Designed by: Anna Knoerlein Group: iGEM15_Marburg (2015-09-18)
This part was characterised from the iGEM Team Marburg 2015 as part of their InterLab Study and Measurement Study. It was named Device 2 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 .
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 2: Growth curve.
The device was also characterized through flow cytometry.
Figure 3: Flow cytometry
Single cell microscopy pictures gave us both an expression on the variability of gene expression levels as well as on the shape of the cells. If the cell shape varies from the empty strain, this usually gives a hint on the burden of a construct.
Figure 4:Microscopy
Evolution is an aspect that is usually not taken into account when characterizing parts and constructs as constructs are normally used for short-term experiments. But as constructs might also be used for different purposes, long-term robustness of genetic devices needs to be ensured. We hence cultured our strains carrying construct 1-3 for 48h and measured the fluorescene intensity by flow cytometry every 12h and inoculated the strains in new flasks. For construct 2, one colony is reduced in fluorescence. The data for the last construct are not shown, as the fluorescence intensity didn’t change over our measurement period. In order to unravel the underlying reason for the decrease in fluorescence we sent extracted plasmids from each construct and replica for sequencing. The promoter sequence of the constructs, that display reduced fluorescence, mutated, which most probably lead to the reduction of fluorescence protein expression. The strains still expression at previously observed levels didn’t show any mutations in the promoter region.
Figure 5:Evolution
