IGEM:IMPERIAL/2006/Protocols/S01656new

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Contents

New Protocol to test aiiA Production

Outline

  • 9am - Make fresh day culture
  • 11am - Add known AHL concs + IPTG
  • Wait 7 hrs
  • 6pm
    • 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 fluoresce.
  • This can be easily adapted to measure 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 different properties to the aiiA we will actually be using This test is solely to see if this gene works.

Controlls

  • -IPTG
  • Non aiia producing cells (gives idea of background breakdown of AHL in medium containing live cells)

Detailed Protocol

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

Protocol

  • Grow cultures of S01656 (4ml) and B0034 (2ml) overnight JS: What is B0034 used for? Isn't B0034 just an RBS?
  • 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 Pre-warmed 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 different concentrations) Use the AHL dilution series below Do we not need to dilute down to OD of 0.1 again to prevent going into the exponential phase?


Sample (ul) Stock Concentration AHL (ul) Final AHL Concentration
990 1000uM 10 10uM
990 100uM

P|10

1uM
990 10uM 10 100nM
990 5uM 10 50nM
990 1uM 10 10nM
990 500nM 10 5nM
990 100nM 10 1nM


  • 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 different AHL concentrations
  • Add 50ul of IPTG to the + IPTG group of S01656 to induce aiiA production
  • Wait for 7 hours JS: what is the justification for keeping at 7 hours? I know that we were doing the experiment at 4 hours, but what is to say that 7 hours will work? Have we gotten any results from this experiment?
  • centrifuge down cells
  • Pipette off supernatant and freeze
  • Tomorrow use T9002 Protocol to measure amount of AHL

Any questions see JohnC

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