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.


~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.