IGEM:IMPERIAL/2006/project/parts/BBa J37022/qualitative
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(Redirected from IGEM:IMPERIAL/2006/project/parts/BBa J37022/qualatatitive)
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