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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
3/16/13
- Did more background reading for T7; literature search for possible plan of attack
- 3.16 Plan of Attack
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.
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