Difference between revisions of "Team:USP-EEL-Brazil/InterLab"

 
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  <img src="https://static.igem.org/mediawiki/2018/0/05/T--USP-EEL-Brazil--interlabbrazil.jpg" width=100% >
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  <img src="https://static.igem.org/mediawiki/2018/b/b1/T--USP-EEL-Brazil--INTERLAB.jpg" width=100% >
 
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<h4>Introduction</h4>
 
<h4>Introduction</h4>
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<p style=" margin-bottom: 50px; text-align: justify;    text-indent: 5%;">IGEM is an international Synthetic Biology competition, which aims to set up standard protocols that work in any laboratory on the planet. To manage this task, Interlab Study was created, inviting all teams to participate by performing the same experiment: making measures of the Green Fluorescent Protein (GFP) to determine the feasible cell population. </p>
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<p style=" margin-bottom: 30px; text-align: justify;    text-indent: 5%;">IGEM is an international Synthetic Biology competition, which aims to set up standard protocols that work in any laboratory on the planet. To manage this task, Interlab Study was created, inviting all teams to participate by performing the same experiment: making measures of the Green Fluorescent Protein (GFP) to determine the feasible cell population. </p>
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<p style=" margin-bottom: 50px; text-align: justify;    text-indent: 5%; ">In order to determine cell population, scientists usually use spectrophotometry to measure cells culture absorbance in 600 nm, and through it, the optical density (OD) can be obtained. So, theoretically, the higher cells number the higher OD. However, the values of OD obtained are under high variability, because of different calibration and precision between labs equipment, different glassware size, and others lab-to-lab factors. In addition, through this methodology, it isn’t possible to separate dead feasible cells.
+
<p style=" margin-bottom: 30px; text-align: justify;    text-indent: 5%; ">In order to determine cell population, scientists usually use spectrophotometry to measure cells culture absorbance in 600 nm, and through it, the optical density (OD) can be obtained. So, theoretically, the higher cells number the higher OD. However, the values of OD obtained are under high variability, because of different calibration and precision between labs equipment, different glassware size, and other lab-to-lab factors. In addition, through this methodology, it isn’t possible to separate dead from feasible cells.
 
</p>
 
</p>
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<p style=" margin-bottom: 50px; text-align: justify;    text-indent: 5%; ">In this sense, to respond to all the problems above, the methodology of measuring GFP is another way to determine feasible cells, once the dead ones will not express GFP. Through the GFP measurement methodology, the Measurement Committee wants our help to answer the following question:  
+
<p style=" margin-bottom: 30px; text-align: justify;    text-indent: 5%; ">In this sense, to respond to all the problems above, the methodology of measuring GFP is another way to determine feasible cells, once the dead ones will not express GFP. Through the GFP measurement methodology, the Measurement Committee wants our help to answer the following question:  
 
</p>
 
</p>
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<p style=" margin-bottom: 50px; text-align:center;"><i>‘’Can we reduce lab-to-lab variability in fluorescence measurements by normalizing to absolute cell count or colony-forming units (CFUs) instead of OD?‘’</i>
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<p style=" margin-bottom: 30px; text-align:center;"><i>‘’Can we reduce lab-to-lab variability in fluorescence measurements by normalizing to absolute cell count or colony-forming units (CFUs) instead of OD?‘’</i>
 
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<p style=" margin-bottom: 50px; text-align: justify;    text-indent: 5%; ">In order to help on the achievement of this goal, Team USP-EEL-Brazil participeted on the Interlab Study, following the standart protocol and sendding the results to iGEM's Measurement committee.</p></div>
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<p style=" margin-bottom: 30px; text-align: justify;    text-indent: 3%; ">In order to help on the achievement of this goal, Team USP-EEL-Brazil participeted on the Interlab Study, following the standart protocol (<a href="https://2018.igem.org/Measurement/InterLab/Plate_Reader">Plate Reader and CFU Protocol</a>) and sendding the results to iGEM's Measurement committee.</p></div>
  
