IGEM:IMPERIAL/2006/project/parts/BBa T9002

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

T9002 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

See T9002 Testing 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