IGEM:IMPERIAL/2007/Projects/Hrp System/Testing/Protocol

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General Protocol

Prepare Cell Cultures

  • Devices are to be constructed in week 6 and transformed into the relevant strains.
  • Remove stored cultures and incubate over night at 37oC with selectable solution and shake. This is to allow amplification of the culture. Grown Overnight


  • Remove cultures and add to a new medium that contains relevant selectable solution (e.g. amplicillin). This allows cells to enter exponential growth phase.
  • This new medium is inncolulated to an O.D.600 of 0.1. This medium is grown for 2 hours.


  • After 2 hours this sample is then used to inoculate a new culture of medium, again to an O.D.600 of 0.1.
  • The result are colonies of a set optical density. In addition the cells are vortexed and the supernaunt removed, this is to help limit any diffusional constraints on addition of the inducer to the relevant promoter.


Fluorometer

~2-4 hours

  • The sample can be loaded into wells (200ul)
  • To this then the specific concentration of inducer is added.
  • Two controls are used, one for the growth medium and one for the fluorescence calibration curve.
  • At set time intervals the absorbance and the fluorescence is measured. Later this will be converted into GFP synthesis cfu -1 sec -1 using the calibration curves.
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