Difference between revisions of "Team:NU Kazakhstan/InterLab"

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<p><b></b> Every year, the Measurement Committee tries to analyze the causes of difference in values of fluorescence measurements obtained in laboratories around the year. Particularly in this year, the main goal of the Fifth International InterLaboratory Measurement Study is to investigate if the normalization of fluorescence measurements to the absolute cell count can contribute to the reduction of lab-to-lab variability in results. Our team, NU_Kazakhstan 2018, used the Varioskan LUX Multimode Microplate Reader to measure the fluorescence of DH 5 𝛼 cells transformed with the GFP inserted in pSB1C3 plasmid with different promoters (BBa_J364000, BBa_J364001, BBa_J364002,  BBa_J364007, BBa_J364008, BBa_J364009, plus negative BBa_R0040 and positive BBa_I20270 controls).
 
<p><b></b> Every year, the Measurement Committee tries to analyze the causes of difference in values of fluorescence measurements obtained in laboratories around the year. Particularly in this year, the main goal of the Fifth International InterLaboratory Measurement Study is to investigate if the normalization of fluorescence measurements to the absolute cell count can contribute to the reduction of lab-to-lab variability in results. Our team, NU_Kazakhstan 2018, used the Varioskan LUX Multimode Microplate Reader to measure the fluorescence of DH 5 𝛼 cells transformed with the GFP inserted in pSB1C3 plasmid with different promoters (BBa_J364000, BBa_J364001, BBa_J364002,  BBa_J364007, BBa_J364008, BBa_J364009, plus negative BBa_R0040 and positive BBa_I20270 controls).
 
<h3><b>Protocol</b></h3>
 
<h3><b>Protocol</b></h3>
βˆ’
We strictly followed the instructions from the protocol provided by iGEM https://static.igem.org/mediawiki/2018/0/09/2018_InterLab_Plate_Reader_Protocol.pdf
+
We strictly followed the instructions from the <a href="https://static.igem.org/mediawiki/2018/0/09/2018_InterLab_Plate_Reader_Protocol.pdf
 +
"> protocol </a> provided by iGEM
  
βˆ’
Plate reader configuration:
+
<h4>Plate reader configuration:</h4>
  
 
<h4><i>Photometric</i></h4>
 
<h4><i>Photometric</i></h4>
βˆ’
Wavelength: 600 nm  
+
<li>Wavelength: 600 nm  
βˆ’
Bandwidth: 5 nm  
+
<li>Bandwidth: 5 nm  
βˆ’
Measurement time: 100 ms
+
<li>Measurement time: 100 ms
  
 
<h4><i>Fluorometric</i></h4>
 
<h4><i>Fluorometric</i></h4>
βˆ’
Excitation Wavelength: 485 nm
+
<li>Excitation Wavelength: 485 nm
βˆ’
Emission Wavelength: 530 nm
+
<li>Emission Wavelength: 530 nm
βˆ’
Excitation bandwidth: 12 nm
+
<li>Excitation bandwidth: 12 nm
βˆ’
Fluorescence reading: top optics
+
<li>Fluorescence reading: top optics
βˆ’
Measurement time: 100 ms
+
<li>Measurement time: 100 ms
 
   
 
   
 
<h3><b>Results</b></h3>
 
<h3><b>Results</b></h3>
  
 
OD600 reference point
 
OD600 reference point
 +
 +
 
   
 
   
 
<img src="https://static.igem.org/mediawiki/2018/thumb/f/fd/T--NU_Kazakhstan--CFUPlates.jpg/450px-T--NU_Kazakhstan--CFUPlates.jpg">
 
<img src="https://static.igem.org/mediawiki/2018/thumb/f/fd/T--NU_Kazakhstan--CFUPlates.jpg/450px-T--NU_Kazakhstan--CFUPlates.jpg">

Revision as of 13:53, 25 July 2018

Bioremediation of Sour Crude Oil Waste using Cyanobacteria




InterLab

Every year, the Measurement Committee tries to analyze the causes of difference in values of fluorescence measurements obtained in laboratories around the year. Particularly in this year, the main goal of the Fifth International InterLaboratory Measurement Study is to investigate if the normalization of fluorescence measurements to the absolute cell count can contribute to the reduction of lab-to-lab variability in results. Our team, NU_Kazakhstan 2018, used the Varioskan LUX Multimode Microplate Reader to measure the fluorescence of DH 5 𝛼 cells transformed with the GFP inserted in pSB1C3 plasmid with different promoters (BBa_J364000, BBa_J364001, BBa_J364002, BBa_J364007, BBa_J364008, BBa_J364009, plus negative BBa_R0040 and positive BBa_I20270 controls).

Protocol

We strictly followed the instructions from the protocol provided by iGEM

Plate reader configuration:

Photometric

  • Wavelength: 600 nm
  • Bandwidth: 5 nm
  • Measurement time: 100 ms

    Fluorometric

  • Excitation Wavelength: 485 nm
  • Emission Wavelength: 530 nm
  • Excitation bandwidth: 12 nm
  • Fluorescence reading: top optics
  • Measurement time: 100 ms

    Results

    OD600 reference point

    Conclusion

    The data from measurements shows that test device 4 (BBa_J364007) has the promoter with the highest fluorescence among the rest of the provided promoters. Its maximum value is 11.84 net fluorescein a.u. after 6 hours of incubation. The weakest promoter was found to be the test device 3 (max. value 0.48 net fluorescein a.u. at 6 hours). Samples transformed with test devices 1,5, and 6 have about similar results (approx. 2 net fluorescein a.u.). The fluorescence of samples transformed with test device 2 is slightly higher than these three. Its maximum value of fluorescence is 5.53 net fluorescein a.u.