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

Aims

 * To show that the aiiA can degrade AHL

Outline

 * Culture J37022 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)

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 shaker
 * Pippette the J27016 cells into a 96 well plate and mesure with flourmeter