Team:ETH Zurich/Optimization

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<h1> Different attempts to reduce the leakiness </h1>
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<h1> Evaluation of the leakiness within the receiver cell system </h1>
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<h1> Optimization of LuxR production regulation to reduce basal reporter activation</h1>
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In a first set of experiments we had to find the source of the leakiness in our basic receiver cell system. In the absence of AHL the expression of GFP can either result from an inducer-independent activation of the P<sub>LuxR</sub> promoter or from an AHL-independent activation of the promoter through the LuxR protein. To test for these two possibilities we tested different constructs (Figure 1) in liquid cultures and analyzed the GFP fluorescence with and without induction by AHL over time (data not shown). We saw that the P<sub>Lac</sub>-LuxR-P<sub>LuxR</sub>-GFP reporter system shows significant leakiness in the absence of AHL. We defined the background using a construct without GFP (Figure 1, first construct) and a simple pLuxR-GFP construct without LuxR protein (Figure 1, second construct) to measure the basal expression resulting from promoter leakiness alone. The measured signal was comparable to background levels, suggesting that P<sub>LuxR</sub> promoter per se is remarkably tight.
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[[File:Luxrleakiness.png|left|375px|thumb|<b>Figure 5: </b> Constructs tested in liquid culture to identify the different sources of leakiness.]][[File:Leakinessgraph.png|right|650px|thumb|<b>Figure 6: </b> Identification of the source of leakiness in the GFP receiver construct.]]
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The complete P<sub>Lac</sub>-LuxR-P<sub>LuxR</sub>-GFP receiver construct (Figure 1, third construct) in the absence of AHL induction shows an increase in the measured GFP levels. Addition of AHL (Figure 1, fourth construct) was used to show the On state of the system. These preliminary results suggested that <b>most of the leakiness comes from an activation of the P<sub>LuxR</sub> promoter through LuxR alone, meaning in the absence of the AHL inducer</b>.  We concluded that a reduction of the LuxR dependent basal activation could significantly improve the On/Off Ratio and tested different strategies to solve the problem.
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[[File:ETHZ luxrleakiness.png|center|850px|thumb|<b>Figure 1: Constructs tested in liquid culture to investigate the different sources of leakiness.</b>]]
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<h1> Introduction of a positive feedback loop to reduce LuxR levels </h1>
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As described in the [https://2013.igem.org/Team:ETH_Zurich/Circuit circuit optimization] part we tried to lower the amount of LuxR present in the uninduced state by testing two different positive feedback loop motives (Figure 2). The constructs were tested in LB liquid culture over time and compared to the basic receiver cell circuit with constitutive expression of LuxR. The experiment was conducted over 16 h using the TECAN plate reader. The values for GFP fluorescence were normalized to the OD<sub>600</sub>.
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[[File:ETHZ feedbackloopstrategies.png|center|600px|thumb|<b>Figure 2: Possible strategies to reduce basal LuxR expression using positive feedback loops.</b>]]
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[[File:ETHZ feedbackgraph.png|center|850px|thumb|<b>Figure 3:  Analysis of the non-optimized circuit and the two feedback strategies in liquid culture experiment.</b> The circuits tested correspond to the ones described in Figure 3. The experiment was conducted over 16 h in liquid cultures with/without induction through 100 nM AHL. The measured GFP fluorescence was normalized to the OD<sub>600</sub>. Experiments were done in triplicates.]]
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[[File:ETHZ feedbackstrategiesgraphOnOff.png|left|600px|thumb|<b>Figure 4:  Comparison of the max. observed On/Off ratios using either one of the positive feedback loop strategies.</b> The On/Off ratios were obtained by dividing the normalized fluorescence in the induced state by the fluorescence of the uninduced circuit (Figure 3). The max. value reached within measuring time was taken for comparison.]]
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We can clearly see from the data that the direct feedback loop were LuxR is put under its own promoter does not work (Figure 3). There is no difference visible between the induced and the uninduced state. We assume that the high plasmid copy number of the construct and the resulting high amount of LuxR could be the reason. The second strategy with the indirect feedback proved to work very well instead. <b>Comparing the maximal On/Off ratios achieved during the time course, we can see a two-fold improvement of the indirect feedback loop over the non-optimized circuit </b>(Figure 4). The reason for the high On/Off ratio is a almost complete reduction of the basal GFP expression.
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The GFP reporter system shows significant leakiness in the absence of OHHL. To pinpoint the source of this basal activation we tested the GFP expression of different constructs in liquid culture over time (Figure 6). We defined the background using a construct without GFP (Figure 5, top left). We used a simple pLuxR-GFP construct without LuxR protein (Figure 5, top right) to measure the basal expression resulting from promoter leakiness alone. The measured signal is comparable to background levels, suggesting that pLuxR promoter per se is remarkably tight.
 
