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Team:Paris Bettencourt/ComS diffusion
From 2011.igem.org
The ComS diffusion system
Introduction to the system
In case of nutriment restriction, B. Subtilis has two way of reacting. One is to soprulate, in order to wait for better times to come back to life again. The second is the competent mechanism. In this state, B. Subtilis try to catch in the medium every pieces of DNA around and make homologue recombination with its genome, and then devide a lot to make a chance for her new genotype to survive the hard life conditions.
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| Fig1: Schematics of the MeKS system | Fig2: Image of a mix between sporulated and competent state during the steady state phase. |
The choice between these two mechanism is controled by a bistable system known as the MeKS system. This system is usually stochastically controled by the apparition of some ComS protein in the cell, that would inhibit the MecA protease and allow the ComK protein to self amplify. But, as ComK inhibit the production of ComS, the system come back to the original state in a few hours.
This phenomenon was completely described by M. Elowitz and al. in this paper [1] from where these figures are extracted.
Introducing the design
The MeKS system is a noise tolerent bi-stable system that regulate the competence. However, this system is working in the stationnary phase, and we want to use it in the expodential phase.
To outsource the system, we have to work with a B. Subtilis strain CodY-, because CodY repress the MeKS system during the expodential phase, as well as thousands of stationnary phase gene. This strategy has been adopted because CodY regulation is so strong and touch so many gene, that it was faster to get rid of the regulator itself. The emittor cell has to be ComK- because we want to avoid the competence in a system that will modify the cell behaviour and may prevent the nanotubes from forming.
The design is the following:
Fig2: Schematic of our ComS design
The use of the rok system has been invented by Elowitz et al. [1]. Adding an additionnal repression help the system to have a shorter period.
The activity of the pComK promoter is monitored by a construction pComK-GFP.
The CodY- strand is obtained thanks to Link Sonenshein [2] and the different constructs will be biobricked from the construction made by the M. Elowitz's laboratory [http://www.elowitz.caltech.edu/index.html] and published in this paper[3]
