Team:USP-Brazil/Results

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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, but at least this indicates that they work. We credit this difference in timing and amount of expression comparing our data and our references to the amount of proteins being expressed. We have a synthase, a receptor, EYFP, ECFP, the araC gene for pBad and two antibiotic resistances, so low signals get closer to unnoticeable.

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, this time in M9 medium, so protein production and population growth were overall slower. We were able to see, as expected, that directly inducing our system with HSL makes for a faster response than inducing with arabinose (EC50 was 2.7x lower), and also reaching a smaller plateau (7x smaller), what leads us to the conclusion that there is a delay due to the need of producing signal synthase to start producing reporter protein and that our auto-inducer system leads to an HSL concentration that allows for a much higher equilibrium, and so increased strength of the promoter. The HSL concentration we used for this assay was 10^-4mM, as literature showed that this concentration already showed the maximum transcription rate by the Lux promoter. We expect the HSL concentration reached using the synthase, then, to be higher than this quantity.

While testing the Las promoter in M9, however, we got an interesting result: activity was only as high as with LB if induced with synthetic HSL. When induced with arabinose, the system presented activity much nearer to controls, what leads us to conclude that in M9 medium the LuxI synthase has trouble reaching HSL concentrations of 10^-4mM.

Las Sender

After finishing building this construct, we tried to do the same experiments as with the Lux Sender, but we didn't get a response for any of the approaches, be it with arabinose or directly with HSLs (hoping that the synthase would be the problem and the receptor would be able to have chemical crosstalk with 3OC6 HSL), despite the construct having correct DNA sizes in analytical digestions along the construction. We still have to sequence our sequences to check for any mutations.

Rhl Sender

When testing with the Rhl system's Sender plasmid, the only promoter that showed significant expression in relation to previously tested controls was pLas (showing that the sender plasmid really works). As with the Lux Sender test, the other promoters could start showing activity at later stages, but we couldn't proceed with the assay at that point. It can be seen, also, that this first promoter showed less activity and sensibility than in the first assay, so if we consider the two synthases doing HSL at the same rate, we should be able to get values for RhlR crosstalk strength and sensibility for the positive promoter. One relevant piece of data here was the lack of activity of pRhl, the Rhl system's native promoter. As with the other promoters in the first assay, we credit this to metabolic burden. This promoter already showed little expression from the part's experience page and, at the levels of HSL concentration that could be deduced from the Lux Sender assay using synthetic HSL, some times higher than 10^-4 mM, this promoter should not show a lot of expression, specially in this conditions and timeframe.

We later tested the activity of this Sender with pLux and pLas in M9 medium, induced with both arabinose (2.5mM) and synthetic HSL (10^-4mM). Again, the only promoter showing activity was pLas, although only when induced by arabinose. This suggests that the Rhl synthase can generate concentrations of HSL higher than 10^-4mM even in M9 medium and that the lack of activity of the other promoters is really due to a lack of crosstalk.

References