Team:Paris Liliane Bettencourt/Project/Population counter


Population counter

This part of our project is dedicated to building a counter and a timer that work on the population level. This system operates on the principle that the statistical occurrence of a rare genetic (excision) event , triggered by an external input can be modeled. The excision culminates in expressing a new phenotype (ie, fluorescent (mRFP) or antibiotic resistance gene (tetR)). Our aim is thus to be able to tell the number of times triggering events took place in the bacterial culture from the moment of its inoculation by simply quantifying the proportion of the new genotype/phenotype in the culture population. For instance, if each triggering event causes the same percentage of cells to change genotype and become red, than the resulting proportion of red cells is an accurate count of the number of input stimuli, which in our case are arabinose pulses.

Apart from counting, another feature of our system is “The 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 (AHL-dependent expression of a second fluorohore, eg GFP). 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 FACS or to simply dilute the culture and plate it. The ratio of red fluorescent colonies to wild-type ones gives you the result (though with a significant delay...).
The Timer sets yet another challenge. 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-dependent as we want it to be. The solution would be to use a chemostat or as we decided, a microfluidic device. Importantly, the microfluidics approach 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.

Our computational model of the above system conveys the essential message that this design can work under a robust set of parameters. To have a look at the model and simulations results, 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.