Team:BYU Provo/Notebook/SmallPhage/Winterexp/Period1/Dailylog

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{| width="100%"
{| width="100%"
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| colspan="3" | <font color="#333399" size="5" font face="Calibri"> '''Phage Purification March - April Notebook: March 15 - March 31 Daily Log'''</font>
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| colspan="3" | <font color="#333399" size="5" font face="Calibri">  
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: '''Small Phage March - April Notebook: March 15 - March 31 Daily Log'''</font>
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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/Phage_Purification|Overview]]
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<font size = "4">
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: [[Team:BYU Provo/Notebook/Phage_Purification/Winterexp|March-April]]
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: <u> '''Small Phage''' </u> </font>
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: [[Team:BYU Provo/Notebook/Phage_Purification/Springexp|May-June]]
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: [[Team:BYU Provo/Notebook/SmallPhage/Winterexp|March-April]]
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: [[Team:BYU Provo/Notebook/Phage_Purification/Summerexp|July-August]]
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: [[Team:BYU Provo/Notebook/SmallPhage/Springexp|May-June]]
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: [[Team:BYU Provo/Notebook/Phage_Purification/Fallexp|September-October]]
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: [[Team:BYU Provo/Notebook/SmallPhage/Summerexp|July-August]]
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: [[Team:BYU Provo/Notebook/SmallPhage/Fallexp|September-October]]
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<font size="4"> '''3/15/13''' </font>
<font size="4"> '''3/15/13''' </font>
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- TODAY MARKS THE START OF THE PHAGE PURIFICATION TEAM!
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-Today we began research on phage purification
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- Today we began researching for a procedure to begin purifying phage. We have found that T7 can self assemble with scaffolding proteins without forming procapsids, and that T4 has only been know to form procapsids. We may not have to worry about T4 if the other group isn't using it.
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-Focused on T4 and T7 as this is what the other groups will be focusing on
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:-T7 can self assemble with the help of scaffolding proteins without forming procapsids
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:-T4 assembles using procapsids
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-Some phage can have their DNA polymerase genes knocked out to make a hollow phage
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:-Possibility for making ghost capsids
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<br>
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- Important findings
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<font size="4"> '''3/16/13''' </font>
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:Phage have been purified before
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:Phage capsids can self assemble
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- Created a priority list of things we need to complete:
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:Phage (amber strain) can have their genes knocked out to make a hollow phage
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:-Find out how drugs are put into capsids
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:-Possibly contact F. W. Studier for his amber T7 phage strain  
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::-This strain had the knocked out DNA polymerase gene
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:-Perform a phage titer on the phage we have to see if we have enough to work with
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:-Find a procedure that we can use to test if our purified phage are viable by filling the capsid with liquid
<br>
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<font size="4"> '''3/18/13''' </font>
<font size="4"> '''3/18/13''' </font>
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- Priorities List:
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- Presented current understanding and plans for the future
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:Find out how to put drugs into the capsid
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: Decided on a two-focus attack
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:Possibly contact F.W. Studier for his amber t7 phage strain
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:: Evolution – selecting naturally occurring smaller phages
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- Next class we plan on growing up phage so that we will have a decent amount of phage to work with.  We have several procedures that we plan on testing so that we can see if we can purify the protein capsid.  We hope to be proceeding with these as soon as we have phage that we can use. 
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:: Site directed mutagenesis – using genome info and comparative studies to identify sites to target
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- Another issue that we need to consider is how to get drugs into the capsid. 
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- More background research
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:We need to be able to test and see if we can actually fill our empty capsids with a material. 
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: Possible ways of making phage smaller
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:We have several procedures found in other papers that could possibly help us with this.
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<font size="4"> '''3/20/13''' </font>
<font size="4"> '''3/20/13''' </font>
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- We spent time trying to find a good procedure to propagate phage in a liquid medium. We finally found one that I think will work well for us.
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- Background research on phiX174: the only phage we have in stock
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- Today we learned a procedure for how to count phage. 
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- We performed a phage titer on the T4 phage to see if we have a high enough concentration to work with.  This is the general procedure:
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- Phage titer: reported in Pfu/mL (Pfu stands for plaque forming unit)
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:mix E. coli (500 microL) with 50 microL of phage lysate
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:incubate for 20 minutes
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:mix with 4 mL top agar and then plate
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:holes will appear in the agar that are called plaques
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- How we followed it:
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:We filled five test tubes full of 90 microliters of Liquid Broth each.
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:We added 10 microliters of our desired phage to the first test tube and then mixed
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:We took 10 microliters of the first tubes mixed solution and added it to tube 2, and followed the same procedure for :each test tube down the line to tube five.
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:We then labeled 6 culture tubes 0 to -5.
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:In the first culture tube, we added 20 microL phage to .5 mL bacteria.
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:In tubes -1 to -5 we took 20 microL from eppendorfs and added to .5 mL bacteria.
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:We then allowed a 20 minute waiting period for the virus to infect the E. coli.
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:5 mL top agar was added to each culture tube.
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:Each tube was then plated and incubated at 37 degrees C.
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-
 
