Team:Chiba/Project

From 2009.igem.org

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== '''Introduction''' ==
== '''Introduction''' ==
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===Implementing a "Timer" Function!===
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=== Implementing a "Timer" Function! ===
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Our project is to make a sort of "timer", where gene activation is triggered after a certain time.
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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.
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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.
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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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*Our project is to make a sort of "timer", where gene activation is triggered after a certain time.
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*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.
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*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.
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*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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Revision as of 08:18, 21 October 2009


E.coli Time Manager -Since 2008-
Logo chiba-u.gif


Home The Team Parts Reference Notebook Protocols Links Acknowledgements Contact

The Project

1, Introduction

2, Project Design

3, Experiments, Results & Discussion

-1, Making LuxR Mutants
-2, Characterization
-3, For improving pictures
-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.

  • ほんとに今のイントロでよいですか？わかりやすさだけを追求して書いたので深みはそんなにないです。質問や付け足したいことあれば言ってください--Maiko 23:45, 20 October 2009 (UTC)
  • タイトルでsince2008とうたっている以上、手術予定にあるように昨年度のこともつけたすべきたと考えます。--Yoshimi 03:17, 21 October 2009 (UTC)
  • あら？じゃあ全部採用しなくて良いのですが...^^;いつの間にか全採用ぽくなっていたので少し吃驚しました。あくまでも案です。でも解りやすいはずだとは思うので使うとこあれば使ってください。--Maiko 06:14, 21 October 2009 (UTC)

(手術予定)

  • 細胞通信を使ったBacterial Timer を創ります。
  • E. coli Timer完成予想図
  • Timerを創る方法は様々（diffusion of molecules, switching（oscillator, communication, arabinose, plac, etc... ））であるが、なぜ細胞通信を選んだのか。

そして、昨年は2段階の通信しか創れなかった。バリエーションが乏しい。

  • 作戦変更!（Sender側をかえたCrosstalk通信にはバリエーションを創るのに限界があるので、今年はReceiver側の調節をする。）目指すは時計の数字分の、１２通りの時間差をつくりたい！

Project Design

（手術予定）

2) Project Design

⑤Receiver側のAHL通信の仕組みを図説する。（1,AHLが入るところ（培地調節）　2,Receiver中のLuxR 3,Receiver中のReporter）


Team:Chiba/Project/Signaling-system

Experiments, Results & Discussion

LuxR mutantづくり

  • directed evolutionで色々な応答速度のLuxR mutantをつくることを目指した。

Experiments

  • 下の絵すこし汚いですね：jpgじゃない？pngとかgifで保存すればもっと絵が綺麗になるはず
Fig. Y Directed evolution to get some delayed-LuxR mutants
  • error-prone PCRでLuxRの変異ライブラリを作製し，発現ベクターに組み込んだ
  • これを，E.coli BW(?) harboring plux-gfpに形質転換し，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


  • genotypeをのせよう

（手術予定） ⑥Mutantを創る。（Error-proneが良い理由：バリエーションを創りやすい。新たなBiobrick作成方法としてError-prone PCRとスクリーニングをセットにしMutant-Partsの作り方を説明する。）

⑦さらに詳しい実験方法（Biobrickからerror-prone PCRをおこなったことなど）

⑧得られたデーター（なぜ8000のライブラリを、200に絞り、どうやって13sampleを選び出したのか説明）

得られたLuxR mutantのcharacterization

⑨LuxR Mutant 個性確定実験の実験方法

Experimental Method


⑩100 nM培地での応答の遅れについての結果



⑪ＡＨＬ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

⑫LuxR Mutantの個性と、変位が入っている部分からの考察



Demonstration

⑯E. coli Timer完成デモ 明日のよるにはあげられます。--Yoshimi 14:21, 20 October 2009 (UTC)

（⑰できればアニメ）

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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