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Team:INSA Toulouse/contenu/project/modelling
From 2013.igem.org
Modelling
N-acyl Homoserine lactone diffusion in agar medium
To build a binary full adder our system must be able to communicate a carry if necessary. We chose to use one of the N-acyl Homoserine Lactone (AHL), 3-oxohexanoyl-homoserine lactone. The AHL is the biological messenger in our system.
Different systems can be devised to implement the expression and diffusion of the AHL.
The system of Figure 1 operates as follows:
A petri dish containing colonies equidistant from each other. The lights provide the information for the addition to perform, and the expression & diffusion of AHL from one colony to another allows the carry propagation.
This system seems quite simple but nevertheless raises a certain number of problems:
- Does a colony produce enough AHL to induce diffusion of the message?
- Do the colonies have to be inoculated all at once or progressively during the calculation step?
- What is the ideal distance between the colonies? How can we avoid excessive AHL diffusion , for example from a colony n to colony n+2 rather than a colony n+1?
Figure 1: Diffusion of AHL through colonies.
Production of AHL
To overcome the problem of the amount of AHL required for a rapid diffusion of the messenger, we can well imagine a system in which liquid precultures may be deposited. So we get a higher cell density and a greater production of AHL.
Figure 2: Bacterial full adder system in wells.
Inoculation of cultures
A gradual inoculation of wells during the addition process allows, first to avoid the direct interference between the wells. Secondly with this method we avoid also the problem of death cell (a death wouldn't be able to answer to the AHL messenger).
The ideal distance between well
In order to find the ideal distance between two colonies we have searched a model which allow us to know: how does AHL diffuse into the medium and how long does the diffusion take to pass from one colony to another.
Modelling steps
Diffusion reminder
Figure 3: Evolution of AHL concentration versus distance.
Figure 3 shows that the largest amount of AHL is between 2.5 and 20 mm. This information is important but we need to develop a model based on time.
Two dimension model:
We realized many experiences in order to measure how AHL diffuse into agar medium. We used a particular bacteria, Chromobacterium violaceum, which is capable to detect presence of AHl by producing a purple pigment, the violacein.
Figure 4 shows petri dishes containing eight colonies of bacteria placed at 5, 10, 15, 20, 25, 30, 35 and 40 mm from the center of the box. With this experience we can track the diffusion of the AHL. Figure 5 represent the evolution of AHL diffusion versus time.
Figure 4: Photographs at 25h of petri dishes containing each 8 Chromobacterium violaceum colonies from 5 mm to 40 mm.
Figure 5: Evolution of AHL diffusion into petri dish.
We can see that the ideal distance between two wells is probably between 15 and 25 mm depending on the amount of AHL produced by the bacteria. Indeed, the AHL diffuses rapidly into the medium during the first 5 hours, then the concentration gradient decreases and the diffusion is slower. However, if the bacteria produce too AHL, the diffusion to colony n+1 will be in the first 5 hours but affect the colony n+2 after 24h. In the ideal case would have bacteria produce about 1 nmol per well of AHL.
Figure 6: Radar graph of AHL diffusion into petri dish.
