"E colock" Chiba 2006
From 2006.igem.org
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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]] | ||
| - | + | = Concept = | |
[[Image:Ecolock model.jpg|left|330px]] | [[Image:Ecolock model.jpg|left|330px]] | ||
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'''Installing Kai system,<br>E.coli starts oscillating!''' | '''Installing Kai system,<br>E.coli starts oscillating!''' | ||
| - | + | = Member = | |
| - | [[Chiba University, Japan 2006|Chiba 2006]] | + | [[Chiba University, Japan 2006|Chiba 2006]] & |
| - | + | [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]] | ||
| - | *[[User:Matsuno|Shinya Matsuno]] | + | *[[User:Matsuno|Shinya Matsuno]] |
| - | + | = Kai system = | |
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[[Image:Kai system.jpg|right|400px]]<br /> | [[Image:Kai system.jpg|right|400px]]<br /> | ||
Kai system is generated by 3 proteins(KaiA,KaiB, and KaiC).<br /> | Kai system is generated by 3 proteins(KaiA,KaiB, and KaiC).<br /> | ||
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SasA is a Sensory Kinase(a kind of Histidine Kinase).<br /> | SasA is a Sensory Kinase(a kind of Histidine Kinase).<br /> | ||
SasA is believed to connect with Kai cycle and transcriptional oscillation of many proteins.<br /> | SasA is believed to connect with Kai cycle and transcriptional oscillation of many proteins.<br /> | ||
| - | RpaA is | + | RpaA is cognate response regulator for SasA, and it's deletion results in the complete loss of transcription oscillation.<br /> |
This is all known about Kai. | This is all known about Kai. | ||
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| + | '''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] | ||
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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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| - | + | = E.colock Project = | |
SasA and RpaA is highly homologous to EnvZ and OmpR,respectively. | 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. | We designed RpaA-OmpR chimera,in hope that we read our Kai oscillation using ''ompC'' promoter. | ||
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| - | + | = Chimera design = | |
In general, Response Regulator has 2 domeins, | In general, Response Regulator has 2 domeins, | ||
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The precedure(↓) | The precedure(↓) | ||
| - | We used 6 excite primers on each RpaA and | + | We used 6 excite primers on each RpaA and OmpR. |
It can make 6 ×6 = 36 different chimeras!! | It can make 6 ×6 = 36 different chimeras!! | ||
| + | [[Image:Excite PCR.JPG|600px]] | ||
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| + | = Next step (＊_＊)/ = | ||
The construct of chimeras is finished! | 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 ''ompC''promoter. | Chimaras along with WT RpaA and OmpR will be tested in their function as response regulator that switch ON/OFF the ''ompC''promoter. | ||
| + | |||
| + | |||
| + | |||
| + | [[Image:Jikkensitai.JPG|600px]] | ||
We will find some chimeras indeed pick up Kai cycle. | We will find some chimeras indeed pick up Kai cycle. | ||
| - | + | = Experiments = | |
| + | *promoter | ||
| + | **pBad/araC promoter | ||
| + | **lacI promoter | ||
| + | **ompC promoter | ||
| + | |||
| + | = Last up date = | ||
| + | *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
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 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]
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.
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!!
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.
We will find some chimeras indeed pick up Kai cycle.
Experiments
- promoter
- pBad/araC promoter
- lacI promoter
- ompC promoter
Last up date
- 11/15
