• July
• August
• September

July 27th

Beginning of the manufacturing project! Let's do it!

Goal

The goal of this project is to create an way to grow and distribute our strains in India, easily and for cheap.
Since our genetically engineered strains will be ready only in september, we will not have time to work on it, so we have to find similar strains, that we can study and select with antibiotics.
We chose Saccharomyces cerevisiae mcherry, wich is resistant to geneticin at a concentration of 200µg/mL, and produce RFP.
Basically, we are going to try different ways to distribute the strains and see how well they survived for each process.
In order to do that, we will have to calculate a survival rate so we have to be able to know how many cells we are putting in the beggining, in the recipe we are trying.

Material

YPD broth

Agar

Geneticin, 100mg/ml

Osmosed water

Glycerol stock of Sc. mcherry

Sterile eppendorf tubes

Sterile plates and beads

Procedure

Make a culture of Sc. mcherry in 10ml of YPD media adding 20µL of geneticin

(We did the culture on the 25th of july)

Two days later, centrifuge the culture tube

Throw the media away and replace it by 10ml of osmosed water

Measure the OD, doing the blank with osmosed water

If the OD is too high to be measured, make a 10 times dilution and measure the OD of the dilution

Make 10^-1 to 10^-5 dilutions of the yeasts/water solution to plate them on YPDagar+GEN(200)

That is what we did today, and we plated the different dilutions.

July 28th

Goal

Our first idea to distribute the yeast is to simply dry them and collect the dry cells.

Procedure

• Centrifuge the culture tube
• Throw the media away
• Add a very small amount of water to detach the yeasts from the tube easily
• Spread on aluminium
• Let it dry for 2 hours
• Scratch the aluminium to detach the dried yeasts
• Plate the powder obtained to see if Sc. M Cherry survived

Results

July 29th

Result of the first OD test:

The 10ml solution of yeasts/water had an OD of 2,557 and 100µL of the 10^-4 dilution grew 359 colonies, so:
1 OD = 1,4x10^7 cells/ml
We launched a similar experience for more repetitions.

July 30th

Results of the drying test:

Yeasts grew in the plate. We can check that they produce RFP, it's Sc. M Cherry who survived.
Let's repeat the experience but drying the yeasts on a plastic dish.
This time, we took the OD and plated a known quantity of the obtained powder to be able to calculate the survival rate.

July 31th

Result of the second OD test:

1 OD = 7,8x10^6 cells/ml

August 1st

Goal

The powder obtained with the drying method is full of static electricity and stick to everything, not very easy to distribute.
We are therefore looking fo other solutions...

Our second idea is to cook a powder

Procedure

(found here: http://www.instructables.com/id/Stop-Paying-for-Yeast-Make-Your-Own/)

• Cook some potatoes
• Save the cooking water, wait till it's not too hot and mix 1cup of it with the yeasts
• Add 1 cup of flour, 1 cup of sugar, 2 cups of mashed potatoes, a teaspoon of ginger
• Add 4 cups of cornmeal

August 3rd

Results of the second drying test:

The survival rate is about 70%.

Procedure

August 4th

Goal

While doing the recipe with the potatoes, we realise it wasn't easy to do and it took a lot of time and a lot of ingredients.
We decided to reduce the number of ingredients, using only water and rice flour.

Procedure

• Mix the yeasts with one cup of osmosed water
• Add 2 cups of rice flour
• Knead till you get a nice paste

At this point, we tought the paste could be shaped, instead of being milled in a powder.

• Shape it in little cubes
• Let it dry for few hours

Result

August 5th

Results of the potato powder:

Survival rate:

• Wheat flour, sugar and ginger: 6,9%
• Rice flour, sugar and ginger: 9,5%
• Rice flour, sugar: 5,7%
• Rice flour: 1,9%

We can see that the more ingredients we put, the more the yeasts survived.

August 6th

Results of the cube test:

August 7th

Goal

Now that we are at ease with the process, we are trying the very same process with lactococcus lactis. We chose MG 13.63, containing the plasmid PSIP411, resistant to erythromycin at a concentration of 10µL/mL, to study it's survival rate. We will name this strain G1513. We also have to correlate the OD of a solution of G1513 to the number of alive cells inside.

August 10th

Results of the OD test for G1513:

1 OD = 6,0x10^4 cells/ml

Procedure

Making new cubes containing Sc. M Cherry and G1513 to calculate the survival rate.

August 11th

Goal

While waiting for the cubes results, we are thinking about the next step: finding a media to grow the strains, easy to give to the population and to use afterward. The perfect media would be an edible one, that we could incorporate to the cube, instead of the water.

Procedure

We tried 2 different medium: potato juice and potato juice with rice flour.

