Difference between revisions of "Team:Heidelberg/InterLab"
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| − | <div style="padding-top: 80px; background-color:white;"> | + | <div style="padding-top: 80px; background-color:white;"> |
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| − | + | <h1>1. Interlab Study</h1> | |
| − | + | <hr> | |
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| − | + | <p>As a first step to contribute to this year's iGEM competition we decided to participate in iGEM's fourth International Interlaboratory Study along with many other teams from around the world. This study is organized by iGEM's measurement committee in an effort to establish standardized, reliable and repeatable measurement tools for the iGEM community and the synthetic biology community as a whole. This year's iGEM InterLab study is about establishing a standardized protocol for the measurement of GFP using a plate reader. To start things off we needed a plate reader that is qualified to measure GFP fluorescence. Namely, Tecan Infinite M200 Pro plate reader. Additionally, we needed competent <i>E.coli</i> DH5α. These were prepared from glycerol stocks. Together with the material iGEM had provided we were ready for work. Throughout our experiments, we tested 8 plasmids (2 controls and 6 test devices) by measuring the OD<sub>600</sub> and the fluorescence of the cells carrying the constructs. | |
| − | + | The workflow can be separated into four segments. The first segment is the transformation of all plasmids into competent DH5α cells. | |
| − | + | <h2>1.1 Transformation of 8 test devices into competent DH5α cells</h2> | |
| − | + | ||
| − | + | <p>The transformation was successful for all the plasmids and resulted in a sufficient amount of colonies on all plates. Therefore, two colonies from each plate were picked and inoculated in LB medium containing chloramphenicol over night for approximately 16-18 hours at 37°C and 220 rpm.</p> | |
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| + | <h2>1.2 Measurement of LUDOX-HS40 OD600 Reference Point</h2> | ||
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| + | <p>The second segment is the measurement of the LUDOX-HS40 solution to obtain a ratiometric conversion factor which allows us to transform the absorbance data into a standard OD<sub>600</sub> measurement. This is necessary because plate readers in general do not have a 1 cm path length which means that measurements are volume dependent. By doing so, we obtained following data.</p> | ||
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| + | <a href="#" target="_blank" ><img src="https://static.igem.org/mediawiki/2017/4/48/T--Heidelberg--LUDOX-HS40.PNG" alt="#"></a> | ||
| + | <p>Figure 1.1: Measurement of the LUDOX-HS40 and water in four replicates and calculation of the ratiometric conversion factor</p> | ||
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| − | + | With these replicates measured, we obtained a ratiometric conversion factor of 4.30 for our specific plate reader when measured 100 μL of the provided LUDOX-HS40 to water.</p> | |
| − | + | <h2>1.3 Graphing a Fluorescein fluorescence standard curve</h2> | |
| − | + | ||
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| − | + | <p>In the third segment we were instructed to measure a standard curve for fluorescence of Fluorescein which can be used to correct our cell based readings to an equivalent Fluorescein concentration. Following the instructions, we could obtain following results for the serial dilution of Fluorescein in phosphate buffered saline. | |
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| − | + | <a href="#" target="_blank" ><img src="https://static.igem.org/mediawiki/2017/a/a0/T--Heidelberg--FluoresceinSerialDilution.PNG" alt="#"></a> | |
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| − | + | <p>Figure 1.2: Measurement of a standard curve with four replicates by serial dilution of Fluorescein in phosphate buffered saline</p> | |
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| + | <a href="#" target="_blank" ><img src="https://static.igem.org/mediawiki/2017/5/57/T--Heidelberg--FluoresceinStandardCurve.png" alt="#"></a> | ||
| + | <div class="caption"> | ||
| + | |||
| + | <p>Figure 1.3: Fluorescence is plotted against the Fluorescein concentration</p> | ||
| + | </div> | ||
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| − | + | <p>Figure 1.4: Fluorescence is plotted against the Fluorescein concentration on a logarithmic scale</p> | |
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| − | + | <p>These data give us an approximate linear regression line when plotted in a logarithmic scale and show an expected exponential course when plotted in a linear form.</p> | |
| − | + | <h2>1.4 Measurement of OD<sub>600</sub> and Fluorescence of Transformed Cells</h2> | |
| + | |||
| + | <p>In the last of the four segments the OD<sub>600</sub> and the fluorescence of the transformed cells were measured according to the protocol after 0, 2, 4 and 6 hours. Measurements of all these time points gave us the following data and calculations which were conducted with the values we obtained from the standard curves and the reference point. </p> | ||
