IGEM:IMPERIAL/2006/Protocols/S01656new
From OpenWetWare
New Protocal to test aiiA Production
Outline
- 9am - Make fresh day culture
- 11am - Add known AHL concs + IPTG
- Wait 7 hrs
- 6pm
- Test Flourescence
- Spin down cells + collect supernatant
- The Next Day - Test frozen supernatant for AHL with T9002
- This will show you that aiiA is being produced and that the cells flouresce.
- This can be easily adapted to mesure AHL conc every hour so that you get a curve. however we will not need to do this for this aiiA part as the aiiA gene is untagged and may have diferent properties to the aiiA we will actually be using This test is solely to see if this gene works.
Detailed Protocal
Equipment/Materials
- Falcon tubes or similar
- Photospectrometer
- Gilson pipettes
- Eppendorf tubes
- Centrifuge
- Plain LB medium
- LB medium with 50 μg/mL Kanamycin
- LB medium with 50 μg/mL Ampicillin
- E. coli DH5a strain with part S01656
- E. coli DH5a strain with part T9002
- Perkin Elmer Victor 3 Fluorimeter
- 96 well plate
Protocal
- Grow cultures of S01656 (4ml) and B0034 (2ml) overnight
- Make Fresh Day Cultures (10ml) - Make 2 S01656 cultures and one B0034 culture they should have an optical density of 0.1 and you should use Prewarmed media to prevent shock. Remember that the S01656 grows in kanomycin media.
- You Should now have three cultures
- Wait for 2 hours
- Add AHL to eppindorf tubes(seven diferent concentrations) Use the AHL dilution series below
|
- You should add 10ul of AHL to each eppindorf tube and 0.99ml of culture to each eppindorf in such a way that you have 21 tubes and each cell type has 7 tubes of diferent AHL concentrations
- Add 50ul of IPTG to the + IPTG group of S01656 to induce aiiA production
- Wait for 7 hours