Difference between revisions of "Team:Pasteur Paris/Interlab study"
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<img src="https://static.igem.org/mediawiki/2015/7/72/InterLab_Study_pasteur2015.jpg" style="width: 100%;"/> | <img src="https://static.igem.org/mediawiki/2015/7/72/InterLab_Study_pasteur2015.jpg" style="width: 100%;"/> | ||
<br/><br/><div class="carregris"> | <br/><br/><div class="carregris"> | ||
| − | <p>After transformation of our Bacteria with each of our construct, we observed | + | <p>After transformation of our Bacteria <i>E.coli</i> DH5-⍺ with each of our construct, we observed the cultures under UV light.</p> |
| − | <center><b>Fluorescence of our Bacteria | + | <center><b>Fluorescence of our Bacteria</b></center> |
<br/> | <br/> | ||
<center> | <center> | ||
| Line 17: | Line 17: | ||
<center><i><p>WT (Non transformed cells) GFP(I13405) </p></i></center> | <center><i><p>WT (Non transformed cells) GFP(I13405) </p></i></center> | ||
<br/> | <br/> | ||
| − | <p style="text-indent:3em;" align="justify">For each clone, we made several | + | <p style="text-indent:3em;" align="justify">For each clone, we made several analyses: |
| − | <p style="text-indent:3em;" align="justify"><span style="color: #004864; font-size: 1.2em;"><b>→ First,</b></span> | + | <ul> |
| + | <li>a quantitative PCR (qPCR) to determine the number of plasmids containing the GFP present in each strain. </li> | ||
| + | <li>a fluorescence test to determine the GFP expression in each 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. In order to do this, we amplified the TyrA gene, a single copy gene in <i>E.coli</i> in genomic DNA (gDNA)(WT strain), and GFP gene in plasmid DNA (pDNA) (GFP in pSB1C3 strain) at various concentrations (dilution series of 1/10). | ||
| + | The results show that the qPCR for TyrA was efficient: ~1ng/µL 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> | + | <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 correct amount of gDNA for each strain, and on another plate the amount of pDNA (GFP+) corresponding to each strain. 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> | ||
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<br/> | <br/> | ||
<br/> | <br/> | ||
| − | <p style="text-indent:3em;" align="justify"><span style="color: #004864; font-size: 1.2em;"><b>→ Finally,</b></span> | + | <p style="text-indent:3em;" align="justify"><span style="color: #004864; font-size: 1.2em;"><b>→ Finally,</b></span> We analyzed the specific fluorescence of each strain and biological clone as a function of its growth since the latest iGEM data suggests that the GFP is expressed differently depending on the growth curve.</p> |
| − | <p>Knowing that the fluorescence depends on | + | <p>Knowing that the fluorescence depends on cell growth, we measured the fluorescence (508 nm) and the OD(600 nm) simultaneously thanks a TECAN robotic station equipped with a spectrophotometer.</p> |
<center><p><img src="https://static.igem.org/mediawiki/2015/c/c3/Fluo_per_plasmid.jpg"></p></center> | <center><p><img src="https://static.igem.org/mediawiki/2015/c/c3/Fluo_per_plasmid.jpg"></p></center> | ||
</br> | </br> | ||
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</table></center> | </table></center> | ||
<br/> | <br/> | ||
| − | <p style="text-indent:3em;" align="justify"><span style="color: #004864; font-size: 1.2em;"><b>→ Thus,</b></span> qPCR ratio values were used to normalize the data obtained from fluorescence | + | <p style="text-indent:3em;" align="justify"><span style="color: #004864; font-size: 1.2em;"><b>→ Thus,</b></span> qPCR ratio values were used to normalize the data obtained from fluorescence and bacterial growth analyses, in order to examine each promoter's strength. 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.</p> |
<br/><br/> | <br/><br/> | ||
<h4><b> Results:</b> </h4> | <h4><b> Results:</b> </h4> | ||
Revision as of 20:39, 18 September 2015
After transformation of our Bacteria E.coli DH5-⍺ with each of our construct, we observed the cultures under UV light.
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) to determine the number of plasmids containing the GFP present in each strain.
- a fluorescence test to determine the GFP expression in each strain
→ First, We have performed an efficacy assay so as to elucidate the settings and the amount of template required for our qPCR. In order to do this, we amplified the TyrA gene, a single copy gene in E.coli in genomic DNA (gDNA)(WT strain), and GFP gene in plasmid DNA (pDNA) (GFP in pSB1C3 strain) at various concentrations (dilution series of 1/10). The results show that the qPCR for TyrA was efficient: ~1ng/µL 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 correct amount of gDNA for each strain, and on another plate the amount of pDNA (GFP+) corresponding to each strain. A ratio of pDNA (GFP+) / gDNA was obtained: it shows the number of pDNA (GFP+) copies per biological clone and strain.
Results:
pDNA / gDNA 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 as a function of its growth since the latest iGEM data suggests that the GFP is expressed differently depending on the growth curve.
Knowing that the fluorescence depends on cell growth, we measured the fluorescence (508 nm) and the OD(600 nm) simultaneously thanks a TECAN robotic station equipped with a spectrophotometer.
Results:
Fluorescence Standard deviation PA 30794.41 670.92 PB 18780.67 772.80 PC 871.42 80.77 GFP 2234.05 85.26 PB 392.75 15.39
→ Thus, qPCR ratio values were used to normalize the data obtained from fluorescence and bacterial growth analyses, in order to examine each promoter's strength. 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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