 
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<h4>Materials and Methods</h4>
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<h4>Our contribution</h4>
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<p style=" margin-bottom: 30px; text-align: justify;    text-indent: 5%;">We couldn’t follow the complete protocol due to the lack of proper equipment. The TECAN in our lab doesn't have the settings for fluorescence measurements and we only discovered that in the middle of our experiments. Fortunately, the Measurement Committee accepted our results.
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<p style=" margin-bottom: 50px; text-align: justify;    text-indent: 5%; ">For the experiments, we followed the InterLab's <a href="https://2018.igem.org/Measurement/InterLab/Plate_Reader"> Plate Reader and CFU Protocol</a>.</p>
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<p style=" margin-bottom: 30px; text-align: justify;    text-indent: 5%;">As for other difficulties we faced, the experiments took longer than we expected and a too much material was used to perform them. In overall, the Interlab worked as a training for our lab team, members that didn’t have much experience in our lab got familiar with the procedures of the place.  </p>
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<p style=" margin-bottom: 50px; text-align: justify;    text-indent: 5%;">Equipment used for measurements: TECAN INFINITY M200 PRO
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    <div style="display: inline-block;">
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<p style=" margin-bottom: 50px; text-align: justify;     text-indent: 5%;">Plate used for measurements: Corning<sup>®</sup> <b>Costar Clear</b> Polystyrene <b>96-Well Plates</b></p>
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        <img src="https://static.igem.org/mediawiki/2018/0/09/T--USP-EEL-Brazil--interlabA.jpg" title="" height="240px"/>  
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<h4>Results</h4>
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<p style=" margin-bottom: 50px; text-align: justify;    text-indent: 5%;">The InterLab study took us longer than we expected. First of all we have to re-do the protocols, we suspected that cells we used were not DH5α as it was supposed to be. We had a partner lab from our university to lend it to us, after obtaining a new DH5α strain we could re-do the procedure and then we got positive fluorescence results.
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        <img src="https://static.igem.org/mediawiki/2018/6/6f/T--USP-EEL-Brazil--interlabB.jpg" title=""height="240px"/>
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At first we weren't able to notice any difference between the blank sample and the positive control samples. With the different new strain we could notice de the difference. Also, we had problems transforming the devices from plate 7, they didn't grow or they didn't exhibits fluorescence, so we had to re-do all the transformations using the devices from plate 6.
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There were a few colonies that didn't grow well, and we believe that didn't help our final results where there are samples with negative fluorescences (less fluorescence than the blank).</p>
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        <img src="https://static.igem.org/mediawiki/2018/d/de/T--USP-EEL-Brazil--interlabC.jpg" title=""height="240px"/>
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        <img src="https://static.igem.org/mediawiki/2018/b/b5/T--USP-EEL-Brazil--interlabD.jpg"height="240px"/>
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        <img src="https://static.igem.org/mediawiki/2018/6/60/T--USP-EEL-Brazil--interlab1.jpg" title=""height="240px"/>
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Latest revision as of 01:02, 18 October 2018

Introduction

IGEM is an international Synthetic Biology competition, which aims to set up standard protocols that work in any laboratory on the planet. To manage this task, Interlab Study was created, inviting all teams to participate by performing the same experiment: making measures of the Green Fluorescent Protein (GFP) to determine the feasible cell population.

In order to determine cell population, scientists usually use spectrophotometry to measure cells culture absorbance in 600 nm, and through it, the optical density (OD) can be obtained. So, theoretically, the higher cells number the higher OD. However, the values of OD obtained are under high variability, because of different calibration and precision between labs equipment, different glassware size, and other lab-to-lab factors. In addition, through this methodology, it isn’t possible to separate dead from feasible cells.

In this sense, to respond to all the problems above, the methodology of measuring GFP is another way to determine feasible cells, once the dead ones will not express GFP. Through the GFP measurement methodology, the Measurement Committee wants our help to answer the following question:

‘’Can we reduce lab-to-lab variability in fluorescence measurements by normalizing to absolute cell count or colony-forming units (CFUs) instead of OD?‘’

In order to help on the achievement of this goal, Team USP-EEL-Brazil participeted on the Interlab Study, following the standart protocol (Plate Reader and CFU Protocol) and sendding the results to iGEM's Measurement committee.

Our contribution

We couldn’t follow the complete protocol due to the lack of proper equipment. The TECAN in our lab doesn't have the settings for fluorescence measurements and we only discovered that in the middle of our experiments. Fortunately, the Measurement Committee accepted our results.

As for other difficulties we faced, the experiments took longer than we expected and a too much material was used to perform them. In overall, the Interlab worked as a training for our lab team, members that didn’t have much experience in our lab got familiar with the procedures of the place.

Go to top