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

From 2013.igem.org

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{| width="100%"
{| width="100%"
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| colspan="3" | <font color="#333399" size="5" font face="Calibri"> '''Large Phage Spring Notebook'''</font>
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| colspan="3" | <font color="#333399" size="5" font face="Calibri"> '''Large Phage May - June Notebook: May 1 - May 12 Daily Log'''</font>
<br>
<br>
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<font color="#333399" size="3" font face="Calibri">
<font color="#333399" size="3" font face="Calibri">
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: [[Team:BYU_Provo/Large_Phage|Overview]]
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<font size = "4">
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: [[Team:BYU Provo/Notebook/LargePhage/Winterexp|Winter]]
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: <u> '''Large Phage''' </u> </font>
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: [[Team:BYU Provo/Notebook/LargePhage/Springexp|Spring]]
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: [[Team:BYU Provo/Notebook/LargePhage/Winterexp|March-April]]
-
: [[Team:BYU Provo/Notebook/LargePhage/Summerexp|Summer]]
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: [[Team:BYU Provo/Notebook/LargePhage/Springexp|May-June]]
-
: [[Team:BYU Provo/Notebook/LargePhage/Fallexp|Fall]]
+
: [[Team:BYU Provo/Notebook/LargePhage/Summerexp|July-August]]
 +
 
 +
: [[Team:BYU Provo/Notebook/LargePhage/Fallexp|September-October]]
</font>
</font>
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| style="width: 50%; background-color: transparent;"|
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| style="width: 82%; background-color: transparent;"|
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<font size="5" font face="Calibri"> '''April 29 - May 12''' </font>
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<font face="Calibri" size="3">
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<br>  
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<font size="4"> '''5/1/13''' </font>
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<font size="3" font face="Calibri"> This is the start of Spring Term at BYU! The weather is finally getting better: no more snowing. And we are ready to do some serious lab work! While we are waiting for our mutgen, 5-bromodeoxyuridine, to arrive, we decided to start this term with establishing the basic procedures for our direct evolution method. Specifically, this involves making sure that we can (1) grow and amplify T7 phage in liquid culture, (2) select for smaller phage using concentrated top agar, and (3) amplify phage from a single plaque. During these two weeks, we also designed primers for cloning and sequencing T7 capsid proteins as well as mapped out detailed procedures for both cloning T7 capsid proteins and applying mutagen. </font>
+
Today we scheduled out the rest of spring semester! See Below!
 +
Results of looking for recipes
 +
M9+ media recipe (grams per liter)
 +
KH,PO4 (3.0 g), Na,HPO4 (7.0 g), NaCl (0.5 g), NH4Cl (1.0 g),
 +
MgSO4.7HOH (0.246 g), CaCl, (0.011 g), ferric citrate trihydrate (0.6 mg), glucose (4.0 g), and Casamino Acids (10 g; Difco).
-
<br>
+
Phage stock preparation
 +
http://myxo.css.msu.edu/ecoli/phagepharm.html
-
<font color="#333399" size="4" font face="Calibri">
 
-
[[Team:BYU_Provo/Notebook/LargePhage/Springexp/Period1/Dailylog|Daily log]]
+
Large Phage Group Supply List:
 +
LB plates
 +
5 mL pipettes
 +
LB top agar
 +
5-bromodeoxyuridine - 1g
-
[[Team:BYU_Provo/Notebook/LargePhage/Springexp/Period1/Explist|Experiment Listing]]
+
M9 minimal salts media 500g - (or wherever else is cheaper)
 +
MgSO4
 +
CaCl
 +
ferric citrate trihydrate - not sure if this is necessary...? we only need 0.6 mg per liter and we can't find where it's sold so we'll probably omit it
 +
glucose
 +
Casamino Acids (500 g)
-
[[Team:BYU_Provo/Notebook/LargePhage/Springexp/Period1/PR| Progress Report]] </font>
+
Adenine (we only need 500 mg or so)
 +
Uracil (we only need 500 mg or so)
 +
 
 +
Magnesium chloride
 +
Chloroform
<br>
<br>
-
| style="width: 20%; background-color: transparent;"| [[File:Spring1.JPG|250px|center]]
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<font size="4"> '''5/2/13''' </font>
-
 
+
Today we created a plan for the next little while:
-
|-
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May 7-8 and 13-14
-
 
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Prepare and mutagenize T4 phage
-
| style="width: 18%; background-color: transparent;"|
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-
 
+
Day 1 Start overnight growth of W3110
-
| <font size="5" font face="Calibri"> '''May 13 - May 26''' </font>
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-
 
+
(http://biology.clc.uc.edu/fankhauser/labs/microbiology/Phage/Phage_Prep.htm)
-
<br>
+
-
 
