Team:ETH Zurich/labblog/20140825

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Latest revision as of 15:10, 11 October 2014

Diffusion Experiments

Monday, 25th August

Now that we tested the different constructs for leakiness, dose response and cross talk, we are investigating in the diffusion rate of AHL. Therefore we designed a chip with chambers connected by channels of different length (see figure). In one chamber we added a strain containing a regulator and a sensor plasmid, to the second chamber we added AHL or an AHL producer strain. As soon as AHL is diffused to the former chamber it induces sfGFP production on the sensor plasmid. The time it takes until we can measure the sfGFP signal and the length of the channel gives us information about the velocity of AHL diffusion.

PDMS millifluidic chip used for diffusion experiments

Three main experiment designs were tested and modified: the agar-design, the liquid-design and the beads-design.

For the agar-design we filled the chamber and channels with LB-agar or in a second attempt with agarose only. Subsequently, we punched a cylindric hole in each chamber. The capacities were filled with regulator/sensor strain culture or AHL/AHL producer strain culture, respectively. After 3 to 4 h at 37 °C we could observe an increase in the sfGFP signal. Since we used comparably high concentration of AHL in our first attempts, we could not detect a delayed sfGFP signal for chambers connected by a longer channel. The experiment will be repeated using lower concentrations. However, conducting the experiment using the agar-design, we encountered a problem: after some hours the LB-agar/agarose started to shrink and dried out. Thus, the agar-design cannot be used without modifications for experiments of longer duration.
In an attempt we only left a small agar fragment in each channel and filled the rest with liquid medium, so as to decrease chance of it drying out, while avoiding at the same time that bacteria from one chamber reach the other chamber.
The bead-design consists of an alginate bead for each chamber and liquid medium in between. The beads either contained regulator/sensor-bacteria or AHL.

Diffusion experiment with agar. Click here to watch the video.

Diffusion experiment with liquid. Click here to watch the video.

Diffusion experiment with beads. Click here to watch the video.

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 == Diffusion Experiments ==
-==== Friday, 15th August ====
+==== Monday, 25th August ====
+Now that we tested the different constructs for leakiness, dose response and cross talk, we are investigating in the diffusion rate of AHL. Therefore we designed a chip with chambers connected by channels of different length (see figure). In one chamber we added a strain containing a regulator and a sensor plasmid, to the second chamber we added AHL or an AHL producer strain. As soon as AHL is diffused to the former chamber it induces sfGFP production on the sensor plasmid. The time it takes until we can measure the sfGFP signal and the length of the channel gives us information about the velocity of AHL diffusion.
+[[File:Channelchip.jpg|center|300px|thumb|PDMS millifluidic chip used for diffusion experiments]]
+Three main experiment designs were tested and modified: the agar-design, the liquid-design and the beads-design.
+* For the agar-design we filled the chamber and channels with LB-agar or in a second attempt with agarose only. Subsequently, we punched a cylindric hole in each chamber. The capacities were filled with regulator/sensor strain culture or AHL/AHL producer strain culture, respectively. After 3 to 4 h at 37 °C we could observe an increase in the sfGFP signal. Since we used comparably high concentration of AHL in our first attempts, we could not detect a delayed sfGFP signal for chambers connected by a longer channel. The experiment will be repeated using lower concentrations. However, conducting the experiment using the agar-design, we encountered a problem: after some hours the LB-agar/agarose started to shrink and dried out. Thus, the agar-design cannot be used without modifications for experiments of longer duration.
+* In an attempt we only left a small agar fragment in each channel and filled the rest with liquid medium, so as to decrease chance of it drying out, while avoiding at the same time that bacteria from one chamber reach the other chamber.
+* The bead-design consists of an alginate bead for each chamber and liquid medium in between. The beads either contained regulator/sensor-bacteria or AHL.
+[[File:ETH_Zurich_Diffusion2.JPG|center|500px|thumb|Diffusion experiment with agar. Click [https://2014.igem.org/Team:ETH_Zurich/Video1 here] to watch the video.]]
+[[File:ETH_Zurich_Diffusion3.JPG|center|500px|thumb|Diffusion experiment with liquid. Click [https://2014.igem.org/Team:ETH_Zurich/Video2 here] to watch the video. ]]
+[[File:ETH_Zurich_Diffusion4.JPG|center|500px|thumb|Diffusion experiment with beads. Click [https://2014.igem.org/Team:ETH_Zurich/Video3 here] to watch the video.]]
-After having constructed the necessary regulator and sensor plasmids we were able to start first cross talk experiments. Therefore we transformed competent cells with different combinations of regulator and sensor plasmids. One example would be the strain siG0024 which contains the regulator plasmid piG0041 producing LuxR under a constitutive promoter and the sensor plasmid piG0051 that encodes for sfGFP. The latter is under control of a pluxR promoter, hence it will be transcribed in the presence of LuxR and Lux.
+{{:Team:ETH_Zurich/tpl/topbutton|green}}
-To assess crosstalk and leakiness we conducted multiple experiments: after adding one of the three AHLs to the cultures we analyzed the raise in sfGFP production using a microtiterplate reader. In a first step we measured the dose response of constructs with different ribosomal binding sites (RBSs) and with or without riboregulator. We could observe differences not only in sensitivity, but also in leakiness; some strains expressed GFP even without AHL induction.
-To give an example: we evaluated the sensitivity of the siG0024 strain to different concentrations of 3OC<sub>6</sub>-HSL (LuxI product) and compared its characteristics to  those of other constructs. Additionally, we measured the sensitivity of siG0024 to 3OC<sub>12</sub>-HSL (LasI product) and C<sub>4</sub>-HSL (RhlI product). Indeed, we could observe the induction of sfGFP transcription by comparably low levels of 3OC<sub>12</sub>-HSL. Crosstalk can be observed between the Las and the Lux system. To simulate a situation most similar to the final experiment, we added the diluted supernatant of AHL producer cells to the test cultures. However, we could not detect any GFP production as reaction to the supernatant. We assume that the AHL concentration is not high enough to induce transcription. By constructing stronger RBSs on the producer plasmids we aim at increasing the AHL production.
-All these informations we will use to design a system tailored to our plans.
-[[File:ETH_Zurich_crosstalk_luxRR12y.png|center|]]
-The figure shows the dose response of xxx to 3OC<sub>12</sub>-HSL, 3OC<sub>6</sub>-HSL and C<sub>4</sub>-HSL. Both 3OC<sub>12</sub>-HSL and 3OC<sub>6</sub>-HSL induce production of GFP, although at different concentrations. This is an example for cross talk between the lux and the las system.
+{{:Team:ETH Zurich/tpl/rmbutton|green|diffusionexp}}
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