Difference between revisions of "Team:Tec-Monterrey/InterLab"
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<div class="body-title">Procedure</div> | <div class="body-title">Procedure</div> | ||
<div class="texto-izquierda"> | <div class="texto-izquierda"> | ||
| − | In order to achieve Bronze requirement #4, the Interlab procedure was successfully carried out, following the iGEM 2018 Interlab Study Protocol. To obtain calibration and experimental data, the following | + | In order to achieve Bronze requirement #4, the Interlab procedure was successfully carried out, following the iGEM 2018 Interlab Study Protocol. To obtain calibration and experimental data, the following materials were used: competent cells from <i>E. coli</i>'s strain DH5a, 96-well plates (black with a clear flat bottom), and the measurement kit, which contains Ludox, silica beads and fluorescein. The first part of the protocol involved generating standard curves for OD measurements, cells and fluorescein. Using a Ludox solution, it was possible to obtain the following reference point for an OD<sub>600</sub>. |
The conversion value obtained was then used to convert any measurement of cell density into OD<sub>600</sub> by multiplying by 2.897, using the same instrument under the same conditions. | The conversion value obtained was then used to convert any measurement of cell density into OD<sub>600</sub> by multiplying by 2.897, using the same instrument under the same conditions. | ||
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<br class="clearBoth"/> | <br class="clearBoth"/> | ||
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| − | <div class=" | + | <div class="articulo"> |
| − | The particle standard curve was obtained using silica microspheres, which have the same size and optical characteristics | + | The particle standard curve was obtained using silica microspheres, which have the same size and optical characteristics as bacteria cells. The standard curve was then used to convert ABS<sub>600</sub> to an estimated number of cells. The following standard curve was obtained: |
</div> | </div> | ||
<div class="imagen-centrada-reducida"> | <div class="imagen-centrada-reducida"> | ||
| − | <img src="https://static.igem.org/mediawiki/ | + | <img src="https://static.igem.org/mediawiki/2018/8/8b/T--Tec-Monterrey--Interlab_Graph_Abs1.png"> |
| − | <div class="leyenda">Figure | + | <div class="leyenda">Figure 2: Calibration curve of particle count from Absorbance at 600 nm.</div> |
</div> | </div> | ||
| − | <div class=" | + | <div class="articulo"> |
| − | Finally, a fluorescein standard curve was made to calculate the fluorescence values of transformed bacteria with GFP. The filter used for the measurements was used according to the recommended values (Bandpass: 530 nm/ 30 nm, width: 25-30 nm, | + | Finally, a fluorescein standard curve was made to calculate the fluorescence values of transformed bacteria with GFP. The filter used for the measurements was used according to the recommended values (Bandpass: 530 nm/ 30 nm, width: 25-30 nm, excitation 485 nm and emission 520-530 nm). The curve displayed in Figure 3 was obtained: |
</div> | </div> | ||
<div class="imagen-centrada-reducida"> | <div class="imagen-centrada-reducida"> | ||
| − | <img src="https://static.igem.org/mediawiki/ | + | <img src="https://static.igem.org/mediawiki/2018/3/3a/T--Tec-Monterrey--Interlab_Graph_Fluor1.png"> |
| − | <div class="leyenda">Figure | + | <div class="leyenda">Figure 3: Calibration curve of Fluorescein</div> |
</div> | </div> | ||
<div class="contenido"> | <div class="contenido"> | ||
| − | Using the previous standard curves, it | + | Using the previous standard curves, it was possible to continue with the cell measurement protocols with transformed <i>E. coli</i> DH5a strains. The following devices from the iGEM kit were transformed: |
</div> | </div> | ||
<div class="imagen-centrada-reducida"> | <div class="imagen-centrada-reducida"> | ||
<img src="https://static.igem.org/mediawiki/2016/7/7f/T--Tec-Monterrey--PM10.png"> | <img src="https://static.igem.org/mediawiki/2016/7/7f/T--Tec-Monterrey--PM10.png"> | ||
| − | <div class="leyenda">Figure | + | <div class="leyenda">Figure 4: ***</div> |
</div> | </div> | ||
</section> | </section> | ||
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<div class="contenido"> | <div class="contenido"> | ||
<div class="body-subtitle">Absorbance</div> | <div class="body-subtitle">Absorbance</div> | ||
| − | Absorbance of cells at t=0 h and t=6 h are shown in the | + | Absorbance of cells at t=0 h and t=6 h are shown in the diagram of Figure 5. |
</div> | </div> | ||
<div class="imagen-centrada-completa"> | <div class="imagen-centrada-completa"> | ||
| − | <img src="https://static.igem.org/mediawiki/ | + | <img src="https://static.igem.org/mediawiki/2018/b/b3/T--Tec-Monterrey--Interlab_Graph_Abs2.png"> |
| − | <div class="leyenda">Figure | + | <div class="leyenda">Figure 5: Absorbance at 600 nm of transformed bacteria with devices from iGEM kit.</div> |
</div> | </div> | ||
<div class="contenido"> | <div class="contenido"> | ||
Revision as of 15:59, 17 October 2018
Home
InterLab
Fifth International InterLab Measurement Study
Procedure
In order to achieve Bronze requirement #4, the Interlab procedure was successfully carried out, following the iGEM 2018 Interlab Study Protocol. To obtain calibration and experimental data, the following materials were used: competent cells from E. coli's strain DH5a, 96-well plates (black with a clear flat bottom), and the measurement kit, which contains Ludox, silica beads and fluorescein. The first part of the protocol involved generating standard curves for OD measurements, cells and fluorescein. Using a Ludox solution, it was possible to obtain the following reference point for an OD600.
The conversion value obtained was then used to convert any measurement of cell density into OD600 by multiplying by 2.897, using the same instrument under the same conditions.
Figure 1: Table for the conversion value of OD600.
The particle standard curve was obtained using silica microspheres, which have the same size and optical characteristics as bacteria cells. The standard curve was then used to convert ABS600 to an estimated number of cells. The following standard curve was obtained:
Figure 2: Calibration curve of particle count from Absorbance at 600 nm.
Finally, a fluorescein standard curve was made to calculate the fluorescence values of transformed bacteria with GFP. The filter used for the measurements was used according to the recommended values (Bandpass: 530 nm/ 30 nm, width: 25-30 nm, excitation 485 nm and emission 520-530 nm). The curve displayed in Figure 3 was obtained:
Figure 3: Calibration curve of Fluorescein
Using the previous standard curves, it was possible to continue with the cell measurement protocols with transformed E. coli DH5a strains. The following devices from the iGEM kit were transformed:
Figure 4: ***
Results
Absorbance
Absorbance of cells at t=0 h and t=6 h are shown in the diagram of Figure 5.
Figure 5: Absorbance at 600 nm of transformed bacteria with devices from iGEM kit.
All transformed bacteria show growth after 6 hours. Some of the devices, such as device 1 and 5, did not grow sufficiently as other transformed bacteria did.
Absorbance
Fluorescence values at t = 0 h and t = 6 h
Figure 1: Normal Table with values
Conclusion
Fluorescence values obtained by GFP show that some devices emit greater fluorescence values than others. The negative device show no to little fluorescence at both times. Device 3 did not show any fluorescence at all, which affected its measurements. The device that shows the greater fluorescence value was device 4.