Difference between revisions of "Team:Pasteur Paris/InterLab"
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| − | <td></td> | + | <td>LUDOX CL-X</td> |
| − | <td></td> | + | <td>H2O</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td></td> | + | <td>Replicate 1</td> |
<td></td> | <td></td> | ||
<td></td> | <td></td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td></td> | + | <td>Replicate 2</td> |
<td></td> | <td></td> | ||
<td></td> | <td></td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td></td> | + | <td>Replicate 3</td> |
<td></td> | <td></td> | ||
<td></td> | <td></td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td></td> | + | <td>Replicate 4</td> |
<td></td> | <td></td> | ||
<td></td> | <td></td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td></td> | + | <td>Arith. Mean</td> |
<td></td> | <td></td> | ||
<td></td> | <td></td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td></td> | + | <td>Corrected Abs600</td> |
<td></td> | <td></td> | ||
<td></td> | <td></td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| + | <td>Reference OD600</td> | ||
<td></td> | <td></td> | ||
| − | <td></td> | + | <td>/</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| + | <td>OD600/Abs600</td> | ||
<td></td> | <td></td> | ||
| − | <td></td> | + | <td>/</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td></td> | <td></td> | ||
<td></td> | <td></td> | ||
| + | <td>/</td> | ||
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</table> | </table> | ||
Revision as of 21:13, 29 September 2018
INTRODUCTION
This year, the iGEM Measurement Committee offered to all teams the possibility to do the Fifth International InterLaboratory Measurement Study in synthetic biology. This work concerns the reliability and repeatability of scientific measurements.
This project involves providing the same protocols and gathering all data from different teams to build a database with reference values.
During this edition, the main objective is to enhance measurements precision in synthetic biology by detecting and correcting sources of errors. Last year, the goal was to reduce variability in fluorescence measurements (GFP) with a normalization of OD. This year, we try to reduce this variability between labs by normalizing to absolute CFUs (colony-forming units).
DEVICES
For this study, we had to transform six plasmids and two controls:
- Negative control BBa_R0040: sequence for pTet inverting regulator, corresponding to TetR repressible promoter.
- Positive control BBa_I20270: promoter and GFP sequence.
- And six GFP expressing constitutive devices:
Device 1 BBa_J364000
Device 2 BBa_J364001
Device 3 BBa_J364002
Device 4 BBa_J364007
Device 5 BBa_J364008
Device 6 BBa_J364009.
FIRST APPROACH
The first approach consists in converting between absorbance of cells to absorbance of a known concentration of beads.
Absorbance of a known concentration of beads
First, we did a calibration, using LUDOX CL-X, to obtain a conversion factor (Figure 1). This factor enables us to transform absorbance data from our plate reader into a basic OD measurement which can be found in a spectrophotometer.
| LUDOX CL-X | H2O | |
| Replicate 1 | ||
| Replicate 2 | ||
| Replicate 3 | ||
| Replicate 4 | ||
| Arith. Mean | ||
| Corrected Abs600 | ||
| Reference OD600 | / | |
| OD600/Abs600 | / | |
| / |
Then, we carried out a second calibration, using silica beads in a microsphere suspension, to convert absorbance measurements into a number of cells. This conversion is based on the plotting of a standard curve of particle concentration (Figure 2) that we have determined during this calibration.
We did the same work creating a standard curve of fluorescence for fluorescein concentration. Then, we had to use this to transform our cell based readings into a fluorescein concentration.
Absorbance of cells
After calibrations, we began cell culture expression measurement. For that, we did overnight cultures of two colonies for each device. 24 hours later, we measured absorbance and fluorescence of growing cultures with our plate reader. Then, we used our fluorescence standard curve to transform our cell measurements into fluorescein concentrations (Figure 4).
Hour 0
| Neg. Control | Pos. Control | Device 1 | Device 2 | Device 3 | Device 4 | Device 5 | Device 6 |
| 104.00 | 165.00 | 496.00 | 237.00 | -13.00 | 444.00 | 237.00 | 216.00 |
| 35.00 | 129.00 | 457.00 | 252.00 | -50.00 | 381.00 | 137.00 | 150.00 |
| 39.00 | 111.00 | 504.00 | 197.00 | -51.00 | 433.00 | 209.00 | 156.00 |
| -24.00 | 118.00 | 426.00 | 156.00 | -17.00 | -92.00 | 156.00 | 170.00 |
| 47.00 | 149.00 | 612.00 | 279.00 | -40 | 581 | 240.00 | 177.00 |
| 61.00 | 122.00 | 588.00 | 258.00 | -34.00 | 564.00 | 166.00 | 132.00 |
| 61.00 | 138.00 | 604.00 | 281.00 | -44.00 | 576.00 | 224.00 | 156.00 |
| 59.00 | 119.00 | 559.00 | 239.00 | -27.00 | 524.00 | 154.00 | 112.00 |
Hour 6
According to those results, we can deduce the strength of each promoter.
Device 1 (BBa_J364000): high strength
Device 2 (BBa_J364001): high strength
Device 3 (BBa_J364002): low strength
Device 4 (BBa_J364007): high strength
Device 5 (BBa_J364008): medium strength
Device 6 (BBa_J364009): medium strength
SECOND APPROACH
This second approach involves counting how many colonies grow on a plate. With this number, we have determined a cell concentration for each sample. Then, with CFU values for positive and negative control samples, we calculated a conversion factor from absorbance to CFU.
Example for a final dilution factor of 8.105
First, we counted the colonies on each plate.
| Sample | Number of colonies |
| 1.1 | 34 |
| 1.2 | 73 |
| 1.3 | 93 |
| 2.1 | 137 |
| 2.2 | 71 |
| 2.3 | 64 |
| 3.1 | 44 |
| 3.2 | 40 |
| 3.3 | 65 |
Secondly, we multiplied the colony count by the final dilution factor. Then, we obtained the CFU/mL.
| Sample | CFU/mL |
| 1.1 | 2.72 107 |
| 1.2 | 5.84 107 |
| 1.3 | 7.44 107 |
| 2.1 | 1.10 108 |
| 2.2 | 5.68 107 |
| 2.3 | 5.12 107 |
| 3.1 | 3.52 107 |
| 3.2 | 3.20 107 |
| 3.3 | 5.20 107 |