Team:UANL Mty-Mexico/Modelling/QS

QS is the ability of cells to sense their own cell density, to communicate with each other through the production of quorum sensing molecules (QSMs), and act like a population instead of individual cells.

The QSMs are small diffusible signaling molecules, which are accumulated in the bacterial habitat and diffuse trough the cell membrane, to form a dimer inside the receptor cell with a quorum sensing protein (QSP). The function of these dimers is to trigger increased expression of specific genes in the bacterium. In our system, the activated gene (TetR) represses another group of genes, thus we say that a cell is “QS-repressed” when is in a QSM-triggered state and we say a cell is “normal” when the cell is not under QSM activation.

In our system this function relies on the basis of the two quorum-sensing systems of Pseudomonas aeruginosa, the las and the rhl systems.

The las system comprehends the lasR gene that codes for transcriptional activator protein lasI and the Autoinducer Synthase gene, which synthetizes 3-oxo-C12-HSL, the QSM (quorum sensing molecule). These gene products form the dimer LasR/3-oxo-C12-HS -the activated form of LasR- which activates the genes under its regulation.

Briefly, after red-light exposure, the red light-induced system will produce QSMs. This will constitute the red light-induced QS system (rQS). The QSMs will be diffused across the cells membranes to finally reach the receiver cell (in this case, the green light-induced system), and form the LasR/3-oxo-C12-HSL dimer, activating pLasR/PAI for the production of TetR. TetR will then inhibit the production of cI on the receiver cell. The figure 1 is a diagram of the circuit in the receiver strain that responds to the rQS produced in the sender strain.

Figure 1. In red is shown the rQS, produced by the red light-sensing cell; in green, the circuitry of the receiver green light-sensing strain. Note that the Biphasic Switch is turned-off, so that there will be no BFP nor RFP signal.

The rhl system is comprised by the rhlR gene –which codes for the transcription factor- and the rhlI gene -coding for the autoinducer C4-HSL (HHL).

In this case, after green-light induction, the green light-induced system will produce C4-HSL molecules (QSM). This will constitute the green light-induced QS system (gQS). The QSM will diffuse across the cells membranes to finally reach the receiver cell (in this case, the red light-induced system), and form the RhlR/C4-HSL dimer, activating pRhlR/HHL for the production of TetR. The figure 2 is a diagram of the circuit in the receiver cell that responds to the gQS produced in the sender cells.

In this way, the rQS will induce TetR production in the green light-induced system, while the gQS will do the same in the red light-induced system. Note that each of these systems is present in separate bacterial strains. TetR will then repress the cI gene, turning off the Biphasic Switch and its output signals. This mutual repression is used as a way to reduce noisy signals –i.e., the Biphasic Switch outputs- that may hinder the AND-gate output and also to get non-overlapping output signals from each of the systems considered.

Figure 2. In green is shown the gQS, produced by the green light-sensing strain; in red, the circuitry of the receiver red light-sensing strain. Note that the Biphasic Switch is turned-off by TetR, so that there will be no YFP nor GFP signal.

Because lack of information regarding the green light-sensing system, we decided to simulate the behavior of the HuBac system considering a red light-sensing strain (clone 1), an IPTG sensing strain (clone 2, similar to the chemically induced system) and a third strain which performs an AND-gate in order to activate a state dependent on the activation of clone 1 and clone 2 and their respective Quorum Sensing mechanisms.

Figure 13. Quorum sensing behavior in response to red-light stimuli. From min 0 to min 90, the system is exposed to darkness; from min 90 to min 600, the system is exposed to the same light cycles as in figure 5 from Biphasic Switch section. Finally, from min 600 to the end of the simulation, the light stimulus is continuously sustained.

When only red light is present, the HuBac system will activate only the Clone 1, i. e. the red light-sensing clone. We simulated the activation of the Biphasic Switch, so GFP and YFP are expressed at different times. Meanwhile, clone 2 is repressed by the Quorum Sensing system produced by clone 1 and clone 3 is in the OFF state, as only one of the inputs needed to trigger the AND-gate is present.

Figure 14. Quorum sensing behavior in response to IPTG stimuli. From minute 0 to minute 250, the IPTG concentration is 285 nM; and finally, from minute 500 to the end of the simulation, the IPTG concentration is 1300 nM.

On the other hand, when only IPTG is present, clone 2 will be the only activated strain in the community. We simulated the Biphasic Switch behavior using the same IPTG concentration changes as in the chemically-induced system, so that RFP and BFP will be expressed at different times. Clone 1 is repressed because of the Quorum Sensing mechanism of clone 2 and clone 3 is in the OFF state.

Figure 15. Quorum sensing behavior in response to both, red light and IPTG stimuli. From min 0 to 1000, the system is exposed to continuous red light and 1300 mM IPTG.

Finally, when the system is exposed to both, red light and IPTG, the Quorum Sensing mechanisms turn off the Biphasic Switch mechanisms on both clone 1 and clone 2. Meanwhile, in the clone 3 the two inputs needed to activate the AND-gate are now present, so it starts expressing CFP.

In conclusion, our mathematical model supports the possibility of controlling the expression of five different outputs using only two inputs: red light and IPTG. Furthermore, this system can be adapted so that green light and red light are used as inputs.

It is important to mention that the system's sensitivity to the Quorum Sensing mechanisms is very sensible to changes of concentration of autoinducer molecules and on the strength of the transcription activation driven by the transcription factor-autoinducer dimer. The parameters governing these phenomena were taken from a model system developed for a general Quorum Sensing mechanism, so our model will be further improved as new data regarding our specific Quorum Sensing mechanisms is available.