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The complete pLac-Lux-pLuxR-GFP receiver construct (Figure 5, bottom left) in the absence of OHHL induction shows a jump in the measured GFP levels. This results suggest that most of the leakiness comes from an activation of the pLux promoter through LuxR alone, meaning in the absence of the OHHL inducer (Figure 3).
 
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Addition of OHHL (Figure 5, bottom right) determines a steep 5-7 times GFP induction. Reduction of the LuxR dependent basal activation could significantly improve the On/Off Ratio.
 
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<h1> Fine-tuning of the P<sub>Lac</sub> driven LuxR using glucose </h1>
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The second approach that we tested experimentally is the use of glucose to repress expression of LuxR. We tested the simple GFP receiver construct under different concentrations of glucose in LB liquid culture with and without induction by 100 nM AHL. The experiment was set up similar to the one described above. From the results we can conclude that the repression of LuxR is increased at higher glucose concentrations. With 1% glucose the repression is so strong, that no full induction of the system is possible any more. We also see that the repression leads to a similar reduction in GFP fluorescence with and without AHL. Therefore the <b>use of glucose does not change the ratio between the On and Off state</b> and can probably not be used to finetune our circuit.
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[[File:ETHZ glucoseleakinessgraph.png|center|850px|thumb|<b>Figure 5:  The influence of different glucose concentrations on the leakiness of the receiver cell circuit.</b> Glucose concentrations range from 0% to 1%. The experiment was conducted over 16 h in liquid cultures with/without induction through 100 nM AHL. The measured GFP fluorescence was normalized to the OD<sub>600</sub>. Experiments were done in triplicates.]]
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[[File:ETHZ glucoseleakinessonoffgraph.png|center|850px|thumb|<b>Figure 6: Comparison of the max. observed On/Off ratios with different concentrations of glucose.</b> The On/Off ratios were obtained by dividing the normalized fluorescence in the induced state by the fluorescence of the uninduced circuit (Figure 5). The max. value reached within measuring time was taken for comparison.]]
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<br>Hydrolase reporters are enzymatic systems. Multiple rounds of catalysis provide intrinsic signal amplification when compared to GFP. Therefore we expect a very high sensitivity to basal expression. Moderate hydrolase expression level could already result in total substrate conversion and signal saturation. Since our model anticipates the potential problem we designed different strategies to reduce the level of LuxR in the uninduced state (Figure 7).  
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The effect of glucose was also tested with a experimental set-up on plates (Figure 7). A P<sub>Lac</sub>-LuxR-P<sub>LuxR</sub>-LacZ receiver construct was used in a DH5α ''E.coli'' strain to avoid background LacZ activity. The plates contain the substrate for LacZ, X-Gal. The influence of different concentrations of glucose was tested in both the induced state with AHL and without induction. The picture was taken after overnight incubation of the plates. Like in the liquid culture experiment described above no difference is visible with or without AHL.
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The overall idea was to replace a constitutive LuxR expression with an OHHL dependent induction. In a self-regulation motif only a residual amount of LuxR is present in the non induced state, minimizing its interaction with the promoter. Upon induction the protein sustain its own production allowing for a full activation of the system. To achieve autoregulation one possibility is to use a direct positive feedback loop in which the luxR gene is placed under it's own pLuxR promoter. Only upon induction with OHHL LuxR would be produced. As an alternative we could use LacI expression to inhibit LuxR production in the uninduced state. By placing the lacI gene under a negatively LuxR/OHHL regulated promoter (pLuxL) an indirect positive autoregulation for LuxR expression is obtained.
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[[File:ETHZ J09855-LacZ Glucose AHL test.png|center|850px|thumb|<b>Figure 7: Test of a P<sub>Lac</sub>-LuxR-P<sub>LuxR</sub>-LacZ construct on plates with different concentrations of glucose.</b> Comparison of the induced (+AHL) and uninduced plates shows no difference of reporter expression. LacZ was visualized using the X-Gal substrate in the plate. The picture was taken after overnight incubation.]]
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[[File:LuxRfeedbackstrategies.png|left|600px|thumb|<b>Figure 7: </b> Possible strategies to reduce basal LuxR expression using positive feedback loops.]]
 
