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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.
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Project Design
- 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
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
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
- 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。
- 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。
- 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。
- 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・・・荒すぎ。余力が有ればなおします。--Yoshimi 18:48, 21 October 2009 (UTC)
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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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