Team:ICT-Mumbai/InterLab
What we learned from the InterLab study
The 2017 InterLab study was initiated to answer two questions: (1) How close can the numbers be when fluorescence is measured all around the world? (2) How reliable will BCD RBS devices turn out to be in labs around the world?
Team ICT-Mumbai participated in the InterLab study and contributed data that will help in answering these two questions. While carrying out this study, we also learned a few things, which we wish to highlight below.
As can be seen from the figure above, the strengths of the promoters tested are as follows: J23101>J23106>J23117.
These three promoters are part of a family of constitutive promoters, of which J23100 is the strongest. The sequences of these promoters (obtained from here) are depicted below. Nucleotides that differ from the those in promoter J23100 are depicted in red.
It is interesting to note that a single base change in J23117 at position 24 (from T to G) leads to a drastic change in promoter strength. The other changes in the sequence of J23117 with respect to J23100 are also found in J23101 and J23106, and as the latter two are quite strong promoters, in the absence of any other information, and taking the liberty of discounting other alternative explanations, it may be presumed that these changes are not responsible for the decreased promoter strength observed in J23117.
The two RBS sequences used here cannot be compared to each other as B0034 is a conventional ‘monocistronic design’ (MCD), while J364100 is a ‘bicistronic design’ (BCD). However, it can be concluded that MCD leads to higher protein synthesis, compared to BCD.
InterLab study measurements
The protocols that we followed are as given in this PDF file.
- Instrument: PerkinElmer EnSpire multimode plate reader
- Excitation filter position: Top
- Excitation wavelength: 485 nm
- Emission wavelength: 521 nm
- Measurement height: 9.5 mm
- Number of flashes: 100
- Reference AD gain: 2
