IGEM:IMPERIAL/2006/project/parts/BBa T9002
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Specification
The motivation to build this part is to allow us to determine how AHL at different levels activate genes, and use this to assay AHL.
This part allows us to investigate three parameters:
- How different AHL levels activate genes at a constant LuxR expression (The transfer function of pLuxR)
- How much AHL is present, by referring to the transfer function
- How LuxR levels effect gene activation
Design
Part Design
T9002 - Link to MIT Parts Registry
Inputs/Outputs
- Part Input = AHL
- Part Output = Green Fluorescent Protein
Comments
- The part responds to AHL levels, via activation of pLuxR
- From this, we can calibrate a AHL assay based on fluorescence
- LuxR is under the control of pTet, allowing different levels of LuxR to be achieved
Modelling
The Model
Variables & Parameters
Variable | Status |
---|---|
AHL | Known |
LuxR | Known |
GFP | Measured |
Paramter | Status | Value in model |
---|---|---|
AHL Degredation | Known | Half-Life = 24 Hours |
GFP Degredation | Known | Half-Life = 45 Minutes |
GFP Vmax | Unknown | 2/sec/plasmid |
LuxR/AHL Ability to Activate Gene (Km) |
Unknown | 100 |
Sensitivty of Key Paramters
- AHL Level
- The model is sensitive to initial AHL level
- As AHL input increase, GFP output increases until saturation
- The relationship between AHL level and GFP output is not linear due to Michaelis-Menten Kinetics
- The exact relationship can be calculated from this assay
- Ability of AHL/LuxR to activate genes (km)
- Increase in Km lowers GFP steady state amplitude. This is due to the decrease in ability of AHL to activate genes, and leading to a reduced apparent AHL concentration
Assumptions
- LuxR is at steady state
- A good approximation if LuxR gene is left in an active state while cells are growing in medium
- The relationship between LuxR and AHL in activating genes is linear
- This is not strictly true as LuxR and AHL bind using Michaelis-Menten kinetics
- Gives a good approximation if LuxR is in excess of AHL (ie not saturated by AHL)
Implementation
- Part is already available in its complete form.
- Well 19B Plate 2
Testing and Validation
Prequisites
- Part xxxxx Characterisation
- Needed to know how LuxR concentration varies with pTet activation
Protocol
Equipment & Materials
- Equipment
- Wallac Victor 3 Multi-Well Fluorimeter (or alternative apparatus)
- Microfuge Tubes
- Gilson Pippettes
- 37oC Shaker
- Materials
- AHL
- aTc
- E.coli Growth Medium w/Ampicilin
- E.coli Culture Containing T9002