Team:ETH Zurich/expresults/qs

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(Quorum Sensing)
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== Quorum Sensing ==
== Quorum Sensing ==
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For our project, we were looking for molecular systems that allow orthogonal cell-to-cell communication to implement connected XOR logic gates (ref project overview). As described above and elsewhere (ref to biotools) we decided to exploited the quorum-sensing systems LuxI/LuxR, LasI/LasR, and RhlI/RhlR (ref to qs parts) in order to achieve this orthogonal cell-to-cell communication. Even though the corresponding inducer molecules are commercially available and the systems often used, in particular in iGEM projects (ref ref), potential cross-talk activity between the different systems may be a severe problem. In order to address that challenge, we have investigated the possible cross-talk on several molecular levels: a) unspecific binding of inducer molecules to one specific regulator (table 1), b) unspecific binding of regulator molecules to one specific promoter sequence (table 2), and c) combinations of both.
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For our Mosia''coli'' project, we were looking for molecular systems that allow orthogonal [https://2014.igem.org/Team:ETH_Zurich/project/background#Quorum_Sensing cell-to-cell communication] in order to implement connected [https://2014.igem.org/Team:ETH_Zurich/modeling/xor#XOR_Logic_Gate XOR logic gates]. We decided to exploit the quorum sensing systems [https://2014.igem.org/Team:ETH_Zurich/data#Gene_Circuit LuxI/LuxR, LasI/LasR, and RhlI/RhlR] in order to achieve the required orthogonal cell-to-cell communication. We developed a [https://2014.igem.org/Team:ETH_Zurich/modeling/qs model] for these cellular information processing. 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 crosstalk activity between the different systems may be a severe problem (e. g. [https://2013.igem.org/Team:Tokyo_Tech/Project/Ninja_State_Switching Tokyo_Tech 2013], [https://2011.igem.org/Team:Peking_S/project/wire/matrix Peking University 2011]).
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PUT TABLE HERE
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In order to address this 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.
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[[File:ETH Zurich Crosstalk.png|1500px|center|thumb|'''Figure 1''' Each quorum sensing system is based on three components: a signaling molecule, a regulatory protein and a promoter. These elements are here ordered into three layers. Cross-talk evaluation can be done by comparing all combinations of those three elements. After collecting the [https://2014.igem.org/Team:ETH_Zurich/expresults experimental data] of all possible pathways, we modeled their influence.]]
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===Summary of experimental results regarding quorum sensing===
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The following matrices serve as an overview summarizing the most significant results of our experiments to characterize crosstalk on different levels.
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On the horizontal top row we see the three different inducer molecules (3OC12-HSL, 3OC6-HSL, C4-HSL). In the top left corner we see the quorum sensing promoter used for all the experiments summarized in this matrix. On the vertical axis we see the three regulators ( [https://2014.igem.org/Team:ETH_Zurich/data#Used_and_Characterized_Pre-Existing_Parts LuxR, LasR, RhlR]).
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These matrices are giving an overview of the experimental results conducted in relation with [https://2014.igem.org/Team:ETH_Zurich/project/background/biotools#Quorum_Sensing 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.
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The solid lines in the graphs show the [https://2014.igem.org/Team:ETH_Zurich/modeling/qs model data], whereas the data points indicated with standard deviation show experimental data in triplicates (mean values of triplicate micro titerplate measurements).
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In the laboratory, 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, and c) a given promoter with both regulator and inducer being unspecific. This gives in total 27 possible combinations. The output was again assessed via sfGFP and measured in terms of fluorescence on microtiter-plate scale. In addition, we assessed both the 'normal' and regulated versions of the quorum sensing modules (see above ref).
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{{:Team:ETH_Zurich/expresults/qs/tab-plux}}
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{{:Team:ETH_Zurich/expresults/qs/tab-plas}}
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{{:Team:ETH_Zurich/expresults/qs/tab-prhl}}
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===Conclusion of crosstalk experiments===
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As shown in the graphs in the matrices above, we found and quantitatively characterized all three levels of crosstalk. The three levels were the following:
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*A given promoter with its corresponding regulator and a different inducer molecule
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*A given promoter with an unspecific regulator and a particular inducer
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*A given promoter with both regulator and inducer being unspecific
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Unspecific inducers binding to the regulators as well as unspecific binding of the regulator to another promoter species was observed in almost all possible combinations.  
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To conclude, we were not able to find an orthogonal quorum sensing pair out of the three systems investigated ([https://2014.igem.org/Team:ETH_Zurich/data#Used_and_Characterized_Pre-Existing_Parts LuxI/LuxR, LasI/LasR, or RhlI/RhlR]). While we see a significant effect when implementing the influence of these crosstalks (on an inducer-, regulator- and promoter-level) in our [https://2014.igem.org/Team:ETH_Zurich/modeling/whole whole cell model], the logic gate still continues to function for a range of inputs at physiological concentrations.
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{{:Team:ETH_Zurich/tpl/topbutton|red}}

Latest revision as of 03:19, 18 October 2014


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. We developed a model for these cellular information processing. 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 crosstalk activity between the different systems may be a severe problem (e. g. Tokyo_Tech 2013, Peking University 2011).


