Difference between revisions of "Team:Tec-Monterrey/InterLab"

Latest revision as of 03:25, 18 October 2018

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 DH5α, 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.

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 1: 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 2 was obtained:

Figure 2: 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 3: Devices used to transform DH5α for fluorescence standardising.

Results

Absorbance

Absorbance of cells at t=0 h and t=6 h are shown in the diagram of Figure 4.

Figure 4: 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.

Fluorescence

Fluorescence values at t = 0 h and t = 6 h

Figure 5: Fluorescence of transformed bacteria with devices from iGEM kit.

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.
From absorbance and fluorescence values it is evident that fluorescence is not directly related to the culture's optical density, which is to say that absolute GFP expression is not directly related to the amount of bacteria, as it depends on each device. It is then possible to standardise each of these GFP devices, comparing the results from the iGEM Teams that carried out this InterLab protocol, so that amount of bacteria can be related to fluorescence produced by each device.

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@@ Line 222: / Line 100: @@
          <ul class="vertical-nav">
              <li><a href="#page-top"><span class="label">Header<br><br></span></a></li>
-             <li><a href="#tec-monterrey-work"><span class="label">What we have done</span></a></li>
+             <li><a href="#procedure-interlab"><span class="label">Procedure<br><br></span></a></li>
-             <li><a href="#general-support"><span class="label">General Support</span></a></li>
+             <li><a href="#results-interlab"><span class="label">Results<br><br></span></a></li>
-             <li><a href="#HP-support"><span class="label">HP Support<br><br></span></a></li>
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    <!-- Header -->
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+        <video autoplay muted loop class="video-header" type="video/mp4">
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+        </video>
        </div>
        <div class="header-bottom">
@@ Line 253: / Line 134: @@
            <div class="div-header-bottom">
            <div class="header-title">
-               Interlab
+               InterLab
            </div>
            <div class="header-subtitle">
@@ Line 264: / Line 145: @@
    <div class="articulo">
      <section id="procedure-interlab" class="seccion-responsiva">
-       <div class="contenido-con-imagen-derecha">
+       <div class="articulo">
          <div class="body-title">Procedure</div>
-          <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 materials were used: competent cells from <i>E. coli 's</i> strain DH5α, 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>.
-             Interlab procedure was made to achieve bronze #4 requirement. To make this, the IGEM 2018 Interlab Study Protocol was followed. To make calibration and experimental data the following material was used: measurement kit which contains Ludox, silica bead and fluorescein, competent cells from <i>E. coli</i> strain DH5a and 96 well plates, black with a clear flat bottom.  The first part of the protocol was to make standard curves for OD measurements, cell standard curves and fluorescein standard curves. Using Ludox solution, it was capable 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 obtain conversion value obtained is 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.
-          </div>
-          <div class="imagen-derecha">
-            <img src="https://static.igem.org/mediawiki/2018/5/53/T--Tec-Monterrey--Imagen_Memo_Pensante.jpeg">
-            <br class="clearBoth"/>
-            <div class="leyenda">Figure 1: Table for the conversion value of OD<sub>600</sub>.</div>
-          </div>
-        <br class="clearBoth"/>
        </div>
-       <div class="contenido">
+       <div class="articulo">
-         The particle standard curve was realized to obtain a standard curve using silica microspheres, which have the same size and optical characteristics of bacteria cells. The standard curve was used to convert ABS<sub>600</sub> to an estimated number of cells. The following standard curve was obtained.
+         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 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/2018/8/8b/T--Tec-Monterrey--Interlab_Graph_Abs1.png">
-         <div class="leyenda">Figure 1: Normal Table with values</div>
+         <div class="leyenda">Figure 1: Calibration curve of particle count from Absorbance at 600 nm.</div>
        </div>
-       <div class="contenido">
-         Finally, a fluorescein standard curve was made to calculate the fluorescence values of transform 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, exicitation 485 nm and emission 520-530 nm). The following curves were obtained.
+       <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, excitation 485 nm and emission 520-530 nm). The curve displayed in Figure 2 was obtained:
        </div>
        <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/2018/3/3a/T--Tec-Monterrey--Interlab_Graph_Fluor1.png">
-         <div class="leyenda">Figure 1: Normal Table with values</div>
+         <div class="leyenda">Figure 2: Calibration curve of Fluorescein</div>
        </div>
        <div class="contenido">
-         Using the previous standard curves it was possible to proceed to the cell measurement protocolos with transform E.coli DH5a strains. The following devices from the IGEM kit were transformed.
+        <br>
+         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:
+        <br>
+        <br>
        </div>
        <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/2018/e/ec/T--Tec-Monterrey--Interlab_Devices.jpeg">
-         <div class="leyenda">Figure 1: Normal Table with values</div>
+         <div class="leyenda">Figure 3: Devices used to transform DH5α for fluorescence standardising.</div>
        </div>
      </section>
@@ Line 305: / Line 183: @@
        <div class="contenido">
          <div class="body-subtitle">Absorbance</div>
-         Absorbance of cells at t=0h and t=6 h are shown in the following diagram.
+         Absorbance of cells at t=0 h and t=6 h are shown in the diagram of Figure 4.
        </div>
-       <div class="imagen-centrada-completa">
+       <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/2018/b/b3/T--Tec-Monterrey--Interlab_Graph_Abs2.png">
-         <div class="leyenda">Figure 1: Normal Table with values</div>
+         <div class="leyenda">Figure 4: Absorbance at 600 nm of transformed bacteria with devices from iGEM kit.</div>
        </div>
        <div class="contenido">
-         It can be seen that for all transform bacteria growth after 6 hours. Some of the devices, such as device 1 and 5 didn´t growth sufficiently as other transform bacteria.
+         <br>
+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.
        </div>
        <div class="contenido">
-         <div class="body-subtitle">Absorbance</div>
+         <div class="body-subtitle">Fluorescence</div>
-           Fluorescences values at t=0h and t=6 h
+           Fluorescence values at t = 0 h and t = 6 h
        </div>
-       <div class="imagen-centrada-completa">
+       <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/2018/a/a5/T--Tec-Monterrey--Interlab_Graph_Fluor2.png">
-         <div class="leyenda">Figure 1: Normal Table with values</div>
+         <div class="leyenda">Figure 5: Fluorescence of transformed bacteria with devices from iGEM kit.</div>
        </div>
      </section>
@@ Line 327: / Line 206: @@
        <div class="conclusion">
          <div class="body-title">Conclusion</div>
-         Fluorescence values obtained by GFP show that some devices emit greater fluorescence values than others.The negative device show no/ little fluorescence at both time. Device 3 didn’t show any fluorescence at all which affect the measurements of it. The device that show the greater fluorescence value was device 4.
+         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.<br>
+        From absorbance and fluorescence values it is evident that fluorescence is not directly related to the culture's optical density, which is to say that absolute GFP expression is not directly related to the amount of bacteria, as it depends on each device. It is then possible to standardise each of these GFP devices, comparing the results from the iGEM Teams that carried out this InterLab protocol, so that amount of bacteria can be related to fluorescence produced by each device.
        </div>
      </section>
@@ Line 334: / Line 214: @@
 </body>
+<div class="clear extra_space"></div>
+<div class="line_divider"></div>
+<div class="clear extra_space"></div>
 </html>
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Difference between revisions of "Team:Tec-Monterrey/InterLab"

Latest revision as of 03:25, 18 October 2018

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