Difference between revisions of "Team:Paris Bettencourt/Notebook/Idli and micro-organisms"

 
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<div id="notebookMenu">
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  <ul>
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    <li><a href="#july">July</a></li>
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    <li><a href="#august">August</a></li>
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    <li><a href="#september">September</a></li>
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  </ul>
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<h2 class="date one">July 2nd to 9th</h2><br><br>
 
<h2 class="date one">July 2nd to 9th</h2><br><br>
  
I experiented different recipes to make idli come from India. I tried to do it with different rice and lentill (called dall in india). Finally, I chose to do  
+
We tested different recipes to make Indian idli. We tried with different rice and lentils (called dall in india). Finally, We chose to do  
<a href="https://2015.igem.org/Team:Paris_Bettencourt/Protocols/Idli_receipe">this recipe</a>
+
<a href="https://2015.igem.org/Team:Paris_Bettencourt/Protocols#FSIdli">this recipe</a>
 
with basmati rice and indian dall. <br><br>
 
with basmati rice and indian dall. <br><br>
  
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<IMG SRC= "https://static.igem.org/mediawiki/2015/5/52/Idli_water.JPG" width=20%>  
 
<IMG SRC= "https://static.igem.org/mediawiki/2015/5/52/Idli_water.JPG" width=20%>  
 
<br>
 
<br>
I did a media from an idli preparation. After fermentation of 18 hours, of ildi composed of 3 glasses of rice (75ml) and 1 glass of dall (25ml) before the soaking phase, I took water and butter mixed it together in 500ml bottle, and did an autoclave. I called this media "idli water", and it look like previously. I will just use the clear phase.<br><br>
+
We did a media from an idli preparation, using 3 glasses of rice (75ml) and 1 glass of dall (25ml) before the soaking phase. After 18 hours of fermentation, W took the water and butter of this idli, mixed it together in a 500ml bottle, and autoclaved it. This media was named "idli water", and it looked like previously. We will just use the clear phase.<br><br>
  
  
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<h2 class="date one">July 12th to 14th and 16th to 18th</h2><br><br>
 
<h2 class="date one">July 12th to 14th and 16th to 18th</h2><br><br>
  
I did grow ''Saccharomyce cerevisiae'' with mCherry and geneticin resistance genes in idli. I added 2 ml of a yeast growth solution (YPD) with an OD600 of 0.455 (around 1.2 x 10<sup>7</sup> cfu) after the soaking phase and the mixing and grinding of rice (3 glasses of rice ~ 75ml) and dall (1 glass of dall ~ 25ml) together.The first try was to see if there are the fermentation process will this laboratory strain. After the fermentation time, I had plated the µorganisms come from the butter phase (middle phase between water on the top, and grind matter in the bottom of the erlenmeyer) on YPD with geneticin. I observed a lot of part of crush rice or dall, and just few colonny, not red like expected with the mCherry gene. The second try was more successful. This time I diluated the butter 10 and 100 time before plating, always on YPD and geneticin. I observed few colonies for the plate with the 100th and around 30 with the 10th. <br><br>
+
We grew ''Saccharomyces cerevisiae'' with mCherry and geneticin resistance genes in idli. We added 2 ml of a yeast growth solution (YPD) with an OD600 of 0.455 (around 1.2 x 10<sup>7</sup> cfu) after the soaking phase and after mixing and grinding the rice (3 glasses of rice ~ 75ml) and dall (1 glass of dall ~ 25ml) together. The first try was to see if there is the fermentation process with these laboratory strains. After the fermentation time, We have plated the µorganisms coming from the butter phase (middle phase between water on the top, and grind matter in the bottom of the erlenmeyer) on YPD with geneticin. We observed a lot of particules of crush rice or dall, and just few colonies, not red like expected with the mCherry gene.<br>
 +
The second try was more successful. This time I diluted the butter 10 and 100 time before plating, still on YPD and geneticin. We could observed only few colonies for the plate with the 100th and around 30 with the 10th. <br><br>
  
  
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<h2 class="date one">July 20th</h2><br><br>
 
