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| | <body> | | <body> |
| | </div> | | </div> |
| | <center> | | <center> |
| − | <img src="https://static.igem.org/mediawiki/2018/0/05/T--USP-EEL-Brazil--interlabbrazil.jpg" width=100% > | + | <img src="https://static.igem.org/mediawiki/2018/b/b1/T--USP-EEL-Brazil--INTERLAB.jpg" width=100% > |
| | </center> | | </center> |
| | | | |
| | <div class="margin" style=" text-align: left; "> | | <div class="margin" style=" text-align: left; "> |
| | <h4>Introduction</h4> | | <h4>Introduction</h4> |
| − | <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> | + | <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> |
| − | <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> |
| − | <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> |
| − | <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> | + | <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> |
| | </p> | | </p> |
| − | <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> | + | <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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| | <div class="margin" style=" text-align: left; "> | | <div class="margin" style=" text-align: left; "> |
| − | <h4>Materials and Methods</h4> | + | <h4>Our contribution</h4> |
| − | | + | <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. |
| − | <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> | + | <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> |
| − | </p> | + | <div style="width: 100%;height: 260px;background-color: white;overflow-x: scroll; white-space:nowrap;"> |
| − | <p>The plasmids used are available in the iGEM kit:</p>
| + | <div style="display: inline-block;"> |
| − | <!-----------------------------TABLE------------------------------------------------------->
| + | <img src="https://static.igem.org/mediawiki/2018/0/09/T--USP-EEL-Brazil--interlabA.jpg" title="" height="240px"/> |
| − | <div style="margin-bottom: 25px;; text-align:center;">
| + | </div> |
| − | <p dir="ltr" style="line-height: 1.38; margin-top: 0pt; margin-bottom: 0pt;"><span style="font-size: 12pt; font-family: Arial; color: #000000; background-color: #ffffff; font-weight: 400; font-style: normal; font-variant: normal; text-decoration: none; vertical-align: baseline; white-space: pre-wrap;">Device Part Number Plate Location</span></p>
| + | <div style="display: inline-block;"> |
| − | <p dir="ltr" style="line-height: 1.38; margin-top: 0pt; margin-bottom: 0pt;"><span style="font-size: 12pt; font-family: Arial; color: #000000; background-color: #ffffff; font-weight: 400; font-style: normal; font-variant: normal; text-decoration: none; vertical-align: baseline; white-space: pre-wrap;">Negative control BBa_R0040 Kit Plate 7 Well 2D</span></p> | + | <img src="https://static.igem.org/mediawiki/2018/6/6f/T--USP-EEL-Brazil--interlabB.jpg" title=""height="240px"/> |
| − | <p dir="ltr" style="line-height: 1.38; margin-top: 0pt; margin-bottom: 0pt;"><span style="font-size: 12pt; font-family: Arial; color: #000000; background-color: #ffffff; font-weight: 400; font-style: normal; font-variant: normal; text-decoration: none; vertical-align: baseline; white-space: pre-wrap;">Positive control BBa_I20270 Kit Plate 7 Well 2B</span></p>
| + | </div> |
| − | <p dir="ltr" style="line-height: 1.38; margin-top: 0pt; margin-bottom: 0pt;"><span style="font-size: 12pt; font-family: Arial; color: #000000; background-color: #ffffff; font-weight: 400; font-style: normal; font-variant: normal; text-decoration: none; vertical-align: baseline; white-space: pre-wrap;">Test Device 1 BBa_J364000 Kit Plate 7 Well 2F</span></p> | + | <div style="display: inline-block;"> |
| − | <p dir="ltr" style="line-height: 1.38; margin-top: 0pt; margin-bottom: 0pt;"><span style="font-size: 12pt; font-family: Arial; color: #000000; background-color: #ffffff; font-weight: 400; font-style: normal; font-variant: normal; text-decoration: none; vertical-align: baseline; white-space: pre-wrap;">Test Device 2 BBa_J364001 Kit Plate 7 Well 2H</span></p>
| + | <img src="https://static.igem.org/mediawiki/2018/d/de/T--USP-EEL-Brazil--interlabC.jpg" title=""height="240px"/> |
| − | <p dir="ltr" style="line-height: 1.38; margin-top: 0pt; margin-bottom: 0pt;"><span style="font-size: 12pt; font-family: Arial; color: #000000; background-color: #ffffff; font-weight: 400; font-style: normal; font-variant: normal; text-decoration: none; vertical-align: baseline; white-space: pre-wrap;">Test Device 3 BBa_J364002 Kit Plate 7 Well 2J</span></p> | + | </div> |
| − | <p dir="ltr" style="line-height: 1.38; margin-top: 0pt; margin-bottom: 0pt;"><span style="font-size: 12pt; font-family: Arial; color: #000000; background-color: #ffffff; font-weight: 400; font-style: normal; font-variant: normal; text-decoration: none; vertical-align: baseline; white-space: pre-wrap;">Test Device 4 BBa_J364007 Kit Plate 7 Well 2L</span></p>
| + | <div style="display: inline-block;"> |
| − | <p dir="ltr" style="line-height: 1.38; margin-top: 0pt; margin-bottom: 0pt;"><span style="font-size: 12pt; font-family: Arial; color: #000000; background-color: #ffffff; font-weight: 400; font-style: normal; font-variant: normal; text-decoration: none; vertical-align: baseline; white-space: pre-wrap;">Test Device 5 BBa_J364008 Kit Plate 7 Well 2N</span></p> | + | <img src="https://static.igem.org/mediawiki/2018/b/b5/T--USP-EEL-Brazil--interlabD.jpg"height="240px"/> |
| − | | + | </div> |
| − | <p dir="ltr" style="line-height: 1.38; margin-top: 0pt; margin-bottom: 0pt;"><span style="font-size: 12pt; font-family: Arial; color: #000000; background-color: #ffffff; font-weight: 400; font-style: normal; font-variant: normal; text-decoration: none; vertical-align: baseline; white-space: pre-wrap;">Test Device 6 BBa_J364009 Kit Plate 7 Well 2P</span></p> | + | <div style="display: inline-block;"> |
| | + | <img src="https://static.igem.org/mediawiki/2018/6/60/T--USP-EEL-Brazil--interlab1.jpg" title=""height="240px"/> |
| | + | </div> |
| | </div> | | </div> |
| − | <!-----------------------------TABLE- END------------------------------------------------------>
| |
| − |
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| − | <p>Equipment used for measurements: TECAN INFINITY M200 PRO
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| − | Plate used for measurements: Corning<sup>®</sup> <b>Costar Clear</b> Polystyrene <b>96-Well Plates</b></p>
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| − |
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| − | <h4>Results</h4>
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| − | <p></p>
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| − | <p></p>
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| − | <p></p>
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| − | <p></p>
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| | | | |
| | </body> | | </body> |
| | </div><center> | | </div><center> |
| − | <a href="#top" >Go to top</a></div></center>
| |
| | | | |
| | </html><div class="margin"> | | </html><div class="margin"> |
| | | | |
| | {{USP-EEL-Brazil/footer}} | | {{USP-EEL-Brazil/footer}} |
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.
