Team:Paris Bettencourt/Notebook/Idli and micro-organisms
Ferment It Yourself
iGEM Paris-Bettencourt 2O15
- Background
- Design
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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 :
Media Dilutions 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
Media Dilutions 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:
Media Dilutions 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
Media Dilutions 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.Area Dilutions 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.