From 2006.igem.org

Contents 1 E colock "self-cycling-E. coli" 2 Concept 3 Member 4 Kai system 5 E.colock Project 6 Chimera design 7 Next step (＊_＊)/ 8 Experiments 9 Last up date

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

• Shinpei Okuyama
• Akinori Katabami
• Sari Sugaya
• Shinya Matsuno

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| 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