In order to address this 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.

Figure 1 Each quorum sensing system is based on three components: a signaling molecule, a regulatory protein and a promoter. These elements are here ordered into three layers. Cross-talk evaluation can be done by comparing all combinations of those three elements. After collecting the experimental data of all possible pathways, we modeled their influence.


Summary of experimental results regarding quorum sensing

The following matrices serve as an overview summarizing the most significant 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). In the top left corner we see the quorum sensing promoter used for all the experiments summarized in this matrix. On the vertical axis we see the three regulators ( LuxR, LasR, RhlR). These matrices are 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 in triplicates (mean values of triplicate micro titerplate measurements).


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.

ETH Zurich 2014 qs-table CornerLux.png ETH Zurich 2014 qs-table 3OC6-HSL.png ETH Zurich 2014 qs-table 3OC12-HSL.png ETH Zurich 2014 qs-table C4-HSL.png
ETH Zurich 2014 qs-table LuxR.png ETH Zurich 2014 qs-table PluxRef.png ETH Zurich 2014 qs-table PluxLuxRLasAHL.png ETH Zurich 2014 qs-table PluxLuxRRhlAHL.png
ETH Zurich 2014 qs-table LasR.png ETH Zurich 2014 qs-table PluxLasRLuxAHL.png ETH Zurich 2014 qs-table PluxLasRLasAHL.png ETH Zurich 2014 qs-table PluxLasRRhlAHL.png
ETH Zurich 2014 qs-table RhlR.png ETH Zurich 2014 qs-table PluxRhlRLuxAHL.png ETH Zurich 2014 qs-table PluxRhlRLasAHL.png ETH Zurich 2014 qs-table PluxRhlRRhlAHL.png


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.

ETH Zurich 2014 qs-table CornerLas.png ETH Zurich 2014 qs-table 3OC12-HSL.png ETH Zurich 2014 qs-table 3OC6-HSL.png ETH Zurich 2014 qs-table C4-HSL.png
ETH Zurich 2014 qs-table LasR.png ETH Zurich 2014 qs-table PlasRef.png ETH Zurich 2014 qs-table PlasLasRLuxAHL.png ETH Zurich 2014 qs-table PlasLasRRhlAHL.png
ETH Zurich 2014 qs-table LuxR.png ETH Zurich 2014 qs-table PlasLuxRLasAHL.png ETH Zurich 2014 qs-table PlasLuxRLuxAHL.png ETH Zurich 2014 qs-table PlasLuxRRhlAHL.png
ETH Zurich 2014 qs-table RhlR.png ETH Zurich 2014 qs-table PlasRhlRLasAHL.png ETH Zurich 2014 qs-table PlasRhlRLuxAHL.png ETH Zurich 2014 qs-table PlasRhlRRhlAHL.png


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.

ETH Zurich 2014 qs-table CornerRhl.png ETH Zurich 2014 qs-table C4-HSL.png ETH Zurich 2014 qs-table 3OC6-HSL.png ETH Zurich 2014 qs-table 3OC12-HSL.png
ETH Zurich 2014 qs-table RhlR.png ETH Zurich 2014 qs-table PrhlRef.png ETH Zurich 2014 qs-table PrhlRhlRLuxAHL.png ETH Zurich 2014 qs-table PrhlRhlRLasAHL.png
ETH Zurich 2014 qs-table LuxR.png ETH Zurich 2014 qs-table PrhlLuxRRhlAHL.png ETH Zurich 2014 qs-table PrhlLuxRLuxAHL.png ETH Zurich 2014 qs-table PrhlLuxRLasAHL.png
ETH Zurich 2014 qs-table LasR.png ETH Zurich 2014 qs-table PrhlLasRRhlAHL.png ETH Zurich 2014 qs-table PrhlLasRLuxAHL.png ETH Zurich 2014 qs-table PrhlLasRLasAHL.png

Conclusion of crosstalk experiments

As shown in the graphs in the matrices above, we found and quantitatively characterized all three levels of crosstalk. The three levels were the following:

  • A given promoter with its corresponding regulator and a different inducer molecule
  • A given promoter with an unspecific regulator and a particular inducer
  • A given promoter with both regulator and inducer being unspecific

Unspecific inducers binding to the regulators as well as unspecific binding of the regulator to another promoter species was observed in almost all possible combinations. To conclude, we were not able to find an orthogonal quorum sensing pair out of the three systems investigated (LuxI/LuxR, LasI/LasR, or RhlI/RhlR). While we see a significant effect when implementing the influence of these crosstalks (on an inducer-, regulator- and promoter-level) in our whole cell model, the logic gate still continues to function for a range of inputs at physiological concentrations.


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