<h2 class="date one">July 20th</h2><br><br>
  
I did a culture on TECAN plate of few species that was ''Saccharomyces cerevisiae'' with mCherry on chromosom, with Lactobacillus plantarum NC8 and Lactococcus Lactis MG1363 on their respective media (YPD, MRS and M17) and on "idli water" and on water, with and without antibiotics (respectivly geneticin disulfate, and erythromycin for the 2 bacteria). <br>
+
We did a culture on a TECAN plate of few species: ''Saccharomyces cerevisiae'' with mCherry on chromosom, Lactobacillus plantarum NC8 and Lactococcus Lactis MG1363 on their respective media (YPD, MRS and M17), then on "idli water" and on water, with and without antibiotics (respectivly geneticin disulfate, and erythromycin for the two bacteria). <br>
I did a TECAN mesure after an over night growth of 18 hours, and we obtain the table followed. We can see that the mesure was nonsens.<br><br>
+
We did a TECAN measure after an overnight growth of 18 hours, and we obtained the table followed. We can see that the results are nonsens.<br><br>
  
  
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Test of the titration protocol with spectrophotometer for the  
 
Test of the titration protocol with spectrophotometer for the  
  <a href="https://2015.igem.org/Team:Paris_Bettencourt/Protocols/Titration_protocols_for_vitamin_A">Vitamin A (protocol titration)</a> <br>
+
  <a href="https://2015.igem.org/Team:Paris_Bettencourt/Protocols#FSVitaminATitration">Vitamin A (protocol titration)</a> <br>
  
I tested this protocol and tried to calibrate it. I used, like food sample, 1g of rice and I did 8 bottles with 8 different concentrations of pure vitamin A from 10 <sup>-1</sup> to 10 <sup>-8</sup> mg.ml<sup>-1</sup>. <br><br>
+
We tested this protocol and tried to calibrate it. We used, like food sample, 1g of rice and We did 8 bottles with 8 different concentrations of pure vitamin A from 10 <sup>-1</sup> to 10 <sup>-8</sup> mg.ml<sup>-1</sup>. <br><br>
  
 
<B>Results :</B> <br>
 
<B>Results :</B> <br>
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<a name="august" class="anchor"><h1></h1></a>
 
<a name="august" class="anchor"><h1></h1></a>
<h2 class="date one">August 10th</h2><br><br>
+
<h2 class="date two">August 10th</h2><br><br>
  
I did electrocompetent cell of ''Lactobacillus plantarum'' with <a href="https://2015.igem.org/Team:Paris_Bettencourt/Protocols/Electrocompetent_Cells_Preparation_for_Lactobacillus_Plantarum"> a particular protocol</a> <br>
+
We did electrocompetent cells of ''Lactobacillus plantarum'' with <a href="https://2015.igem.org/Team:Paris_Bettencourt/Protocols#FSElectrocompetentLactobacillus"> a particular protocol</a> <br>
 
It worked very well.<br><br>
 
It worked very well.<br><br>
  
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<a name="august" class="anchor"><h1></h1></a>
 
<a name="august" class="anchor"><h1></h1></a>
<h2 class="date one">August 14th</h2><br><br>
+
<h2 class="date two">August 14th</h2><br><br>
  
  
I had done <a href="https://2015.igem.org/Team:Paris_Bettencourt/Protocols/Idli_receipe">idli protocol</a>, with 75 ml of rice, for 25 ml of dall. After grinding and mixing, I added 2ml of ''Saccharomyce cerevisiae'' (with mCherry gene and geneticin resistance) that contained 10<sup>8</sup> cells. I had plated at t0 and 2h, 4h, 6h, 19h30, 21h30 after t0 three different positons (middle top, lateral middle and bottom middle) at three different concentrations (10<sup>0</sup>, 10<sup>-2</sup> and 10<sup>-3</sup>). <br>
+
We did the <a href="https://2015.igem.org/Team:Paris_Bettencourt/Protocols#FSIdli">idli protocol</a>, with 75 ml of rice, for 25 ml of dall. After grinding and mixing, we added 2ml of ''Saccharomyces cerevisiae'' (with mCherry gene and geneticin resistance) that contained 10<sup>8</sup> cells. We plated at t0 and 2h, 4h, 6h, 19h30, 21h30 after t0 three different positons (middle top, lateral middle and bottom middle) at three different concentrations (10<sup>0</sup>, 10<sup>-2</sup> and 10<sup>-3</sup>). <br>
  
