Difference between revisions of "Team:TU Darmstadt/judging/interlab"

 
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<span class="image avatar"><a href="https://2017.igem.org/Team:TU_Darmstadt"><img src="https://static.igem.org/mediawiki/2017/3/3d/LogoOWL.png" alt="home" /></a></span>
 
<h1 id="logo"><a href="https://2017.igem.org/Team:TU_Darmstadt" alt="home">ChiTUcare</a></h1>
 
<h1 id="logo"><a href="https://2017.igem.org/Team:TU_Darmstadt" alt="home">ChiTUcare</a></h1>
 
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<p>iGEM is coping with the problem of different groups measuring fluorescence differently, in different units and evaluating the results non-identically. Therefore, the InterLab Study aims to develop a standardized protocol for repeatable fluorescence measurements of GFP.  
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<p>Since iGEM is an international competition with many teams from all over the world, it is inevitably coping with the problem of unreproducible scientific measurement results. Multiple teams are measuring fluorescence differently, in different units and are evaluating the results non-identically. Therefore, the InterLab Study aims to develop a standardized protocol for repeatable fluorescence measurements of GFP.  
 
The study should be carried out and examined in different laboratories all over the world.  
 
The study should be carried out and examined in different laboratories all over the world.  
 
Our team followed the standardized procedure guiding us through transformation, inoculation and measurement process. <br>For more information: <a href="https://2017.igem.org/Competition/InterLab_Study">Fourth International InterLab Measurement Study</a> <br>
 
Our team followed the standardized procedure guiding us through transformation, inoculation and measurement process. <br>For more information: <a href="https://2017.igem.org/Competition/InterLab_Study">Fourth International InterLab Measurement Study</a> <br>
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                                                 <tr>
 
                                                 <tr>
 
                                             <td>Replicate 1</td>
 
                                             <td>Replicate 1</td>
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                                                     <td>0,041700002</td>
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                                                     <td>0.041700002</td>
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                                                     <td>0,035300002</td>
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                                                     <td>0.035300002</td>
 
                                                 </tr>
 
                                                 </tr>
 
                                                 <tr>
 
                                                 <tr>
 
                                                     <td>Replicate 2</td>
 
                                                     <td>Replicate 2</td>
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                                                     <td>0,042399999</td>
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                                                     <td>0.042399999</td>
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                                                     <td>0,035</td>
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                                                     <td>0.035</td>
 
                                                 </tr>
 
                                                 </tr>
 
                                                 <tr>
 
                                                 <tr>
 
                                                     <td>Replicate 3</td>
 
                                                     <td>Replicate 3</td>
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                                                     <td>0,043000001</td>
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                                                     <td>0.043000001</td>
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                                                     <td>0,035700001</td>
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                                                     <td>0.035700001</td>
 
                                                 </tr>
 
                                                 </tr>
 
                                                 <tr>
 
                                                 <tr>
 
                                                     <td>Replicate 4</td>
 
                                                     <td>Replicate 4</td>
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                                                     <td>0,043000001</td>
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                                                     <td>0.043000001</td>
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                                                     <td>0,034699999</td>
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                                                     <td>0.034699999</td>
 
                                                 </tr>
 
                                                 </tr>
 
                                                 <tr>
 
                                                 <tr>
 
                                                     <td>Arithmetic Mean</td>
 
                                                     <td>Arithmetic Mean</td>
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                                                     <td>0,042525001</td>
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                                                     <td>0.042525001</td>
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                                                     <td>0,035175</td>
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                                                     <td>0.035175</td>
 
                                                 </tr>
 
                                                 </tr>
 
                                                 <tr>
 
                                                 <tr>
 
                                                     <td>Corrected Absorbance 600</td>
 
                                                     <td>Corrected Absorbance 600</td>
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                                                     <td>0,007350001</td>
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                                                     <td>0.007350001</td>
 
                                                 </tr>
 
                                                 </tr>
 
                                                 <tr>
 
                                                 <tr>
 
                                                     <td>Reference OD<sub>600</sub> </td>
 
                                                     <td>Reference OD<sub>600</sub> </td>
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                                                     <td>0,0425</td>
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                                                     <td>0.0425</td>
 
