IGEM:IMPERIAL/2006/project/parts/BBa J37022/qualitative

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Contents

Aims

  • To show that the aiiA can degrade AHL

Outline

  • Culture <bbpart>J37022</bbpart> overnight with AHL
  • The next day mesure AHL content of the media.
  • Observe reduction in AHLconc as a result of the aiiA being produced by J37022
  • Controls
    • Un-modified DH5a
    • Blank media
    • No AHL (+ and - J37022 cells)

Protocal

Evening of Day 1

  • Make up all tubes to 2ml in BIG tubes according to this table
  • Culture (8ml) J37106 to test the AHL tomorrow
  • Those + AHL should be made to a final concentration of 1 micromolar AHL
  • Thos + IPTG should have 100ul of IPTG added

Image:Table.JPG

Day 2

  • Make fresh Day culture of J37016, OD = 0.1, (25ml)
    • Wait 2 hrs
  • Pipette 1.5ml of each culture into labeled eppindorphs.
    • Centrafuge down the cells for 3 min
  • Pippette 1.5ml of J37016 cells into 15 labeled eppendorfs
    • centrafuge down the cells for 3 min
  • Disgard J37016 supernatent
  • Transfer the supernatent from the test tubes into the now empty J37016 tubes
  • Resuspend the j37016 cells in the test supernatents
  • Incubate for four hours. in the 37[[:Category:{{{1}}}|{{{1}}}]] shaker
  • Pippette the J27016 cells into a 96 well plate and mesure with flourmeter
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