Team:BIOTEC Dresden/Results

From 2010.igem.org

(Difference between revisions)
Line 21: Line 21:
<h3>Materials and methods</h3>
<h3>Materials and methods</h3>
<p>The characterization was performed in a fluorescence plate reader using a 96 well plate, Which was maintained at 37°C during the whole measurement.In order to quantify and compare the amount of AHL produced by the sender, the amount of these bacteria as well as the final volume are kept constant while the amount of receiver cells are varied. The bacterial suspensions were both set to an optical density of 0.4 at 600nm. Furthermore, we detected the induction of the receiver cells with known concentrations of AHL to have a calibrating system. As a negative control and as an information source of the cell growth we also measured the sender and the receiver alone. As an additional negative control, we also measured the fluorescence of only the sender, of only the receiver, as well of the pure medium.</p>
<p>The characterization was performed in a fluorescence plate reader using a 96 well plate, Which was maintained at 37°C during the whole measurement.In order to quantify and compare the amount of AHL produced by the sender, the amount of these bacteria as well as the final volume are kept constant while the amount of receiver cells are varied. The bacterial suspensions were both set to an optical density of 0.4 at 600nm. Furthermore, we detected the induction of the receiver cells with known concentrations of AHL to have a calibrating system. As a negative control and as an information source of the cell growth we also measured the sender and the receiver alone. As an additional negative control, we also measured the fluorescence of only the sender, of only the receiver, as well of the pure medium.</p>
-
<p>For a more detailed description of the experimental setup, please have a look at our protocols page (link).</p>
+
<p>For a more detailed description of the experimental setup, please have a look at our protocols page <a href="http://2010.igem.org/Team:BIOTEC_Dresden/Protocols:Cocultivation_assay"> here.</a.</p>
<div class="visualClear"></div>
<div class="visualClear"></div>
</div>
</div>

Revision as of 01:05, 28 October 2010

Results

Cocultivation assay

As a proof of concept it is shown possible to detect a fluorescence signal when cocultivating one bacterial strain producing AHL and one strain detecting it. For this purpose we supplied one strain with a plasmid coding for the part BBa_J23039 which leads to the constitutive expression of LuxI, the enzyme that converts SAM into AHL. This part functions as a AHL sender part in the assay. Another strain was prepared containing a plasmid with the part reporter system BBa_I13263 that produces YFP when AHL is around. This part is considered to be the AHL-detector in the assay.

Theoretically, by cocultivating sender and receiver strains, the AHL is being produced by the sender cells and diffuses through the membrane into the surrounding where it finally can be taken-up by the receiver cells and lead to the development of a fluorescence signal.

Results

In figure 1 the fluorescence signal of YFP is shown. As already noticed in many of our characterization assays, the LB shows background fluorescence. The error bars show the standard deviation of average values of eight measurements we run. The fluorescence of the sender (BBa_J23039 ) and the receiver (BBa_I13263) alone is not much higher than the one of the LB and the cells can thus be considered as non-fluorescent. The system was set to 100% fluorescence. To calibrate the system, the receiver strain was induced with 500nM AHL concentration. Relative to this fluorescent signal, the four dilutions can be discussed. The ratio depicted at the x-axis is the ratio of receiver (volume kept constant) to the volume of sender.

Discussion

The fact that we can detect a fluorescent signal that is proportional to the amount of sender cells confirms the hypothesis that LuxI catalytically converts SAM to AHL and that the messenger molecule AHL can diffuse through the membrane of the sender cells into the solution and finally into the receiver cells, where it binds LuxR and then induces the transcription and expression of YFP. It is clearly visible, that the induction of YFP transcription depends on the amount of sender cells producing LuxI.

Materials and methods

The characterization was performed in a fluorescence plate reader using a 96 well plate, Which was maintained at 37°C during the whole measurement.In order to quantify and compare the amount of AHL produced by the sender, the amount of these bacteria as well as the final volume are kept constant while the amount of receiver cells are varied. The bacterial suspensions were both set to an optical density of 0.4 at 600nm. Furthermore, we detected the induction of the receiver cells with known concentrations of AHL to have a calibrating system. As a negative control and as an information source of the cell growth we also measured the sender and the receiver alone. As an additional negative control, we also measured the fluorescence of only the sender, of only the receiver, as well of the pure medium.

For a more detailed description of the experimental setup, please have a look at our protocols page here.