Team:Chiba/jk/γ/Trl

From 2008.igem.org

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*PBS
*PBS
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===Protocol===
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===Method===
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*pre培養(Day,1:pre culture)
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[[Image:Reporter method Chiba.jpg|rifht|thumb|'''Fig. '''レポーターのタイムレスポンスの実験方法]]
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:*LB培養液(2ml)に固体培地から大腸菌をpickし３７℃で12時間培養(48穴Deep Well)
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Strain:XL10G Kan<sup>R</sup>
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:*pick colony(E.coli) from solid culture medium
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:*into LB culture fluid(2 ml)
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*Liquid medium experiment
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:*cultivate for 12hours in shaking incubator(37℃)
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#Pre-culture
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*本培(Day,2:main culture)
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##Picked and cultured the following plate in 2mL of LB:
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:*measure OD values and dilution with LB culture fluid to 0.01(OD values)
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###LB-Amp+0.2 % Glucose, (pGFPuv, pLac-Venus YFP, pLac-mCherry, pUC19)
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:*cultivate to 1.0(OD values)
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###LB-Amp, ([http://partsregistry.org/Part:BBa_T9002 BBa_T9002], [http://partsregistry.org/Part:BBa_K084003 BBa_K084003])
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:*wash with physiological saline (2.0min 8,000rpm)x2
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##Cultured at 37°C for 12h.
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:*culture in M9 or PBS and add IPTG or AHL
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#Culture
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:*measure OD values ,fluorecent intensity
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##Dilute pre-cultures and add to new LB medium.
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:*judge whether we can see the color
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###LB-Amp+0.2 % Glucose, (pGFPuv, pLac-Venus YFP, pLac-mCherry, pUC19)
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###LB-Amp, ([http://partsregistry.org/Part:BBa_T9002 BBa_T9002], [http://partsregistry.org/Part:BBa_K084003 BBa_K084003])
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##Cultured at 37°C for  about 6 h
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#Wash
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##Transfer 10mL each of the culture to 50mL centrifuge tubes.
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##Centrifuged for 6min at 3600rpm,20°C and discarded the supernatant.
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##Add physiological saline and resuspention
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##Repeated wash twice.
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##Add M9 minimal medium.
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#Mix
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##Dispens each culture into 48-well deep well or 96-well deep well
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##Add IPTG fainal conc. 0.2 mM (pGFPuv, pLac-Venus YFP, pLac-mCherry, pUC19)
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##Add AHL fainal conc. 100nM ([http://partsregistry.org/Part:BBa_T9002 BBa_T9002], [http://partsregistry.org/Part:BBa_K084003 BBa_K084003])
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#Measure fluorescence intensity every 1 h.
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 +
 
 +
 
 +
 
 +
 
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:ODを計測し1/200に希釈しOD=0.01から1.0になるまで培養
 
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:OD=1.0になったら生理食塩水でwash(2.0min 8,000rpm)x2
 
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:M9またはPBSで培養し、IPTG,AHLを加え計測する。
 
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*計測(measurements)
 
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37°Cでしんとう培養 <br>
 
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96 shallow well plateに100μLずつ分注し、蛍光強度を測定する。<br>
 
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2h後、3h後…に、100μLずつ分注し、蛍光強度を測定する。 <br>
 
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Beam width:Normal Beam <br>
 
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Wavelength pair = 485nm(excitation) and 527nm(emission) <br>
 
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Incubate testing cultures with shaking at 37°C. <br>
 
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After some intervals,aliqupte 100μL of testing cultures into a 96-well plate(shallow well).<br>
 
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Measure fluorescence intensity. <br>
 
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conditions <br>
 
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Beam width:Normal Beam<br>
 
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Wavelength pair = 485nm(excitation) and 527nm(emission)<br>
 
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Retrieved from "https://2008.igem.org/Team:Chiba/protocol/phenotype/timedelay/j"
 
<br>'''Result'''
<br>'''Result'''

Revision as of 23:51, 29 October 2008

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, (BBa_T9002, 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, (BBa_T9002, 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 (BBa_T9002, 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
Retrieved from "http://2008.igem.org/Team:Chiba/jk/%CE%B3/Trl"