IGEM:IMPERIAL/2007/Projects/Hrp System

Hrp Characterisation

• Specifications
• Design
• Modelling
• Implementation
• Testing/Validation
• Notes
• References

Characterisation Tasks

• 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

Techniques for measuring PoPS

• Link to the techniques used for the characterisation of the BBa_F2620
• There are several screening vectors available that can be used when measuring PoPS. An example are the BBa_13450 to 13458

Characterisation Specification

All characterisation will be done in terms of PoPS, initially using indirect measurements relative to GFP/RFP synthesis per CFU per second. If time and resources permit, a more direct method will be used. Some of the characteristics will be gleaned from literature in order to reduce the number of required experiments.

Experiments will attempt to fulfil an objective within a certain context, and with variation of a single parameter for the listed components. Since there are several objectives, contexts, and parameters, the number of data points to be obtained is very large. Therefore, priorities were established for each list in order to aid scheduling of experiments.

Parts and Devices

This is a prioritised list of parts and devices using the Hrp system that we would like to characterise.

1. 

   Follower: pHrpL promoter - RS (single operon) only

   
   

2. 

   AND with one inversion: pHrpL promoter - RS and V

   
   

3. 

   AND Gate: pHrpL promoter - R and S (separate operons) only

   
   

4. 

   Pulse generator (RS -> pHrpL expressing HrpV)

   
   

5. 

   3-port AND with one inversion: pHrpL promoter - R and S and V

   
   

6. 

   HrpBOX and σ_L (HrpL)

   
   

7. 

   The full HRP system

   
   

Objectives

This is a prioritised list of experimental objectives - the quantities we are trying to find:

1. Strength of response (ratio of PoPS out to PoPS in)
2. Minimum/maximum PoPS output and input
3. Signal fluctuation and noise (base level of PoPS out)
4. Leakiness
5. Response time
6. Half-life of proteins (stability)
   • Recovery of response
7. Input ratio effects on output
   • How R and S affect activity separately
   • Having both R and S under same promoter
8. Interaction with other parts
9. Energy requirements
10. Enhancer length effects

Contexts

This is a prioritised list of the experimental contexts that we would like to perform characterisation in.

1. E.Coli K12 strain - BBa_V1002
2. In Vitro
3. E.Coli K12 strain - BBa_V1002
   • With the following parts from the registry: (Note, check that the last four items are useful)
   1. The F2620 Promoter
   2. Plasmid pSB1A3
   3. Plasmid pSB3K3
   4. BBa_E0430
   5. BBa_E0434
   6. BBa_E0240
   7. BBa_C0040
4. E.Coli DH5a strain - BBa_V1001
5. E.Coli MG1655 strain - BBa_V1000

Parameters

1. Promoters
2. Ribosome Binding Sites
3. Temperature
4. Medium
5. pH
6. PspF (alter the expression of PspF - this is a source of noise)
7. LoN protease (LoN is known to break down HrpR)
8. Other σ₅₄ regulators