 
<IMG SRC= "https://static.igem.org/mediawiki/2015/2/26/Cfu_number_evolution.png" width=50%> <br>
 
<IMG SRC= "https://static.igem.org/mediawiki/2015/2/26/Cfu_number_evolution.png" width=50%> <br>
Like we can observe, the areas where I take sample for plating don't matter, and the yeast did't grow very well : just a doubling size of population during 22 hours of culture. <br><br>
+
We can see that the area from where we sample for plating doesn't matter, and the yeast didn't grow very well : just a doubling size of population in 22 hours of culture. <br><br>
  
 
<div class="column-left"align="center">
 
<div class="column-left"align="center">
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<!-- 19 - 20 Août -->
  
  
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<a name="august" class="anchor"><h1></h1></a>
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<h2 class="date two">August 19th and 20th</h2><br><br>
  
<!--  -->
+
We started an <a href="https://2015.igem.org/Team:Paris_Bettencourt/Protocols#FSIdli">idli</a> and We put for the fermentation Sc SK1, ScBY4743 mCherry, Sc SK1 with PHO 80 avoid by geneticinR, Sc SK1 with PHO 85 avoid by geneticinR and Sc SK1 with PHO 85 avoid by FRT-geneticinR-FRT. <br>
 +
After the right time of fermentation, we took samples from each idli in each conditions (different µorganisms) from 3 diffenrent area of the idli, and we used the <a href="https://2015.igem.org/Team:Paris_Bettencourt/Protocols#FSTitrationacidphytic">Phytic acid assay kit</a> on the samples.<br>
 +
<br>
 +
The analysis of the phyticacid with the kit said us that ther are no phytic acid in the samples. (cf. <a href="https://2015.igem.org/Team:Paris_Bettencourt/Notebook/Phytase">Phytase Notebook</a>)
  
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+
 
 +
<!-- 20 Août -->
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 +
<a name="august" class="anchor"><h1></h1></a>
 +
<h2 class="date two">August 20th</h2><br><br>
 +
 
 +
We did again the experiment where we added 10<sup>8</sup> cells for the fermentation step of the idli recipe. We used <i>S. cerevisiae</i> BY4743 mCherry and <i>S. cerevisiae</i> SK1 with the gene PHO80 or PHO85 were avoid, <i>Lactobacillus plantarum</i> NC8, and <i>Lactococcus lactis</i> MG1363. With a plate counter, we obtained this numbers of colonies in function of the dilution for the plating. <br>
 +
From the media in the top of the idli :<br>
 +
 
 +
<table>
 +
<tr> <td><b>Media</b></td><td><b>Dilutions</b> </td><td> <b>Number of colonies of Yeast </b></td> </tr>
 +
<tr> <td>YPD+gen</td> <td>10<sup>-1</sup>  </td><td> 79</td>  </tr>
 +
<tr>  <td>YPD+gen</td> <td>  10<sup>-2</sup> </td><td>  32  </td>  </tr>
 +
<tr>  <td>YPD+gen</td> <td> 10<sup>-3</sup>  </td><td> 10 </td>  </tr>
 +
<tr>  <td>MRS</td> <td>  10<sup>-1</sup>  </td><td> 1 </td>  </tr>
 +
<tr>  <td>MRS</td> <td>  10<sup>-2</sup>  </td><td> 0  </td> </tr>
 +
<tr>  <td>MRS</td>  <td>  10<sup>-3</sup>  </td><td> 0 </td>  </tr>
 +
<tr>  <td>LB</td> <td>  10<sup>-1</sup>  </td><td> 0 </td>  </tr>
 +
<tr>  <td>LB</td> <td>  10<sup>-2</sup>  </td><td> 0 </td>  </tr>
 +
<tr>  <td>LB</td>  <td>  10<sup>-3</sup>  </td><td> 1 </td>  </tr>
 +
<tr>  <td>M17</td> <td>  10<sup>-1</sup>  </td><td> 0 </td>  </tr>
 +
<tr>  <td>M17</td> <td>  10<sup>-2</sup>  </td><td> 0</td>  </tr>
 +
<tr>  <td>M17</td>  <td>  10<sup>-3</sup>  </td><td> 0 </td>  </tr>
 +
</table>
 +
<br>
 +
 
