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3/15/13
- TODAY MARKS THE START OF THE PHAGE PURIFICATION TEAM!
- 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.
- Important findings
- Phage have been purified before
- Phage capsids can self assemble
- Phage (amber strain) can have their genes knocked out to make a hollow phage
3/18/13
-Priorities List:
- Find out how to put drugs into the capsid
- Possibly contact F.W. Studier for his amber t7 phage strain
-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.
-Another issue that we need to consider is how to get drugs into the capsid.
- We need to be able to test and see if we can actually fill our empty capsids with a material.
- We have several procedures found in other papers that could possibly help us with this.
3/20/13
-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.
-Today we learned a procedure for how to count phage.
-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:
-Phage titer: reported in Pfu/mL (Pfu stands for plaque forming unit)
- mix E. coli (500 microL) with 50 microL of phage lysate
- incubate for 20 minutes
- mix with 4 mL top agar and then plate
- holes will appear in the agar that are called plaques
-How we followed it:
- We filled five test tubes full of 90 microliters of Liquid Broth each.
- We added 10 microliters of our desired phage to the first test tube and then mixed
- 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.
- We then labeled 6 culture tubes 0 to -5.
- In the first culture tube, we added 20 microL phage to .5 mL bacteria.
- In tubes -1 to -5 we took 20 microL from eppendorfs and added to .5 mL bacteria.
- We then allowed a 20 minute waiting period for the virus to infect the E. coli.
- 5 mL top agar was added to each culture tube.
- Each tube was then plated and incubated at 37 degrees C.
-The following phage was worked on by the following:
- Amber - T2
- Arick - T5
- Darren - T3
-Results:
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.
RESULTS: 3/22 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.
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.
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