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From 2014.igem.org

Quorum Sensing

For our Mosiacoli project, we were looking for molecular systems that allow orthogonal cell-to-cell communication in order to implement connected XOR logic gates. We decided to exploit the quorum-sensing systems LuxI/LuxR, LasI/LasR, and RhlI/RhlR in order to achieve the required orthogonal cell-to-cell communication. Even though the corresponding inducer molecules are commercially available and the systems often used, in particular in iGEM projects (e.g. [http://parts.igem.org/Part:BBa_R0062 pLux (BBa_R0062)], '[http://parts.igem.org/Frequently_Used_Parts Top 10 Most used promoters]' with 246 uses), potential cross-talk activity between the different systems may be a severe problem (e.g. Tokyo_Tech 2013).

In order to address that challenge, we measured a) a given promoter with its corresponding regulator and a different inducer molecule, b) a given promoter with an unspecific regulator and a particular inducer, c) a given promoter with both regulator and inducer being unspecific, and always included the correct combination of inducer molecule, regulator and promoter as a positive control. This gives in total 27 possible combinations. The output was assessed via sfGFP and measured in terms of fluorescence on microtiter-plate scale.

Summary of experimental results regarding quorum sensing

The following matrices serve as on overview summarizing the results of our experiments to characterize crosstalk on different levels. On the horizontal top row we see the three different inducer molecules (3OC12-HSL, 3OC6-HSL, C4-HSL). On the vertical axis we see the three regulators (LuxR, LasR, RhlR). These matrices aim at giving an overview of the experimental results conducted in relation with quorum sensing and crosstalk. The graph shown in each matrix on the very top left describes the situation where the correct autoinducer molecule has bound the corresponding regulator and this complex has then induced the correct promoter. The solid lines in the graphs show the model data whereas the data points indicated with standard deviation show experimental data.

Table 1 Crosstalk matrix for the promoter pLux ([http://parts.igem.org/Part:BBa_R0062:Experience BBa_R0062])

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.

In all the measurements conducted to create this matrix the 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 pLux is induced by 3OC6-HSL (Lux-AHL) binding to the corresponding LuxR regulator. The observed transition occurs at a concentration of approximately 1 nM 3OC6-HSL. The light-blue curve plotted shows pLux induced by 3OC6-HSL (Lux-AHL) binding to the corresponding LuxR regulator from modeling data. This curve from the model and the dark blue data point gained in experiments were plotted as a reference in all the other graphs describing pLux.

Crosstalk can be observed for the cases where the 3OC12-HSL (Las-AHL) binds the LuxR regulator. Additionally for 3OC12-HSL binding to its corresponding regulator LasR and then binding to the pLux as seen in the middle of the top row and center of the matrix. For the case of Las-AHL binding the LasR and subsequently the promoter pLux, the transition occurs at 1 nM and reaches 0.5 fold the fluorescence as pLux induced by 3OC6-HSL binding LuxR. In the case of 3OC12-HSL binding LuxR and inducing the promoter pLux, the transition is observed at approximately 100nM and severe crosstalk is observed.

The promoter pLux was not tested in combination with the RhlR regulator and the C4-HSL.

Observation of C4-HSL has shown, that there is no significant crosstalk with the LuxR and LasR regulators binding C4-HSL and subsequently to pLux. This is indicated on top right and middle right graphs.

Table 2 Crosstalk matrix for the promoter plas ([http://parts.igem.org/Part:BBa_R0079:Experience BBa_R0079])

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.

The promoter of interest of the experiments summarized in this matrix is pLas. The graph on top left corner shows the induction of pLux by 3OC12-HSL binding the corresponding LasR. The red line shows the model whereas the cross shown in red represent the experimental data. The transition can be observed at a concentration of Las-AHL of about 5 nM.

For C4-HSL binding the three regulators LasR, LuxR and RhlR and then the pLas no crosstalk can be observed. Also 3OC6-HSL binding RhlR does not induce the pLas. For the binding of 3OC12-HSL to RhlR a negligible increase of fluorescence can be observed. The same can be observed for 3OC12-HSL binding to the LuxR as this combination is to a small degree inducing pLas.

The interesting case of crosstalk when observing 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 LuxR regulator is able to induce pLas to fluorescence values of about 250. This is the phenomenon of severe crosstalk as the induction of pLas by the corresponding inducer and regulator molecule is nearly on the same level measured by fluorescence as induction by Lux-AHL binding the LuxR and subsequently pLas.

Table 3 Crosstalk matrix for the promoter prhl ([http://parts.igem.org/Part:BBa_I14017:Experience BBa_I14017])

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.

In this matrix the results of experiments with the promoter pRhl are summarized. The solid green line shows the modeling data whereas the cross represent experimental data. In the top left corner we see the pRhl induced by the corresponding C4-HSL bound to RhlR. Transition occurs at a concentration of 1 micromolar of CS4-HSL. When observing pRhl we see crosstalk when inducing the promoter with 3OC6-HSL binding to RhlR for induction of pRhl. In this case the switching behaviour is observable at a concentration of 1 micro molar of 3OC6-HSL. The other observed crosstalk is the binding of 3OC12-HSL to LasR with subsequent induction of the promoter pRhl with a transition at 1nM of 3OC12-HSL.

Conclusion of crosstalk experiments

As shown in the graphs in the matrices above, we found and quantitatively characterized all three levels of crosstalk described previously. Unspecific inducer binding to the regulators as well as unspecific binding of the regulator to the promoter occurred in almost all possible combinations. To conclude, we were not able to find an orthogonal quorum sensing pair due to inevitable crosstalk between the systems employing LuxI/LuxR, LasI/LasR, or RhlI/RhlR.