Team:Chiba/Project

The Project

1. Introduction
2. Project Design
3. Experiments, Results & Discussion
   1. Making LuxR Mutants
   2. Characterization
   3. For improving pictures
   4. Demonstration
4. Conclusion

Introduction

It has been demanded in biological engineering that making the function that organisms sense the passage of time. We can utilize the excellent functions of organisms such as material synthesis or sensing as a device for drug delivery system or physical exam when these functions have been time-controlled as we like. However we can control cells individually, without method to control them all together, advantage of excellent functions may attenuate. Then we want to make the timer that cells works concurrently.

(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（画像作成中です--Yoshimi 09:12, 20 October 2009 (UTC)）

• 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

For making some timers without need of synchronizer system and that can be used in a single system we try to make following 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）

Signaling System

In this project, we use acylated homoserine lactones (AHLs), signaling molecules used for quorum sensing in gram negative bacteria. Senders express LuxI or similar enzymes, which catalyze the production of AHLs, under the control of a constitutive (Tet) promoter. Each cell thus generates AHL more or less at a constant rate. AHL can freely permeate cell membranes and are detected by neighboring cells. Receivers constitutively express LuxR proteins (or a similar ortholog), the protein that detects AHL concentrations. When AHLs bind LuxR proteins, the AHL-LuxR complex activates the Lux promoter. The threshold [AHL] at which switching occurs is determined by the affinity of AHL for the particulr LuxR ortholog. about quorum sensing)

Constructing A Delay Switch (Since 2008)

We are constructing delay switches to control/preset the timing of target gene expression. Our project uses two classes of bacteria: senders and receivers. Senders produce signaling molecules, and receivers are activated only after a particular concentration of this molecule is reached. The combinatorial use of senders/receivers allows us to make a‘switching consortium’which activates different genes at the preset times.

Constructing A Delay Switch, Multiple Ways

In principle, there are three ways to delay the activation of chemical communications;

1. Silencing the Speakers: Rate of signal accumulation down-regulated, for instance, by slowing down the signal generators.
2. Desensitize Receivers: Switching threshold elevated, for instance, by using insensitive receiver/ reporter systems.
3. Partial Blocking: Decreasing the by chewing the signal up.

Inter-species communications!

We decided to go for the strategy inspired by Japanese-classic experience; Whenever we speak to somebody in English, we often experience a certain delay in activating the communication. We though this is exactly what we pursued in. See Exp #4. Same applies to the reverse, too. When somebody speaks to us, we definitely need some time (sometime infinite) to get activated. This is all in spite that he/ she was loud and clear enough. The less affinity (perception) we have to English, the longer we need to activate them.See exp #5.

-> Get more information about a Delay Switch!

Experiments, Results & Discussion

LuxR mutantづくり

（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

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

より良い絵を描くために

⑬培地を振った実験について。実験方法と結果。

⑭Reporter毎の応答の差についての実験と結果。ＧＦＰuvがＤｅｌａｙを見やすい。

⑮Reporter毎の応答についての考察。

Demonstration

⑯E. coli Timer完成デモ

（⑰できればアニメ）

Conclusions

手術予定

18・Biobrickを使って（iGEM的には）新たな方法（error-prone PCR）を用い、新パーツをつくりました。

・何通り（ただいま確認中）のバリエーションの遅れを生み出せました。（Mutant+WT:4~5種、gel調節：2種、Reporters: 4通り・・・これらの組み合わせの分（タイミングがかぶるのは除く）だけ時間差をうみだせた！）

・demonstrationをおこないました。

⑲ Future Works : Whisper down the lane

時計というものに、時間だけでなく順番も入れられる。

例えば培地中に拡散するＡＨＬにムラがあったとしても、ちゃんと順番通りに動くシステム、

バリエーションは作れないが順番ならば得意。

成長するアニメーションを作るには、この順番通りに動くシステムが必要。

アニメを作るときは時間の刻み方が人にとって意味を持つようなものであることが大切。