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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
- 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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