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| Small Phage March - April Notebook: Experiments
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- Overview
- March-April
- May-June
- July-August
- September-October
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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?
- 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
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