"E colock" Chiba 2006

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[[Image:Hyousi.jpg|650px]]
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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|'''⇒Go to "E colock" Japanese page??'''<br>"E colock"日本語ページ]]
[[E colock|'''⇒Go to "E colock" Japanese page??'''<br>"E colock"日本語ページ]]
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= Concept =
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[[Image:Ecolock model.jpg|left|330px]]
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== Member ==
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'''Installing Kai system,<br>E.coli starts oscillating!'''
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[[Chiba University, Japan 2006|Chiba 2006]]  
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= Member =
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[[http://chem.tf.chiba-u.jp/igem/ iGEM Chiba]]
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[[Chiba University, Japan 2006|Chiba 2006]] &
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[http://chem.tf.chiba-u.jp/igem/|iGEM Chiba official site (Japanese)]
'''『mu』team'''
'''『mu』team'''
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*[[User:Katabami|Akinori Katabami]]
*[[User:Katabami|Akinori Katabami]]
*[[User:Sugaya|Sari Sugaya]]  
*[[User:Sugaya|Sari Sugaya]]  
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*[[User:Matsuno|Shinya Matsuno]]  
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*[[User:Matsuno|Shinya Matsuno]]
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= Kai system =
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[[Image:Kai system.jpg|right|400px]]<br />
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Kai system is generated by 3 proteins(KaiA,KaiB, and KaiC).<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 />
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RpaA is cognate 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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:*SasA : [http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WSN-4195FK3-10&_coverDate=04%2F14%2F2000&_alid=435215541&_rdoc=1&_fmt=&_orig=search&_qd=1&_cdi=7051&_sort=d&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=e171c9aaba0717231fe1d4c3400fcff0| H.Iwasaki'' et al.''  Cell, Vol 101, 223-233, 2000]
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:*RpaA : [http://www.pnas.org/cgi/content/abstract/103/32/12109?maxtoshow=&HITS=10&hits=10&RESULTFORMAT=&fulltext=RpaA&searchid=1&FIRSTINDEX=0&resourcetype=HWCIT| N.Takai ''et al.'' PNAS.2006; 103: 12109-12114]
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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.
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We convinced that SasA is communicating with Kai oscillator.
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We convinced that RpaA communicate with SasA.
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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.
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We create 36 chimera of RpaA-OmpR.
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== Concept ==
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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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[[Image:CONCEPT.jpg|650px]]
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== How It Works ==
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'''Kai system'''  
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Kai system：the simplest and the most well known clock-system with a period of about 24h.
 
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Kai system is driven by phosphorylation cascade composed of three proteins, such as KaiA,KaiB, and KaiC.
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The precedure(↓)
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The oscillation of Kai system is converted to transcription cycle by SasA and RpaA.  
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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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This cycle is observable by produced fluorescent proteins.
 
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[[
 
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== Techinical Issues ==
 
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'''1.Installing Kai Operon'''
 
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The DNA is installable, whether the Kai system in cyanobacteria is function in E.coli or not?
 
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'''2.Lack of Synchronization Mechanism'''
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= Next step (＊_＊)/ =
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Cyanobacteria use light for “reseting” the cycle, thus its population keeps synchronizing.  
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The construct of chimeras is finished!
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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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However the genes for “reset bottom” has not been identified.
 
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→→①Placing Lac represser under KaiA and KaiBC promoter,Kai oscillation is all stopped.
 
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The population is synchronized.
 
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→→②Can we use AI??
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[[Image:Jikkensitai.JPG|600px]]
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== Option ==
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We will find some chimeras indeed pick up Kai cycle.
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'''Control of cycle duration'''
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There have been KaiA mutants with altered cycle periods.
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= Experiments =
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We want to do a similar to create “E clock” s with various cycle periods on demand.
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*promoter
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**pBad/araC promoter
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**lacI promoter
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**ompC promoter
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== Work Schedule and Progress ==
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= Last up date  =
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;08.06 :１.Seek　for　an appropriate　promoter　and　fluoresceint　protein　for this project.[[『mu』team activities|←NOW!!]]<br>２.Obtain　KaiABC　and　SasA.
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*11/15

Latest revision as of 01:45, 16 November 2006

Contents

E colock "self-cycling-E. coli"

⇒Go to "E colock" Japanese page??
"E colock"日本語ページ

Concept

Ecolock model.jpg


Installing Kai system,
E.coli starts oscillating!

Member

Chiba 2006 & [http://chem.tf.chiba-u.jp/igem/|iGEM Chiba official site (Japanese)]

『mu』team

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 cognate response regulator for SasA, and it's deletion results in the complete loss of transcription oscillation.
This is all known about Kai.



reference

  • SasA : [http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WSN-4195FK3-10&_coverDate=04%2F14%2F2000&_alid=435215541&_rdoc=1&_fmt=&_orig=search&_qd=1&_cdi=7051&_sort=d&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=e171c9aaba0717231fe1d4c3400fcff0| H.Iwasaki et al. Cell, Vol 101, 223-233, 2000]
  • RpaA : [http://www.pnas.org/cgi/content/abstract/103/32/12109?maxtoshow=&HITS=10&hits=10&RESULTFORMAT=&fulltext=RpaA&searchid=1&FIRSTINDEX=0&resourcetype=HWCIT| N.Takai et al. PNAS.2006; 103: 12109-12114]


Cyanobavteria.JPG






E.colock Project

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

We convinced that SasA is communicating with Kai oscillator.

We convinced 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.

Experiments

  • promoter
    • pBad/araC promoter
    • lacI promoter
    • ompC promoter

Last up date

  • 11/15
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