Week 6: Project planning, Gibson assemblies planning, First Matlab simulations

Wednesday, July 2nd

• We have a clear organization of the different experiments we would like to perform.

We want to optimize quorum sensing to have non-leaky non-cross-talking constructs. We want to prevent cross-talk between integrases and check their dynamics with different quorum sensing promoters. Then we will test the XOR gate without production of LuxI, to check how it works without the loop, and we will finally test our final construct with the loop, hoping that the delay between GFP production and the possible second switching due to AHL production is long enough to have enough fluorescence.

The red line is our critical timeline. Numbers of days are very optimistic,therefore we multiply the whole timeline by 2. The stars stand for modeling inputs.

Tasks organization with critical timeline in red.

• We have also clear plans for the Gibson assemblies to perform. As an example, here is the plan for regulator plasmids :

Exemple of Gibson assembly plan for regulator constructs

• We have a facebook page !

• We simulated quorum sensing without leakiness on Matlab, with parameters from the literature.

Week 1 : Project selected

Wednesday, May 28th

After more than one month of endless meetings and passionate debates, we finally chose the project that will keep us occupied in the next five months. From the beautiful pattern made by the Sierpinski triangles, we will focus on cellular automata and try to implement one.

Sierpinski triangles appear when the rule 90 is followed by every cell on the grid :

Pattern given by rule 90 if only one cell is switched on in the first row at the beginning. The pattern appears row by row.

Ideally we will use a microfluidic chip. We could also use a 3D printed agar plate like this one to load the colonies. On this grid we can implement the rule 6, which is the simplification of rule 90 considered as a rule with 2 inputs : each cell computes a simple XOR gate of its two parents.

Arrangement of p and q cells on the chip

Pattern given by rule 6 if only one cell is switched on in the first row at the beginning. The pattern appears row by row.

The logic part will be built with integrases and the colony-to-colony communication will use quorum sensing.

Every colony will receive two quorum sensing signals (QSp and QSq) from the two cells above it. These two signals trigger the production of two different integrases r and s in the colony. Integrases enable to build biological XOR logic gates by switching twice a terminator. Indeed, every integrase can switch the terminator only once. Thus if the colony produces only r or only s, the terminator is switched only once, so the terminator is OFF, and GFP and QS1 or QS2 are produced (depending on the colony). If the colony produces r and s, the terminator is switched twice, so it is ON and it blocks expression of GFP and of the quorum sensing molecule.

Rule that will be computed by all cells : an XOR gate between the signals from the 2 cells above. Biologically, integrases p and q are able to bind sites around a terminator and switch this terminator, thereby turning off the effect of the terminator and enabling gfp transcription. If p and q integrases are both present, the terminator is switched twice and comes back to its initial state, gfp is not transcribed.

We need to  :

• find orthogonal quorum sensing molecules and orthogonal integrases
• discuss with microfluidics experts to check if using microfluidics is possible and presents advantages in our case
• find possible parts in the registry for integrases, and design plasmids