"
Team:Paris Bettencourt/ComS diffusion
From 2011.igem.org
The ComS diffusion system
Introduction to the system
When it faces nutriment restriction, B.subtilis has two way of reacting. One is to sporulate in order to wait for better times and come back to "life" again. The second is the competence mechanism. In this state, B.subtilis tries to catch in the medium every piece of DNA around and make homologous recombination with its genome. It then divides a lot to give a chance to its new genotype to survive the harsh conditions.
 |
 |
| Fig1: Schematics of the MeKS system [1] | Fig2: Image of a mix between sporulated and competent state during the steady state phase. [1] |
The choice between these two mechanisms is controled by a bistable system known as the MeKS system. This system is usually stochastically controled by the apparition of ComS proteins in the cell. ComS inhibits the MecA protease and allows the ComK protein to self-amplify. But as ComK inhibits the production of ComS, the system comes back to the original state within a few hours.
A comprehensive study of this phenomenon has been conducted by M. Elowitz and al. [1].
Idea behind the design
The idea behind this design is to pass ComS proteins through the nanotube. This protein is very small (40 amino-acids) and it is expected to pass quite efficiently. We also know from the M. elowitz paper [1] that very few proteins are required to trigger the switch (around 200). This makes this system a very good candidate for what we want to do.
We contacted M. Elowitz and he kindly send us a strain containing a chromosomally integrated reporter that monitors the level of ComK and ComS produced by a cell (pComG-cfp/pComS-yfp). This construct could in theory directly be used as a receiver cell, but the MeKS system is known to be repressed in exponential phase. We explain later how to avoid this problem.
The design is summed up in the following picture:
Fig3: Schematic of the ComS design
How this design works
Explanation step by step:
In standard conditions, the ComK protein in late expodential phase is destroyed by the protease MecA. In the emitter cell, the system is repressed because codY is active.
Fig4: Step one, the system is repressed and there is no ComK.
A nanotube is etablished, some ComS diffuses from the first cell to the second one, and blocks the protease by affinity inhibtion. The ComK can start amplifying, and activates the ComG promoter.
Fig5: The ComK protein activates the ComG promoter.
The ComG promoter produces CFP that report the cell has entered a competence phase. The ComS promoter is giving us the quantity of ComS that have passed through the nanotubes.
Fig6: Schematic of the simplified general principle of the ComS design
Then, if the nanotubes break, the ComS start to disapears and several hours later, the cell come back to its original state.
Problems linked to the growth phase
The MeKS system is a noise tolerent bi-stable system that regulate the competence of the cell. 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 to bind, that is known to repress many genes of the steady state phase during the expodential phase.
In order to avoid this problem, we have created a B. Subtilis strain ∆CodY. This will allow the MeKS system to be active during the expodential growth phase, as well as thousands of stationnary phase gene. This mutation is not lethal although it reduces the growth speed significantly.
A CodY- strand is obtained thanks to Link Sonenshein [2] and this strain was crossed with the reporter strain from M. Elowitz's laboratory [2] (see the paper[3]) using DNA extraction and competence of the late expodential phase competence.
We sucessfully managed to get this strain. See the experiment page for details.
Modeling and experiments
To know more about what we have done on this system and in the experiments, we invite you to visit the corresponding modeling and experiment pages:
