IGEM:The University of Sheffield/2009/Notebook/Sheffield iGEM 2010/2010/09/24

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Friday 24th September

  • Two experiments were set up to test the function of the BarA KO E.coli the contained the cholera system and HapR-GFP promoter in response to the cholera auto-inducer CAI-1:

1.) Simple test:

  • For a simple test of the cholera system based approach the BarA KO strain which contained both the cholera system and the HapR promoter(with GFP) was grown for 1 hour at 37°C in 5ml LB (containing Cm and Amp). Then 5μL of the auto-inducer CAI-1 was added and the E.coli was left to grow again for 2 hours at 37°C.
  • After the 2 hours the sample was centrifuged at 13,000rpm for 1min and excess LB was removed from the top of the sample to concentrate the sample.
  • The concentrated sample was staged on a slide and looked at under a fluorescent microscope which was successfully able to identify bacteria producing GFP.

2.) Characterisation:

  • An overnight flouresomter kinetic was set up for the co-transformed E.coli in a 96 well plate as shown bellow:
  Row 1 - LB control
  Row 2 - No CAI-1
  Row 3 - 0.5μL CAI-1
  Row 4 - 1.0μL CAI-1
  Row 5 - 5μL CAI-1
  Row 6 - 10μL CAI-1
  Row 7 - MG1655       



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