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 experiment 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 over time with the TECAN plate reader. The experiments were conducted in triplicates and for activation 100nM AHL was used. Levels of fluorescence were normalized to the OD600.
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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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[[File:ETHZ luxrleakiness.png|center|850px|thumb|<b>Figure 1: </b> Constructs tested in liquid culture to investigate the different sources of leakiness.]]
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<br><br>The pLac-LuxR-pLuxR-GFP reporter system showed significant leakiness in the absence of AHL. We defined the background using a construct without GFP (Figure 1, first construct). We used 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 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 1, third construct) in the absence of AHL induction shows a jump in the measured GFP levels. This results suggest that <b>most of the leakiness comes from an activation of the pLux promoter through LuxR alone, meaning in the absence of the AHL inducer</b> (Figure 3).
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Addition of AHL (Figure 1, fourth construct) determines a steep 5-7 times GFP induction. We concluded, that a reduction of the LuxR dependent basal activation could significantly improve the On/Off Ratio.
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[[File:Leakinessgraph.png|center|850px|thumb|<b>Figure 2: </b> Identification of the source of leakiness in the GFP receiver construct.]]
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<h1> Introduction of a negative 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 testing two different positive feedback loop motives.
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[[File:LuxRfeedbackstrategies.png|center|600px|thumb|<b>Figure 3: </b> Possible strategies to reduce basal LuxR expression using positive feedback loops.]]
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[[File:ETHZ feedbackgraph.png|center|850px|thumb|<b>Figure 4: </b> Analysis of the two feedback strategies in liquid culture experiment.]]
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<h1> Fine-tuning of the pLac driven LuxR using glucose </h1>
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<h1> Destabilization of the enzymatic reporters with degradation tags </h1>
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The GFP reporter system shows significant leakiness in the absence of AHL. 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 AHL 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 AHL inducer (Figure 3).
 
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Addition of AHL (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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<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 overall idea was to replace a constitutive LuxR expression with an AHL 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 AHL 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/AHL regulated promoter (pLuxL) an indirect positive autoregulation for LuxR expression is obtained.
 
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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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Revision as of 20:33, 25 October 2013

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Contents

Evaluation of the leakiness within the receiver cell system

In a first experiment 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 over time with the TECAN plate reader. The experiments were conducted in triplicates and for activation 100nM AHL was used. Levels of fluorescence were normalized to the OD600.

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



The pLac-LuxR-pLuxR-GFP reporter system showed significant leakiness in the absence of AHL. We defined the background using a construct without GFP (Figure 1, first construct). We used 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 is comparable to background levels, suggesting that pLuxR promoter per se is remarkably tight. The complete pLac-Lux-pLuxR-GFP receiver construct (Figure 1, third construct) in the absence of AHL 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 AHL inducer (Figure 3). Addition of AHL (Figure 1, fourth construct) determines a steep 5-7 times GFP induction. We concluded, that a reduction of the LuxR dependent basal activation could significantly improve the On/Off Ratio.

Figure 2: Identification of the source of leakiness in the GFP receiver construct.

Introduction of a negative 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 testing two different positive feedback loop motives.

Figure 3: Possible strategies to reduce basal LuxR expression using positive feedback loops.


Figure 4: Analysis of the two feedback strategies in liquid culture experiment.

Fine-tuning of the pLac driven LuxR using glucose

Destabilization of the enzymatic reporters with degradation tags





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