+
Day 2 before class:
-
<font size="3" font face="Calibri"> In these two weeks, we performed our first round of mutagenesis and first PCR. Although we are still testing the results to optimize our protocol, we are optimistic about the outcome!</font>
+
Grow E. coli W3110 in H-broth --find substitute-- (supplemented with 20 ug of adenine per ml) to a density of 3 x 10^8 cells/ml
 +
- How do we know it's at this density?
 +
 +
Day 2 during class:
 +
Chill and pellet bacteria
 +
 +
Resuspend in at concentration of 1.5 x 10^8 cells/ml in M9+ medium with 5-bromodeoxyuridine (mutagen) and other supplements
 +
 +
Add 2 x 10^9 phage particles to 30 mL of bacteria and aerate at 37C until cleared (a few hours).  Then add Chloroform.
 +
- Good stopping point here
 +
 +
May 15
 +
Titer mutagenized phage stocks
 +
 +
May 17, 20
 +
Liquid culture of giant phage
 +
 +
Day 1 - Start overnight growth of W3110 in M9+
 +
 +
Day 2 -
 +
Start mid-log to to density of 4e8 cells/mL.
 +
 +
Add mutagenized phages to each culture (MOI of 0.5)
 +
- wait 7 minutes -
 +
 +
Add ten times the amount of T1 (compared to T4 mutagenized phage) to lyse uninfected cells (preventing giant phage recombination and loss).
 +
- T1 has short latent period
 +
- Wait seven minutes
 +
 +
Find a way to inhibit lysis...
 +
 +
Pellet bacteria, resuspend in 25 mL, lyse with Chloroform, treat with DNase and Mg+
 +
Pellet debris
 +
 +
May 21
 +
Start centrifugation
 +
 +
May 22
 +
Work with small plaques
 +
Mutagenize more phage
 +
 +
May 24
 +
Titer centrifugation fractions
 +
 +
May 29 – UV  and plate large phage fractions
 +
 +
May 30
 +
Purify large phage further
 +
 +
First week of June – take EMs of putative giant phage
 +
Second week of June – Plate out lots of samples, look for small plaques, try to purify large phage
<br>
<br>
-
<font color="#333399" size="4" font face="Calibri">
+
<font size="4"> '''5/3/13''' </font>
-
 
+
-
[[Team:BYU_Provo/Notebook/LargePhage/Springexp/Period2/Dailylog|Daily log]]
+
-
 
+
-
[[Team:BYU_Provo/Notebook/LargePhage/Springexp/Period2/Explist|Experiment Listing]]
+
-
[[Team:BYU_Provo/Notebook/LargePhage/Springexp/Period2/PR| Progress Report]] </font>
+
-Today we archived our phage stock of T4 Do. Dr. Grose said she couldn’t find T1 easily available so now we are going to look into alternative e.coli phage that has a short latent period.
<br>
<br>
 +
<font size="4"> '''5/6/13''' </font>
-
| style="width: 20%; background-color: transparent;"| [[File:Spring2.JPG|250px|center]]
+
- Today we did a phage titer of 0,-3,-6,-7,-8,-9,-10,-11 We started by putting 1 ul of phage into 1ml of bacteria, we went down from there. We infected each test tube with 10 uL from each dilution level.
 +
We also did a UV pre-test of sorts. We put 20ul of phage onto parafilm and exposed it to UV light in the hood of Dr. Breakwell’s lab. At 30 second intervals we took the 20 ul’s off and put it in a half ml of e.coli W3110. We tested every 30 seconds up to 3 minutes.
-
|-
+
We also streaked out the W3110 bacteria from freezer stock onto an LB plate so we can use it to start future bacterial cultures.
-
| style="width: 18%; background-color: transparent;"|
+
[[File:Ex.jpg|400px|center|]]
-
 
+
[[File:Ex1.jpg|400px|center|]]
-
| <font size="5" font face="Calibri"> '''May 27 - June 9''' </font>
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[[File:Ex2.jpg|400px|center|]]
-
 
