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

From 2013.igem.org

(Difference between revisions)
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==May==
==May==
===5/1/13===
===5/1/13===
 +
1 May 2013 KS- Today we scheduled out the rest of spring semester! See Below!
 +
�
 +
1 May 2013 - BDM
 +
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).
-
===5/3/13===
+
Phage stock preparation
 +
http://myxo.css.msu.edu/ecoli/phagepharm.html
-
===5/6/13===
 
-
===5/8/13===
+
Large Phage Group Supply List:
 +
LB plates
 +
5 mL pipettes
 +
LB top agar
 +
5-bromodeoxyuridine - 1g
-
===5/10/13===
+
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)
-
===5/13/13===
+
Adenine (we only need 500 mg or so)
 +
Uracil (we only need 500 mg or so)
-
===5/15/13===
+
Magnesium chloride
 +
Chloroform
-
===5/17/13===
+
Calendar/Schedule:
 +
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
-
===5/20/13===
 
-
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.
+
===5/3/13===
 +
5/3/13 KS and BDM
 +
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.
-
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.
+
===5/6/13===
 +
5/6/13- 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.
-
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.
+
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.
-
UV tests were done by placing 20 uL spots on parafilm and placed in a BSL-2 hood with the UV light turned on.
+
We also streaked out the W3110 bacteria from freezer stock onto an LB plate so we can use it to start future bacterial cultures.
-
===5/22/13===
+
[[File:Example.jpg|400px|center|]]
-
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.
+
[[File:Example.jpg|400px|center|]]
-
(We infected with ___ mL of phage at ___ titer in ____ vol of resuspended bacteria.)
+
[[File:Example.jpg|400px|center|]]
 +
[[File:Example.jpg|400px|center|]]
 +
===5/8/13===
 +
5/8/13- Results from Monday KS BDM
-
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).
+
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.
-
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 calculated pfus/mL by doing ( # of plaques ) / (dilution level x mLs infected with).  Our stock is between 3x10^9 and 5x10^9 pfus/mL.
-
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.
+
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.
-
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.
+
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.
-
[[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===
+
We also streaked out the W3110 bacteria from freezer stock onto an LB plate so we can use it to start future bacterial cultures.
-
===5/27/13===
+
plaques
 +
60 sec = ~300 plaques
 +
30 sec =  
 +
0 sec = ~300-500 plaques
-
===5/29/13===
+
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
-
===5/31/13===
+
===5/10/13===
-
 
+
10 May 2013 - Friday BDM KS
-
==June==
+
Phage similar to T1:
-
===6/3/13===
+
Rtp
-
 
+
46219
-
===6/5/13===
+
NC_007603
-
 
+
E. coli
-
===6/7/13===
+
JBACT 188:1419
-
 
+
Rogue1
-
===6/10/13===
+
45805
-
 
+
JQ182736
-
===6/12/13===
+
E. coli
-
 
+
VirolJ 9:2-7
-
===6/14/13===
+
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.
-
 
+
-
===6/17/13===
+
-
 
+
-
===6/19/13===
+
-
 
+
-
===6/21/13===
+
-
 
+
-
===6/24/13===
+
-
 
+
-
===6/26/13===
+
-
 
+
-
===6/28/13===
+
-
 
+
-
==July==
+
-
===7/1/13===
+
-
 
+
-
===7/3/13===
+
-
 
+
-
===7/5/13===
+
-
 
+
-
===7/8/13===
+
-
 
+
-
===7/10/13===
+
-
 
+
-
===7/12/13===
+
-
 
+
-
===7/15/13===
+
-
 
+
-
===7/17/13===
+
-
 
+
-
===7/19/13===
+
-
 
+
-
===7/22/13===
+
-
 
+
-
===7/24/13===
+
-
 
+
-
===7/26/13===
+
-
 
+
-
===7/29/13===
+
-
 
+
-
===7/31/13===
+
-
 
+
-
==August==
+
-
===8/2/13===
+
-
 
+
-
===8/5/13===
+
-
 
+
-
===8/7/13===
+
-
 
+
-
===8/9/13===
+
-
 
+
-
===8/12/13===
+
-
 
+
-
===8/14/13===
+
-
 
+
-
===8/16/13===
+
-
 
+
-
===8/19/13===
+
-
 
+
-
===8/21/13===
+
-
 
+
-
===8/23/13===
+
-
 
+
-
===8/26/13===
+
-
 
+
-
===8/28/13===
+
-
 
+
-
===8/30/13===
+
-
 
+
-
==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===
+
13 May 2013 - KS and BDM
 +
Today we made stocks for our M9+ media.  We made 1 L of 50X E-salts media that is stored in Dr. Grose’s lab.  We also made a 100x stock of CaCl2 and NaCl which is stored in the iGEM classroom.
 +
To make M9+ media, mix:  (makes 1 L)
 +
20 mL 50X E-salts
 +
10 mL 20% glucose (in Dr. Grose’s lab)
 +
2.5 mL Ca/Na (iGEM room)
 +
10g Casamino acids (need to order)
 +
Fill jar to 1 L with dH20
 +
Autoclave
-
===9/27/13===
 
-
===9/30/13===
 
{{TeamBYUProvoFooter}}
{{TeamBYUProvoFooter}}

Revision as of 20:12, 3 June 2013


Contents

May

5/1/13

1 May 2013 KS- Today we scheduled out the rest of spring semester! See Below! � 1 May 2013 - BDM 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

Calendar/Schedule: 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

5/3/13 KS and BDM 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

5/6/13- 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.

Example.jpg
Example.jpg
Example.jpg
Example.jpg

5/8/13

5/8/13- Results from Monday KS BDM

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

10 May 2013 - Friday BDM KS 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.

13 May 2013 - KS and BDM Today we made stocks for our M9+ media. We made 1 L of 50X E-salts media that is stored in Dr. Grose’s lab. We also made a 100x stock of CaCl2 and NaCl which is stored in the iGEM classroom. To make M9+ media, mix: (makes 1 L) 20 mL 50X E-salts 10 mL 20% glucose (in Dr. Grose’s lab) 2.5 mL Ca/Na (iGEM room) 10g Casamino acids (need to order) Fill jar to 1 L with dH20 Autoclave


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