Team:Chiba/protocol/phenotype/timedelay

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Contents

Time-delay check

Purpose

To Confirm that communication using non-endogenous molecules results in slower activation of receivers.

Equipments　and Materials

Equipment

  • shaking incubator(37°C,30°C)
    • Innova 4200 Benchtop or Floor-Stackable Incubator Shaker(37°C)
    • Taitec BioShaker BR-33FM(30°C,200rpm)
  • 46-well plate(deep well)
  • Fluoroskan Ascent 2.5(program:Ascent Software Version 2.6)
  • Beckman Allegratm X-12R Centrifuga(Beckman Coulter)

Materials

  • AHL(100μM)
  • E.coli Culture Containing BBa_T9002
  • E.coli Culture Containing plasmids you will testing
ex.)[http://partsregistry.org/Part:BBa_K084007 BBa_K084007]([http://partsregistry.org/Part:BBa_J04500 plac+rbs]+[http://partsregistry.org/wiki/index.php?title=Part:BBa_C0178 LasI(no LVA)])
[http://partsregistry.org/Part:BBa_K084008 BBa_K084008]([http://partsregistry.org/Part:BBa_J04500 plac+rbs]+[http://partsregistry.org/wiki/index.php?title=Part:BBa_C0170 RhlI(no LVA)])

Protocol

Culturing overnight (before the testing day):

  1. Inoculate all cultures you will testing from gloycerol stocks into 2 mL of LB-ampicillin liquid medium.
  2. Also inoculate a culture containing BBa_T9002 into 2 mL of LB-ampicillin liquid medium.
  3. Incubate all cultures with shaking at 37°C(O/N).

Following day

  • cultures containing BBa_T9002
  1. Inoculate a culture by adding 100 μL of the cultures into 40mL of LB-ampicillin medium.(in a flask).
  2. Incubate a culture for 6-8 hours with shaking at 37°C.
  3. Wash
    1. Aliquote 10mL of the culture into 50 mL four 50 mL falcon tubes.
    2. Cultures are centrifuged at 3500 rpm for 6 minutes.
    3. Dinspense supernatant.
    4. Add 10mL of new LB-ampicillin medium and resuspense with pipetting.
  4. aliquote 1mL of the cultures into a 48-deep well plate(deep well).
  • cultures containing plasmids you will testing
  1. Inoculate cultures by adding 12.5 μL of the cultures into 5 mL of LB-ampicillin medium.
  2. Incubate cultures for 6-8 hours with shaking at 37°C.
  3. Wash
    1. Cultures are centrifuged at 3500　rpm for 6 minutes.
    2. Dinspense supernatant.
    3. Add 3mL of new LB-ampicillin medium and resuspense with pipetting.
  4. repeat washing process twice.
  5. Cultures are centrifuged at 3500　rpm for 6 minutes.
  6. Dinspense supernatant.
  7. Add 5　mL of new LB-ampicillin medium and resuspense with pipetting.
  8. aliquote cultures into a 48-deep well plate(deep well).

Measurement

  • Mix senders culture and receiver culture(Prepare three replicate cultures).

ex.)sender culture:500μL,receiver culture:500μL

Positive control:receiver culture with 100μM AHL solution.
Negative control:receiver culture without sender culture and AHL solution.
  1. Incubate testing cultures with shaking at 37°C.
  2. After some intervals,aliqupte 100μL of testing cultures into a 96-well plate(shallow well).
  3. Measure fluorescence intensity.
  • conditions
    • shaking(before measurement):On time = 1min,Off time = 10 sec,
    • integration time = 1000 ms
    • Beam width:Normal Beam
    • Wavelength pair = 485 nm(excitation) and 527 nm(emission)

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