For the potato juice:

• Heat 3 potatoes in 750ml of water
• Let the water boil (sterilisation)
• Save the water and stock it in a bottle
• Put 10ml of this potatoe water in a culture tube
• Add 20μL of geneticin (100mg/ml)
• Inoculate with Sc. M Cherry
• Put in the incubator at 30°C for 2 days

For the mix potato juice with rice flour:

• Put 5ml of the potatoe water made previously in a culture tube
• Inoculate with Sc. M Cherry
• Add 20μL of geneticin (100mg/ml)
• Add 5ml of rice flour
• Put in the incubator at 30°C for 2 days

August 12th

Results of the cube tests

The yeasts survived well: 42%.
Seven days later, we plated again cubes from this test, and obtained a survival rate of 21%.
But strangely, the number of cells in the plates of M17 glucose with erythromycin, which are supposed to be G1513, is the same as in the plates with YPD and geneticin, selecting the yeasts.
Therefore, we checked the production of RFP, since Sc. M Cherry produces RFP but not G1513.
Unfortunately, we came to the conclusion that the cells in the M17 plates were yeasts. Actually, Sc. M Cherry resist to erythromycin and is therefore growing in the plate.

Goal

We need a chemical that will allow us to select G1513 and kill Sc. M Cherry.
After some researchs, we found out that cycloheximide could be helpful and ordered it. While waiting for this chemical, we can't work with G1513.

August 13th

Results of the media test

Nothing grew on the potato juice media.
We had the idea of adding sugar, to see if it changes something.
The potato juice/rice flour media strongly smells yeasts. We therefore admit the yeast grew in this media but decided that the aspect wasn't very attractive and the smell so so bad that indian population would be disgusted and won't use it.

August 17th

Procedure

Making new cubes with Sc. M Cherry alone, G1513 alone, and a mix of both strains.
Measuring the OD and plating to count the cells

Results of the media test

With sugar, we can see there are cells growing in the potato juice.
It's a very small growth compared to the one in YPD but it's still a growth.
We will work more precisely on this media, trying to deduce the influence of the concentration in potato and in sugar.

August 18th

Results of the OD test

For Sc. M Cherry:
1 OD = 3,8x10^6 cells/ml
For G1513:
1 OD = 1,1x10^8 cells/ml

August 19th

Results of the cube test

• Sc. M Cherry alone: 39%
• Sc. M Cherry in the mix: 26%
• G1513 alone: 1,8%

We kept samples of the mix in the fridge to be able to study it later, when we will have the cycloheximide.

August 20th

Procedure

Making more cubes with Sc. M Cherry alone, G1513 alone, and a mix of both.
Making more OD correlations.

August 24th

Procedure

Making more cubes...

Results of the OD test

For Sc. M Cherry:
1 OD = 3,1x10^6 cells/ml
For G1513:
Nothing... It seems that the colony used was dead so the cubes will not contained any G1513.

Results of the cube test

• Sc. M Cherry alone: 92%
• Sc. M Cherry in the mix (but dead G1513): 96%
• G1513 alone: Nothing

August 25th

Results of the OD test

For Sc. M Cherry:
1 OD = 2,6x10^6 cells/mL
For G1513:
Nothing... We used cells from the previous culture, wich was dead, so it seems normal.

August 26th

Results of the cube test

Sc. M Cherry alone: 122% et 162%

Sc. M Cherry in the mix (but dead G1513): 178% et 217%

G1513 alone: Nothing

August 27th

Goal

We want to find a media that could be use in India to grow the yeasts and the Lactococcus lactis for cheap.

Procedure

• Make 3 different potato juice solutions: low, medium and high concentration in potatoes
• For each one of these solutions, make samples with different concentration of sugar
• Inoculate the strains separetely in these media, with the appropriate antibiotics
• Put 150μL of the solutions in a Tecan plate well and add 50μL of oil
• Put in the Tecan at 30°C, measuring the OD every 15minutes, for 2 days

August 28th

Goal

We want to see how the strains in the cubes react when put in the idli.

Procedure

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August 31th

Results of the media test

It's a fail, the OD was too high so we can't really see the growth.
We did it again, diluting the media and strain 200times in osmosed water.

Results of the idli test

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September 2nd

Results of the second media test

It's a fail, there was almost only water in the well so nothing grew.
We will do it again, asking for a real protocol to some experts.

September 4th

Procedure

Trying the new method for the Tecan test.
This time, we did an overnight culture for each media and each strain, that we diluted 200times (till we get an OD smaller than 0,01)

September 7th

Results of the Tecan test

Third fail, because one parameter was changed and we didn't noticed: the Tecan measured the OD for only few hours, not enough to see the growth...

September 8th

Procedure

We made fresh new culture of Sc. M Cherry and G1513, that we used today to make lots of new cubes and OD test. We planned on making many cubes to plate one each day and follow the survival rate during the week.

September 9th

September 10th