Team:Chiba/protocol/phenotype/timedelay/j

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Contents

Time-delay check(冨永)

目的 Purpose

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

装置 and 試薬 Equipments　and Materials

装置 Equipment

  • shaking incubator(37°C,30°C)
    • Innova 4200 Benchtop or Floor-Stackable Incubator Shaker(37°C)
    • タイテック 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)

試薬 Materials

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

プロトコル Protocol

プレ培（O/N,測定日の前日）,Culturing overnight (before the testing day):

  • グリセロールストックからpickして、37℃しんとう培養器で一晩培養する(LB-Amp液体培地,2mL,37°C )。
  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).

翌日,Following day

日本語

  • T9002
  1. 培養液100μLを、40mlのLB-Amp液体培地に加え、37℃で6-8時間しんとう培養する。
  2. Wash(T9002)-->培養液を50mlファルコンチューブに、10mlずつ分注。
-->3500rpmで6分間遠心。上澄みを捨てる。
  1. 新しいLB-Amp培地を10mlずつ加え（1倍希釈）、pipettingにより再懸濁。
  2. 48　deep well plateに、1mlずつ分注。

English:

  • 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).

日本語

  • cultures containing plasmids you will testing
  1. 培養液12.5μLを、5mlのLB-Amp液体培地に加え、37°Cで6-8時間しんとう培養する。
  2. Wash-->3500rpmで6分間遠心。上澄みを捨てる。
-->新しいLB-Amp培地を3mlずつ加え、pipettingにより再懸濁。

-->この操作を二回繰り返す

  1. 新しいLB-Amp培地を10mlずつ加え（1倍希釈）、pipettingにより再懸濁。
  2. 48　deep well plateに、所定量分注。


English

  • 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).

測定,Measurement

日本語:

  1. 37°Cでしんとう培養
  2. 96 shallow well plateに100μLずつ分注し、蛍光強度を測定する。
  3. 2h後、3h後…に、100μLずつ分注し、蛍光強度を測定する。
  • 測定条件
測定前-->shake:On time = 1min,Off time = 10 sec,
測定-->integration time = 1000ms
Beam width:Normal Beam
Wavelength pair = 485nm(excitation) and 527nm(emission)

English:

  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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