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− | <span class="image avatar"><img src="https://static.igem.org/mediawiki/2017/3/3d/LogoOWL.png" alt="" /></span> | + | <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>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. | <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. | ||
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<tr> | <tr> | ||
<td>Replicate 1</td> | <td>Replicate 1</td> | ||
− | <td>0 | + | <td>0.041700002</td> |
− | <td>0 | + | <td>0.035300002</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td>Replicate 2</td> | <td>Replicate 2</td> | ||
− | <td>0 | + | <td>0.042399999</td> |
− | <td>0 | + | <td>0.035</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td>Replicate 3</td> | <td>Replicate 3</td> | ||
− | <td>0 | + | <td>0.043000001</td> |
− | <td>0 | + | <td>0.035700001</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td>Replicate 4</td> | <td>Replicate 4</td> | ||
− | <td>0 | + | <td>0.043000001</td> |
− | <td>0 | + | <td>0.034699999</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td>Arithmetic Mean</td> | <td>Arithmetic Mean</td> | ||
− | <td>0 | + | <td>0.042525001</td> |
− | <td>0 | + | <td>0.035175</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td>Corrected Absorbance 600</td> | <td>Corrected Absorbance 600</td> | ||
− | <td>0 | + | <td>0.007350001</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td>Reference OD<sub>600</sub> </td> | <td>Reference OD<sub>600</sub> </td> | ||
− | <td>0 | + | <td>0.0425</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td>Correction Factor</td> | <td>Correction Factor</td> | ||
− | <td>5 | + | <td>5.78231249</td> |
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</table> | </table> | ||
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<figcaption> Figure 1. Data from table 1 exported in beam chart. </figcaption></center> | <figcaption> Figure 1. Data from table 1 exported in beam chart. </figcaption></center> | ||
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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 nm, Emission 530 nm, 50 gains and 20 flashes. </figcaption></center> | + | <figcaption> 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. </figcaption></center> |
</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> | <figure><center> | ||
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<figcaption> Figure 2. Calibration curve of fluorescein: fluorescence measurement of different concentrations (µM). </figcaption></center> | <figcaption> Figure 2. Calibration curve of fluorescein: 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> | <figcaption> Figure 3. Calibration curve of fluorescein (log scale): fluorescence measurement of different concentrations (µM). </figcaption></center> | ||
</figure> | </figure> | ||
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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> | ||
− | <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 | + | <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 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. | ||
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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%,> | ||
− | <figcaption> 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. </figcaption></center> | + | <figcaption> 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. </figcaption></center> |
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− | <figcaption> Figure 5. Absorbance measurement ( | + | <figcaption> Figure 5. Absorbance measurement (600 nm) of six different 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> | <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
ChiTUcare
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 |
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).
Generated standard curve of fluorescence for different fluorescein concentrations can be found below.
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.
Fluorescein / OD600
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.