Difference between revisions of "Team:Freiburg/InterLab"

 
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<h1 align="center">Interlab Study</h1>       
 
<h1 align="center">Interlab Study</h1>       
 
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<h2>Background</h2>
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<h2>Introduction</h2>
 
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The aim of this year’s Interlab Study was to compare fluorescence measured in different labs using the exact same protocol, trying to exclude any deviations in the experimental procedure and to standardize all measurements so that they are no longer machine-dependent. Afterwards, it should be possible to evaluate if this approach is sufficient to generate a comparability between different measurements.
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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.<br>
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The Interlab Study was divided into one part for standardization and one part for the cell measurements. The standardization part consisted of two experiments, firstly the OD600 calibration and secondly the standard curve of fluorescein.
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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>
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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>Methods and Results</h2>
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<h2>Materials and Methods</h2>
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<h3>Single Point Reference</h3>
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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>As absorbance is instrument-dependent, it was necessary to perform an OD600 calibration with LUDOX-S40 as a single point reference. Therefore, the absorbance at 600 nm of 100&nbsp;µl LUDOX-S40 and of 100&nbsp;µl H<sub>2</sub>O was measured with a spectrometer. Four replicates were done and the automatic path length correction was turned off. The respective results can be seen in the table below.
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After subtracting the values for H<sub>2</sub>O from the values for LUDOX-S40, a correction factor could be obtained which made the transformation of Abs600 measurements into OD600 measurements possible.
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<h3>Calibration</h3>
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<h4>OD<sub>600</sub>  Reference Point</h4>
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<p>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 OD<sub>600</sub> 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 OD<sub>600</sub>  reference point calibration.  
 
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<p><strong>Table 1: Results of the OD<sub>600</sub>  reference point calibration. </strong></p>
  
 
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<td>Corrected Abs600</td>
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<td>Corrected Abs<sub>600</sub></td>
 
<td>0.0125</td>
 
<td>0.0125</td>
  
 
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<td>Reference OD600</td>
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<td>Reference OD<sub>600</sub></td>
 
<td>0.0425</td>
 
<td>0.0425</td>
  
 
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<td>OD600/Abs600</td>
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<td>OD<sub>600</sub>/Abs<sub>600</sub></td>
 
<td>3.4</td>
 
<td>3.4</td>
  
 
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<h3>Fluorescein Standard Curve</h3>
 
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<p>For standardization, a dilution series of fluorescein was prepared. Fluorescein was diluted in PBS to a final concentration of 50&nbsp;µM. For the standard curve, a 96 well plate was used. 100&nbsp;µl PBS were added into well 2-12 of each row. Well 1 was filled with 200&nbsp;µl of the diluted fluorescein (50&nbsp;µM). For the dilution series, 100&nbsp;µl were taken from well 1 and pipetted into well 2, then from well 2 into well 3. This step was repeated till the end of the row. Measuring the fluorescence (excitation at 485&nbsp;nm, emission at 530/30&nbsp;nm), a curve with an exponential increase could be obtained. The following graph shows the fluorescence intensity plotted against the fluorescein concentration.
 
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                    <p><strong>Figure 1: Standardization curve of fluorescein.</strong><br>
 
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<p>To obtain the standardization, the fluorescence was plotted against the fluorescein concentration on a logarithmic scale.
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<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 and 2.
 
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                    <p><strong>Figure 2: Logarithmic scale of standardization curve.</strong><br>
 
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            <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>
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<h3>Cell Measurements</h3>
 
<h3>Cell Measurements</h3>
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<p><i>E.coli</i> (DH5-α) were transformed with each of the 8 plasmids provided by iGEM. The plasmids contained the following devices:
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<p>After the calibration steps were successfully established, the test devices were transformed into <i>E. coli</i> K12 DH5-alpha and characterized. The results of this characterization are shown in figure 3.
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<p>1. Positive Control<br>
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2. Negative Control<br>
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3. Test Device 1<br>
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4. Test Device 2<br>
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5. Test Device 3<br>
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6. Test Device 4<br>
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7. Test Device 5<br>
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8. Test Device 6<br>
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<p>Every plasmid (except of the negative control) contained GFP as well as a chloramphenicol resistance. The bacteria were grown in LB medium with chloramphenicol (final concentration of 25 µg/ml).
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One day after the transformation, two colonies of each plate were picked and were grown overnight in 10 ml medium with chloramphenicol (37°C, 220&nbsp;rpm). In the following, the respective first colonies are named 1.1, 2.1, etc. The second ones are named 1.2, 2.2, etc.
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The next day, OD600 of the bacteria was measured. As the final OD600 should be adjusted to 0.02, all samples had to be diluted in the respective amount of medium with chloramphenicol. Then, the bacteria were grown again at 37°C and 220 rpm. After 0, 2, 4 and 6 hours 0.5&nbsp;ml of each sample were taken and put on ice. In the end, OD600 and the fluorescence of each sample for each time point was measured with the plate reader. Therefore, four replicates of each 100&nbsp;µl were used.
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The values were converted from relative fluorescence units to [µM fluorescein/OD600], so they become comparable to the fluorescein standard curve measurements. <b>Figure 3</b> shows the fluorescence of each approach plotted against the time.
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<h2 style="text-align:left">Results</h2>
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                     <p><strong>Figure 3: Relative fluorescence intensities of the bacteria, each containing a plasmids with one of the test devices.</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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<p>For each device, the fluorescence goes against 0. As most of the initial values are lower than 0, it is likely that this is only background fluorescence. The bacteria do not seem to express GFP at all.
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<h2>Conclusion</h2>
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<h2>Discussion</h2>
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<p>The expectation of this experiment was an increase in the fluorescence intensity over the time. For the different devices, curves with various intensities were expected.
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<p>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.
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The fluorescence measurements from the Interlab Study cannot be compared at all because the whole experiment failed. There were several problems which prevented getting GFP expression. The low concentration of chloramphenicol was a problem which we initially missed. Additionally, the use of chloramphenicol-resistant bacteria hindered to get GFP expression. Nevertheless, these two problems did not seem to be the only ones. Also the repetition of the Interlab Study with new DH5-α and a final chloramphenicol concentration of 34&nbsp;µg/ml could not produce a significant increase in the fluorescence intensity. The actual problem could not be identified. The results cannot become involved in the evaluation of the comparability of measurements. To evaluate the results of the Interlab Study as a whole, you would have to consider all data from many laboratories and statistically analyze them.
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After looking for possible error sources, chloramphenicol stock solutions were exchanged.
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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 
  
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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.