Team:BYU Provo/Notebook/SmallPhage/Winterexp/Period1/Exp/PlanOfAttack

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Small Phage March - April Notebook: Experiments

3.16 Plan of Attack

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?

Stability?

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