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

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

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