 +
<table>
 +
<tr> <td><b>Media</b></td><td><b>Dilutions</b> </td><td> <b>Number of colonies of <i>L. Plantarum</i>/<i>L. Lactis</i> </b></td> </tr>
 +
<tr> <td>YPD+gen</td> <td>10<sup>-1</sup>  </td><td> 0</td>  </tr>
 +
<tr>  <td>YPD+gen</td> <td>  10<sup>-2</sup> </td><td>  0  </td>  </tr>
 +
<tr>  <td>YPD+gen</td> <td> 10<sup>-3</sup>  </td><td> 0 </td>  </tr>
 +
<tr>  <td>MRS</td> <td>  10<sup>-1</sup>  </td><td> 465 </td>  </tr>
 +
<tr>  <td>MRS</td> <td>  10<sup>-2</sup>  </td><td>  99 </td> </tr>
 +
<tr>  <td>MRS</td>  <td>  10<sup>-3</sup>  </td><td> 6 </td>  </tr>
 +
<tr>  <td>LB</td> <td>  10<sup>-1</sup>  </td><td> 96 </td>  </tr>
 +
<tr>  <td>LB</td> <td>  10<sup>-2</sup>  </td><td> 51 </td>  </tr>
 +
<tr>  <td>LB</td>  <td>  10<sup>-3</sup>  </td><td> 15 </td>  </tr>
 +
<tr>  <td>M17</td> <td>  10<sup>-1</sup>  </td><td> 123 </td>  </tr>
 +
<tr>  <td>M17</td> <td>  10<sup>-2</sup>  </td><td> 3</td>  </tr>
 +
<tr>  <td>M17</td>  <td>  10<sup>-3</sup>  </td><td> 7 </td>  </tr>
 +
</table>
 +
<br>
 +
 
 +
From the batter phase of the idli:<br>
 +
 
 +
 
 +
<table>
 +
<tr> <td><b>Media</b></td><td><b>Dilutions</b> </td><td> <b>Number of colonies of Yeast </b></td> </tr>
 +
<tr> <td>YPD+gen</td> <td>10<sup>-1</sup>  </td><td> 217</td>  </tr>
 +
<tr>  <td>YPD+gen</td> <td>  10<sup>-2</sup> </td><td>  150  </td>  </tr>
 +
<tr>  <td>YPD+gen</td> <td> 10<sup>-3</sup>  </td><td> 19 </td>  </tr>
 +
<tr>  <td>MRS</td> <td>  10<sup>-1</sup>  </td><td> 6 </td>  </tr>
 +
<tr>  <td>MRS</td> <td>  10<sup>-2</sup>  </td><td> 1  </td> </tr>
 +
<tr>  <td>MRS</td>  <td>  10<sup>-3</sup>  </td><td> 1 </td>  </tr>
 +
<tr>  <td>LB</td> <td>  10<sup>-1</sup>  </td><td> 2 </td>  </tr>
 +
<tr>  <td>LB</td> <td>  10<sup>-2</sup>  </td><td> 0 </td>  </tr>
 +
<tr>  <td>LB</td>  <td>  10<sup>-3</sup>  </td><td> 0 </td>  </tr>
 +
<tr>  <td>M17</td> <td>  10<sup>-1</sup>  </td><td> 0 </td>  </tr>
 +
<tr>  <td>M17</td> <td>  10<sup>-2</sup>  </td><td> 0</td>  </tr>
 +
<tr>  <td>M17</td>  <td>  10<sup>-3</sup>  </td><td> 0 </td>  </tr>
 +
</table>
 +
<br>
 +
 
