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. Our Rhl Sender also worked, as reported in our Results page.
Proposed system
Correct expected Result
With this system in hands, we were able to assess the sensibility and strength of these 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 of the crosstalk experimentally, by changing the medium in which we were doing our assays. In M9 medium, pLux still showed significative (although reduced) activity when being induced by 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, in M9 medium
Same experiment in M9 medium, with decreased scale; pLux has normal but reduced activity, while pLas induced by arabinose has very reduced expression
Our system was designed in a way that, after induced with arabinose, the bacteria would produce the HSL synthase and receptor, ruling out the need to use synthetic HSL. By comparing our measurements using this strategy and with synthetic HSL, we could see that even with the burden of producing synthases, the cells reached a higher plateau for reporter expression. Although the induction with synthetic HSL leads to a faster response, the plateau level for this case was not expected, because we applied a concentration of HSL known to activate the promoter in a very strong manner. So we can say that the synthase is capable of maintaining a higher concentration of HSL for a longer time. Considering the price of synthetic HSL, this pathway could be a better choice when working in larger scale projects. These assays were able to validate our strategy, with its perks and limitations, which should be taken into account when designing other projects.
