Team:ETH Zurich/expresults/qs

In all the measurements conducted to create this matrix the [http://parts.igem.org/Part:BBa_R0062 promoter pLux] was the basis and was induced in six different variations shown. The dark blue points in the graph top left show the activation of gene expression when [http://parts.igem.org/Part:BBa_R0062 pLux] is induced by 3OC6-HSL (Lux-AHL) binding to the corresponding [http://parts.igem.org/Part:BBa_C0062 LuxR regulator]. The observed transition occurs at a concentration of approximately 1 nM of 3OC6-HSL. The light-blue curve plotted shows modeling data of [http://parts.igem.org/Part:BBa_R0062 pLux] induced by 3OC6-HSL (Lux-AHL) binding to the corresponding [http://parts.igem.org/Part:BBa_C0062 LuxR regulator]. This curve from the model and the dark blue data points obtained from experiments were plotted as a reference in all the other graphs describing [http://parts.igem.org/Part:BBa_R0062 pLux]. Crosstalk can be observed for the cases where the 3OC12-HSL (Las-AHL) binds the [http://parts.igem.org/Part:BBa_C0062 LuxR regulator]. Additionally for 3OC12-HSL binding to its corresponding [http://parts.igem.org/Part:BBa_C0179 regulator LasR] and then binding to the [http://parts.igem.org/Part:BBa_R0062 pLux] as seen in the middle of the top row and center of the matrix. For the case of Las-AHL binding the [http://parts.igem.org/Part:BBa_C0179 regulator LasR] and subsequently the [http://parts.igem.org/Part:BBa_R0062 promoter pLux], the transition occurs at 1 nM and reaches 0.5 fold the fluorescence as [http://parts.igem.org/Part:BBa_R0062 pLux] induced by 3OC6-HSL binding [http://parts.igem.org/Part:BBa_C0062 LuxR]. In the case of 3OC12-HSL binding [http://parts.igem.org/Part:BBa_C0062 LuxR] and inducing the promoter [http://parts.igem.org/Part:BBa_R0062 pLux], the transition is observed at approximately 100 nM and severe crosstalk is observed, meaning that the ON-OFF-ratio is not significantly different from the reference curve.

Observation of C4-HSL has shown, that there is no significant crosstalk with the [http://parts.igem.org/Part:BBa_C0062 LuxR regulator] and [http://parts.igem.org/Part:BBa_C0179 LasR regulator] binding C4-HSL and subsequently to [http://parts.igem.org/Part:BBa_R0062 pLux]. This is indicated on top right and middle right graphs. However, [http://parts.igem.org/Part:BBa_C0171 RhlR] induced with its corresponding inducer (C4-HSL) binds to [http://parts.igem.org/Part:BBa_R0062 pLux] and activates expression of GFP at about 100 nM.

The promoter of interest in this matrix is [http://parts.igem.org/Part:BBa_R0079 pLas]. The graph on top left corner shows the induction of [http://parts.igem.org/Part:BBa_R0079 pLas] by its corresponding inducer (3OC12-HSL) binding the corresponding [http://parts.igem.org/Part:BBa_C0179 LasR]. The red line shows the model whereas the datapoints shown in red represent the experimental results. The transition can be observed at a concentration of Las-AHL of about 2 nM. 3OC6-HSL binding [http://parts.igem.org/Part:BBa_C0171 RhlR] does not induce the [http://parts.igem.org/Part:BBa_R0079 pLas]. For the binding of 3OC12-HSL to [http://parts.igem.org/Part:BBa_C0171 RhlR] a minor increase of fluorescence can be observed. The same can be observed for 3OC12-HSL binding to the [http://parts.igem.org/Part:BBa_C0062 LuxR] as this combination is to a small degree inducing [http://parts.igem.org/Part:BBa_R0079 pLas]. The most significant case of crosstalk when observing [http://parts.igem.org/Part:BBa_R0079 pLas] is shown in the graph in the center of the matrix. It is clearly shown that 3OC6-HSL (Lux-AHL) binding to the corresponding [http://parts.igem.org/Part:BBa_C0062 LuxR] regulator is able to induce [http://parts.igem.org/Part:BBa_R0079 pLas], resulting in fluorescence values of about 250 a.u.. This is the most severe case of crosstalk observed as the induction of [http://parts.igem.org/Part:BBa_R0079 pLas] by the corresponding inducer and regulator molecule is not significantly different measured by fluorescence as induction by Lux-AHL binding the [http://parts.igem.org/Part:BBa_C0062 LuxR] and subsequently [http://parts.igem.org/Part:BBa_R0079 pLas]. For C4-HSL binding the three regulators [http://parts.igem.org/Part:BBa_C0179 LasR], [http://parts.igem.org/Part:BBa_C0062 LuxR] and [http://parts.igem.org/Part:BBa_C0171 RhlR] and then the [http://parts.igem.org/Part:BBa_R0079 pLas] no crosstalk can be observed.

In this set of experiments the promoter [http://parts.igem.org/Part:BBa_R0071 pRhl] was tested for potential crosstalk. In the top left position we observe the induction of [http://parts.igem.org/Part:BBa_R0071 pRhl] by C4-HSL bound to the [http://parts.igem.org/Part:BBa_C0171 regulator RhlR]. The switching behaviour was observed at a C4-HSL concentration of 1 μM. In the case of 3OC12-HSL binding the [http://parts.igem.org/Part:BBa_C0171 RhlR regulator] and subsequently the [http://parts.igem.org/Part:BBa_R0071 promoter pRhl]insignificant crosstalk has been observed. Severe crosstalk was observed in the case of 3OC6-HSL binding the [http://parts.igem.org/Part:BBa_C0171 RhlR regulator] followed by induction of [http://parts.igem.org/Part:BBa_R0071 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 [http://parts.igem.org/Part:BBa_R0071 pRhl] was detected with 3OC12-HSL binding to the corresponding [http://parts.igem.org/Part:BBa_C0179 LasR regulator] followed by inducing the promoter [http://parts.igem.org/Part:BBa_R0071 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.