 +
<table>
 +
<tr> <td><b>Media</b></td><td><b>Dilutions</b> </td><td> <b>Number of colonies of <i>L. Plantarum</i>/<i>L. Lactis</i> </b></td> </tr>
 +
<tr> <td>YPD+gen</td> <td>10<sup>-1</sup>  </td><td> 0</td>  </tr>
 +
<tr>  <td>YPD+gen</td> <td>  10<sup>-2</sup> </td><td>  0  </td>  </tr>
 +
<tr>  <td>YPD+gen</td> <td> 10<sup>-3</sup>  </td><td> 0 </td>  </tr>
 +
<tr>  <td>MRS</td> <td>  10<sup>-1</sup>  </td><td> 396 </td>  </tr>
 +
<tr>  <td>MRS</td> <td>  10<sup>-2</sup>  </td><td> 51  </td> </tr>
 +
<tr>  <td>MRS</td>  <td>  10<sup>-3</sup>  </td><td> 6 </td>  </tr>
 +
<tr>  <td>LB</td> <td>  10<sup>-1</sup>  </td><td> 125 </td>  </tr>
 +
<tr>  <td>LB</td> <td>  10<sup>-2</sup>  </td><td> 13 </td>  </tr>
 +
<tr>  <td>LB</td>  <td>  10<sup>-3</sup>  </td><td> 8 </td>  </tr>
 +
<tr>  <td>M17</td> <td>  10<sup>-1</sup>  </td><td> 503 </td>  </tr>
 +
<tr>  <td>M17</td> <td>  10<sup>-2</sup>  </td><td> 111</td>  </tr>
 +
<tr>  <td>M17</td>  <td>  10<sup>-3</sup>  </td><td> 29 </td>  </tr>
 +
</table>
 +
<br>
 +
 
 +
We osberved that the µorganisms was in higher number near in the batter of the idli than in the idli water or in the grind rice and dall phase, where nothing has grew.
 +
 
 +
<br><br>
 +
<!-- 27 Août -->
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 +
<a name="august" class="anchor"><h1></h1></a>
 +
<h2 class="date two">August 27th</h2><br><br>
 +
 
 +
 +
We did idli with <i>S. cerevisiae</i> SK1 where PHO85 were avoid by a genetic system  composed by two FRT sequences and the gene of the geneticin resistance. And also, the <i>S. cerevisiae</i> SK1 PHO80 avoid, the <i>S. cerevisiae</i> SK1 PHO85 avoid, Sc BY4743 mCherry and the first <i>S. cerevisiae</i> SK1 together with the <i>S. cerevisiae</i> with the mCherry gene. <br>
 +
To plate on YPD and geneticin (at 200 µg.µl<sup>-1</sup>), We took the sample from 3 area of the idli : top, middle and bottom of the idli, and we did 3 dilutions (from 10<sup>-1</sup> to 10<sup>-3</sup>). Below, we can see the picture of each plates for each conditions of dilution, area and yeast strains that we used.<br><br>
 +
 
 +
<div class="column-right"align="center">
 +
<IMG SRC= "https://static.igem.org/mediawiki/2015/b/b0/Tube1%262.png” width=50%>
 +
</div><br>
 +
 
 +
<div class="column-left"align="center">
 +
<IMG SRC= "https://static.igem.org/mediawiki/2015/a/a4/Tube3%264.png” width=50%>
 +
</div>
 +
<br><br>
 +
 
 +
<div align="left">
 +
<IMG SRC= "https://static.igem.org/mediawiki/2015/e/e6/Tube5.png" width=50%>
 +
</div><br>
 +
 
 +
 
 +
The number of yeast was around the same for each strains, and we obtained the next table, where we did a mean of the yeast that we counted, with a plate counter.
 +
 
 +
<table>
 +
<tr> <td><b>Area</b></td><td><b>Dilutions</b> </td><td> <b>Number of colonies of Yeast </b></td> </tr>
 +
<tr> <td>Top</td> <td>10<sup>-1</sup>  </td><td> 285</td>  </tr>
 +
<tr>  <td>Top</td> <td>  10<sup>-2</sup> </td><td>  11  </td>  </tr>
 +
<tr>  <td>Top</td> <td> 10<sup>-3</sup>  </td><td> 2 </td>  </tr>
 +
<tr>  <td>Middle</td> <td>  10<sup>-1</sup>  </td><td> 362 </td>  </tr>
 +
<tr>  <td>Middle</td> <td>  10<sup>-2</sup>  </td><td> 15  </td> </tr>
 +
<tr>  <td>Middle</td>  <td>  10<sup>-3</sup>  </td><td> 2 </td>  </tr>
 +
<tr>  <td>Bottom</td> <td>  10<sup>-1</sup>  </td><td> 357 </td>  </tr>
 +
<tr>  <td>Bottom</td> <td>  10<sup>-2</sup>  </td><td> 16 </td>  </tr>
 +
<tr>  <td>Bottom</td>  <td>  10<sup>-3</sup>  </td><td> 1 </td>  </tr>
 +
</table>
 +
<br>
 +
 
