Difference between revisions of "Team:Freiburg/InterLab"

 
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This year’s Interlab Study tried to determine the absolute fluorescence units of eight plasmids either in a plate reader or by FACS analysis. These set of plasmids consisted of one positive control, one negative control and 6 test devices containing different ribosome binding sites (RBS) and promoters of different strengths.<br>
 
This year’s Interlab Study tried to determine the absolute fluorescence units of eight plasmids either in a plate reader or by FACS analysis. These set of plasmids consisted of one positive control, one negative control and 6 test devices containing different ribosome binding sites (RBS) and promoters of different strengths.<br>
  
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For a more detailed description visit the homepage of the iGEM's measurement committee.  
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For a more detailed description visit the homepage of the <a href="https://2017.igem.org/Competition/InterLab_Study"target="_blank">iGEM's measurement committee. </a>
 
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<h2>Materials and Methods</h2>
 
<h2>Materials and Methods</h2>
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<p>All steps of the OD<sup>600</sup> reference point calibration, the fluorescein fluorescence standard curve and the cell measurements were performed according to the protocol provided by iGEM's measurement committee. For the measurements Corning 96-well flat clear bottom black polystyrol plates and a Synergy H4 from Biotek plate reader were used. Competent <i>E. coli</i> K-12 DH5-alpha cells were produced using the Zymo Research Mix & Go <i>E. coli</i> Transformation Kit.  
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<p>All steps of the OD<sub>600</sub> reference point calibration, the fluorescein fluorescence standard curve and the cell measurements were performed according to the <a href="https://2017.igem.org/Competition/InterLab_Study"target="_blank">protocol</a> provided by iGEM's measurement committee. For the measurements Corning 96-well flat white polystyrol plates and a <a href="https://www.biotek.com/resources/application-notes/utility-of-synergy-h4/"target="_blank">Synergy H4 from Biotek plate reader</a> were used. Competent <i>E. coli</i> K-12 DH5-alpha cells were produced using the <a href="https://www.zymoresearch.com/e-coli/transformation-kits-accessories/transformation-kit-buffer-set"target="_blank">Zymo Research Mix & Go <i>E. coli</i> Transformation Kit</a>.  
 
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<p><strong>Table 1: Results of the OD<sub>600</sub>  reference point calibration. </strong></p>
  
 
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<h4>Fluorescein Fluorescence Standard Curve</h4>
 
<h4>Fluorescein Fluorescence Standard Curve</h4>
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<p>In the second step, a standard curve of fluorescence intensity dependent on the fluorescein concentration had to be determined to convert cell-based readings to an equivalent fluorescein concentration. Afterwards, the fluorescein concentration can be converted into the GFP concentration. Results for the fluorescein fluorescence standard curve are depicted in figure 1.
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<p>In the second step, a standard curve of fluorescence intensity dependent on the fluorescein concentration had to be determined to convert cell-based readings to an equivalent fluorescein concentration. Afterwards, the fluorescein concentration can be converted into the GFP concentration. Results for the fluorescein fluorescence standard curve are depicted in figure 1 and 2.
 
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             <p><strong>Fig. 1: Fluorescein Fluorescence Standard Curve on a linear scale</strong></p>
 
             <p><strong>Fig. 1: Fluorescein Fluorescence Standard Curve on a linear scale</strong></p>
 
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             <p><strong>Fig. 2: Fluorescein Fluorescence Standard Curve on a log scale</strong></p>
 
             <p><strong>Fig. 2: Fluorescein Fluorescence Standard Curve on a log scale</strong></p>
 
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                 <img src="https://static.igem.org/mediawiki/2017/0/0b/T-Freiburg-Interlab_Study_3.png"="100%" style="width:100%;height:100%">
 
