IGEM:Cambridge/2008/Turing Pattern Formation/Experiments/Testing AHL degradation in B. subtilis

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Testing degradation of AHL by bacillus aiiA

  • 5 plates without selection with E.coli receiver overlay in soft agar. Center of plate spotted with:
    • B subtilis in LB and AHL in EA medium without incubation
    • B subtilis in EA medium only (control for competition between B.subtilis and E.coli)
    • AHL only
    • B.subtilis and AHL incubated for 90 minutes at room temperature
    • B.subtilis in overnight growth medium (test for effect of acidity on AHL) and AHL in EA.
  • Plates grown overnight
  • Result: inconclusive. All plates except plate 2 (no AHL) had similar levels of fluorescence. No fluorescence in plate 2. This may because our Bacillus strain (168) is a aiiA knockout, as no aiiA gene was found in the genome. However, at 5 hours after incubation, only plate 3 (no Bacillus) had detectable levels of fluoresence.

Testing different volumes of AHL

Our previous experiments were inconclusive because we believed that we possibly swamped our Bacillus with excess amounts of AHL so that no appreciable degradation was seen. In this experiment we tested and compared different concentrations of AHL.

Fluorescence with 1 uM AHL

  • Without Bacillus: 2457
  • With Bacillus: 2497

Fluorescence with 10nM AHL

  • Without Bacillus: 1424
  • With Bacillus: 1318

Fluorescence with 1nM AHL

  • not enough for good readings

Images of 1uM AHL plates with accompanying plot profiles

The graphs demonstrate fluorescence change across the plate from left to right.

AHL without Bacillus

Plot profile of AHL without Bacillus

AHL with Bacillus

Plot profile of AHL with Bacillus