 +
 +
 
 +
<!-- 10 Septembre -->
 +
 
 +
<a name="september" class="anchor"><h1></h1></a>
 +
<h2 class="date three">September 10th</h2><br><br>
 +
 
 +
We started, using a TECAN, to find any informations on our strains that we want to use in fine. We  did in a first time an experiment at 30°C, with <i>S. cerevisiae</i> yPH151, <i>S. cerevisiae</i> SK1 PHO 80 deleted, <i>S. cerevisiae</i> SK1 PHO 85 deleted, <i>S. cerevisiae</i> SK1 PHO 80 and PHO 85 deleted, and <i>Propioni f.</i> on 8 different media (Water, Idli batter/100, Idli water, M17, MRS, URA3free media, YPD and LB. We obtained this growth curve : <br>
 +
 
 +
<div class="column-left"align="center">
 +
<IMG SRC= "https://static.igem.org/mediawiki/2015/b/be/Mean_Control_10sep.png" width=50%>
 +
</div>
 +
<div class="column-right"align="center">
 +
<IMG SRC= "https://static.igem.org/mediawiki/2015/6/6d/Mean_PHO80_10sep.png" width=50%>
 +
</div><br>
 +
<div class="column-left"align="center">
 +
<IMG SRC= "https://static.igem.org/mediawiki/2015/4/42/Mean_PHO85_10sep.png" width=50%>
 +
</div>
 +
<div class="column-right"align="center">
 +
<IMG SRC= "https://static.igem.org/mediawiki/2015/0/0a/Mean_PHOavoid_10sep.png" width=50%>
 +
</div><br>
 +
<div class="column-left"align="center">
 +
<IMG SRC= "https://static.igem.org/mediawiki/2015/c/ca/Mean_Propioni_10sep.png" width=50%>
 +
</div>
 +
<div class="column-right"align="center">
 +
<IMG SRC= "https://static.igem.org/mediawiki/2015/f/fd/Sc_yPH151_10sep.png" width=50%>
 +
</div><br><br>
 +
 
 +
And particulary, the growth curve of the strains on idli water :<br>
 +
<IMG SRC= "https://static.igem.org/mediawiki/2015/9/97/Mean_Idliwater_10sep.png" width=50%>
 +
<br><br>
 +
 
 +
<!-- 11 Septembre -->
 +
 
 +
<a name="september" class="anchor"><h1></h1></a>
 +
<h2 class="date three">September 11th</h2><br><br>
 +
 
 +
We  did in a first time an experiment at 37°C, with <i>E. coli</i> who product vitamin B2, <i>Propioni f.</i> who product vitamin B12 and <i>Lactobacillus plantarum</i> NC8 who should have obtained the vitamin B2 pathway that we engineered.
 +
 
 +
<div class="column-left"align="center">
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<IMG SRC= "https://static.igem.org/mediawiki/2015/d/df/Mean_Control_11sep.png" width=50%>
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</div>
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<div class="column-right"align="center">
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<IMG SRC= "https://static.igem.org/mediawiki/2015/9/9f/Mean_EcoliB2_11sep.png" width=50%>
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</div><br>
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<div class="column-left"align="center">
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<IMG SRC= "https://static.igem.org/mediawiki/2015/6/6b/Mean_Lactobacillus_11sep.png" width=50%>
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</div>
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<div class="column-right"align="center">
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<IMG SRC= "https://static.igem.org/mediawiki/2015/6/6c/Mean_Propionif_11sep.png" width=50%>
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</div><br><br>
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And particulary, the growth curve of the strains on idli water :<br>
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<IMG SRC= "https://static.igem.org/mediawiki/2015/3/3a/Mean_Idliwater_11sep.png" width=50%>
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<br><br>
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With this two last experiments, we can say that the yeasts and the bacteria that we engineered, can grow in idli water, and with the data coming from the idli fermentation with our micro-organisms, it suggests that our micro-organisms grow well in idli in the higher phase of the idli.
 +
<br>

Latest revision as of 18:51, 20 November 2015

July 2nd to 9th



We tested different recipes to make Indian idli. We tried with different rice and lentils (called dall in india). Finally, We chose to do this recipe with basmati rice and indian dall.


