ETH Zurich 2014

Characterization of two-order crosstalk on the promoter

Background information

The E. coli strain used and the experimental set-up are described above. However, here we focus on the characterization of crosstalk and as a result we used only one, strong promoter (BBa_J23100) controlling the three different regulators (LuxR, LasR, and RhlR) used in the experiments in order to quantify crosstalk with pRhl. In the following, we describe all the different levels of crosstalk we have assessed.

First-order crosstalk

In the first order crosstalk section we describe crosstalk of pRhl due to RhlR binding to inducers different from C4-HSL or pRhl itself binding a regulator-inducer pair different from RhlR-C4-HSL.

First Level crosstalk: RhlR binds to different HSL and activates the promoter

In the conventional system C4-HSL binds to its corresponding regulator, RhlR, and activates the pRhl promoter (figure 2, green). However, RhlR can potentially also bind other AHLs and then activate pRhl (figure 2, 3OC12-HSL in red and 3OC6-HSL in blue).

Second Level crosstalk: other regulatory proteins, like LuxR, bind to their natural HSL substrate and activates the promoter

Second order crosstalk: Combination of both cross-talk levels

The second order crosstalk describes unintended activation of pLux by a mixture of both the levels described above. The regulator and inducer are being different from LuxR and 3OC6-HSL, respectively, and at the same time they do not belong to the same module. For example, the inducer C4-HSL (green), usually binding to the regulator RhlR, could potentially interact with LasR regulator (red) and together activate pLux (light blue). This kind of crosstalk is explained in figure 4.

Figure 4 Overview of possible crosstalk of the pLux promoter with both the regulator and inducer being unrelated to the promoter and each other. Usually, LuxR together with inducer 3OC6-HSL activate their corresponding promoter pLux (light blue). However, pLux may also be activated by another regulator together with an unrelated inducer. For example, the inducer C4-HSL (green) may interact with the LasR regulator (red) and together activate pLux (light blue).

Results

Table 1 Crosstalk matrix for the promoter prhl (BBa_I14017)

In this set of experiments the promoter pRhl was tested for potential crosstalk. In the top left position we observe the induction of pRhl by C4-HSL bound to the regulator RhlR. The switching behaviour was observed at a C4-HSL concentration of 1 μM. In the case of 3OC12-HSL binding the RhlR regulator and subsequently the promoter pRhlinsignificant crosstalk has been observed. Severe crosstalk was observed in the case of 3OC6-HSL binding the RhlR regulator followed by induction of pRhl. The transition occurred at a concentration of the inducer molecule of 1 μM but compared to the reference curve a lower value of fluorescence per OD was observed (1000 a.u.). Another case of crosstalk with the pRhl was detected with 3OC12-HSL binding to the corresponding LasR regulator followed by inducing the promoter pRhl. Here switching occurred at a concentration 1 nM of 3OC12-HSL and reached fluorescence per OD of 750 a.u.. This is approximately 0.5 fold the value of the fluorescence per OD shown by the reference curve indicated in green.

Modeling crosstalk

Each experimental data set was fitted to an Hill function using the Least Absolute Residual method.

The fitting of the graphs was performed using the following equation :

rFluo = the relative fluorescence (absolute measured fluorescence value over OD)[a.u.]
a = basal expression rate [a.u.](“leakiness”)
b = maximum expression rate [a.u.]("full induction")
n = Hill coefficient (“cooperativity”)
K_m = Half-maximal effective concentration (“sensitivity”)
[AHL] = AHL concentration [nM]

Antiquity

This review comes from the old result system and indicates that this part did not work in some test.