Designed by: Anna Knoerlein Group: iGEM15_Marburg (2015-09-18)
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Applications of BBa_K1650006
This part was characterised from the iGEM Team Marburg 2015 as part of their InterLab Study and Measurement Study. It was named Device 7 in the further characterisation.
First, the fluorescence was measured in the stationary phase, diluted to an OD of 0,5. As described on the registry page of the constitutive promoter family we used construct 1 to 3 with decreasing promoter activity. In most cases, the normalized promoter activity was higher in MG1655 than in DH5alpha.Taken together, we show that the chassis plays an important role for the characterization of a construct. In order to provide a comprehensive characterization/analysis we used both, the commonly used cloning strain DH5alpha as well as the wildtype strain MG1655 for our further studies.
Figure 1: Fluorescence at the stationary phase .Figure 2: Fluorescence at the stationary phase .
The timelapse measurement of our constructs reveals that fluorescence intensity increases over time. The used fluorescent proteins are not fused to a degradation tag, which may have led to an accumulation of GFP and RFP and hence to an increase of fluorescence intensity. In accordance with the one-point measurements in the plate reader, we observed a variation between the two different chassis. Also the expression levels and the ratios of fluorescence intensities are consistent.
Figure 3: GFP over time .Figure 4: RFP over time.
The device was also characterisated throught flow cytometry.
Figure 5: Flow cytometry
Usually, platereader results are normalized over the OD. A more prize method is the normalization of the fluorescence intensities over an internal standard. With the internal standard we can reduce the variation of metabolic state of the cells, cell age and plasmid copy number of our fluorescent output. Therefore we constructed our double constructs 6 and 7, which have both the promoters of construct 1 and 2 for the expression of GFP and an additional promoter from the same promoter family, expressing RFP. The results for this normalization can be found in Fig. 8. Construct 6 showed reduced fluorescence of RFP, therefore we only focused on construct 7. This construct is a composite part and can be used as a standard for promoter characterization, as it allows a more precise normalization of fluorescence intensities of the promoter of interest and generate more reliable data. You can find some of our recommendations below. Here we show as an example the differences in normalization over OD and an internal standard for construct 7.
Figure 6: Fluorescence normalized over RFP and OD.