+
[[File:Ex3.jpg|400px|center|]]
-
<br>
+
-
 
+
-
<font size="3" font face="Calibri"> Add description! </font>
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<br>
<br>
-
<font color="#333399" size="4" font face="Calibri">
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<font size="4"> '''5/8/13''' </font>
-
[[Team:BYU_Provo/Notebook/LargePhage/Springexp/Period3/Dailylog|Daily log]]
+
-[[File:hey1.jpeg|400px|thumb|center|]]
 +
[[File:hey2.jpeg|400px|thumb|center|]]
 +
[[File:hey3.jpeg|400px|thumb|center|]]
 +
[[File:hey4.jpeg|400px|thumb|center|]]
-
[[Team:BYU_Provo/Notebook/LargePhage/Springexp/Period3/Explist|Experiment Listing]]
+
We looked at the results for the dilution series (titer) we did on the phage stock we made from the liquid culture and found that there were 5 plaques on the 10^-8 plate.
-
[[Team:BYU_Provo/Notebook/LargePhage/Springexp/Period3/PR| Progress Report]] </font>
+
We calculated pfus/mL by doing ( # of plaques ) / (dilution level x mLs infected with).  Our stock is between 3x10^9 and 5x10^9 pfus/mL.
 +
180 sec = ~50 plaques
 +
150 sec = ~102 plaques
 +
120 sec = ~170 plaques
 +
90 sec = ~2805/6/13- KS, BDM
 +
Today we did a phage titer of 0,-3,-6,-7,-8,-9,-10,-11 We started by putting 1 ul of phage into 1ml of bacteria, we went down from there. We infected each test tube with 10 uL from each dilution level.
-
<br>
+
We also did a UV pre-test of sorts. We put 20ul of phage onto parafilm and exposed it to UV light in the hood of Dr. Breakwell’s lab. At 30 second intervals we took the 20 ul’s off and put it in a half ml of e.coli W3110. We tested every 30 seconds up to 3 minutes.
-
 
+
-
 
+
-
| style="width: 20%; background-color: transparent;"| [[File:Spring3.JPG|250px|center]]
+
-
 
+
-
 
+
-
|-
+
-
 
+
-
| style="width: 18%; background-color: transparent;"|
+
-
| <font size="5" font face="Calibri"> '''June 10 - June 23''' </font>
+
We also streaked out the W3110 bacteria from freezer stock onto an LB plate so we can use it to start future bacterial cultures.
-
<br>
+
plaques
 +
60 sec = ~300 plaques
 +
30 sec =
 +
0 sec = ~300-500 plaques
-
<font size="3" font face="Calibri"> Add description! </font>
+
Goals soon:
 +
Refine liquid culture technique - read online procedure
 +
Pick plaques - what titer do we get on plates from picked plaques?
 +
Make top agar (1.5x to dilute to 0.75x)
 +
Pour LB plates
 +
Purify small plaques from UV test
<br>
<br>
-
<font color="#333399" size="4" font face="Calibri">
+
<font size="4"> '''5/10/13''' </font>
-
[[Team:BYU_Provo/Notebook/LargePhage/Springexp/Period4/Dailylog|Daily log]]
+
- Phage similar to T1:
-
 
+
Rtp
-
[[Team:BYU_Provo/Notebook/LargePhage/Springexp/Period4/Explist|Experiment Listing]]
+
46219
-
 
+
NC_007603
-
[[Team:BYU_Provo/Notebook/LargePhage/Springexp/Period4/PR| Progress Report]] </font>
+
E. coli
 +
JBACT 188:1419
 +
Rogue1
 +
45805
 +
JQ182736
 +
E. coli
 +
VirolJ 9:2-7
 +
We found that these two phage have homologous genes to T1, but the nucleotide sequences are not significantly similar. We looked into more T1 articles to try to find someone we can ask to send us a sample, and found this article from 2004 when it was sequenced (http://www.sciencedirect.com/science/article/pii/S0042682203007153).  We checked at picultures.com before placing the email, and found that they have both T1 and E. coli B that we can order. We will do this so we can use T1 in our mutagenesis procedure.
<br>
<br>
-
 
+
</font>
-
| style="width: 20%; background-color: transparent;"| [[File:Spring4.JPG|250px|center]]
+
-
 
+
|}
|}
{{TeamBYUProvoFooter}}
{{TeamBYUProvoFooter}}

Latest revision as of 17:35, 27 September 2013


Large Phage May - June Notebook: May 1 - May 12 Daily Log



Large Phage
March-April
May-June
July-August
September-October

5/1/13

Today we scheduled out the rest of spring semester! See Below! Results of looking for recipes M9+ media recipe (grams per liter) KH,PO4 (3.0 g), Na,HPO4 (7.0 g), NaCl (0.5 g), NH4Cl (1.0 g), MgSO4.7HOH (0.246 g), CaCl, (0.011 g), ferric citrate trihydrate (0.6 mg), glucose (4.0 g), and Casamino Acids (10 g; Difco).

Phage stock preparation http://myxo.css.msu.edu/ecoli/phagepharm.html


Large Phage Group Supply List: LB plates 5 mL pipettes LB top agar 5-bromodeoxyuridine - 1g

M9 minimal salts media 500g - (or wherever else is cheaper) MgSO4 CaCl ferric citrate trihydrate - not sure if this is necessary...? we only need 0.6 mg per liter and we can't find where it's sold so we'll probably omit it glucose Casamino Acids (500 g)

Adenine (we only need 500 mg or so) Uracil (we only need 500 mg or so)

Magnesium chloride Chloroform


5/2/13

Today we created a plan for the next little while: May 7-8 and 13-14 Prepare and mutagenize T4 phage

Day 1 Start overnight growth of W3110 

(http://biology.clc.uc.edu/fankhauser/labs/microbiology/Phage/Phage_Prep.htm)

Day 2 before class: Grow E. coli W3110 in H-broth --find substitute-- (supplemented with 20 ug of adenine per ml) to a density of 3 x 10^8 cells/ml 

- How do we know it's at this density?

