Team:IISER-Bhopal-India/InterLab
Introduction
Over the last several years the Measurement committee has hosted Interlab studies to help develop a robust measurement procedure for Green Fluorescent Protein (GFP). To be a part of such a huge international collaborative experiment has been a steep learning curve for all of us. It helped us not only understand the collective might of science, but also gave a sense of pride to have contributed our bit towards the scientific community’s progress. The key to any experiment is reliable and reproducible measurements, which however is not an easy task. Ranging over issues like varying ambient conditions to usage of different measurement units altogether, reliable reproduction of data across labs has always proven to be a great challenge. Fluorescence measurement happens to be one such problem.
Aim
The Interlab study has already reduced the variability amongst different sets of readings by measuring GFP expression in absolute fluorescence units, and calibrating it against known concentrations of a fluorescent molecule. This year we are a part of the attempt to answer- "Can we reduce lab-to-lab variability in fluorescence measurements by normalizing to absolute cell count or colony-forming units (CFUs) instead of OD?”
Protocols
The parts provided to us for transformation were (in the plasmid pSB1C3) as follows:
We diligently followed the protocol provided to us by the measurement committee to ensure uniformity was maintained, thereby reducing chances of unpredicted variability in our data. Except for preparation of competent E. Coli cells, we followed all the protocols to the word as provided by iGEM. Even though our competent cell preparation differed from the iGEM protocol, the transformation efficiency achieved was in the power of 10^8 which is ideal for any transformation experiment. We would be glad to share our preparation protocol, so kindly contact us regarding the same.
Parts
The parts provided to us for transformation were (in the plasmid pSB1C3) as follows:
| Part Designation | Part Name |
|---|---|
| Negative control | BBa_R0040 |
| Positive control | BBa_I20270 |
| Test Device 1 | BBa_J364000 |
| Test Device 2 | BBa_J364001 |
| Test Device 3 | BBa_J364002 |
| Test Device 4 | BBa_J364007 |
| Test Device 5 | BBa_J364008 |
| Test Device 6 | BBa_J364009 |
All the above mentioned parts had GFP expression at varied levels due to the presence of different promoters. The mutations in the test devices 1-6 when compared to the positive control were seen in the first 57 base pairs of the 919 bp long promoter.
Results
Our Interlab data can be accessed from here.
The only trouble we faced during the Interlab study was for the growth of test device 1 (BBa_J364000) and Test Device 5 (BBa_J364008), marked with red in the chart above. These devices displayed growth during the overnight incubation, but the OD for the corresponding test devices barely increased as compared to others during the final 6 hours incubation stage. We repeated the experiment using different set of hands in order to account for and eliminate human error, but were stumped to obtain similar results. It might be so that these devices affect the growth rate of the bacteria. After having attempted to troubleshoot the aforementioned problem not only with our PI but also in collaboration with other iGEM teams, we decided to report all the results including Test device 1 and Test device 5.
Our Take
The Interlab study was a wonderful experience for us. We refreshed numerous concepts, along with learning some novel ones as well. We became well acquainted with the usage of plate readers, which would definitely be of help to us in our very own project, during which we would be employing RFP as reporter gene. It wouldn’t be an exaggeration to say that this Interlab study challenged us to think critically and inspired us to explore and devise more accurate procedures to standardize such varied data procuring methods.