Team:Chiba/protocol/phenotype/timedelay

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Contents

Time-delay check(冨永)

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(100uM)
  • E.coli Culture Containing T9002
  • E.coli Culture Containing plasmids you will testing

Protocol

Culturing overnight (before the testing day):

  1. Inoculate all cultures you will testing from gloycerol stocks into 2mL of LB-ampicillin liquid medium.
  2. Also inoculate a culture containing T9002 into 2mL of LB-ampicillin liquid medium.
  3. Incubate all cultures with shaking at 37°C(O/N).

Following day

  • cultures containing T9002
  1. Inoculate a culture by adding 100μL 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 50mL four 50mL falcon tubes.
    2. Cultures are centrifuged at 3500rpm 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 12.5μL of the cultures into 5mL of LB-ampicillin medium.
  2. Incubate cultures for 6-8 hours with shaking at 37°C.
  3. Wash
    1. Cultures are centrifuged at 3500rpm 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 3500rpm for 6 minutes.
  6. Dinspense supernatant.
  7. Add 5mL of new LB-ampicillin medium and resuspense with pipetting.
  8. aliquote cultures into a 48-deep well plate(deep well).

Measurement

  • Mix senders and receiver as shown in Table below:
Senders culture(μL)T9002 culture(μL)Receiver cells/Sender cellsAHL(100nM)
5005001-
100100010-
101000100-
-1000-1
-1000--


  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 = 1000ms
    • Beam width:Normal Beam
    • Wavelength pair = 485nm(excitation) and 527nm(emission)

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