The Interlab Measurement Study
The characterization of standard parts has always been one of the main concerns in Synthetic Biology. For this very same reason, iGEM teams from all around the World were suggested to take part in the biggest Measurement Interlab Study ever conducted and the 2015 UNITN iGEM Team answered the call. The goal of this Second International Measurement Interlab Study is to assemble three different devices, each one containing a promoter with a screening plasmid intermediate and collect as many fluorescence data as possible. The three different promoters will differently affect the GFP production and thating plasmid intermediate and collect as iGEM teams are free to use any technique to measure their devices as long the obtained data are solid and reproducible.
Experimental Design
We used the three mandatory devices for the measurement study:
- Device 1: BBa_J23101 + BBa_I13504 in pSB1C3
- Device 2: BBa_J23106 + BBa_I13504 in pSB1C3
- Device 3: BBa_J23117 + BBa_I13504 in pSB1C3
- Negative control: BBa_R0040 in psB1C3
- Positive control: BBa_I20270 in pSB1C3.
The measurement devices were prepared by amplifying the reporter (BBa_I20270) by PCR. The amplified insert was then cut with XbaI and PstI and ligated into the plasmid containing the promoter previously cut with SpEI and PstI. All the devices were confirmed by restriction digestion as well as DNA sequencing.
In-vivo Measurements
The confirmed devices were then transformed in different bacterial strains of E. Coli, NEB10β, NEB Express, and JM109. Each measurement was taken at the same optical density to allow a more precise comparison of the data. For each device we have 3 biological and 3 technical measurements for each used technique. We measured in vivo fluorescence emission in different ways using Tecan Infinite 200 PRO plate reader, Varian Cary Eclipse spectrofluorimeter, and BD FACSCanto FACS.
In-vitro Measurements
We also focused of transcription since the characterization is about promoters. To do so we we performed RT-qPCR using a BioRad CFX96 Touch™ Real-Time PCR Detection System. Additionally, we performed an in vitro characterization study, by measuring the fluorescence intensities of each device with a Cell Free E. coli S30 Extract System with a Circular DNA Real Time PCR.