Day 2 during class: Chill and pellet bacteria

Resuspend in at concentration of 1.5 x 10^8 cells/ml in M9+ medium with 5-bromodeoxyuridine (mutagen) and other supplements

Add 2 x 10^9 phage particles to 30 mL of bacteria and aerate at 37C until cleared (a few hours). Then add Chloroform. 

- Good stopping point here

May 15 Titer mutagenized phage stocks

May 17, 20 Liquid culture of giant phage

Day 1 - Start overnight growth of W3110 in M9+

Day 2 - Start mid-log to to density of 4e8 cells/mL.

Add mutagenized phages to each culture (MOI of 0.5) 

- wait 7 minutes -

Add ten times the amount of T1 (compared to T4 mutagenized phage) to lyse uninfected cells (preventing giant phage recombination and loss). 

- T1 has short latent period
- Wait seven minutes

Find a way to inhibit lysis...

Pellet bacteria, resuspend in 25 mL, lyse with Chloroform, treat with DNase and Mg+ Pellet debris

May 21 Start centrifugation

May 22 Work with small plaques Mutagenize more phage

May 24 Titer centrifugation fractions

May 29 – UV and plate large phage fractions

May 30 Purify large phage further

First week of June – take EMs of putative giant phage Second week of June – Plate out lots of samples, look for small plaques, try to purify large phage


5/3/13

-Today we archived our phage stock of T4 Do. Dr. Grose said she couldn’t find T1 easily available so now we are going to look into alternative e.coli phage that has a short latent period.


5/6/13

- Today we did a phage titer of 0,-3,-6,-7,-8,-9,-10,-11 We started by putting 1 ul of phage into 1ml of bacteria, we went down from there. We infected each test tube with 10 uL from each dilution level.

We also did a UV pre-test of sorts. We put 20ul of phage onto parafilm and exposed it to UV light in the hood of Dr. Breakwell’s lab. At 30 second intervals we took the 20 ul’s off and put it in a half ml of e.coli W3110. We tested every 30 seconds up to 3 minutes.

We also streaked out the W3110 bacteria from freezer stock onto an LB plate so we can use it to start future bacterial cultures.

Ex.jpg
Ex1.jpg
Ex2.jpg
File:Ex3.jpg


5/8/13

-
Hey1.jpeg
Hey2.jpeg
Hey3.jpeg
Hey4.jpeg

We looked at the results for the dilution series (titer) we did on the phage stock we made from the liquid culture and found that there were 5 plaques on the 10^-8 plate.

We calculated pfus/mL by doing ( # of plaques ) / (dilution level x mLs infected with). Our stock is between 3x10^9 and 5x10^9 pfus/mL. 180 sec = ~50 plaques 150 sec = ~102 plaques 120 sec = ~170 plaques 90 sec = ~2805/6/13- KS, BDM Today we did a phage titer of 0,-3,-6,-7,-8,-9,-10,-11 We started by putting 1 ul of phage into 1ml of bacteria, we went down from there. We infected each test tube with 10 uL from each dilution level.

We also did a UV pre-test of sorts. We put 20ul of phage onto parafilm and exposed it to UV light in the hood of Dr. Breakwell’s lab. At 30 second intervals we took the 20 ul’s off and put it in a half ml of e.coli W3110. We tested every 30 seconds up to 3 minutes.

We also streaked out the W3110 bacteria from freezer stock onto an LB plate so we can use it to start future bacterial cultures. 

plaques

60 sec = ~300 plaques 30 sec = 0 sec = ~300-500 plaques

Goals soon: Refine liquid culture technique - read online procedure Pick plaques - what titer do we get on plates from picked plaques? Make top agar (1.5x to dilute to 0.75x) Pour LB plates Purify small plaques from UV test


5/10/13

- Phage similar to T1: Rtp 46219 NC_007603 E. coli JBACT 188:1419 Rogue1 45805 JQ182736 E. coli VirolJ 9:2-7 We found that these two phage have homologous genes to T1, but the nucleotide sequences are not significantly similar. We looked into more T1 articles to try to find someone we can ask to send us a sample, and found this article from 2004 when it was sequenced (http://www.sciencedirect.com/science/article/pii/S0042682203007153). We checked at picultures.com before placing the email, and found that they have both T1 and E. coli B that we can order. We will do this so we can use T1 in our mutagenesis procedure.


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