Team:Aberdeen Scotland/parameters/invest 5

= The Amended Model =

In the V.fischeri; where quorum sensing occurs naturally, the production of LuxI is on a very low level before quorum sensing occurs. This means that the HSL concentration is low in the cell, and thus is more likely to diffuse out of the cell rather than combining with LuxR. This results in a low level of HSL-LuxR which is never at a high enough concentration to activate the lux-box. Mimicking the natural system, we suggest putting the LuxI gene on a different promoter additionally regulating LuxR and that the promoter for the LuxI gene by a lux-box.



The model we now look at is the final step in our progression. In the natural system, it is suggested that the lux box constitutively transcripts LuxI at a rate of 1.5*10-4 pops and a maximal transcription 2.0*10-3 pops[1]. Using these values for our system including an amplifying loop for LuxI, we are left with:



The graph shows again an increase of GFP when IPTG enters the cell. However, in the beginning the amount of HSL produced in cell is less than the critical concentration required to activate the lux-box. When HSL is present, the increase in the production of GFP becomes even more pronounced. This graph shows that changing the system to mimick V.fischeri, gives us the behaviour that we want for our Pico Plumber.

= Sensitivity Analysis =

We next investigate the robustness of our system by performing a sensitivity analysis on KCI and KTetR. We see in the next few plots that the values of KCI and KTetR are very robust in this model:



We know from the literature that KCI is expected to be greater than KTetR. This means that we can assume that in a worst case scenario KCI might be equal to KTetR. We use this worst case assumption now to check the relationship between KCI (= KtetR) VS. KP. This is what we find:



So our system is also very robust for different values of KP.

The required levels of HSL outside and IPTG outside
The only area left to explore is how the levels of the input molecules affect the output. We do this here in the same manner as was done for previous versions of the system.



= Graphs =

Here is how our system functions with the parameters we believe to be correct:





We can conclude that this system is very robust for K CI, KTetR, KlacI and KP. It can be activated by lower levels of HSL and IPGT than its predecessors and consistently produces large quantities of the glue molecule, molecule X. We have also shown that it is capable of latching behaviour for quorum sensing, given the right promoter strengths on the luxI feedback lux box. The following graphs shows that the levels of HSL leaving the cell when it is not in quorum sensing state should be high enough to activate quorum sensing when the bacteria group closely enough together for long periods of time.



Conclusion
The original design for the system triggers itself because of a higher production of LuxI than in the natural system. Having LuxI and LuxR both on the same amplifying loop results in a shortage of LuxR that can combine to HSL and start full production of the AND – gate as we can see here

In conclusion, changing the system to have LuxI on an amplifying loop will change the behaviour such that we have an AND-gate to trigger glue production and will result in a far more robust system.

We can see a complete comparison between the quorum sensing systems in this final summary diagram: