"E colock" Chiba 2006

From 2006.igem.org

Contents 1 E colock "self-cycling-E. coli" 2 Member 3 Concept 4 How It Works 5 E.colock Project 6 Chimera design 7 Next step (＊_＊)/

E colock "self-cycling-E. coli"

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"E colock"日本語ページ

Member

Chiba 2006 [iGEM Chiba]

『mu』team

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

Concept

Installing Kai system,
E.coli starts oscillating!

How It Works

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 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.

E.colock Project

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.

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(→)

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.