Team:Chiba/jk/γ/trs

From 2008.igem.org

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===Discussion===
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'''Discussion'''
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このプロジェクトでは、発現するまでの時間を変えることが目的である
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Discussion
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*X-galの濃度を高くすると発現を確認できるまでの時間は短くなったが、蛍光たんぱくで発現を確認できるまでの時間より遅い
 
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*X-galを基質とするβ-galでは、時間がたつごとに基質濃度が下がり、出力までの時間が長くなってしまうことも考えられる
 
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以上の2つの理由から蛍光タンパク質が適している
 
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またGFP,YFPの発現時間までのタイムラグが一番少なかった
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The purpose of this project is to alter the time required for expression.
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*目視で確認した際、GFPのほうがYFPよりも目視で確認するのが容易であった
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* By increasing the concentration of X-gal, the time required for
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以上の理由から、出力はGFPとする。
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expression to be observable decreased, but this still takes longer
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than the time required when using fluorescent proteins.
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*Using β-gal, which uses X-gal as substrate, the time required for
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output may increase because the substrate concentration decreases with
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time.
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For the above reasons, fluorescence proteins are more suited for our purposes.
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Of the fluorescent proteins, GFP and YFP fluorescence was observable
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at earlier stages.
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* But to the naked eye, GFP could be observed more easily.
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 +
Therefore, we chose GFP as our output reporter.

Revision as of 04:40, 30 October 2008

Contents

Time Responce Liquid

Reporter

  • Fluorescent Protein
  • GFP
  • pGFPuv
BBa_T9002
  • Venus YFP
  • BBa_K084003
  • pLac-Venus YFP
  • mCherry

pLac-mCherry

  • β-gal (X-gal assay)
  • pUC19(plac-LacZα)

Equipment

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


Method

Fig. レポーターのタイムレスポンスの実験方法

Strain:XL10G KanR

  • Agar plate experiment
  1. Pre-culture
    1. Picked and cultured the following plate stocks 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. Spread on plate
    1. Spread on new plate
      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.
  3. Colony lift
    1. Colony lift to inducible agar plate (containing IPTG or AHL)
      1. LB-Amp+0.2 mM IPTG agar plate, (pGFPuv, pLac-Venus YFP, pLac-mCherry, pUC19)
      2. LB-Amp+100 nM AHL agar plate, (BBa_T9002, BBa_K084003)
    2. Incubate at 37 °C
  4. Check expression every 30 min.


Result


Discussion Discussion


The purpose of this project is to alter the time required for expression.

  • By increasing the concentration of X-gal, the time required for

expression to be observable decreased, but this still takes longer than the time required when using fluorescent proteins.


  • Using β-gal, which uses X-gal as substrate, the time required for

output may increase because the substrate concentration decreases with time.

For the above reasons, fluorescence proteins are more suited for our purposes.

Of the fluorescent proteins, GFP and YFP fluorescence was observable at earlier stages.


  • But to the naked eye, GFP could be observed more easily.

Therefore, we chose GFP as our output reporter.




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