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Latest revision as of 02:35, 29 October 2013

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Contents

Evaluation of the leakiness within the receiver cell system

In a first set of experiments we had to find the source of the leakiness in our basic receiver cell system. In the absence of AHL the expression of GFP can either result from an inducer-independent activation of the PLuxR promoter or from an AHL-independent activation of the promoter through the LuxR protein. To test for these two possibilities we tested different constructs (Figure 1) in liquid cultures and analyzed the GFP fluorescence with and without induction by AHL over time (data not shown). We saw that the PLac-LuxR-PLuxR-GFP reporter system shows significant leakiness in the absence of AHL. We defined the background using a construct without GFP (Figure 1, first construct) and a simple pLuxR-GFP construct without LuxR protein (Figure 1, second construct) to measure the basal expression resulting from promoter leakiness alone. The measured signal was comparable to background levels, suggesting that PLuxR promoter per se is remarkably tight. The complete PLac-LuxR-PLuxR-GFP receiver construct (Figure 1, third construct) in the absence of AHL induction shows an increase in the measured GFP levels. Addition of AHL (Figure 1, fourth construct) was used to show the On state of the system. These preliminary results suggested that most of the leakiness comes from an activation of the PLuxR promoter through LuxR alone, meaning in the absence of the AHL inducer. We concluded that a reduction of the LuxR dependent basal activation could significantly improve the On/Off Ratio and tested different strategies to solve the problem.

Figure 1: Constructs tested in liquid culture to investigate the different sources of leakiness.

Introduction of a positive feedback loop to reduce LuxR levels

As described in the circuit optimization part we tried to lower the amount of LuxR present in the uninduced state by testing two different positive feedback loop motives (Figure 2). The constructs were tested in LB liquid culture over time and compared to the basic receiver cell circuit with constitutive expression of LuxR. The experiment was conducted over 16 h using the TECAN plate reader. The values for GFP fluorescence were normalized to the OD600.

Figure 2: Possible strategies to reduce basal LuxR expression using positive feedback loops.
Figure 3: Analysis of the non-optimized circuit and the two feedback strategies in liquid culture experiment. The circuits tested correspond to the ones described in Figure 3. The experiment was conducted over 16 h in liquid cultures with/without induction through 100 nM AHL. The measured GFP fluorescence was normalized to the OD600. Experiments were done in triplicates.


Figure 4: Comparison of the max. observed On/Off ratios using either one of the positive feedback loop strategies. The On/Off ratios were obtained by dividing the normalized fluorescence in the induced state by the fluorescence of the uninduced circuit (Figure 3). The max. value reached within measuring time was taken for comparison.

We can clearly see from the data that the direct feedback loop were LuxR is put under its own promoter does not work (Figure 3). There is no difference visible between the induced and the uninduced state. We assume that the high plasmid copy number of the construct and the resulting high amount of LuxR could be the reason. The second strategy with the indirect feedback proved to work very well instead. Comparing the maximal On/Off ratios achieved during the time course, we can see a two-fold improvement of the indirect feedback loop over the non-optimized circuit (Figure 4). The reason for the high On/Off ratio is a almost complete reduction of the basal GFP expression.


Fine-tuning of the PLac driven LuxR using glucose

The second approach that we tested experimentally is the use of glucose to repress expression of LuxR. We tested the simple GFP receiver construct under different concentrations of glucose in LB liquid culture with and without induction by 100 nM AHL. The experiment was set up similar to the one described above. From the results we can conclude that the repression of LuxR is increased at higher glucose concentrations. With 1% glucose the repression is so strong, that no full induction of the system is possible any more. We also see that the repression leads to a similar reduction in GFP fluorescence with and without AHL. Therefore the use of glucose does not change the ratio between the On and Off state and can probably not be used to finetune our circuit.

Figure 5: The influence of different glucose concentrations on the leakiness of the receiver cell circuit. Glucose concentrations range from 0% to 1%. The experiment was conducted over 16 h in liquid cultures with/without induction through 100 nM AHL. The measured GFP fluorescence was normalized to the OD600. Experiments were done in triplicates.
Figure 6: Comparison of the max. observed On/Off ratios with different concentrations of glucose. The On/Off ratios were obtained by dividing the normalized fluorescence in the induced state by the fluorescence of the uninduced circuit (Figure 5). The max. value reached within measuring time was taken for comparison.

The effect of glucose was also tested with a experimental set-up on plates (Figure 7). A PLac-LuxR-PLuxR-LacZ receiver construct was used in a DH5α E.coli strain to avoid background LacZ activity. The plates contain the substrate for LacZ, X-Gal. The influence of different concentrations of glucose was tested in both the induced state with AHL and without induction. The picture was taken after overnight incubation of the plates. Like in the liquid culture experiment described above no difference is visible with or without AHL.

Figure 7: Test of a PLac-LuxR-PLuxR-LacZ construct on plates with different concentrations of glucose. Comparison of the induced (+AHL) and uninduced plates shows no difference of reporter expression. LacZ was visualized using the X-Gal substrate in the plate. The picture was taken after overnight incubation.