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

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Contents

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

image:T9002.JPG

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

image:T9002_Model.JPG

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 1000


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

None

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

Predicted Experimental Results

Image:GraphT9002.JPG

The image shows the expected form of the results as predicted by the model with a varying steady-state of LuxR.

Value Extraction

The experiment will allow us to extract the values of Km and Vmax of GFP expression, with AHL as the activator.

We will still have to borrow the following values from the literature:

  • AHL degredation rate
  • GFP degredation rate

This is how to do it

  • At equlibrium the rate of degredation = rate of synthesis
  • We know the degredation rate of GFP
    • Therefore conc GFP x Degredation Rate = rate of synthesis (V).
  • We know the concentration of substrate
  • Plot a graph of 1/V against 1/[S]

[:Lineweaver_Buke_Plot.JPG]

  • We can read Km and Vm off the Graph or solve it mathamatically
    • The Km and Vm of published results (Km and Vm were not publsihed I worked them out from the raw data) are
      • Km = 5x10 -9
      • Vm = 5x10 -15

JohnChattaway

Potential Issues

A potential issue with the experiment is regarding using pTet as the promoter for LuxR expression. It may be necessary to add a gene for TetR expression to ensure control over this promoter.

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