useful data.  We saw that LB broth worked better for growing phage than M9 in this experiment.  By comparing the supernatants of the liquid cultures, LB showed one plaque at 10^-8, and M9 showed zero plaques below 10^-6.  However, the lysed bacterial pellet for LB had many plaques at 10^-9, while the lysed M9 pellet had 2 plaques at 10^-9.  This shows that there are more than 10 times the amount of phage inside the bacteria than outside.  This is good, because any phage inside the bacteria are progeny phage which must have been exposed to the mutagen as they were assembling.
+| style="width: 82%; background-color: transparent;"|
-Next, we will run the phage through a sucrose or CsCl to try to isolate fractions of large phage and image them.
-We also need to refine our EM prep technique.
-==August==
+<font face="Calibri" size="3">
-===8/2/13===
-===8/5/13===
+<font size="4"> '''9/2/13''' </font>
-===8/7/13===
+Results from 8/28/13: Fractions 20 and 21 produced clearage on 10^0, and many small plaques at 10^-2. #4 had one plaque on 10^-2, and #1 had one plaque on 10^0.
-===8/9/13===
+Today we infected 0.5 mL of E. coli with: 100 uL of #1 10^0, 100 uL of #4 10^0, 10 uL of #20 10^-2, 10 uL of #21 10^-2, and for controls we did 10 uL of T4 wild type 10^-6 and 1 uL of T4 10^-7. We also plated a lawn of E. coli as a control.
-===8/12/13===
-===8/14/13===
+[[File:September1-0.jpg|400px]]
-===8/16/13===
+[[File:September1-1.jpg|400px]]
-===8/19/13===
+BDM KW
-===8/21/13===
+<br>
-===8/23/13===
+<font size="4"> '''9/6/13''' </font>
+Results from 9/2:
-===8/26/13===
-===8/28/13===
+[[File:September2-0.jpg|400px]]
-===8/30/13===
+[[File:September2-1.jpg|400px]]
-==September==
+[[File:September2-2.jpg|400px]]
-===9/2/13===
-===9/4/13===
+[[File:September2-3.jpg|400px]]
-===9/6/13===
-===9/9/13===
+In general, the plaques for the mutagenized phage seem to be smaller than the pre-mutagenized phage. From this it would appear that we have large phage! But, it's important to make sure that we have a stable phage, so from here we will be picking the small plaques from the mutant plate and plating them out so we can observe if there is a steady result of small plaques.
+BDM,KW
-===9/11/13===
+<br>
-===9/13/13===
+<font size="4"> '''9/9/13''' </font>
-===9/16/13===
+Today we picked plaques from phages found in the 20 and 21 fractions of the Cesium Chloride gradient that had the smallest plaques. We plated them out with .075 agarose in hopes that the plaques would be bigger on the whole, so differences would be more easily seen.
-===9/18/13===
+The results were somewhat disappointing, it appeared that each plaques was smeared either because we allowed the phage to infect e.coli too long before we plated them, because the concentration was not high enough for the agarose to solidify, or a combination of the two.
-===9/20/13===
-===9/23/13===
+[[File:September9.jpg|400px]]
-===9/25/13===
+[[File:September9-1.jpg|400px]]
-===9/27/13===
+[[File:September9-2.jpg|400px]]
-===9/30/13===
+[[File:September9-3.jpg|400px]]
+[[File:September9-4.jpg|400px]]
+<font size="4"> '''9/11/13''' </font>
+[[File:September11.jpg|400px]]
+[[File:September11-1.jpg|400px]]
+[[File:September11-2.jpg|400px]]
+[[File:September11-3.jpg|400px]]
+[[File:September11-4.jpg|400px]]
+[[File:September11-5.jpg|400px]]
+[[File:September11-6.jpg|400px]]
+<br>
+<font size="4"> '''9/13/13''' </font>
+We picked plaques and put them in eppendorf tubes numbered:
+#1: T4 control phage
+#2: 2 on 20-7
+#3: 3 on 20-7
+#4: 4 on 20-7
+We grew liquid cultures of #1 and #2 (10 uL in .5 mL of E. coli B) for 3 hours. We then added 100 uL of chloroform and pelleted the bacteria.
+We prepared seven EM grids (300 mesh). Each grid was prepared by adding 10 uL of sample to the grid for 1 minute, wicking off the droplet with filter paper, adding 10 uL of 2% phosphotungstic acid for 1 minute, wicking off the droplet, and placing back in the grid box.
+Grid label   Sample description
+These next two samples were prepared by inserting a 23.5 gauge needle in a plaque on a plate and rinsing it in 50 uL of LB broth.
+B7 - T4 fraction 21 plaque 7-1
+B8 - T4 fraction 21 plaque 7-2
+These three samples were prepared by incubating E. coli and T4 together to try to get pictures of both.
+B6 - T4 + E. coli incubated together for 2 minutes
+B9?- T4 incubated with E. coli for 10 mins
+C7 - T4 incubated with E. coli for 40 mins
+These two samples were prepared by trying to propagate 10 uL of the samples used in B7 and B8 using a liquid culture technique as described above.
+B10 - T4 fraction 21 plaque 7-1 liquid culture
+C6 - T4 fraction 21 plaque 7-2 liquid culture
+We imaged all grids except B10.  Also, B9 may be mislabeled as some of the grids fell out of the box as the technician was loading the grid into the machine.
+KW BDM
+<br>
 {{TeamBYUProvoFooter}}