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-The following teammates were assigned phage as follows:
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:Amber - T2
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:Arick - T5
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:Darren - T3
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-Results:
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:We ran into several problems while doing the titer.  After we had completed the titer, we found out that the pipet tips we had used were contaminated.  When preparing the top agar, we had to melt it in the microwave which caused it to boil over.  This could have caused some contamination.  While filling my -5 plate with top agar, there was only enough to put in 4mL of agar instead of the 5mL that was called for in our procedure. 
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:None of the plates had any phage. There was just a lawn of bacteria growing.  This could either be because of the problems mentioned above or because the source of T3 was bad.  Seeing as nobody else was able to grow any phage we believe that the source was old.
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- Performed [[Team:BYU_Provo/Notebook/SmallPhage/Winterexp/Period1/Exp/3.20 Phage Viability Test|3.20 Phage Viability Test]]
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- Outlined protocol for producing stock top agar
- Outlined protocol for producing stock top agar
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: [[Team:BYU_Provo/Notebook/SmallPhage/Winterexp/Period1/Exp/4.3 Top Agar Stock Preparation|4.3 Top Agar Stock Preparation]]
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: [[Team:BYU_Provo/Notebook/SmallPhage/Winterexp/Period3/Exp/4.3 Top Agar Stock Preparation|4.3 Top Agar Stock Preparation]]
<br>
<br>

Latest revision as of 13:23, 9 September 2013


Small Phage March - April Notebook: March 15 - March 31 Daily Log



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

3/15/13

-Today we began research on phage purification

-Focused on T4 and T7 as this is what the other groups will be focusing on

-T7 can self assemble with the help of scaffolding proteins without forming procapsids
-T4 assembles using procapsids

-Some phage can have their DNA polymerase genes knocked out to make a hollow phage

-Possibility for making ghost capsids


3/16/13

- Created a priority list of things we need to complete:

-Find out how drugs are put into capsids
-Possibly contact F. W. Studier for his amber T7 phage strain
-This strain had the knocked out DNA polymerase gene
-Perform a phage titer on the phage we have to see if we have enough to work with
-Find a procedure that we can use to test if our purified phage are viable by filling the capsid with liquid


3/18/13

- Presented current understanding and plans for the future

Decided on a two-focus attack
Evolution – selecting naturally occurring smaller phages
Site directed mutagenesis – using genome info and comparative studies to identify sites to target

- More background research

Possible ways of making phage smaller


3/20/13

- Background research on phiX174: the only phage we have in stock

- Performed 3.20 Phage Viability Test


3/21/13

- Checked up on results for 3.20 Phage Viability Test


3/22/13

- Discussed results from tittering experiment (preliminary experiment 1)

Contamination mostly likely resulted from the step involving suspending phage in LB – obvious contamination in the LB bottle
One of the stock phage solution had contamination as well

- Discussed step of attack with Dr. Grose

Decided to go with T7, if necessary Qbeta
Need to learn to make top agar at various concentrations
Need to do more background research and decide whether to assemble phage capsid in vitro (plasmid + E coli) or do direct mutagenesis of phage genome
Need to correspond with the isolation team

- Sequencing will be for individual genes to cut down cost

Need to design primers and get to know the genome of the phage

- Learnt about Mega5 to compare genome and protein sequence


3/25/13

- Reported on past week and plans for this week

From last week: titering experiment
This week
Learn to make top agar at various concentrations
Background research to determine in vitro assembly vs altering genome – look into specific techniques
Comparing genome of phage and decide on possible site-directed mutagenesis options

- Start working on designing our site directed mutagenesis

Qbeta vs MS2
Look for places where sequences are significantly different
Might be worthwhile to look at capsid structure to identify the regions where interactions between subunits take place
Qbeta vs T7 major
No real consensus – more worthwhile to compare capsid protein sequence of T7 with those that have similar size to it
T7 major vs minor
Minor is longer, but not necessary – the tail overhang is due to ribosome moving two codons downstream instead of three
Suggest we can direct mutation to the poly-U site and prevent ribosome slippage
Qbeta major vs minor
Just continue transcribing after it reaches the stop codon. What does the stop codon code for?

- Research into in-vitro assembly vs direct mutation of phage genome

It seems that we’ll need to clone the genome of the phage into a plasmid and let it assemble in an E coli
Using chemicals we can induce random mutations in phage – might be worthwhile if selection in agar is not working as well.


3/27/13

- More research on genome of enterobacteria phage

Generation of the major and minor capsid in Q beta
Capsid protein information research
Capsid protein sequence comparison

- Outlined protocol for producing stock top agar

4.3 Top Agar Stock Preparation


3/29/13

- Worked on our first team presentation.