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 5: Assembly strategy and steady states derivation

Wednesday, June 25th

• We will use Gibson assembly to assemble our constructs, and have now a construct assembly strategy. We know the list of experiments we want to carry (cross-talk between quorum sensing molecules, quorum sensing leakiness with or without riboswitch, cross-talk between integrases, integrase activity and logic gate behaviour) and the exact sequences of base units we will assemble (lux, rhl, las promoter and repressor units, reporter units, logic units with 3 different integrases, constitutive AHL production units for diffusion test, backbone units).

Exemple of sequences of base units.

• From the modeling side, we calculated analytically the steady state of the integrase and terminator modules, and it appears that the deterministic model gives a binary steady state corresponding to an XOR gate. This is what we expected, it confirms that deterministic model in our case is interesting for the study of dynamics of the whole system, and not for steady state calculations. So we are now moving to dynamic studies.

Week 4: Improving plasmid design, Modularization of the whole model

Wednesday, June 18th

• We improved a little bit the plasmid design, determined which parts will be taken from the iGEM registry, and which parts we have to synthesize.

Second draft of plasmids. The repressors production genes have been moved to the sensor plasmid in order to distribute more evenly the number of kb.

Sensor plasmids will be approx. 9.3kb long, and logic plasmids will be approx. 5,2kb long.

• From the modeling side, the whole model has been modularized and we have calculated and simulated steady state for the quorum sensing module.
• GeneScript, Microsynth, EraSynbio are willing to support us !

Week 3: Plasmid design started, Modeling started

Wednesday, June 11th

• We started plasmid design :

First draft of plasmids. The sensor plasmid is the same in every cell. Different logic + repressors plasmids are present in cells p and q. The logic + repressors plasmid from cells p is producing the quorum sensing molecule p, and another version is present in cells q which produces QSq. Colonies of p and q cells will be arranged in an alternate way on the millifluidic chip. Here as an example we use the lux system for p cells and the rhl system for q cells.

ΦC31 and Bxb1 are integrases. LuxR an RhlR are quorum sensing repressors. A riboswitch construct is placed around quorum sensing constructs to prevent leakiness of lux and rhl promoters. Type p colonies produce AHL by expressing the enzyme LuxI. Type q colonies produce Rhl by expressing the enzyme RhlI. In fact, we don't know yet which quorum sensing systems we will use. We will have to perform cross-talk experimetns in order to choose the ones that are the most orthogonal.

• We tried to print our first agar millifluidic chip : we printed it too small, and the printer had resolution problems.
• We wrote all reactions and found parameters from the literature for our model.

Week 2: Investigating microfluidics, Writing a modeling pipeline

Wednesday, June 4th

This week, we have talked with people from the microfluidics group and it came out that we should be able to use beads to encapsulate our cells and use a microfluidic chip. Therefore we will try to develop this possibility in parallel with the 3D-printed agar chip.
We have set up a modeling pipeline. We divided the modeling project into 3 parts :

• diffusion,
• parameter fitting,
• modeling of the genetic circuit itself, which comprises
  • a deterministic model
  • a stochastic model.

We know that the modeling challenges will be to model how integrases work, and to model the delay between reception of a quorum sensing signal and production of a quorum sensing signal by the receiver cell. Indeed, a colony could switch itself OFF when it should be ON, if it receives QS1 and produces QS2, for example. If a delay is present between reception of QS1 and production of QS2, the colony will produce GFP before it is switched OFF, and as GFP is stable enough, it will stay visible during a long time and the self-switching OFF won't be observed. This delay should be modeled.

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