Team:BYU Provo/Notebook/Phage Purification/Winterexp/Period1/Dailylog

Overview

March-April

May-June

July-August

September-October

-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.