Team:REC-CHENNAI/InterLab
INTRODUCTION
InterLab Measurement Study, an important element under the Bronze Medal criteria of the iGEM competition strives to standardize measurements and data processing across various lab environments thus addressing one of the major hurdles in the field of synthetic biology and engineering. This enables scientists to calibrate the defect in engineered biological construct, to share construct between labs and to analyse experimental results across different lab conditions. The iGEM Measurement Committee, through the InterLab study, has been developing a robust and resolute measurement procedure for the Green Fluorescent Protein (GFP) over the span of three years. GFP is widely used owing to its ability to report gene expression and successful transformation. Our role in this study required that we submit measurement data dealing with the fluorescence of GFP and Optical Density(OD) associated with cells transformed with different test devices.
TEST DEVICES
6 Test Devices, a Positive Control and a Negative Control were provided, all of which contained the Chloramphenicol resistance genebuilt in a pSB1C3 vector. Additionally all devices except the negative control had built-in GFP. The negative control had Tet R which is non-fluorescent. The devices contained 3 different promoters of the Anderson family. Those were,
- Bba_J23101 which has high expression
- Bba_J23106 which has medium expression
- Bba_J23117 which has low expression.
Test Device 1
pSB1C3 backbone containing J23101 (Promoter), B0034 (Ribosome binding site), B0010 (Terminator) and B0012 (Double terminator)
Test Device 2
pSB1C3 backbone containing J23106 (Promoter), B0034 (Ribosome binding site), B0010 (Terminator) and B0012 (Double terminator)
Test Device 3
pSB1C3 backbone containing J23117 (Promoter), B0034 (Ribosome binding site), B0010 (Terminator) and B0012 (Double terminator)
Test Device 4
pSB1C3 backbone containing J23101 (Promoter), J364100 (Ribosome binding site), B0010 (Terminator) and B0012 (Double terminator)
Test Device 5
pSB1C3 backbone containing J23106 (Promoter), J364100 (Ribosome binding site), B0010 (Terminator) and B0012 (Double terminator)
Test Device 6
pSB1C3 backbone containing J23117 (Promoter), J364100 (Ribosome binding site), B0010 (Terminator) and B0012 (Double terminator)
Positive control
pSB1C3 backbone containing J23151 (Promoter), B0032 (Ribosome binding site), B0010 (Terminator) and B0012 (Double terminator)
Negative control
pSB1C3 backbone containing R0040(no GFP expression device)
Fluorescence Raw Readings
Results
CONCLUSION
The Plate Reader results indicate that Test Device 4 showed highest GFP expression and thus it has the strongest promoter and the best RBS. This is in accordance with Anderson promoter collections which establishes that Bba_J23101 has the highest GFP expression of the three promoters provided. Test Devices 2 and 6 also displayed good level of GFP expression. The other devices produced results (absence of significant GFP expression) which indicates weak or non-functional promoters and RBS.