Team:BIOTEC Dresden/Characterized Parts/BBa K407014

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Part BBa_K407014

This part was created by the team BIOTEC_Dresden 2010 as a detection device for AHL, which directly includes a normalization system. Basically, this part consists of the part BBa_I13263, in which 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. Bacteria supplied with this plasmid express RFP and after induction with AHL and LuxR, they can additionally produce YFP. By dividing the fluorescent signal of YFP by the fluorescent signal of RFP, one is able to normalize the fluorescence data without considering the cell density.

Results

Figure 1 shows the fluorescence signal of YFP plotted against an increasing AHL concentration. It can be seen that the signal intensity stays constant over the whole time.

Figure 1: The fluorescence of RFP is shown over increasing

AHL concentrations after 2 hours of incubation

Figure 2 is the same as figure 1 except that the fluorescence of YFP is depicted. A logarithmic trend line can be fit to the data with a fair correlation. Anyhow, after normalizing the YFP data with the RFP data, simply by division, we still are able to fit the curve logarithmic but the correlation improves remarkably through normalization, as can be seen in figure 3.

Figure 2: The fluorescence of YFP is shown over increasing

AHL concentrations after 2 hours of incubation

Figure 3: The fluorescence of YFP normalized by division through the fluorescence of

RFP is shown over increasing AHL concentrations after 2 hours of incubation

Figure 4 shows the normalized YFP fluorescence over time for different AHL concentrations. It can be seen that the rate of induction is determined by the AHL concentration and that even concentration of only 50nM already leads to a significant increase in YFP fluorescence.

Figure 4: The fluorescence of YFP normalized by RFP is shown over time for different

AHL concentrations ranging from 0 to 2000nM

Discussion:

The stagnation of the fluorescence signal of the RFP was not expected. Actually, it was considered to rise with the amount of bacteria as they divide and grow during the measurement. The fact that the fluorescence does not increase during the measurement must mean that the cells are not dividing. This leads to the conclusion that the metabolic load through the production of the fluorescent proteins is too high for the cells to divide and the cell number stays almost constant over the two hours that the kinetic measurement takes.

The YFP signal depicted in figure 2 on the other hand behaved as expected. The same behavior could already be detected for the part I13263.

The fact that the correlation coefficient R² improves after the normalization of the fluorescence signal of YFP by division through those of RFP shows that the system our team created works and is useful.

Figure 4 shows that, using this part for the detection of AHL and the novel normalization approach, a sensitive detection system is accomplished. After 2 hours of incubation it can clearly be seen, that different initial concentrations of AHL result in different fluorescece intensities and even such low concentrations as 50nM can be detected.

Materials and methods:

The characterization was performed using a 96-well plate and a fluorescence plate reader, which was kept at 37°C during the whole measurement.. Bacteria supplied with the part BBa_K407014 were suspended in medium of a certain concentration of AHL ranging from 0.01 to 2000nM. The fluorescence of both RFP and YFP was measured every 5 minutes. While for RFP an excitation wavelength of 562 nm and an emission wavelength of 612 nm were used, those for YFP were 485nm and 535nm respectively. As controls the optical density at 612nm and the RFP/YFP fluorescence of uninduced bacteria and LB-medium without cells were measured.

Additional information

For detailed information of the experimental set-up, please have a look at the protocols on our team-wiki. (Link) In case you want to know more about the normalization and negative controls, please have a look at this page: (Link)