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                     <p><strong>Figure 3: Fluorescence quantification of test devices. Plate reader measurements of transformed <i>E. coli</i> K12 DH5-alpha converted to µM Fluorescein/OD<sub>600</sub>.</strong><br>
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                     <p><strong>Fig. 3: Fluorescence quantification of test devices.</strong><br> Plate reader measurements of transformed <i>E. coli</i> K12 DH5-alpha converted to µM Fluorescein/OD<sub>600</sub>.<br>
 
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Additionally, new competent <i>E. coli</i> K12 DH5-alpha cells were produced. Afterwards the InterLab study was performed for a last time but still no differences were observable   
 
Additionally, new competent <i>E. coli</i> K12 DH5-alpha cells were produced. Afterwards the InterLab study was performed for a last time but still no differences were observable   
  
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In conclusion, no meaningful data could be obtained. Since both positive and negative control did not perform as expected the problem could not be identified.  
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In conclusion, no meaningful data could be obtained. Since both positive and negative control did not perform as expected, the problem could not be identified.  
  
 
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Latest revision as of 02:54, 2 November 2017

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Interlab Study

Introduction

Reproduction of experimental data remains one of the big challenges in synthetic biology. In order to overcome this problem, three years ago the iGEM's measurement committee started to develop a robust measurement procedure for green fluorescent protein (GFP), in the form of the Interlab Study.
This year’s Interlab Study tried to determine the absolute fluorescence units of eight plasmids either in a plate reader or by FACS analysis. These set of plasmids consisted of one positive control, one negative control and 6 test devices containing different ribosome binding sites (RBS) and promoters of different strengths.
For a more detailed description visit the homepage of the iGEM's measurement committee.

Materials and Methods

All steps of the OD600 reference point calibration, the fluorescein fluorescence standard curve and the cell measurements were performed according to the protocol provided by iGEM's measurement committee. For the measurements Corning 96-well flat white polystyrol plates and a Synergy H4 from Biotek plate reader were used. Competent E. coli K-12 DH5-alpha cells were produced using the Zymo Research Mix & Go E. coli Transformation Kit.

Calibration

OD600 Reference Point

Because plate readers do not have standardized path length, the first step of the InterLab study was to obtain a ratiometric conversion factor to transform absorbance data into a standard OD600 measurement. This was achieved by using LUDOX-HS40 as a single point reference. After data analysis a ratiometric conversion factor of 3.4 was calculated. The following table shows the results of the OD600 reference point calibration.

Table 1: Results of the OD600 reference point calibration.

Replicate LUDOX-S40 H2O
1 0.043 0.035
2 0.049 0.036
3 0.047 0.035
4 0.052 0.035
Arith. Mean 0.04775 0.03525
Corrected Abs600 0.0125
Reference OD600 0.0425
OD600/Abs600 3.4

Fluorescein Fluorescence Standard Curve

In the second step, a standard curve of fluorescence intensity dependent on the fluorescein concentration had to be determined to convert cell-based readings to an equivalent fluorescein concentration. Afterwards, the fluorescein concentration can be converted into the GFP concentration. Results for the fluorescein fluorescence standard curve are depicted in figure 1 and 2.

Fig. 1: Fluorescein Fluorescence Standard Curve on a linear scale

Fig. 2: Fluorescein Fluorescence Standard Curve on a log scale

Cell Measurements

After the calibration steps were successfully established, the test devices were transformed into E. coli K12 DH5-alpha and characterized. The results of this characterization are shown in figure 3.

Results

Fig. 3: Fluorescence quantification of test devices.
Plate reader measurements of transformed E. coli K12 DH5-alpha converted to µM Fluorescein/OD600.

Discussion

Expecting an increase in the fluorescence intensity over the time for the different test devices, it was difficult to evaluate the obtained data. Repetition with the initial conditions showed no effect. After looking for possible error sources, chloramphenicol stock solutions were exchanged. Additionally, new competent E. coli K12 DH5-alpha cells were produced. Afterwards the InterLab study was performed for a last time but still no differences were observable In conclusion, no meaningful data could be obtained. Since both positive and negative control did not perform as expected, the problem could not be identified.