Team:BYU Provo/Notebook/Phage Purification/Winterexp/Period1/Exp/PlanOfAttack


Small Phage March - April Notebook: Experiments


6.3 CsCl Gradient Phage Purification

1. Primary ideas

Direct evolution method
Separation method: ultracentrifugation sedimentation with sucrose gradient, size exclusion chromatography, TEM
Environment that would hasten evolution?
Limit nutrient as Dr. Kooyman suggested – affect host more than phage?
Different strains of E Coli – will phage still infect?
Selected phage can self replicate – storage
Purified product and DNA sequence can direct further engineering for drug delivery purpose (protein team’s job)
Site-directed mutagenesis
Identifying sites to mutate
Modeling: compare CP and scaffolding protein sequence of similar phage that has slightly different size, deduce from this possible sites that can be mutated to produce smaller phage
Capsid production
Introduce genes into E coli for expression and assembly – producing a novel machine that is not naturally found
How many genes to introduce into E coli?
In vitro assembly?
Has been done with T7 genome in E coli extract (no cell)
Site directed mutagenesis of a genome (40kbp)
Our parts would be plasmid introduced into E coli, but capsid produced this way won’t need extracting DNA/RNA from capsid to make an empty carrier

2. Refined plans

Get the E coli and T7/Q beta to grow
Test with different Agar concentration – see if selection method works (evolution)
Sequencing if evolution occurs
Compare genome to find targets for point mutation
Playing around with phage genome
Knocking out the polymerase
Change genome size - experiment – could go bigger or smaller
T7 specific
Play around with minor and major capsid protein – CCC site