                                                 </tr>
 
                                                 </tr>
 
                                                 <tr>
 
                                                 <tr>
 
                                                     <td>Correction Factor</td>
 
                                                     <td>Correction Factor</td>
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                                                     <td>5,78231249</td>
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                                                     <td>5.78231249</td>
 
                                                 </tr>
 
                                                 </tr>
 
                                             </table>
 
                                             </table>
−
<figcaption> Tab. 1: Absorbance measurement of LUDOX-S40 and H<sub style="font-size: 0.9em;">²</sub>O of four different replicates.</figcaption></center>
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<figcaption> Table 1. Absorbance measurement of LUDOX-S40 and H<sub style="font-size: 0.9em;">²</sub>O of four different replicates.</figcaption></center>
 
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<img src="https://static.igem.org/mediawiki/2017/1/1b/T--TU_Darmstadt--Interlab1.gif", alt="hier fehlt was", width=50%,>
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<img src="https://static.igem.org/mediawiki/2017/4/46/T--TU_Darmstadt--graph1.png", alt="hier fehlt was", width=70%,>
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<figcaption> Fig. 1: Data from table 1 exported in beam chart. </figcaption></center>
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<figcaption> Figure 1. Data from table 1 exported in beam chart. </figcaption></center>
 
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<p>
 
<p>
 
<h5>Fluorescein standard curve</h5><br>
 
<h5>Fluorescein standard curve</h5><br>
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<p>The fluorescence of a dilution series, fluorescein in four replicates were measured in standard modes in our Tecan plate reader (infinite 200Pro).</p>
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<p>The fluorescence of a dilution series, fluorescein in four replicates, were measured in standard modes in our tecan plate reader (infinite 200Pro).</p>
 
<br>
 
<br>
 
<p>
 
<p>
 
<figure><center>
 
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<img src="https://static.igem.org/mediawiki/2017/9/9f/T--TU_Darmstadt--Interlab2.gif", alt="hier fehlt was", width=100%,>
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<figcaption> Tab. 2: Fluorescence measurement of fluorescein in eleven dilutions and one control containing PBS.<br>
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<figcaption> Table 2. Fluorescence measurement of fluorescein in eleven dilutions and one control containing PBS. Measurement settings for the plate reader: Excitation 485&nbsp;nm, Emission 530&nbsp;nm, 50&nbsp;gains and 20&nbsp;flashes. </figcaption></center>
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Measurement settings for the plate reader: Excitation 485 nm, Emission 530 nm, 50 gains and 20 flashes. </figcaption></center>
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</figure></p><br>
 
</figure></p><br>
 
<p>Generated standard curve of fluorescence for different fluorescein concentrations can be found below.</p><br>
 
<p>Generated standard curve of fluorescence for different fluorescein concentrations can be found below.</p><br>
 
<p>
 
<p>
 
<figure><center>
 
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<figcaption> Fig. 2: Calibration curve of fluorescein: fluorescence measurement of different concentrations (µM). </figcaption></center>
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<figcaption> Figure 2. Calibration curve of fluorescein: fluorescence measurement of different concentrations (µM). </figcaption></center>
 
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<figcaption> Fig. 3: Calibration curve of fluorescein (log scale): fluorescence measurement of different concentrations (µM). </figcaption></center>
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<figcaption> Figure 3. Calibration curve of fluorescein (log scale): fluorescence measurement of different concentrations (µM). </figcaption></center>
 
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<h3>Cell Measurement</h3><br>
 
<h3>Cell Measurement</h3><br>
 
<h5>Fluorescence and Absorbance 600 Measurement</h5><br>
 
<h5>Fluorescence and Absorbance 600 Measurement</h5><br>
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<p>Fluorescence and absorbance was measured from six different devices, positive and negative control which are built in pSB1C3 containing chloramphenicol resistance. Samples were taken after 0,2,4,6 hours.<br>   
+
<p>Fluorescence and absorbance was measured from six different devices, positive and negative control which are built in pSB1C3 containing chloramphenicol resistance. Samples were taken after 0, 2, 4, 6&nbsp;h.<br>   
 
The arithmetic mean from two colonies was calculated and can be found below.
 
The arithmetic mean from two colonies was calculated and can be found below.
 
