Difference between revisions of "Team:Pasteur Paris/Interlab study"
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<br/> | <br/> | ||
<p style="text-indent:3em;" align="justify">For each clone, we made several analyses : a quantitative PCR (qPCR) for the determination of the plasmid/strain ratio, and a fluorescence test for the determination of the GFP expression by strain.</p> | <p style="text-indent:3em;" align="justify">For each clone, we made several analyses : a quantitative PCR (qPCR) for the determination of the plasmid/strain ratio, and a fluorescence test for the determination of the GFP expression by strain.</p> | ||
| − | <p style="text-indent:3em;" align="justify"><span style="color: #004864; font-size: 1.2em;"><b>→ First,</b></span> we have performed an efficacy assay so as to elucidate the settings and the amount of template required for our qPCR. For this, we amplified TyrA gene (a single copy gene) in gDNA (WT strain), and GFP gene in plasmid DNA (GFP in pSB1C3 strain) at different concentrations (dilution series of 1:10). The results show that the qPCR for TyrA was efficient so the parameters can be used; ≈1ng/uL is the amount of template to use.</p> | + | <p style="text-indent:3em;" align="justify"><span style="color: #004864; font-size: 1.2em;"><b>→ First,</b></span> we have performed an efficacy assay so as to elucidate the settings and the amount of template required for our qPCR. For this, we amplified TyrA gene (a single copy gene) in genomic DNA (gDNA)(WT strain), and GFP gene in plasmid DNA (pDNA) (GFP in pSB1C3 strain) at different concentrations (dilution series of 1:10). The results show that the qPCR for TyrA was efficient so the parameters can be used; ≈1ng/uL is the amount of template to use.</p> |
</br> | </br> | ||
| − | <p style="text-indent:3em;" align="justify"><span style="color: #004864; font-size: 1.2em;"><b>→ Afterwards,</b></span> we performed a parallel qPCR of our templates in order to amplify in one plate the amount of gDNA per strain, and | + | <p style="text-indent:3em;" align="justify"><span style="color: #004864; font-size: 1.2em;"><b>→ Afterwards,</b></span> we performed a parallel qPCR of our templates in order to amplify in one 96-well plate the amount of gDNA per strain, and on another plate the amount of pDNA (GFP+) corresponding to each strain. Thanks to this, a ratio of pDNA (GFP+) / gDNA was obtained; it shows the number of pDNA (GFP+) copies per biological clone and strain.</p> |
</br> | </br> | ||
<center><p><img src="https://static.igem.org/mediawiki/2015/2/24/Plasmid_copy_per_strain.jpg"></p></center> | <center><p><img src="https://static.igem.org/mediawiki/2015/2/24/Plasmid_copy_per_strain.jpg"></p></center> | ||
| Line 33: | Line 33: | ||
<tr> | <tr> | ||
<td> PA </td> | <td> PA </td> | ||
| − | <td>1 | + | <td>1.96</td> |
| − | <td>0 | + | <td>0.24</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td> PB </td> | <td> PB </td> | ||
| − | <td>2 | + | <td>2.62</td> |
| − | <td>0 | + | <td>0.83</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td> PC </td> | <td> PC </td> | ||
| − | <td>2 | + | <td>2.85</td> |
| − | <td>1 | + | <td>1.58</td> |
<tr> | <tr> | ||
</table></center> | </table></center> | ||
Revision as of 22:05, 17 September 2015
Promoter A(J23101) + GFP Promoter B(J23106) + GFP Promoter C(J23117) + GFP
WT (Non transformed cells) GFP(I13405)
For each clone, we made several analyses : a quantitative PCR (qPCR) for the determination of the plasmid/strain ratio, and a fluorescence test for the determination of the GFP expression by strain.
→ First, we have performed an efficacy assay so as to elucidate the settings and the amount of template required for our qPCR. For this, we amplified TyrA gene (a single copy gene) in genomic DNA (gDNA)(WT strain), and GFP gene in plasmid DNA (pDNA) (GFP in pSB1C3 strain) at different concentrations (dilution series of 1:10). The results show that the qPCR for TyrA was efficient so the parameters can be used; ≈1ng/uL is the amount of template to use.
→ Afterwards, we performed a parallel qPCR of our templates in order to amplify in one 96-well plate the amount of gDNA per strain, and on another plate the amount of pDNA (GFP+) corresponding to each strain. Thanks to this, a ratio of pDNA (GFP+) / gDNA was obtained; it shows the number of pDNA (GFP+) copies per biological clone and strain.
Results:
| pDNA/gNDA | Standard deviation | |
| PA | 1.96 | 0.24 |
| PB | 2.62 | 0.83 |
| PC | 2.85 | 1.58 |
→ Finally, we analyzed the specific fluorescence of each strain and biological clone in function of its growth (latest iGEM data suggest that GFP is expressed differently depending on the growth curve).
Knowing that the fluorescence depends on the cell growth, we measured the fluorescence (508nm) and the cell growth (600nm) in parallel thanks to the spectrophotometer TECAN.
Results:
| Fluorescence | Standard deviation | |
| PA | 30794,4063 | 670,922388 |
| PB | 18780,6735 | 772, 800562 |
| PC | 871,420085 | 80,7704353 |
| GFP | 2234,04915 | 85,2624456 |
| PB | 392, 751863 | 15,3897067 |
→ Thus, qPCR ratio values were used to normalize the data obtained from fluorescence/growth analyzes, with the purpose of examining the promoter strength on expressing the GFP protein in our different biological clones and strains. In fact, the fluorescence obtained on each biological clone over time is divided by its qPCR ratio, in order to set the specific fluorescence value to a single pDNA (GFP+) devise. Therefore, by analyzing the differential of expression between strains and their biological clones, we determined which promoter triggered a higher GFP expression.
Results:
Conclusion
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