Specification, design and testing document for part j37016

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Contents

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SPECIFICATIONS

  • To measure the rate of enzyme production as a function of the amount of HSL present in the medium
  • To measure the Km and Vm of LuxR in our cells.

Total amount of HSL = Amount of HSL we add to the medium = Free HSL + HSL bound to LuxR


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Design

  • Kosak's laws were neglected in the design of this part so it needs to be re-designed

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MODDELLING

[:Model_J37016.JPG]

  • Input = AHL
  • Output = GFP

Values

[:Paremeter_table.JPG]

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Parameters

[:Perameter_table.JPG]

Key paremeter - Km of LuxR

This must be high. We can increace the apparant Km in real cells using recombinant plasmids.


Assume HSL is constant

[:Model_J37016_output.JPG]

As transcription at lux pR is proportional to the amount of LuxR+HSL which is dependent on the amount of HSL added the rate of GFP production will be proportional to the amount of HSL added.

If we know the Rate of GFP degradation and the equilibrium conc of GFP then we can work out the rate of GFP synthesis for that amount of HSL as degradation = synthesis at equilibrium.

If we know the rates and the substrate conc for those rates we can make a lineweaver burke plot. Plot 1/v against 1/[s].

1 / V = (Km / Vm)(1 / [S]) + (1 / Vm)

Y = M X + C

[:Lineweaver_Buke_Plot.JPG]

This will allow us to work out the real values of Km and Vm for LuxR+AHL when it binds LuxPr,:-D

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