Team:Chiba/Project

From 2009.igem.org

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(→Characterization of LuxR Mutants)
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=== Characterization of LuxR Mutants ===
=== Characterization of LuxR Mutants ===
[[Image:Chiba-LuxRs characterization.png|frame|right|Fig. Characterization of LuxR Mutants]]
[[Image:Chiba-LuxRs characterization.png|frame|right|Fig. Characterization of LuxR Mutants]]
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==== Experiments ====
==== Experiments ====
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#AHLの濃度を0, 1, 10, 100, 1000 nMでふった固体培地に、NCフィルタを移動。
#AHLの濃度を0, 1, 10, 100, 1000 nMでふった固体培地に、NCフィルタを移動。
#この時を測定開始点とし、30分毎に4時間蛍光の上がり方を目視で確認。
#この時を測定開始点とし、30分毎に4時間蛍光の上がり方を目視で確認。
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*フナコシのUVGL-58のlong waveは365 nm
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*UVの波長かく！--[[User:Yoshimi|Yoshimi]] 08:57, 21 October 2009 (UTC)
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*フナコシのUVGL-58のlong waveは365 nmです--[[User:Masahiro|Masahiro]] 14:18, 21 October 2009 (UTC)
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===== 100 nM培地での応答の遅れについての結果 =====
===== 100 nM培地での応答の遅れについての結果 =====
[[Image:Chiba-Mutants4.png|frame|Fig.時間依存]]
[[Image:Chiba-Mutants4.png|frame|Fig.時間依存]]
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===== ＡＨＬ濃度を振った場合の、６ｈ後の蛍光強度の差についての結果=====
===== ＡＨＬ濃度を振った場合の、６ｈ後の蛍光強度の差についての結果=====
[[Image:Chiba-Mutants3.png|frame|center|Fig.濃度依存]]
[[Image:Chiba-Mutants3.png|frame|center|Fig.濃度依存]]
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==== Results and Discuss ====
==== Results and Discuss ====
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**AHL応答感度が低いもの
**AHL応答感度が低いもの
**AHL応答速度のおそいもの
**AHL応答速度のおそいもの
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=== Demonstration ===
=== Demonstration ===

Revision as of 20:00, 21 October 2009

E.coli Time Manager


Home The Team Parts Reference Notebook Protocols Links Acknowledgements Contact

The Project

1, Introduction

2, Project Design

3, Experiments, Results & Discussion

3-1, Making LuxR Mutants

3-2, Characterization

3-3, For improving pictures

3-4, Demonstration

4, Conclusions


Introduction

Implementing a "Timer" Function!

  • Our project is to make a sort of "timer", where gene activation is triggered after a certain time.
  • Our approach to this goal is to create a series of transcription factors (TFs) that can be activated by the same inducer molecule but with a different sensitivity. In an environment where the inducer concentration gradually levels up, these TFs switches on one by one according to the order of sensitivity.
  • We believe such collection of such TF variants would be useful for the timing control in biological function at will, and thereby contribute to the synthetic biology & iGEM community.
  • Using a set of TF variants, we aimed to draw an "animated picture": a picture that pop up one by one.

Project Design

Fig. LuxRの機能について:(1)LuxRの生産, (2)LuxRとAHL分子の結合, (3)二量化 , (4)DNAとの結合

  • 私たちはタイマーを創るためにLuxRを利用する。
  • LuxRはcell-cellcommunicationで使われるタンパクである。
  • LuxRは細胞内で右図にしめすとおりに働く。
  • 私たちはLuxRに変異を入れることで、スイッチングのdelayを数種類入れられると考えた。

Experiments, Results & Discussion

Making delayed-LuxR mutants

Experiments

Fig. Y Directed evolution to get some delayed-LuxR mutants
  • error-prone PCRでLuxRの変異ライブラリ(ライブラリサイズ=8000)を作製し，発現ベクターに組み込んだ
  • これを，E.coli BW1226 harboring plux-gfpに形質転換し，200のコロニーを形成させた。
  • コロニーをニトロセルロース膜でリフトし，AHL入りのプレートにのせ，gfpの蛍光の経時変化をみた（Fig. X)。
  • 蛍光しはじめるのが遅いものを13個pickし，delayed-LuxR変異体を得た。
  • pickした13個のクローンの転写活性化速度を，再度transformして確認し，最終的に○個の速度バリエーションのluxR変異体を得た。
Fig. X LuxRライブラリによるのPlux-gfp蛍光経時変化


  • 変異が入った場所は以下の通り。
Fig.
  • リガンド（AHL）結合ドメイン／DNA結合ドメイン、どちらに変異が入っても、Delay変異体の表現型を与えた。

Characterization of LuxR Mutants

Fig. Characterization of LuxR Mutants


Experiments

  1. LuxR WT, Mutant（13種）とpLux-GFPのプラスミドが2つトランスフォーメーションされたJW1226株を12h@37℃液体培養。
  2. Cultureを48ピンでNCフィルタへ植菌し、フィルタを固体培地にのせて12h@37℃で培養。
  3. AHLの濃度を0, 1, 10, 100, 1000 nMでふった固体培地に、NCフィルタを移動。
  4. この時を測定開始点とし、30分毎に4時間蛍光の上がり方を目視で確認。
  • フナコシのUVGL-58のlong waveは365 nm


100 nM培地での応答の遅れについての結果
Fig.時間依存


ＡＨＬ濃度を振った場合の、６ｈ後の蛍光強度の差についての結果
Fig.濃度依存


Results and Discuss

  • Delay変異体には大きく分けて以下の２通りのタイプがみられた。
    • AHL応答感度が低いもの
    • AHL応答速度のおそいもの


Demonstration

⑯E. coli Timer完成デモ

  • 写真あげました。--Kaoru
  • どうがつくります・・--Yoshimi 16:17, 21 October 2009 (UTC)
  • この時計細胞分裂しそう・・・余力が有ればなおします。--Yoshimi 18:48, 21 October 2009 (UTC)



T=0

T=130 min

T=150 min

T=180 min

T=230 min

T=260 min

T=300 min

T=360 min


For the determination of best condition for E coli painting, Click here!

Conclusions

  • By using error-prone PCR, we have created a LuxR library.
  • With a simple and convenient screening method, we have isolated various LuxR mutants which confer delayed switching behavior in GFP signals.
  • By introducing these LuxR variants together with reporter genes (such as GFP) under the control of Lux promoter, we created bacteria 'ink's that develop their color with unique delay-time.
  • We conducted painting with these bacteria inks thereby created animated pictures.
  • Characterization of LuxR mutants is ongoing. But our preliminary data showed that some of the variants turned out to be the one with less sensitivity to AHLs, and others seemed to be as sensitive as wild-type LuxR but seemed less efficient somewhere in the downstream process.
  • We created variety of Biobricks during the course of this projects. Some of them are characterized and sent to the HQ, and many more are almost ready for shipping!
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