Results
After some trouble with gene synthesis and building our constructs, we got to doing experiments with the first three candidates for Sender systems (Lux, Las, Rhl) and all of the six Receiver systems. In this page, we will go through each one of the Sender systems and report how our reporters responded to the signal.
Our experiments were normally done in 15mL falcon tubes or 24-well plates (with admitted differences in aeration) and in 2 mL LB or M9 (for kinetic experiments) medium, at 37ºC and shaking at 180~200RPM. As the auto-induced quorum sensing system should be kickstarted by arabinose (see Design), after growing an overnight culture diluted 1:10 for one hour, we induced our cells with 2.5mM L-arabinose and started measurements.
Lux Sender
This was our first system to get results. As it had the most straightforward cloning steps from our Distribution Kit parts. To test if the system would respond, we first plated our co-transformations in solid medium, slathered with 20uL of 1M arabinose. Also, to check if it was really our quorum sensing signal inducing the fluorescence, we tested inducing with arabinose in a single point of the petri dish.
After seeing that our systems worked, we proceeded to the liquid medium assays. They were done as reported above, then fitted for a dose-response curve, assuming an increasing amount of HSL signal over time. The promoters that showed the most significant response were pLux and pLas, with the latter having a lower sensitivity (~3.3x pLux EC50) but plateauing at a higher expression (~1.7x pLux expression). The other promoters only showed significant expression at later stages of our experiment, and also at a very lower strength.
Team Düsseldorf helped us by providing some 3OC6 HSL, that we could use in an assay to discern the amount of HSL in our auto-inducer assays.