We wanted to incorporate quorum sensing to make Mantis more robust and increase sensitivity to be able to sense very low antigen concentrations. By using quorum sensing, we aim to convert the antigen concentration into a singaling molecule concentration. Every biosensor cell in our Mantis system will then be able to detect the signaling molecule. To do this, we need a quorum sensing system maybe link to background info on LuxR, LuxI, AHL, [LuxR-AHL], dimer, etc that can self-activate, but only does so upon induction. As a starting point we took the BBa_K1913005 biobrick. This contains the LuxI and LuxR proteins from Alivibrio fischeri, each with their native promoter. It also contains the GFP protein under control of a promoter identical to the promoter of LuxI. LuxI can auto-activate itself while GFP can report on the amount of auto-activation.

Previous experiments have shown that this construct produces fluorescence even at low cell densities. This suggests that leaky expression of LuxI and high basal levels of LuxR will result in a significant amount of GFP production. The goal is to construct a variation on this part that no longer has spontaneous activation, but can be induced by an outside mechanism, for example a two-component system.

Natalia says something about her initial BBa_K1913005 experiments

In paralel to the lab experiments, we developed a model of our quorum sensing system. The global parameter space was explored showing the important role of certain parameters in preventing spontaneous auto-activation.

The model showed that in simulations, a low rate for the cellular AHL degradation is associated with spontaneous auto-activation. The role of using aiiA to combat leaky expression has been explored by previous iGEM teams; ETH

The next step was to add aiiA to the spatial model of the entire system. This includes both the quorum sensing system, as well as the lytic mechanism responsible for producing a fluorescent signal. In the spatial model, parameter sets that result in spontaneous auto-activation of the system were simulated after including a production term of aiiA. Now, cells induced by antigen detection stop producing aiiA. In the model, aiiA is able to suppress auto-activation. Then, when a signal is detected and aiiA production is halted, levels of aiiA will drop until that specific cell starts auto-activation spontaneously, similar to how all cells behaved before addition of aiiA. Auto-activation of a small number of cells would eventually lead to auto-activation of all cells in the system. After adding aiiA to our model, we simulated a model where cells are able to communicate the presence of antigen, resulting in a population-wide signal. But in the absence of antigen, cells remain inactive and no fluorescence is generated.

These modeling results support the strategy of using aiiA to tune the sensitivity of the quorum sensing construct and indicate that in principle the functionality we want to engineer can be obtained using only the components we are using.

Natalia says something about how after adding aiiA, the construct produces significant fluorescence after inducing the system.