InterLab

Objectives

While our BacMan was gearing up to protect people from arsenic , we decided to pay a visit to the GFP expressing bacteria. It is no doubt that measuring flourescence seemed exciting, but even more appealing was the opportunity to be a part of a large community and share our data for collective benefit.

So we prepared ourselves to address the question that formed the core theme of the Interlab experiments: Can we reduce lab-to-lab variability in fluorescence measurements by normalizing to absolute cell count or colony-forming units (CFUs) instead of OD?

And then we started :
Day 1 : Transforming E coli DH5 alpha cells with the required biobr/icks.
Day 2 : Checking plates . Some biobricks didn’t give enough colonies.
Day 3 : Transforming bacteria with 1.5 uL of biobricks. Checking plates. We had beautiful and ample colonies this time.
Day 4 : Performing all calibration protocols with ludox , silica beads , fluorescein.
Day 5 : Performed the cell measurements.
Day 6 : Repeated cell measurements.
Day 7 : Performing CFU protocol.
Day 8 : Counting colonies and updating excel files.

Results

The above image represents the silica bead standard curve

The above image represents the fluorescein standard curve

Fluoroscence Raw Readings

The above graph depicts the tabular data for Fluorescence Raw Readings

Abs600 Raw Readings

The above graph depicts the tabular data for Absorbance (at 600nm wavelength) Raw Readings

Observations

• The test device 3 doesn’t show a great increase in fluorescence though its absorbance is increasing. Its expression level thus must be low.
• The test device 2 has shown difference florescence and absorbance for the two difference colonies. This could be indication of improper growth in one colony.
• The test device 1 has shown very low growth after 6 hrs. This could be due to slow growth of bacteria in set conditions and time.

Comments

At the end of interLab we learnt the importance of standardizing protocols in setting up experiments and adopting measures to make data more robust by minimizing errors. We are glad that we did participate in this global collective effort to make fluorescence quantification techniques more immune to spatial and temporal variabilities.

Final Results

Team IISER Kolkata successfully completed the Interlab exercise contributing positively to the international collaboration by submitting meaningful data.