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Team:BIOTEC Dresden/Results
From 2010.igem.org
1. 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..
2. Novel Normalization method
The detection of AHL for SensorBricks was accomplished using fluorescent proteins. As further described in the protocols, we measured the development of fluorescence within the bacteria carrying a AHL detection device on a plasmid over 2 hours at 37°C. At this temperature the bacteria are expected to grow and divide. Since every bacteria develops fluorescence when AHL is present, a normalization of the fluorescence by the amount of cells is required. Otherwise it is not clear if a raise in signal is due to an induction of transcription or is just based on the increasing amount of cells.That is why after every measurement of the fluorescence, the optical density of the bacteria solution at 612nm was taken that can be correlated to the amount of bacteria within the solution. In the beginning, the fluorescence raw data were divided by the optical density for normalization. This calculation led often to a negative slope of the fluorescence intensity over time, that could not be logically explained. Using this normalization method, no useful information about the transcriptional induction by AHL could be achieved.
Therefore a new normalization method was introduced based on the following system: The newly assembled part consists of the part BBa_I13263 fused to BBa_J04450. In the first one, LuxR is constitutively expressed. In the presence of AHL a LuxR-AHL complex is formed activating lux pR and thereby the expression of eyfp. Our team added the part BBa_J04450 to this part, which constitutively expresses RFP in the absence of lactose. Bacteria supplied with this plasmid express RFP and after induction with AHL and LuxR, they can additionally produce YFP. The amount of RFP fluorescence is expected to bedirectly proportional to the amount of bacteria. Consequently, by dividing the fluorescence signal of YFP by the fluorescent signal of RFP, one is able to normalize the fluorescence data without considering the cell density.
To learn more about this designed BioBrick have a look at the characterization of this part here.
