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Revision as of 00:00, 1 November 2017
![](https://static.igem.org/mediawiki/2017/6/6e/T--Wageningen_UR--HP_MobileIcon.png)
Cell signaling
We wanted to incorporate quorum sensing (QS) to make Mantis more robust and increase sensitivity to be able to sense very low antigen concentrations. By using QS, we aim to amplify the response to antigen presence in a tested sample. To achieve this, we use a QS system that allows cells to signal the presence of antigen to the entire cell population. After detecting the antigen, a cell will start producing LuxI, the enzyme able to generate the signaling molecule AHL. AHL will diffuse out of the cell into the external medium. When it enters a neighboring cell, AHL can bind with LuxR to form an active transcription factor (TF). The TF factor activates production of the lysis protein COLE7, causing the cell to lyse. There are two subpopulations of cells; one containing TEV protease and the other containing a quenched form of GFP. The GFP domain in this protein is fused to a REACh domain, preventing fluorescence. Upon release, TEV protease cuts the GFP free from the REACh domain, leading to production of fluorescence. An overview is visualized in Figure 1. More details are available on the wet-lab results page.
![](https://static.igem.org/mediawiki/2017/6/61/T--Wageningen_UR--Results_Quorum_Sensing_Figure_1.png)
Existing biobrick
We selected an existing QS biobrick containing a LuxI in a positive feedback motive as the starting point for our project. This biobrick, BBa_K1913005, also contains GFP under the same promoter as LuxI. The amount of fluorescence is therefore a reporter for the degree of LuxI production. The more the cells engage in QS, the more fluorescence they produce. Previous experiments have shown that BBa_K1913005 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.
Phase 1: Initial testing
To confirm the properties of BBa_K1913005 reported in the repository, we first analyzed the fluorescence produced by cells containing this biobrick. As a negative control, cells containing pLuxR-GFP, as well as cells containing pLuxR-GFP and pLuxL-LuxR (i.e. BBa_K1913005 without LuxI) were also analyzed (Figure 2). The BBa_K1913005 biobrick functions properly only as a reporter for QS. As it always engages in QS, it cannot be used in systems that require QS in specific cases. Our goal is to fix the behavior of BBa_K1913005 so that the QS mechanism can be applied in a useful manner.
![](https://static.igem.org/mediawiki/2017/8/85/T--Wageningen_UR--Model-ReporterQS.png)
This indicates that cells containing BBa_K1913005 auto-activate and produce the active transcription factor [LuxR-AHL} dimer. Without LuxI, there is no AHL production and there is no active form of the LuxR transcription factor. Our goal now is to make an inducible version of BBa_K1913005 that initially produces no GFP, but that can be activated.
Phase 2: Modeling insights
In parallel to the wet-lab experiments, we developed a mathematical modelof our quorum sensing system. In the model, certain parameters resulted in behavior identical to our wet-lab results (Video 1). Leaky expression of LuxI would result in fluorescence despite the fact that LuxI production was never induced.
The parameter space showed 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. A potential method of controlling this parameter in the wet-lab is to use aiiA. The role of using aiiA to combat leaky expression has been explored by previous iGEM teams; ETH.
We first explored the effect of adding aiiA to our spatiotemoral model. Kinetic parameters for aiiA were added to the model and parameter sets that resulted in spontaneous lysis were simulated. The result is visualised in video 2. This demonstrates that aiiA can prevent fluorescence from being generated in the absence of GFP while still maintaining the generation of fluorescence in the presence of antigen.
We were able to tune the kinetic parameters of aiiA so that aiiA would prevent spontaneous auto-activation caused by leaky expression while not preventing the detection of low levels of AHL produced by neighboring cells.
Table 3: A system with negatively regulation of LuxR that self-activated spontaneously was modified by adding aiiA or increasing the degradation rate of LuxR, resulting in the desired behavior. | |||
---|---|---|---|
Score | Animation, no antigen | Animation, antigen | |
Original set | 99.97 | link | link |
Original Set & aiiA | 0.059 | link | link |
Original Set & increased LuxR deg. | 0.084 | link | link |
These modeling results support the strategy of using aiiA to tune the sensitivity of the QS construct and indicate that, in principle, the functionality we want to engineer can be obtained using only the components we are using.
Phase 3: Lab implementation
Inspired by the modeling result, we decided to add aiiA to BBa_K1913005. The aiiA gene had to be under an inducible and tight promoter. We used the pTet promoter to regulate aiiA, causing aiiA to be produced constitutively. To turn on fluorescence, aiiA production can be halted by introducing the TetR protein, which represses production from the pTet promoter. This construct was submitted as a new biobrick BBa_K2387070. Production of TetR is regulated by the inducible promoter pBAD. Addition of arabinose should lead to production of TetR. TetR can then bind and repress pTed, halting aiiA production. Lower levels of aiiA in the cells would mean higher levels of AHL, which should then lead to auto-activation and production of GFP. In this way, addition of arabinose should induce a fluorescent signal.
The fluorescence of this new construct was measured alongside the old construct and the negative controls (Figure 3).
![](https://static.igem.org/mediawiki/2017/0/0e/T--Wageningen_UR--Model-InducibleQS.png)
Conclusion
For Mantis to amplify responses to antigen, we need a system that can produce fluorescence, but only in response to detection of antigen. We determined that QS could be a good strategy to achieve this. Our goal was to engineer a construct where QS could be induced, for example by a two-component system or by addition of arabinose. We started off with a QS system that always produces fluorescence. The goal was to reduce the sensitivity to AHL. Through the mathematical model of the QS module of Mantis we discovered that aiiA can be used to tune the sensitivity of the QS system. Inspired by the model, a biobrick that allows for inducible repression of aiiA production was engineered. Combining our biobrick with an existing QS reporter biobrick, we managed to only produce fluorescence after addition of arabinose. Our module was therefore able to detect arabinose and turn on QS. Thus, GFP was produced when required.
Our biobrick allows the engineering of whole-cell biosensors that incorporate QS as a means of signal amplification or to synchronize the reporter system across an entire population. For this biobrick to work with a detection module, that module has to trigger TetR production upon detection. In doing so, QS will be triggered, resulting in a fluorescent signal.
References