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| + | <a href="#" target="_blank" ><img src="https://static.igem.org/mediawiki/2017/2/2e/T--Heidelberg--RawPlateReadings.PNG" alt="#"></a> | ||
| + | <div class="caption"> | ||
| + | |||
| + | |||
| + | <p>Figure 1.5: Raw data obtained from measurements</p> | ||
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| + | <div class="row" style="padding-top: 30px;padding-bottom: 30px;"> | ||
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| + | <a href="#" target="_blank" ><img src="https://static.igem.org/mediawiki/2017/8/8e/T--Heidelberg--ExperimentalValues0-2.PNG" alt="#"></a> | ||
| + | <div class="caption"> | ||
| + | |||
| + | |||
| + | <p>Figure 1.6: Raw data and measurements for the 0 and 2 hour time points</p> | ||
| + | </div> | ||
| + | |||
| + | </div> | ||
| + | </div> | ||
| + | <div class="row" style="padding-top: 30px;padding-bottom: 30px;"> | ||
| + | <div class="col-lg-offset-1 col-md-offset-1 col-sm-offset-1 col-xs-offset-1 col-lg-11 col-md-11 col-sm-11 col-xs-11"> | ||
| + | |||
| + | <a href="#" target="_blank" ><img src="https://static.igem.org/mediawiki/2017/4/4d/T--Heidelberg--ExperimentalValues4-6.PNG" alt="#"></a> | ||
| + | <div class="caption"> | ||
| + | |||
| + | |||
| + | <p>Figure 1.7: Raw data and measurements for the 4 and 6 hour time points</p> | ||
| + | </div> | ||
| + | |||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | <h2>2. Discussion</h2> | ||
| + | <p>The protocol was easy to follow and the constructs were nicely expressed which allowed reliable and comparable measurements. In the process of obtaining transformed DH5α cells we did not encounter any problems with transformation as bacteria showed exceptional growth. The specific ratiometric conversion factor of our plate reader could be determined without further adjustments using the LUDOX-HS40 solution. When measuring the Fluorescein standard curve on a logarithmic scale the data points showed a linear distribution. The final step, the measurement of the fluorescence emission and optical density of the cells, resulted in the raw data shown in figure 1.5. To make this data visually accessible the mean values for OD and fluorescence were calculated and plotted against their respective time points which is seen in the following graphs. | ||
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| + | |||
| + | <a href="#" target="_blank" ><img src="https://static.igem.org/mediawiki/2017/e/eb/T--Heidelberg--ODGraph.png" alt="#"></a> | ||
| + | <div class="caption"> | ||
| + | |||
| + | |||
| + | <p>Figure 1.8: Calculated means from both replicates for the OD<sub>600</sub> after each time point</p> | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | <div class="row" style="padding-top: 30px;padding-bottom: 30px;"> | ||
| + | <div class="col-lg-offset-1 col-md-offset-1 col-sm-offset-1 col-xs-offset-1 col-lg-11 col-md-11 col-sm-11 col-xs-11"> | ||
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| + | <a href="#" target="_blank" ><img src="https://static.igem.org/mediawiki/2017/0/07/T--Heidelberg--FluorescenceGraph.png" alt="#"></a> | ||
| + | <div class="caption"> | ||
| + | |||
| + | |||
| + | <p>Figure 1.9: Calculated means from both replicates for the fluorescence after each time point</p> | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | |||
| + | <p>Conclusively, these data suggest that the Interlab study was successful and that the provided protocol can be shared in the community for standardized fluorescence measurement with a plate reader.</p> | ||
| + | </div> | ||
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Revision as of 08:08, 26 September 2017
Team:Heidelberg/InterLab
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From 2014.igem.org
1. Interlab Study
As a first step to contribute to this year's iGEM competition we decided to participate in iGEM's fourth International Interlaboratory Study along with many other teams from around the world. This study is organized by iGEM's measurement committee in an effort to establish standardized, reliable and repeatable measurement tools for the iGEM community and the synthetic biology community as a whole. This year's iGEM InterLab study is about establishing a standardized protocol for the measurement of GFP using a plate reader. To start things off we needed a plate reader that is qualified to measure GFP fluorescence. Namely, Tecan Infinite M200 Pro plate reader. Additionally, we needed competent E.coli DH5α. These were prepared from glycerol stocks. Together with the material iGEM had provided we were ready for work. Throughout our experiments, we tested 8 plasmids (2 controls and 6 test devices) by measuring the OD600 and the fluorescence of the cells carrying the constructs. The workflow can be separated into four segments. The first segment is the transformation of all plasmids into competent DH5α cells.
