Demonstrate
Using this project as a foundation other groups should be able to take a step further with their quorum sensing projects' design, by having the possibility of quantifying more precisely the interaction between different systems, and use it to improve their modelling. Furthermore, we took into account the time needed to produce the components of the system, generating a more realistic result. The next step must be to consider the cross talk between systems that was measured and work to minimize them, creating increasingly orthogonal circuits.
We have been successful in our initial proposition, of making a modular construct thas was capable of measuring quorum sensing interactions. Our Lux Sender system (BBa_K2771030) worked as expected, generating a Homoserine Lactone signal capable of activating a collection of quorum sensing promoters (parts BBa_K2771000, BBa_K2771001, BBa_R0071, BBa_R0078, BBa_K2771002 and BBa_K2771003). We also established a reliable method for measuring this activity, with a ratiometric activity reporter that gives us an adimensional, robust measurement, by utilizing EYFP as a reporter (BBa_K2771020) and CFP as normalization control.
Proposed system
Correct expected Result
With this system in hands, we were able to make assays to assess the sensibility and strength of this promoters, getting results for crosstalk with pLux and pLas. This results enabled us to use our modelling to estimate relative values of crosstalk strength, when joining our data with what we have seen from literature. The other promoters showed little expression. In one of our assays, they showed minimal activity torwards the end of the experiment, but nothing significative considering our timeframe and conditions.
We were also capable of reducing some amount of crosstalk experimentally, by changing the medium in which we were doing our assays. In M9 medium, pLux still showed significative (although reduced) activity being induced bý arabinose, while pLas could only show activity similar to what was seen in LB when being induced by synthetic HSL. This leads us to believe that the concentration reached by LuxI synthase in M9 medium is in the range between the threshold of activation of pLux and pLas.
pLas showing traditional activity only with synthetic HSL
