Difference between revisions of "Team:Paris Bettencourt/Notebook/Idli and micro-organisms"
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{{Paris_Bettencourt/menu}} | {{Paris_Bettencourt/menu}} | ||
− | {{Paris_Bettencourt/ | + | {{Paris_Bettencourt/banner|page_id=notebook|page_name=Notebook - Idli and Micro-organisms}} |
<html> | <html> | ||
+ | |||
+ | |||
+ | <div id="notebookMenu"> | ||
+ | <ul> | ||
+ | <li><a href="#july">July</a></li> | ||
+ | <li><a href="#august">August</a></li> | ||
+ | <li><a href="#september">September</a></li> | ||
+ | </ul> | ||
+ | </div> | ||
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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> | ||
− | + | 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 | + | <a href="https://2015.igem.org/Team:Paris_Bettencourt/Protocols#FSIdli">this recipe</a> |
− | with basmati rice and indian dall. <br> | + | with basmati rice and indian dall. <br><br> |
+ | <IMG SRC= "https://static.igem.org/mediawiki/2015/1/1b/Schema_idli.png" width=50%> | ||
+ | <IMG SRC= "https://static.igem.org/mediawiki/2015/5/52/Idli_water.JPG" width=20%> | ||
+ | <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> | ||
− | + | 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> | ||
+ | |||
+ | |||
+ | |||
+ | <!-- 20 juillet --> | ||
+ | |||
+ | <a name="july" class="anchor"><h1></h1></a> | ||
+ | <h2 class="date one">July 20th</h2><br><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> | ||
+ | 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> | ||
+ | |||
+ | |||
Line 33: | Line 59: | ||
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 | + | <a href="https://2015.igem.org/Team:Paris_Bettencourt/Protocols#FSVitaminATitration">Vitamin A (protocol titration)</a> <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> | ||
− | We can just observe concentrations higher than 10<sup>-3</sup>mg.ml<sup>-1</sup>. <br><br> | + | We can just observe concentrations higher than 10<sup>-3</sup>mg.ml<sup>-1</sup>. <br> |
+ | |||
+ | <table> | ||
+ | <tr> <td><b>dilution of the initial concentration</b> </td><td> <b>Value of the spectrophotometer </b></td> </tr> | ||
+ | <tr> <td>10<sup>-1</sup> </td><td> 1.625</td> </tr> | ||
+ | <tr> <td> 10<sup>-2</sup> </td><td> 0.078 </td> </tr> | ||
+ | <tr> <td> 10<sup>-3</sup> </td><td> 0.034 </td> </tr> | ||
+ | <tr> <td> 10<sup>-4</sup> </td><td> 0.008 </td> </tr> | ||
+ | <tr> <td> 10<sup>-5</sup> </td><td> 0.002 </td> </tr> | ||
+ | <tr> <td> 10<sup>-6</sup> </td><td> 0.005 </td> </tr> | ||
+ | <tr> <td> 10<sup>-7</sup> </td><td> -0.001 </td> </tr> | ||
+ | <tr> <td> 10<sup>-8</sup> </td><td> 0.000 </td> </tr> | ||
+ | </table> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | <!-- 10 Août --> | ||
+ | |||
+ | |||
+ | <a name="august" class="anchor"><h1></h1></a> | ||
+ | <h2 class="date two">August 10th</h2><br><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> | ||
+ | |||
+ | |||
+ | |||
+ | <!-- 14 Août --> | ||
+ | |||
+ | <a name="august" class="anchor"><h1></h1></a> | ||
+ | <h2 class="date two">August 14th</h2><br><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> | ||
+ | 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"> | ||
+ | <IMG SRC= "https://static.igem.org/mediawiki/2015/3/31/Sc_growth_in_LB.png" width = 75%> | ||
+ | </div> | ||
+ | |||
+ | <div class="column-right"align="center"> | ||
+ | <IMG SRC= "https://static.igem.org/mediawiki/2015/2/29/Sc_growth_in_YPD.png" width = 75%> | ||
+ | </div> <br> | ||
+ | |||
+ | <center> | ||
+ | <IMG SRC= "https://static.igem.org/mediawiki/2015/8/8d/Sc_growth_in_water.png" width = 40%><br> | ||
+ | </center> | ||
+ | <IMG SRC= "https://static.igem.org/mediawiki/2015/8/8d/YPD_precise_strain.png" width = 70%><br> | ||
+ | |||
+ | |||
+ | |||
+ | <!-- 19 - 20 Août --> | ||
+ | |||
+ | |||
+ | <a name="august" class="anchor"><h1></h1></a> | ||
+ | <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>) | ||
+ | |||
+ | |||
+ | <!-- 20 Août --> | ||
+ | |||
+ | <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 --> | ||
+ | |||
+ | <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"> |
+ | <IMG SRC= "https://static.igem.org/mediawiki/2015/d/df/Mean_Control_11sep.png" width=50%> | ||
+ | </div> | ||
+ | <div class="column-right"align="center"> | ||
+ | <IMG SRC= "https://static.igem.org/mediawiki/2015/9/9f/Mean_EcoliB2_11sep.png" width=50%> | ||
+ | </div><br> | ||
+ | <div class="column-left"align="center"> | ||
+ | <IMG SRC= "https://static.igem.org/mediawiki/2015/6/6b/Mean_Lactobacillus_11sep.png" width=50%> | ||
+ | </div> | ||
+ | <div class="column-right"align="center"> | ||
+ | <IMG SRC= "https://static.igem.org/mediawiki/2015/6/6c/Mean_Propionif_11sep.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/2015/3/3a/Mean_Idliwater_11sep.png" width=50%> | ||
− | < | + | <br><br> |
+ | 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