We did a media from an idli preparation, using 3 glasses of rice (75ml) and 1 glass of dall (25ml) before the soaking phase. After 18 hours of fermentation, W took the water and butter of this idli, mixed it together in a 500ml bottle, and autoclaved it. This media was named "idli water", and it looked like previously. We will just use the clear phase.

July 12th to 14th and 16th to 18th



We grew ''Saccharomyces cerevisiae'' with mCherry and geneticin resistance genes in idli. We added 2 ml of a yeast growth solution (YPD) with an OD600 of 0.455 (around 1.2 x 107 cfu) after the soaking phase and after mixing and grinding the rice (3 glasses of rice ~ 75ml) and dall (1 glass of dall ~ 25ml) together. The first try was to see if there is the fermentation process with these laboratory strains. After the fermentation time, We have plated the µorganisms coming from the butter phase (middle phase between water on the top, and grind matter in the bottom of the erlenmeyer) on YPD with geneticin. We observed a lot of particules of crush rice or dall, and just few colonies, not red like expected with the mCherry gene.
The second try was more successful. This time I diluted the butter 10 and 100 time before plating, still on YPD and geneticin. We could observed only few colonies for the plate with the 100th and around 30 with the 10th.

July 20th



We did a culture on a TECAN plate of few species: ''Saccharomyces cerevisiae'' with mCherry on chromosom, Lactobacillus plantarum NC8 and Lactococcus Lactis MG1363 on their respective media (YPD, MRS and M17), then on "idli water" and on water, with and without antibiotics (respectivly geneticin disulfate, and erythromycin for the two bacteria).
We did a TECAN measure after an overnight growth of 18 hours, and we obtained the table followed. We can see that the results are nonsens.

July 29th



Test of the titration protocol with spectrophotometer for the Vitamin A (protocol titration)
We tested this protocol and tried to calibrate it. We used, like food sample, 1g of rice and We did 8 bottles with 8 different concentrations of pure vitamin A from 10 -1 to 10 -8 mg.ml-1.

Results :
We can just observe concentrations higher than 10-3mg.ml-1.
dilution of the initial concentration Value of the spectrophotometer
10-1 1.625
10-2 0.078
10-3 0.034
10-4 0.008
10-5 0.002
10-6 0.005
10-7 -0.001
10-8 0.000

August 10th



We did electrocompetent cells of ''Lactobacillus plantarum'' with a particular protocol
It worked very well.

August 14th



We did the idli protocol, with 75 ml of rice, for 25 ml of dall. After grinding and mixing, we added 2ml of ''Saccharomyces cerevisiae'' (with mCherry gene and geneticin resistance) that contained 108 cells. We plated at t0 and 2h, 4h, 6h, 19h30, 21h30 after t0 three different positons (middle top, lateral middle and bottom middle) at three different concentrations (100, 10-2 and 10-3).

We can see that the area from where we sample for plating doesn't matter, and the yeast didn't grow very well : just a doubling size of population in 22 hours of culture.




August 19th and 20th



We started an idli and We put for the fermentation Sc SK1, ScBY4743 mCherry, Sc SK1 with PHO 80 avoid by geneticinR, Sc SK1 with PHO 85 avoid by geneticinR and Sc SK1 with PHO 85 avoid by FRT-geneticinR-FRT.
After the right time of fermentation, we took samples from each idli in each conditions (different µorganisms) from 3 diffenrent area of the idli, and we used the Phytic acid assay kit on the samples.

