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

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This year's InterLab study was conducted to answer the following question:  
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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:
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https://2018.igem.org/Measurement/InterLab
 
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<B>Can we reduce lab-to-lab variability in fluorescence measurements by normalizing to absolute cell count or colony-forming units (CFUs) instead of OD?</B>
 
 
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Team ICT-Mumbai participated in the interlab study this year. The plate reader protocol of the plate reader study as given on the measurements page of the iGEM website was strictly followed (the protocol can be downloaded <a href='https://static.igem.org/mediawiki/2018/0/09/2018_InterLab_Plate_Reader_Protocol.pdf'><b><u>here</u></b></a>) and resulting data added to the provided Excel sheet which was subsequently uploaded (the Excel sheet can be downloaded <a href='https://static.igem.org/mediawiki/2018/2/2a/T--ICT-Mumbai--InterLab.xlsx'><b><u>here</u></b></a>
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Team ICT-Mumbai has participated in the InterLab study this year.
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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 <a href='https://static.igem.org/mediawiki/2018/0/09/2018_InterLab_Plate_Reader_Protocol.pdf'><b><u>here</u></b></a>) and resulting data added to the provided Excel sheet (the Excel sheet can be downloaded <a href='https://static.igem.org/mediawiki/2018/2/2a/T--ICT-Mumbai--InterLab.xlsx'><b><u>here</u></b></a>
 
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The figures given below show the obtained results for the cell measurement protocol of this year’s interlab study.  
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The InterLab study uses the following 6 test devices:
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<li>BBa_R0040 (Negative control)</li>
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<li>BBa_I20270 (Positive control)</li>
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<li>BBa_J364000 (Test Device 1)</li>
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<li>BBa_J364001 (Test Device 2)</li>
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<li>BBa_J364002 (Test Device 3)</li>
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<li>BBa_J364007 (Test Device 4)</li>
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<li>BBa_J364008 (Test Device 5)</li>
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<li>BBa_J364009 (Test Device 6)</li>
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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.
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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.
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The instrument settings used for this study are:
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<li>Model – PerkinElmer EnSpire Multimode Plate Reader</li>
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<li>Absorbance measurement wavelength – 600 nm</li>
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<li>Fluorescence measurement wavelengths – 485 nm (absorption) / 525 nm (emission)</li>
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The results obtained are shown in the charts given below.  
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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.
 
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The number of CFUs obtained are shown below:
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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. Test device 1 worked better than expected whereas test device 5 failed to work as expected. It is seen that the common sense criteria are being followed, i.e., both the fluorescence and absorbance values are increasing with time for all devices and the positive control exhibits a better fluorescence and absorbance than the negative control. It was also observed that both, the fluorescence/OD and fluorescence/particle values decreased after 6 hours.
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The results of the CFU protocol were uploaded in the form given on the interlab page of the iGEM website. The results obtained are shown below.
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Revision as of 13:31, 22 September 2018

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InterLab Study

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:

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










    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: