Difference between revisions of "Team:Amsterdam/test/thijs/interlab"

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           Interlab Study
 
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         The aim for the iGEM InterLab study was to test the comparability in fluorescence measurements between labs. To do this, Green Fluorescent Protein (GFP) was used as a fluorescent marker, because this is the most frequently used marker in synthetic biology. Every team that joined this study followed exactly the same protocol, provided by the iGEM Measurement Committee.
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          The aim for the iGEM InterLab study was to compare the measurement tools in order to compare data more directly. To do this, the Green Fluorescent Protein (GFP) was used, because this is the most frequently used marker in synthetic biology. Every team that joined this study followed exactly the same protocol, provided by the iGEM Measurement Committee.
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          Before the GFP measurement, the OD
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          600
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          reference point needed to be determined. This was done by measuring LUDOX in the plate reader and calculate the correction factor of our instrument. For the calculation of our cell based readings to the right GFP concentration a fluorescein fluorescent standard curve was made. This curve was made by reading out serial dilutions of fluorescein with PBS.
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          Multiple vectors were transformed in
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          E. Coli
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          to insert different genes which would have different effects on the amount of GFP produced. After the transformation, two colonies of each strain were inoculated overnight. Then the samples were put in a plate and measured in the Fluorstar Optima plate reader, this was repeated every two hours for six hours.
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         Results
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          Figure 1. Raw fluorescence plate reading measurements.
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        The intensity of the GFP for the different conditions were measured over time and every one of them was measured at the same moment with the same settings.
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         <img class="interlab-graph" src="https://static.igem.org/mediawiki/2017/d/de/Abs_test.png"/>
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        Before the GFP measurement, the a correction factor needed to be determined, so the ABS
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        600
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           Figure 2. Raw ABS
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        can be calculated correctly into the OD
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          plate reading measurements.
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        . This was done by measuring LUDOX in the plate reader and compare this data with the OD
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        This shows the measurements of the optical density at 600nm over a period of time. All the conditions were measured at the same moments and with the same settings.
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        600
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        reference point. For the calculation of our cell based readings to the right GFP concentration a fluorescein fluorescent standard curve was made. This curve was made by reading out serial dilutions of fluorescein with phosphate buffered saline (PBS). The correction factor and the fluorescent standard curve are used to standardize the measurements for comparison between the different labs.
 +
      </p>
 +
      <p class="inter-text">
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        Multiple vectors, Test Devices, were transformed in
 +
        <i>
 +
        E. Coli
 +
        </i>
 +
        to insert different genes which would lead to different GFP expression. After transformation, two colonies of each strain were inoculated overnight. Then the samples were put in a 96-well plate and measured in the Fluorstar Optima (Software 2.20 Filmware 1.26) plate reader, this was repeated every two hours for six hours.
 +
        <b>
 +
        Figure 1
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        </b>
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        shows the repeated measurements of the fluorescence for the different Test Devices and
 +
        <b>
 +
        Figure 2
 +
        </b>
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        shows the absorbance for the test devices. After that, the data was filled in in the excel sheet provided by iGEM. This document devided the fluorescence by the absorbance in order to compare the data between labs equally.
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      </p>
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        <img class="interlab-graph" src="https://static.igem.org/mediawiki/2017/4/41/Flu_final.png"/>
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        <p class="interlab-graph-description">
 +
          <b>
 +
          Figure 1. Raw fluorescence plate reading measurements.
 +
           </b>
 +
          The intensity of the GFP for the different conditions were measured over time and every one of them was measured at the same moment with the same settings.
 +
        </p>
 +
        </div>
 +
        <div class="graph-description">
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        <img class="interlab-graph" src="https://static.igem.org/mediawiki/2017/6/69/Abs_final.png"/>
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          <b>
 +
          Figure 2. Raw ABS
 +
          <sub>
 +
            600
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          </sub>
 +
          plate reading measurements.
 +
          </b>
 +
          This shows the measurements of the optical density at 600nm over a period of time. All the conditions were measured at the same moments and with the same settings.
 +
        </p>
 +
         </div>
 +
      </div>
 +
      <p class="inter-text">
 +
        Due to the fact that the results cannot be compared with results from the other iGEM teams. The iGEM Measurement Committee will analyse all the results and publish them. We are looking forward to the results and their interpretation of the results!
 +
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Revision as of 12:36, 5 October 2017

InterLab

The aim for the iGEM InterLab study was to test the comparability in fluorescence measurements between labs. To do this, Green Fluorescent Protein (GFP) was used as a fluorescent marker, because this is the most frequently used marker in synthetic biology. Every team that joined this study followed exactly the same protocol, provided by the iGEM Measurement Committee.

INTERLAB

Results

Before the GFP measurement, the a correction factor needed to be determined, so the ABS 600 can be calculated correctly into the OD 600 . This was done by measuring LUDOX in the plate reader and compare this data with the OD 600 reference point. For the calculation of our cell based readings to the right GFP concentration a fluorescein fluorescent standard curve was made. This curve was made by reading out serial dilutions of fluorescein with phosphate buffered saline (PBS). The correction factor and the fluorescent standard curve are used to standardize the measurements for comparison between the different labs.

Multiple vectors, Test Devices, were transformed in E. Coli to insert different genes which would lead to different GFP expression. After transformation, two colonies of each strain were inoculated overnight. Then the samples were put in a 96-well plate and measured in the Fluorstar Optima (Software 2.20 Filmware 1.26) plate reader, this was repeated every two hours for six hours. Figure 1 shows the repeated measurements of the fluorescence for the different Test Devices and Figure 2 shows the absorbance for the test devices. After that, the data was filled in in the excel sheet provided by iGEM. This document devided the fluorescence by the absorbance in order to compare the data between labs equally.

Figure 1. Raw fluorescence plate reading measurements. The intensity of the GFP for the different conditions were measured over time and every one of them was measured at the same moment with the same settings.

Figure 2. Raw ABS 600 plate reading measurements. This shows the measurements of the optical density at 600nm over a period of time. All the conditions were measured at the same moments and with the same settings.

Due to the fact that the results cannot be compared with results from the other iGEM teams. The iGEM Measurement Committee will analyse all the results and publish them. We are looking forward to the results and their interpretation of the results!

INTERLAB

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