Designed by: Anna Knoerlein Group: iGEM15_Marburg (2015-09-18)
Constitutive promoter expressing GFP and constitutive promoter expressing RFP
Two Constitutive Promoters from promoter family of the Berkeley Team 2006 expressing GFP and RFP.
===Usage and Biology===
This part was characterised from the iGEM Team Marburg 2015 as part of their InterLab Study and Measurement Study. It was named Device 6 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 construct 4+5 we used the same promotor as in construct 1+2, they just expressed RFP instead GFP 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
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Usage and Biology
Sequence and Features
Assembly Compatibility:
10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 7 Illegal NheI site found at 30 Illegal NheI site found at 933 Illegal NheI site found at 956
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal AgeI site found at 1542 Illegal AgeI site found at 1654
