Team:Bielefeld-CeBiTec/InterlabStudy
Measurement Interlab Study
Here you find our results of the Interlab Study.
This year we took part in the iGEM 2015 Measurement Interlab Study. The aim of this study is to obtain data about the fluorescence of three devices which express GFP from iGEM teams all around the world. Each team had to built these three required devices using the given Biobricks and measure the fluorescence.
Chassi and Devices
As chassi we used Escherichia coli KRX. The required devices were created by Standard BioBrick Assembly (Suffix Insertion). As backbone we used pSB1C3. For the measurement, the following devices and controls were used:
- Device 1: GFP Generator (BBa_I13504) under control of the Anderson promoter J23101 (BBa_K823005)
- Device 2: GFP Generator (BBa_I13504) under control of the Anderson promoter J23106 (BBa_K823008)
- Device 3: GFP Generator (BBa_I13504) under control of the Anderson promoter J23117 (BBa_K823013)
- Positive control (PC): GFP expression device (BBa_I20270)
- Negative control 1 (NC 1): ptetR (BBa_R0040)
- Negative control 2 (NC 2): E. coli KRX with no plasmid
The final devices were validated by DNA Sequencing. You can find the protocol for growing cells for the measurement here: 2015 Interlab Protocol.
Results
The sequencing confirmed the accuracy of the devices. At first a contig (black) was assembled from the sequencing data. The forward sequencing reads are shown in blue and the reverse sequencing reads are shown in rose. To see the alignment of the resulting contig and the device sequence (green) click the image.
Sequence assembly for device 1:
Sequence assembly for device 2:
Sequence assembly for device 3:
Moreover, after transformation of the devices 1-3 we could already see some fluorescence on the plates (Fig. 2). To confirm the GFP fluorescence we took a photo with a smartphone and our filter combination for GFP detection.
The fluorescence was measured with a plate reader (Tecan Infinite M200). The excitation wavelength was 475 nm and the emission wavelength 509 nm. As expected, there was almost no fluorescence for the negative controls and about 14,000 RFU for the positive control (Fig. 3). The highest fluorescence was measured for device 1 (47,000 RFU) confirming the expectation as device 1 carries the strongest promoter. Unexpectedly, one biological replicate of device 1 showed a much lower fluorescence (Fig. 3). A reason for that could be a dilution error. To review this possibility the dilutions were prepared again. However, the results were the same. At a second glance on the plates it could be determined that there are already colonies with different levels of staining although they were plated out from one colony. To see all readings click here.
