Team:Cambridge/Signalling/Experiments/AHL test



Testing AHL sender and receiver

Testing AHL receiver (T9002)

  • Spot 10 μl 10μM AHL on receiver-soft agar overlay and test for fluorescece. Result: postive response.
  • Spot a range of AHL concentrations (10μM, 1μM, 10nM, 1nM)for a qualitative dose-response test. Result: can detect down to 1μM AHL using UV lamp, but response is poor.
  • The above test is re-run this time using fresh, log-phase receiver cells (2 hours incubation after inoculation) and a different set of AHL concentrations (10μM, 1μM, 100nM, 10nM). Detection much higher for 10μM and 1μM. Observable response to 10nM although signal-to-noise ratio is poor.

Testing AHL sender (I13202)

  • Sender grown overnight from plate in 2*10 ml LB. Receiver grown overnight from plate in 10 ml LB.
  • 3 Amp plates made with receiver-SA overlay (see above). In addition, the SA overlay of the 4th plate contains 1mM IPTG.
  • 10 ml of sender culture used for plates 1,2 and 4.
    • Plate 1: spot 10μL sender on the center of the plate.
    • Plate 2: Spot 10μL sender premixed with IPTG (final concentration of 1mM). Spot immediately after mixing.
    • Plate 4: Spot 10μL sender (plate contains IPTG)
  • 10 ml of sender culture pelleted and resuspended in LB containing 1mM IPTG. Grow for 2 hours.
    • Plate 3: Culture pelleted again and 10μL supernatant, which contains AHL, spotted onto plate.
  • Result
    • All plates, including plate 1, had high levels of fluorescence, detectable by a UV lamp. This confirms that the lacI+pL hybrid promoter (R0011) is constitutively on at a high level ( However, there is no evidence for higher fluorescence in the presence of IPTG despite claims that a 600 fold increase in expression should occur. This may be because the concentration of sender is too high.
    • The properties of lacI+pL, though interesting, is not of concern for our project, as we intend to use another Bacillus compatible promoter anyways.
    • This experiment has confirmed that the sender is definitely capable of secreting AHL, which is sufficient for this stage.
  • Future work:
    • A lower concentration of sender could be used either by dilution or lower growth times.
    • for better quantification, we could try a protocol similar to the one used by the NCBS 2007 team.