Team:Chiba/jk/γ/Trl

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Time Responce Liquid

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

shaking incubator

Innova 4200 Benchtop or Floor-Stackable Incubator Shaker(37°C)

46-well plate(deep well)

96-well plate(deep well)

Fluoroskan Ascent 2.5(program:Ascent Software Version 2.6)

Beckman Allegratm X-12R Centrifuga(Beckman Coulter)

試薬(reagents)

Fig. レポーターのタイムレスポンスの実験方法

Strain:XL10G Kan^R

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.
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
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.
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])
Measure fluorescence intensity every 1 h.

Result

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