We achieved

Pre-Processing:

- to characterize the AHL diffusion on agar plates and define, with predictions from the model and the experimental results, the distance between colonies, the strengh of the promoter controlling LuxI production and the incubation time needed to establish a proper AHL gradient.
- to establish several AHL gradients from different mines on one plate, intersections of gradients result in higher AHL levels for the detection of 1, 2 or 3 mines.
- to make a 2D spatio-temporal model for AHL, that encompasses local reactions and diffusion.

Processing:

- to design a P_LuxR promoter 1st libary by site directed mutagenisis and a 2nd on by rational design with different EC₅₀.
- to measured the dose-response of those promoters by single cell analysis and determined the EC₅₀ going from 0.02 nM to 6482 nM in liquid culture and from 4.45 nM to 12'555 nM on agar plates.
- to find a analytical solution for the EC₅₀ of the promoter we need and therby choose the right candidate from the libary.

⇒ a proof-of-principle using a GFP reporter.

Optimization:

- the identification of the leakiness source.
- to model different solution to reduce the leakiness, such as destabilization of proteins and double negative feedback loop.
- to optimize the circuit to reduce the leakiness by introduction of a negative feedback loop.

Output:

- to implement several reporters (hydrolases and fluorescent proteins) in one construct.
- to show different colorimetric responses (yellow, salmon, /magenta/violet, green, blue) for five hydrolases.
- to show the orthogonality between hydrolase activities.
- to characterize the hydrolases with Michaelis-Menten kinetics.


A preliminary game with the hydrolases.

Information processing:

- to establish a spatio-temporal model of the proof-of-principle and the final game.

We thank our sponsors: