Team:ETH Zurich/Experimentalresults

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<h1> Experimental results overview </h1>
<h1> Experimental results overview </h1>
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<p = align"justify">In our game, we use the LuxI-LuxR quorum sensing system to [https://2013.igem.org/Team:ETH_Zurich/Modeling/Reaction_Diffusion_OOHL transport the signal] from the sender (mine) to the receiver (non-mine) cells. We started experiments using LuxI producing sender cells and OHHL inducible GFP expressing receiver cells. We used this sender-receive module to characterize [https://2013.igem.org/Team:ETH_Zurich/Experiments_2 OHHL diffusion] in agar plates. The main goal of these experiments was to find conditions regarding time and distance for the design of the game grid. Having set these parameters, we tried to construct a library of pLux promoters with OHHL sensitivities differing from those of the wild type pLux. Through site-saturation [https://2013.igem.org/Team:ETH_Zurich/Experiments_5#high-pass-filter_mutagenesis promoter mutagenesis] we changed the LuxR binding sites. The promoter variants were characterized studying OHHL dose response curves and we were able to select for two mutant variants.</p>
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<p = align"justify">In our game, we use the <b>LuxI-LuxR quorum sensing system</b> to [https://2013.igem.org/Team:ETH_Zurich/Modeling/Reaction_Diffusion_OOHL transport the signal] from the sender (mine) to the receiver (non-mine) cells. We started experiments using LuxI producing sender cells and AHL inducible GFP expressing receiver cells. We used this sender-receive module to characterize [https://2013.igem.org/Team:ETH_Zurich/Experiments_2 AHL diffusion] in agar plates. The main goal of these experiments was to find conditions regarding <b>time and distance</b> for the design of the game grid. Having set these parameters, we tried to construct a <b>library of P<sub>LuxR</sub></b> promoters with AHL sensitivities differing from those of the wild type P<sub>LuxR</sub>. Through site-saturation [https://2013.igem.org/Team:ETH_Zurich/Experiments_5#high-pass-filter_mutagenesis promoter mutagenesis] we changed the LuxR binding sites. The promoter variants were characterized studying AHL <b>dose response curves</b> and we were able to select for several mutant variants.</p>
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<p>In parallel we characterized our hydrolase [https://2013.igem.org/Team:ETH_Zurich/Experiments_3 reporter system] by testing different substrates and investigating kinetics. During all of these experiments we encountered problems with the leakiness of the pLux promoter meaning that there was basal expression of reporter even in the absence of OHHL. By testing different LuxR generating constructs we tried to minimize the [https://2013.igem.org/Team:ETH_Zurich/Experiments_6 leakiness] of the system, because we expect our enzymatic reporter system to be even more sensitive than the fluorescent protein reporters. So far we have successfully set up the game structure with the wild type pLux promoter and a GFP reporter. We have characterized one possible promoter variant with a much lower sensitivity for OHHL and we successfully used five different hydrolases without observing crosstalk. Using a positive feedback loop we could also reduce the basal expression of LuxR and therefore the leakiness of the system</p>
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<p>In parallel <b>characterized our hydrolases</b> [https://2013.igem.org/Team:ETH_Zurich/Experiments_3 reporter system] by testing [https://2013.igem.org/Team:ETH_Zurich/Experiments_3 different substrates and investigating kinetics]. During all of these experiments we encountered problems with the <b>leakiness of the P<sub>LuxR</sub> promoter</b>, meaning that there was basal expression of reporter even in the absence of AHL. By testing different LuxR generating constructs we tried to <b>minimize the [https://2013.igem.org/Team:ETH_Zurich/Experiments_6 leakiness]</b> of the system, because we expect our enzymatic reporter system to be even more sensitive than the fluorescent protein reporters. So far we have [https://2013.igem.org/Team:ETH_Zurich/Experiments_6 successfully set up] the game structure with the wild type P<sub>LuxR</sub> promoter and a GFP reporter and also a <b>GFP-RFP based game</b>. We have characterized one possible promoter variant with a much lower sensitivity for AHL and we successfully used five different hydrolases without observing crosstalk. For the presentation we will have the final game with the hydrolases.</p>
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Latest revision as of 19:12, 28 October 2013

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Experimental results overview

In our game, we use the LuxI-LuxR quorum sensing system to transport the signal from the sender (mine) to the receiver (non-mine) cells. We started experiments using LuxI producing sender cells and AHL inducible GFP expressing receiver cells. We used this sender-receive module to characterize AHL diffusion in agar plates. The main goal of these experiments was to find conditions regarding time and distance for the design of the game grid. Having set these parameters, we tried to construct a library of PLuxR promoters with AHL sensitivities differing from those of the wild type PLuxR. Through site-saturation promoter mutagenesis we changed the LuxR binding sites. The promoter variants were characterized studying AHL dose response curves and we were able to select for several mutant variants.


In parallel characterized our hydrolases reporter system by testing different substrates and investigating kinetics. During all of these experiments we encountered problems with the leakiness of the PLuxR promoter, meaning that there was basal expression of reporter even in the absence of AHL. By testing different LuxR generating constructs we tried to minimize the leakiness of the system, because we expect our enzymatic reporter system to be even more sensitive than the fluorescent protein reporters. So far we have successfully set up the game structure with the wild type PLuxR promoter and a GFP reporter and also a GFP-RFP based game. We have characterized one possible promoter variant with a much lower sensitivity for AHL and we successfully used five different hydrolases without observing crosstalk. For the presentation we will have the final game with the hydrolases.