Difference between revisions of "Team:Paris Bettencourt/Notebook/Phytase"
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{{Paris_Bettencourt/header}} | {{Paris_Bettencourt/header}} | ||
{{Paris_Bettencourt/menu}} | {{Paris_Bettencourt/menu}} | ||
− | {{Paris_Bettencourt/ | + | {{Paris_Bettencourt/banner|page_id=notebook|page_name=Notebook - Phytase}} |
<html> | <html> | ||
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<h2>Result of colony PCR (August 19th)</h2> | <h2>Result of colony PCR (August 19th)</h2> | ||
<div class="column-left"><br><br><p> | <div class="column-left"><br><br><p> | ||
− | We | + | We see bands smaller than 500bp, but not the fragments we expected : this is probably contaminations. We start again the same PCR colony. There is DNA in holes too, which didn't migrate, probably the genomic DNA. |
</p></div> | </p></div> | ||
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<div class="column-right"><p> | <div class="column-right"><p> | ||
The DNA ladder migrate, but there was any amplification of the both genes.<br> | The DNA ladder migrate, but there was any amplification of the both genes.<br> | ||
− | We tested two PCR mix : the first did not work. The positive control worked with the second PCR mix. It is composed of | + | We tested two PCR mix : the first did not work. The positive control worked with the second PCR mix. It is composed of HO plasmid and the oligos using for PHO85 gene. |
</p></div> | </p></div> | ||
<div style="clear:both"></div> | <div style="clear:both"></div> | ||
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<h2>Phytic acid dosage</h2> | <h2>Phytic acid dosage</h2> | ||
<div class="column-right"><p> | <div class="column-right"><p> | ||
− | We dose the phytic acid in fermented rice.</p></div> | + | We dose the phytic acid in fermented rice.<br> |
+ | After analysis, the fermented rice have very little quantity of phosphorus and of phytic acid.<br> | ||
+ | <b>[Phosphorus] :</b> 0.0758 mg/100g of rice.<br> | ||
+ | <b>[Phytic acid] :</b> 0.269 mg/100g of rice.</p></div> | ||
<div class="column-left"> | <div class="column-left"> | ||
− | <img src="https://static.igem.org/mediawiki/2015/ | + | <img src="https://static.igem.org/mediawiki/2015/0/09/ParisBettencourtPhyticAcidTitration1.png" width="550px"> |
− | <p class="legend"><b>Figure 11 :</b> | + | <p class="legend"><b>Figure 11 :</b> Phosphorus calibration curve for titration of phytic acid in fermented rice</p></div> |
<div style="clear:both"></div> | <div style="clear:both"></div> | ||
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<br><h1 class="date one">August 28th</h1> | <br><h1 class="date one">August 28th</h1> | ||
<h2>Phytic acid dosage in different strains</h2> | <h2>Phytic acid dosage in different strains</h2> | ||
− | < | + | |
− | <img src="https://static.igem.org/mediawiki/2015/ | + | <img src="https://static.igem.org/mediawiki/2015/3/3c/ParisBettencourtTitrationPhyticAcidCurve2.png" width="750px"><br><br> |
− | <p class="legend"><b>Figure 13 :</b> | + | <img src="https://static.igem.org/mediawiki/2015/b/bb/ParisBettencourtTitrationPhyticAcidResultsOD2.png" width="1000px"> |
− | <img src="https://static.igem.org/mediawiki/2015/ | + | <p class="legend"><b>Figure 13 :</b> Phosphorus calibration curve + results of OD in some strains</p><br> |
− | <p class="legend"><b>Figure 14 :</b> Results of acid phytic dosage on fermented rice</p | + | <img src="https://static.igem.org/mediawiki/2015/8/85/ParisBettencourtTitrationPhyticAcidResultsConcentration2.png" width="850px"> |
− | + | <p class="legend"><b>Figure 14 :</b> Results of acid phytic dosage on fermented rice</p> | |
<h2>Results of PCR</h2> | <h2>Results of PCR</h2> | ||
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</div> | </div> | ||
<div class="column-right"><p>We watch bands for the both of genes, but not at the same temperatures. Thanks to the gradient, we can suppose that oligos have not exactly the same melting temperature, and it may be the reason why the previous PCR failed.<br> | <div class="column-right"><p>We watch bands for the both of genes, but not at the same temperatures. Thanks to the gradient, we can suppose that oligos have not exactly the same melting temperature, and it may be the reason why the previous PCR failed.<br> | ||
− | The controle postive with the | + | The controle postive with the HO plasmid worked, the band matches with the PHO85 gene size (1020bp). |
</p></div> | </p></div> | ||
<div style="clear:both"></div> | <div style="clear:both"></div> | ||
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<br><h1 class="date two">September 4th</h1> | <br><h1 class="date two">September 4th</h1> | ||
− | After the transformation of September 2nd, any culture is observable on plates. The tansformation are not function. We restart transformation, but we transform with <i>Cre< | + | After the transformation of September 2nd, any culture is observable on plates. The tansformation are not function. We restart transformation, but we transform with <i>Cre</i> and <i>RFP</i> genes. It is more interesting because we not forced to simply remove the resistance gene, we can make a double identification. It helps to identify yeast that is already integrated in place of the resistance gene PHO85 (they cultivate on agar with geneticin) and we also integrate the RFP in place of PHO80 gene (they will be red). |
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We start to do the phosphorus scale.<br><br> | We start to do the phosphorus scale.<br><br> | ||
− | + | ||
− | <img src="https://static.igem.org/mediawiki/2015/ | + | <img src="https://static.igem.org/mediawiki/2015/c/cb/ParisBettencourtTitrationPhyticAcidCurve3.png" width="500px"> |
− | + | <p class="legend"><b>Figure 20 :</b> Phosphorus calibration curve</p> | |
− | + | ||
− | + | ||
− | <p class="legend"><b>Figure 20 :</b> Phosphorus calibration curve</p | + | |
− | + | ||
<br> | <br> | ||
With calculations of the protocol, we can determine the concentration in our samples.<br> | With calculations of the protocol, we can determine the concentration in our samples.<br> | ||
− | <b>Protocol :</b><a href="https://2015.igem.org/Team:Paris_Bettencourt/Protocols | + | <b>Protocol :</b><a href="https://2015.igem.org/Team:Paris_Bettencourt/Protocols#FSTitrationacidphytic"> Titration of phytic acid</a> |
<br><br> | <br><br> | ||
Latest revision as of 14:04, 30 October 2015