The analysis of the phyticacid with the kit said us that ther are no phytic acid in the samples. (cf. Phytase Notebook)

August 20th



We did again the experiment where we added 108 cells for the fermentation step of the idli recipe. We used S. cerevisiae BY4743 mCherry and S. cerevisiae SK1 with the gene PHO80 or PHO85 were avoid, Lactobacillus plantarum NC8, and Lactococcus lactis MG1363. With a plate counter, we obtained this numbers of colonies in function of the dilution for the plating.
From the media in the top of the idli :
MediaDilutions Number of colonies of Yeast
YPD+gen 10-1 79
YPD+gen 10-2 32
YPD+gen 10-3 10
MRS 10-1 1
MRS 10-2 0
MRS 10-3 0
LB 10-1 0
LB 10-2 0
LB 10-3 1
M17 10-1 0
M17 10-2 0
M17 10-3 0

MediaDilutions Number of colonies of L. Plantarum/L. Lactis
YPD+gen 10-1 0
YPD+gen 10-2 0
YPD+gen 10-3 0
MRS 10-1 465
MRS 10-2 99
MRS 10-3 6
LB 10-1 96
LB 10-2 51
LB 10-3 15
M17 10-1 123
M17 10-2 3
M17 10-3 7

From the batter phase of the idli:
MediaDilutions Number of colonies of Yeast
YPD+gen 10-1 217
YPD+gen 10-2 150
YPD+gen 10-3 19
MRS 10-1 6
MRS 10-2 1
MRS 10-3 1
LB 10-1 2
LB 10-2 0
LB 10-3 0
M17 10-1 0
M17 10-2 0
M17 10-3 0

MediaDilutions Number of colonies of L. Plantarum/L. Lactis
YPD+gen 10-1 0
YPD+gen 10-2 0
YPD+gen 10-3 0
MRS 10-1 396
MRS 10-2 51
MRS 10-3 6
LB 10-1 125
LB 10-2 13
LB 10-3 8
M17 10-1 503
M17 10-2 111
M17 10-3 29

We osberved that the µorganisms was in higher number near in the batter of the idli than in the idli water or in the grind rice and dall phase, where nothing has grew.

August 27th



We did idli with S. cerevisiae SK1 where PHO85 were avoid by a genetic system composed by two FRT sequences and the gene of the geneticin resistance. And also, the S. cerevisiae SK1 PHO80 avoid, the S. cerevisiae SK1 PHO85 avoid, Sc BY4743 mCherry and the first S. cerevisiae SK1 together with the S. cerevisiae with the mCherry gene.
To plate on YPD and geneticin (at 200 µg.µl-1), We took the sample from 3 area of the idli : top, middle and bottom of the idli, and we did 3 dilutions (from 10-1 to 10-3). Below, we can see the picture of each plates for each conditions of dilution, area and yeast strains that we used.


The number of yeast was around the same for each strains, and we obtained the next table, where we did a mean of the yeast that we counted, with a plate counter.
AreaDilutions Number of colonies of Yeast
Top 10-1 285
Top 10-2 11
Top 10-3 2
Middle 10-1 362
Middle 10-2 15
Middle 10-3 2
Bottom 10-1 357
Bottom 10-2 16
Bottom 10-3 1

September 10th



We started, using a TECAN, to find any informations on our strains that we want to use in fine. We did in a first time an experiment at 30°C, with S. cerevisiae yPH151, S. cerevisiae SK1 PHO 80 deleted, S. cerevisiae SK1 PHO 85 deleted, S. cerevisiae SK1 PHO 80 and PHO 85 deleted, and Propioni f. on 8 different media (Water, Idli batter/100, Idli water, M17, MRS, URA3free media, YPD and LB. We obtained this growth curve :




And particulary, the growth curve of the strains on idli water :


September 11th



We did in a first time an experiment at 37°C, with E. coli who product vitamin B2, Propioni f. who product vitamin B12 and Lactobacillus plantarum NC8 who should have obtained the vitamin B2 pathway that we engineered.



And particulary, the growth curve of the strains on idli water :


With this two last experiments, we can say that the yeasts and the bacteria that we engineered, can grow in idli water, and with the data coming from the idli fermentation with our micro-organisms, it suggests that our micro-organisms grow well in idli in the higher phase of the idli.