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Population counter

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Microfluidic devices

We have used microfluidics for the population counter project. Microfluidic devices are small polymeric chips with a hollow circuit imprinted inside.

When a microfluidic device is connected to a pump, it creates a liquid flow in the circuit channels. The liquid can be water or, as in our case, bacterial media. The channels are connected to small chambers.

When the media flowing through the device contains bacteria, they can be trapped into these chambers by a pulse. The trapped cells can divide inside the chamber, feeding on the nutriments that diffuse into the chamber from the channel where the flux is constant.
As the cells divide, they can fill up the chamber. In this case some of the cells will be pushed out of the chamber and eluted by the flux. This means that the rest of the cells can continue dividing instead of entering a stationary phase.

The microfluidic device can be observed under a microscope. The kinetics of individual chambers or even cells can then be tracked. Also, fluorescence of cells can be detected.





Microfluidic devices have several features that are important for our project :

• The diameter of the channels is so small that physical laws that apply to the flow are different from those used on a usual scale. In fact, the flow is laminar and exchanges of molecules between two adjacent fluxes or compartments happens only by diffusion. This means that we can induce the cells in the chambers by adding arabinose to the flowing media and predict its concentration in the chambers by modeling its diffusion.
• Cells can be trapped in small chambers so that they can be observed under a microscope. In gives us a possibility to see the phenotype of single cells. This means that if a cell has performed an excision and started producing RFP, we will be able to see this event among all other cells in the chamber.
• The flux of nutriments is kept constant by the pump. At the same time, as the cells in the chamber divide, they are eluted into the channel. This creates an analogue to a chemostat when cells are kept in an exponential phase. This is crucial in order to be able to induce the integrase. In fact, the induction works best when the cells grow and divide, so the microfluidic device is a perfect solution for our construct.

In fact, while using the timer, we expect the concentration of AHL rise with 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. Our solution is to use 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.

For our system, we thought of a microfluidic device design that would fit our needs. It would look like as a series of chambers connected to a channel. The media would flow along the channel supplying nutriments to the chambers and elute excessive cells from the chambers. After a bibliographic research we found an existing chip has already been made and used for in-vitro studies (reference below). Dr. Baigl (ENS) Kindly provided us with the chip from which we casted our PDMS chemostats. Here is the design taken from the paper and slightly modified for our needs, as well as a photo of the circuit taken under a microscope with a 5 objective.



References :
Oil micro-sealing: a robust micro-compartmentalization method for on-chip chemical and biological assays.
A. Yamada, F. Barbaud, L. Cinque, L. Wang, Q. Zeng, Y. Chen, D. Baigl Small, 2010, 6, 2169-2174