Team:Chiba/jk/γ/Trl

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実験ログ
出力班


Contents

Time Responce Liquid

purpose

インダクションをかけてからいち早く、確認できる出力を見つけること
To find the earliest output gene after induction

装置&試薬(apparatus&reagents)

装置（apparatus)

  • しんとう培養器(shaking incubator)(37℃,30℃)
  • 48 well plate(deep well)
  • Fluoroskan Ascent 2.5(program:Ascent Software Version 2.6)

試薬(reagents)

  • AHL(100uM,5uM,100nM)
  • IPTG(100nM)
  • X-gal
  • M9
  • PBS

Method

Fig. レポーターのタイムレスポンスの実験方法

Strain:XL10G KanR

  • Liquid medium experiment
  1. Pre-culture
    1. Picked and cultured the following plate in 2mL of LB:
      1. LB-Amp+0.2 % Glucose, (pGFPuv, pLac-Venus YFP, pLac-mCherry, pUC19)
      2. LB-Amp, ([http://partsregistry.org/Part:BBa_T9002 BBa_T9002], [http://partsregistry.org/Part:BBa_K084003 BBa_K084003])
    2. Cultured at 37°C for 12h.
  2. Culture
    1. Dilute pre-cultures and add to new LB medium.
      1. LB-Amp+0.2 % Glucose, (pGFPuv, pLac-Venus YFP, pLac-mCherry, pUC19)
      2. LB-Amp, ([http://partsregistry.org/Part:BBa_T9002 BBa_T9002], [http://partsregistry.org/Part:BBa_K084003 BBa_K084003])
    2. Cultured at 37°C for about 6 h
  3. Wash
    1. Transfer 10mL each of the culture to 50mL centrifuge tubes.
    2. Centrifuged for 6min at 3600rpm,20°C and discarded the supernatant.
    3. Add physiological saline and resuspention
    4. Repeated wash twice.
    5. Add M9 minimal medium.
  4. Mix
    1. Dispens each culture into 48-well deep well or 96-well deep well
    2. Add IPTG fainal conc. 0.2 mM (pGFPuv, pLac-Venus YFP, pLac-mCherry, pUC19)
    3. Add AHL fainal conc. 100nM ([http://partsregistry.org/Part:BBa_T9002 BBa_T9002], [http://partsregistry.org/Part:BBa_K084003 BBa_K084003])
  5. Measure fluorescence intensity every 1 h.






Result

ホーム メンバー紹介 プロジェクト紹介 Parts Submitted to the Registry モデリング ノート
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