E colock

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[[Image:Ecolock Model.jpg|right|450px]]
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= '''E colock'''    ''"self-cycling-E. coli"'' =
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== '''E colock'''    ''"self-cycling-E. coli"'' ==
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[["E colock" Chiba 2006|'''⇒Go to "E colock" English page??'''<br>wikiページ(英語)]]
[["E colock" Chiba 2006|'''⇒Go to "E colock" English page??'''<br>wikiページ(英語)]]
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= Concept =
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[[Image:Ecolock model.jpg|left|330px]]
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== '''メンバー''' ==
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'''Installing Kai system,<br>E.coli starts oscillating!'''
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= Member =
[[Chiba University, Japan 2006|Chiba 2006 wikiページ(英語)]] <br>
[[Chiba University, Japan 2006|Chiba 2006 wikiページ(英語)]] <br>
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*[[User:Matsuno|松野 伸哉]]  
*[[User:Matsuno|松野 伸哉]]  
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== '''コンセプト''' ==
 
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シアノバクテリアのもつ、生物時計システムKaiを大腸菌に導入し、周期的な動作をさせる。
 
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== '''原理''' ==
 
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[[Image:E colock How it works.jpg|right|550px]]
 
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'''Kaiシステムの導入'''
 
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Kaiシステムは最も単純で分子レベルでの解析が進んでいる、24時間周期の生体時計システムである。<br>
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= Kai system =
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Kaiシステムは3つのタンパク質KaiA、KaiB、KaiCの行うリン酸化サイクルであり、KaiCとリン酸化KaiCの量により周期が発生する。<br>
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[[Image:Kai system.jpg|right|400px]]<br />
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この振動は特定部位の転写を活性化するセンサーキナーゼSasAにより伝えられる。<br>
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Kai system is generated by 3 proteins(KaiA,KaiB, and KaiC).<br />
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Kaiシステムのサイクルを蛍光たんぱく質の発現量により観測する。<br>
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This is the most simplest circardian oscillation system.<br />
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But,it is not known how this oscillation regulate global expression of many proteins in cyanobacteria.
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<br />
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SasA is a Sensory Kinase(a kind of Histidine Kinase).<br />
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SasA is believed to connect with Kai cycle and transcriptional oscillation of many proteins.<br />
 +
RpaA is coganate response regulator for SasA, and it's deletion results in the complete loss of transcription oscillation.<br />
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This is all known about Kai.
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'''reference '''
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:*
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:*
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[[image:Cyanobavteria.JPG|right|210px]]
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= E.colock Project =
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SasA and RpaA is highly homologous to EnvZ and OmpR,respectively.
 +
 
 +
Believing that SasA is communicating with Kai oscillator.
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 +
Believing that RpaA communicate with SasA.
 +
 
 +
We designed RpaA-OmpR chimera,in hope that we read our Kai oscillation using ''ompC'' promoter.
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[[Image:Ecolock.jpg|right|400px]]<br />
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= Chimera design =
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In general, Response Regulator has 2 domeins,
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 +
regulator domein receives phospate from Hiatidine Kinase and effector domein bind to specific sequence of DNA.
 +
 
 +
We create 36 chimera of RpaA-OmpR.
 +
 
 +
There are different in cutting point, but all of them have recerver domein of RpaA to communicate
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SasA,and DNA-binding domein of OmpR to bind and activete right to ''OmpC'' promoter.
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 +
 
 +
The precedure(↓)
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We used 6 excite primers on each RpaA and OmpR,
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It can make 6 ×6 = 36 different chimeras!!
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[[Image:Excite PCR.JPG|600px]]
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== '''問題点''' ==
 
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'''1.Kaiオペロンの導入'''
 
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Kai遺伝子は導入が可能だが、シアノバクテリア由来なので大腸菌内でKaiシステムが機能するだろうか?
 
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'''2.同調機能の欠如'''
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= Next step (*_*)/ =
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シアノバクテリアは光合成により、その周期をリセットしている。これにより全体の振動が同調している。<br>
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The construct of chimeras is finished!
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しかし、このリセットを行っている遺伝子は未だ同定されていないので、大腸菌の振動がバラバラになることが予想される。<br>
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Chimaras along with WT RpaA and OmpR will be tested in their function as response regulator that switch ON/OFF the ''ompC''promoter.
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→→①LacリプレッサーをKaiAプロモーター、KaiBCプロモーターの下流に置き、Kaiの振動を止める。<br>
 
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これにより全体が同調する。
 
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→→②AIが使える??
 
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[[Image:Jikkensitai.JPG|600px]]
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== '''オプション''' ==
 
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'''周期の制御'''
 
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Kaiタンパク質の変異株はその周期が様々であることが報告されている。<br>
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We will find some chimeras indeed pick up Kai cycle.
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同様に変異株を作成し、任意の周期を持った“E clock”を作成したい。
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== '''実験計画''' ==
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= Last up date  =
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[[『mu』team activities|'''活動報告:8月''']]<br>
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*11/15

Revision as of 13:34, 15 November 2006

Contents

E colock "self-cycling-E. coli"

⇒Go to "E colock" English page??
wikiページ(英語)

Concept

Ecolock model.jpg


Installing Kai system,
E.coli starts oscillating!

Member

Chiba 2006 wikiページ(英語)
[http://chem.tf.chiba-u.jp/igem/ iGEM Chiba オフィシャルページ(日本語)]

『む』チーム



Kai system

Kai system.jpg

Kai system is generated by 3 proteins(KaiA,KaiB, and KaiC).
This is the most simplest circardian oscillation system.
But,it is not known how this oscillation regulate global expression of many proteins in cyanobacteria.
SasA is a Sensory Kinase(a kind of Histidine Kinase).
SasA is believed to connect with Kai cycle and transcriptional oscillation of many proteins.
RpaA is coganate response regulator for SasA, and it's deletion results in the complete loss of transcription oscillation.
This is all known about Kai.



reference


Cyanobavteria.JPG






E.colock Project

SasA and RpaA is highly homologous to EnvZ and OmpR,respectively.

Believing that SasA is communicating with Kai oscillator.

Believing that RpaA communicate with SasA.

We designed RpaA-OmpR chimera,in hope that we read our Kai oscillation using ompC promoter.

Ecolock.jpg







Chimera design

In general, Response Regulator has 2 domeins,

regulator domein receives phospate from Hiatidine Kinase and effector domein bind to specific sequence of DNA.

We create 36 chimera of RpaA-OmpR.

There are different in cutting point, but all of them have recerver domein of RpaA to communicate SasA,and DNA-binding domein of OmpR to bind and activete right to OmpC promoter.


The precedure(↓)

We used 6 excite primers on each RpaA and OmpR, It can make 6 ×6 = 36 different chimeras!! Excite PCR.JPG



Next step (*_*)/

The construct of chimeras is finished! Chimaras along with WT RpaA and OmpR will be tested in their function as response regulator that switch ON/OFF the ompCpromoter.


Jikkensitai.JPG


We will find some chimeras indeed pick up Kai cycle.


Last up date

  • 11/15
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