1.1 Transformation of 8 test devices into competent DH5α cells
The transformation was successful for all the plasmids and resulted in a sufficient amount of colonies on all plates. Therefore, two colonies from each plate were picked and inoculated in LB medium containing chloramphenicol over night for approximately 16-18 hours at 37°C and 220 rpm.
1.2 Measurement of LUDOX-HS40 OD600 Reference Point
The second segment is the measurement of the LUDOX-HS40 solution to obtain a ratiometric conversion factor which allows us to transform the absorbance data into a standard OD600 measurement. This is necessary because plate readers in general do not have a 1 cm path length which means that measurements are volume dependent. By doing so, we obtained following data.
Figure 1.1: Measurement of the LUDOX-HS40 and water in four replicates and calculation of the ratiometric conversion factor
With these replicates measured, we obtained a ratiometric conversion factor of 4.30 for our specific plate reader when measured 100 μL of the provided LUDOX-HS40 to water.
1.3 Graphing a Fluorescein fluorescence standard curve
In the third segment we were instructed to measure a standard curve for fluorescence of Fluorescein which can be used to correct our cell based readings to an equivalent Fluorescein concentration. Following the instructions, we could obtain following results for the serial dilution of Fluorescein in phosphate buffered saline.
Figure 1.2: Measurement of a standard curve with four replicates by serial dilution of Fluorescein in phosphate buffered saline
Figure 1.3: Fluorescence is plotted against the Fluorescein concentration
Figure 1.4: Fluorescence is plotted against the Fluorescein concentration on a logarithmic scale
These data give us an approximate linear regression line when plotted in a logarithmic scale and show an expected exponential course when plotted in a linear form.
1.4 Measurement of OD600 and Fluorescence of Transformed Cells
In the last of the four segments the OD600 and the fluorescence of the transformed cells were measured according to the protocol after 0, 2, 4 and 6 hours. Measurements of all these time points gave us the following data and calculations which were conducted with the values we obtained from the standard curves and the reference point.
Figure 1.5: Raw data obtained from measurements
Figure 1.6: Raw data and measurements for the 0 and 2 hour time points
Figure 1.7: Raw data and measurements for the 4 and 6 hour time points
2. Discussion
The protocol was easy to follow and the constructs were nicely expressed which allowed reliable and comparable measurements. In the process of obtaining transformed DH5α cells we did not encounter any problems with transformation as bacteria showed exceptional growth. The specific ratiometric conversion factor of our plate reader could be determined without further adjustments using the LUDOX-HS40 solution. When measuring the Fluorescein standard curve on a logarithmic scale the data points showed a linear distribution. The final step, the measurement of the fluorescence emission and optical density of the cells, resulted in the raw data shown in figure 1.5. To make this data visually accessible the mean values for OD and fluorescence were calculated and plotted against their respective time points which is seen in the following graphs.
Figure 1.8: Calculated means from both replicates for the OD600 after each time point
Figure 1.9: Calculated means from both replicates for the fluorescence after each time point
Conclusively, these data suggest that the Interlab study was successful and that the provided protocol can be shared in the community for standardized fluorescence measurement with a plate reader.
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