IGEM:IMPERIAL/2007/Projects/Hrp System

HRP Characterization

Things to Characterize

What we want to know, in order of importance (Most of these are performed using PoPS):

1. Strength of response
2. Minimum/maximum PoPS output
3. Signal fluctuation and noise
4. Leakiness
5. Response times
6. Stability of proteins (half life)
   • Recovery of response
7. Ratio effects on output
8. Interaction with other parts

• How R and S affect activity separately
  • Having both R and S under same promoter
• Energy requirements
• Enhancer length effects

We still need to:

• Find literature and protocols for each question
• Design experiments for each question
  • Note down the time requirements
• Plan the schedule
• Look for alternatives to PoPS (RiPs?)
• Schedule meeting with instructors

Measuring PoPS

PoPS depends on:

1. Temperature
2. Medium
3. pH

• IHF
• PspF
• LoN protease
• Other regulators like σ₅₄
• Host cell

Other things that affect PoPS:

• Promoter
• Enhancer length

How do we measure PoPS?
Can we do it using the screening plasmid?

Contexts

Measuring transfer function in E.coli

1. Normal genome E.coli in vivo
2. in vitro
3. E.coli with other parts
4. Minimal genome E.coli in vivo

Experiments

Promoters

List input promoters, find/measure PoPS in of the following promoters:

• Ara
• Lac
• Tet
• cI
• pHrpL
• HrpBox

Measuring PoPS of Promoters

We need to measure the following:

1. Parts HrpR, HrpS on PoPS of pHrpL
2. Parts HrpR, HrpS, HrpV on PoPS of pHrpL
3. Part HrpL on PoPS of HrpBox
4. Parts HrpR, HrpS, HrpL on PoPS of HrpBox
5. Parts HrpR, HrpS, HrpV, HrpL on PoPS of HrpBox

Note: Standard RBS meeds to be cloned in Pilot experiment done before full experiment