</p>
 
</p>
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<img src="https://static.igem.org/mediawiki/2017/b/bd/T--TU_Darmstadt--Interlab5.gif", alt="hier fehlt was", width=70%,>
 
<img src="https://static.igem.org/mediawiki/2017/b/bd/T--TU_Darmstadt--Interlab5.gif", alt="hier fehlt was", width=70%,>
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<figcaption> Fig. 4: Fluorescence measurement of six different devices, positive and negative control.<br>
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<figcaption> Figure 4. Fluorescence measurement of six different devices, positive and negative control. Measurement settings for the plate reader: Excitation 485&nbsp;nm, Emission 530&nbsp;nm, 50&nbsp;gains and 20&nbsp;flashes. </figcaption></center>
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Measurement settings for the plate reader: Excitation 485 nm, Emission 530 nm, 50 gains and 20 flashes. </figcaption></center>
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<img src="https://static.igem.org/mediawiki/2017/9/90/T--TU_Darmstadt--graph3.png", alt="hier fehlt was", width=70%,>
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<figcaption> Fig. 5: Absorbance measurement (600nm) of six different devices, positive and negative control. </figcaption></center>
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<figcaption> Figure 5. Absorbance measurement (600&nbsp;nm) of six different devices, positive and negative control. </figcaption></center>
 
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<figcaption> Fig. 6: Average level of the six devices, positive and negative control. </figcaption></center>
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<figcaption> Figure 6. Average level of the six devices, positive and negative control. </figcaption></center>
 
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Latest revision as of 17:08, 1 November 2017

MainPage

InterLab Measurement Studies

Since iGEM is an international competition with many teams from all over the world, it is inevitably coping with the problem of unreproducible scientific measurement results. Multiple teams are measuring fluorescence differently, in different units and are evaluating the results non-identically. Therefore, the InterLab Study aims to develop a standardized protocol for repeatable fluorescence measurements of GFP. The study should be carried out and examined in different laboratories all over the world. Our team followed the standardized procedure guiding us through transformation, inoculation and measurement process.
For more information: Fourth International InterLab Measurement Study
Following below you can find our results.

Calibration

OD600 reference point

Several measurements of LUDOX-S40 and H²O were spectrometric analyzed below.

LUDOX-S40 H²O
Replicate 1 0.041700002 0.035300002
Replicate 2 0.042399999 0.035
Replicate 3 0.043000001 0.035700001
Replicate 4 0.043000001 0.034699999
Arithmetic Mean 0.042525001 0.035175
Corrected Absorbance 600 0.007350001
Reference OD600 0.0425
Correction Factor 5.78231249
Table 1. Absorbance measurement of LUDOX-S40 and H²O of four different replicates.



hier fehlt was
Figure 1. Data from table 1 exported in beam chart.



Fluorescein standard curve

The fluorescence of a dilution series, fluorescein in four replicates, were measured in standard modes in our tecan plate reader (infinite 200Pro).


hier fehlt was
Table 2. Fluorescence measurement of fluorescein in eleven dilutions and one control containing PBS. Measurement settings for the plate reader: Excitation 485 nm, Emission 530 nm, 50 gains and 20 flashes.


Generated standard curve of fluorescence for different fluorescein concentrations can be found below.


hier fehlt was
Figure 2. Calibration curve of fluorescein: fluorescence measurement of different concentrations (µM).


hier fehlt was
Figure 3. Calibration curve of fluorescein (log scale): fluorescence measurement of different concentrations (µM).

Cell Measurement


Fluorescence and Absorbance 600 Measurement

Fluorescence and absorbance was measured from six different devices, positive and negative control which are built in pSB1C3 containing chloramphenicol resistance. Samples were taken after 0, 2, 4, 6 h.
The arithmetic mean from two colonies was calculated and can be found below.


hier fehlt was
Figure 4. Fluorescence measurement of six different devices, positive and negative control. Measurement settings for the plate reader: Excitation 485 nm, Emission 530 nm, 50 gains and 20 flashes.



hier fehlt was
Figure 5. Absorbance measurement (600 nm) of six different devices, positive and negative control.


Fluorescein / OD600

hier fehlt was
Figure 6. Average level of the six devices, positive and negative control.


Conclusion


Examining the measurement results: device 1,2 and 4 are similar to the positive control, these express GFP. In comparison, device 3 and 6 are similar to the negative control and do not express GFP.
During measurements we differed the gains up to 70 in plate reader settings and noticed a hundredfold higher fluorescence data.
This could influence the reproducibility from laboratory to laboratory.