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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 competence 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.
A comprehensive study of this phenomenon has been done by M. Elowitz and al. and published in this paper [1] from where these figures are extracted.
Ideas behind the design
The ideas behind this design is to pass through the nanotube some ComS proteins. This protein is very small (40 amino-acids) and so is expected to pass very efficiently. It is also known from the M. elowitz paper [1] that very few proteins are required to trigger the switch, which makes this system a very good candidate for what we want to do.
We contacted M. Elowitz, and he kindly sensed us a strain containing a chromosomally integrated reporter that monitors the level of ComK and ComS in the cell, with the construction pComG-cfp/pComS-yfp inside. This construct could in theory directly be used as a receiver cell, but the MeKS system is known to be repressed in expodential phase. We explain later how we dodged this pitfall.
The design can simply be summed up by the following picture:
Fig3: Schematic summing up the ComS diffusion design.
Problems linked to the growth phase
The MeKS system is a noise tolerent bi-stable system that regulate the competence. This system is working in the stationnary phase and is theorically repressed during the expodential phase. We investigated the issue using computer assisted sequence homology analysis, and we found 3 locuses in which we expect the protein CodY, that is known to repress many genes of the steady state phase during the expodential phase and we want to use it in the expodential phase.
In order to avoid theproblem, we have to create a B. Subtilis strain CodY- for the MeKS system to be active during the expodential growth 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 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]
