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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 to avoid this problem.
The design can simply be summed up by the following picture:
Fig3: Schematic of the simplified general principle of the ComS design
How this design works
Here is the explanation step by step:
In standard conditions, the ComK protein, in late expodential phase, is destroyed by the protease MecA. In the emmitor cell, the system is repressed because codY is active.
Fig4: Step one, the system is repressed, and there is no ComK.
Is a nanotube is etablished, some ComS will diffuse from the first cell to the second one, and block the protease by affinity inhibtion. The ComK can start amplifying, and activate the ComG promoter.
Fig5: The ComK protein activates the ComG promoter.
Fig6: Schematic of the simplified general principle of the ComS design
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.
