Difference between revisions of "Team:ICT-Mumbai/InterLab"

The InterLab study is conducted by the iGEM organization and is an attempt at reducing variability in measurements taken in different laboratories, thus facilitating easier sharing of data between laboratories. More specifically, the Measurement Committee at iGEM is attempting to develop a robust method for fluorescence measurements of Green Fluorescent Protein (GFP) that can minimize lab-to-lab variations in readings taken by a standard plate reader. For more details on the InterLab study please visit the following URL: https://2018.igem.org/Measurement/InterLab

Team ICT-Mumbai has participated in the InterLab study this year. Team ICT-Mumbai participated in the interlab study this year. The plate reader protocol as given on the iGEM website has been strictly followed (the protocol can be downloaded here) and resulting data added to the provided Excel sheet (the Excel sheet can be downloaded here ).
The InterLab study uses the following 6 test devices:

1. BBa_R0040 (Negative control)
2. BBa_I20270 (Positive control)
3. BBa_J364000 (Test Device 1)
4. BBa_J364001 (Test Device 2)
5. BBa_J364002 (Test Device 3)
6. BBa_J364007 (Test Device 4)
7. BBa_J364008 (Test Device 5)
8. BBa_J364009 (Test Device 6)

Each test device used in the study comprises of the same gene and RBS, but has a different constitutive promoter from the Anderson family of promoters. Each promoter thus has a different level of expression of the GFP protein leading to the different values of absorbance and fluorescence.
The 6 test devices, positive control and negative control were all transformed into E. coli DH5α cells. The transformed cells were plated onto LB + chloramphenicol plates. Two colonies were taken from each plate and inoculated into LB + chloramphenicol medium and measurements of absorbance and fluorescence were taken for each colony as per the protocol given.
The instrument settings used for this study are:

• Model – PerkinElmer EnSpire Multimode Plate Reader
• Absorbance measurement wavelength – 600 nm
• Fluorescence measurement wavelengths – 485 nm (absorption) / 525 nm (emission)

The results obtained are shown in the charts given below.

Figure 1: Absorbance measurement for all devices at 0 hours as defined in the protocol

Figure 2: Absorbance measurement for all devices at 6 hours as defined in the protocol

Figure 3: Fluorescence measurement for all devices at 0 hours as defined in the protocol

Figure 4: Fluorescence measurement for all devices at 6 hours as defined in the protocol

For the second part of the InterLab study, i.e., the CFU measurements, the dilution series was created according to the given protocol, and the number of colonies that grew on each plate were counted.
The number of CFUs obtained are shown below:

Figure 5: Number of CFUs observed for each dilution as mentioned in the protocol