Team:Bielefeld-CeBiTec/InterlabStudy

Measurement Interlab Study

Here you find our results of the Interlab Study.

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.

Device 1:

Device 2:

Device 3:

Fig. 2: A: Photo of KRX colonies after transformation with device 1. B: The same plate (KRX transformation with device 1) photographed with a smartphone and a filter combination for GFP detection.

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 Reader). 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 14000 RFU for the positive control (Fig. 3). The highest fluorescence was measured for device 1 (47000 RFU) confirming the expectation as device 1 carries the stronges promoter.
Unexpectedly, one biological replicate of device 1 shows 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 steaked out from one colonie.

Fig. 3: Results of the GFP fluorescence measurement with a plate reader (excitation wavelength : 475 nm, emission wavelength: 509 nm; three biological and three technical replicates). PC: positive control (KRX-BBa_I20270); NC1: negative control 1 (KRX with no plasmid); NC2: negative control 2 (KRX-BBa_R0040); Device 1: KRX-J23101+I13504; Device 2: KRX-J23106+I13504; Device 3: KRX-J23117+I13504.

Modeling was an important part of our project as well.