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Team:Paris Liliane Bettencourt/Project/Population counter
From 2010.igem.org
Population counter
This part of our project is dedicated to building a counter and a timer that work on a population level. This system operates on the principle that the statistical occurrence of a rare event in a large population can be modeled. Our aim is to be able to tell the number of events that have happened to the bacterial culture from the moment of its inoculation by simply looking at the culture and calculating the proportion of red fluorescent cells in it. If each event causes the same percentage of cells to change phenotype and become red, than the resulting proportion of red cells is an accurate count of the number of input stimuli, or in our case arabinose pulses.
Apart from the counter, another feature of our system is a timer designed as a threshold-detecting device. This is achieved by quorum-sensing : every bacteria that has turned red also starts producing AHL that diffuses into the media. The more events occur, the more cells produce AHL. After a certain number of events, the concentration of AHL reaches the threshold and becomes sufficient for triggering the timer. No matter if the cell has the wild-type or the red phenotype, they all sense high AHL concentration and start producing GFP. So all of the culture starts emitting green fluorescence. Thus, it’s an event-dependent rather that a time-dependent timer. Please refer to our design page for further information.
In order to use the counter, you need to simply dilute the culture and plate it. The ratio of red fluorescent colonies to wild-type ones gives you the result, even though you need to wait at least 1 day before you can count the colonies.
It is more difficult to make sure the timer works correctly. We expect the concentration of AHL to reflect the number of events, but we must take into account that AHL can accumulate in the media. This would make our timer time-dependent rather that event-depended as we want it to be. The solution would be to use a chemostat or as we decided, a microfluidic device.
It also allows us to better use the counter by calculating the ratio of red fluorescent to wild-type cells directly in the microfluidic device. This way we have the results on the go, without having to plate and wait for the colonies to grow.
Please refer to our microfluidics page for further information.
We have also worked on the modelling of this system. To have a look at the model and some simulations, please refer to our modelling page.
We invite you to see our results page to know how far we have gotten in testing of our counter, as well as our Parts page to see which biobricks we've submitted and characterized in the parts registry.
