IGEM:Cambridge/2008/Turing Pattern Formation/Experiments

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Finding and Biobricking proper Ribosomal Binding Sites

• Solving Biobrick RBS issues

Testing Sender and Reciever

• We can test one, then use to test the other, or we can test them in parallel

Testing AgrC Reciever

• Use purified AIP
  • Synthesized
  • Sourced
    • Possibly from Tom Muir at Rochester
  • S. aureus purified supernatant (requires Pathology department personnel)
    • Guangyong Ji at Catholic University has a protocol for purification

Testing AgrA Transcription Factor and P2 promoter

• Can we get an 'always on' mutant my modifying some residues? (Jim's Idea)

Testing AgrB Exporter

• Membrane Localization Test
  • Can use membrane fractionation
  • Can use antibody tags with EM and gold

Testing AgrD Prepeptide/AIP

• Mass Spec Assay
  • Daniel Goodman 11:47, 31 July 2008 (UTC):Speaking to Kathryn Lilley in Proteomics
• S. aureus Reporter Strain
  • Daniel Goodman 11:47, 31 July 2008 (UTC):We cannot use staph ourselves, checking to see if we can get someone in Pathology department to test for us

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 (http://partsregistry.org/wiki/index.php?title=Part:BBa_R0011). 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.

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

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