Team:Paris Bettencourt/Notebook/Manufacturing

Ferment It Yourself

iGEM Paris-Bettencourt 2O15

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Notebook - Manufacturing

• 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. Therefore, it's easy to select and characterize it.
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

• Glycerol stock of Sc. mcherry
• YPD broth
• Agar
• Geneticin, 100mg/ml
• Osmosed water
• Sterile culture tubes
• 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 cultures 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
• In the eppendorf tubes, 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, thanks to the beads.

July 28th

Goal

Our first idea to distribute the yeast was to simply dry them and collect the dry cells, without adding anything.
Today is a first try, to have an idea of how to do it.

Material

• Culture of Sc. mcherry
• YPD broth
• Agar
• Geneticin, 100mg/ml
• Osmosed water
• Sterile eppendorf tubes
• Sterile plates and beads

Procedure

• Centrifuge a culture tube
• Throw the media away
• Add 300µL of water to detach the yeasts from the tube easily
• Spread on aluminium
• Let it dry for 2 hours in the open air or in the incubator (2 different tests)
• Scratch the aluminium to detach the dried yeasts
• Put the powder in 10ml of osmosed water
• Plate the solution to see if Sc. mcherry survived

Results and discussion

We obtained this powder:

We realised the aluminium was not a good idea:
The yeasts stick to it and it gets destroyed when we scratch.
We have to find another drying paper, more resistant.

July 29th

Result of the OD test made on the 27th of July:

The 10ml solution of Sc. mcherry/water had an OD of 2,557.
100µL of the 10^-4 dilution grew 359 colonies, so:
1 A = 1,4x10⁷ cells/ml
We repeated the very same OD experiment today, for more repetitions.

July 30th

Results of the drying test made on the 28th of July:

Yeasts grew in the plate.
We can check that they produce RFP, it's Sc. mcherry who survived.

Procedure

Today, we are repeating the drying experiment (see July 28th) but drying the yeasts on a plastic dish.

• Take 100µL of the culture solution of Sc. mcherry to make dilutions (10^-1 to 10^-5)
We remind that the culture tube contain 10ml of media.
• Plate 100µL of each dilutions to deduce the number of cells in the culture tube
• Centrifuge the culture tube
• Throw the media away
• Add 300µL 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
• Put the powder in 10ml of osmosed water
• Take 100µL of the solution to make dilutions (10^-1 to 10^-5)
• Thanks to the beads, plate 100µL of each dilution

July 31th

Result of the OD test made on the 29th of July:

The 10ml solution of Sc. mcherry/water had an OD of 2,635.
100µL of the 10^-4 dilution grew 205 colonies, so:
1 A = 7,8x10⁶ cells/ml

August 1st

Result of the drying test made on the 30th of July:

100µL of the 10^-5 dilution of the initial 10ml culture solution grew 106 colonies, so we initially dried 1,06x10⁹ cells.
100µL of the 10^-4 dilution of the powder mixed with 30ml of osmosed water grew 123 colonies, so 8,4x10⁸ cells survived.
We therefore have a survival rate of 79%.

Discussion and New Goal

The powder we obtained with the drying method stick to everything, because of static electricity and therefore, it is not very easy to distribute and manipulate.
Consequently, we are looking fo other solutions...

Our second idea was to cook a homemade powder with edible ingredients easily found in India.
We found a recipe that inspired us here.
We decided to compare the initial recipe to our modifications, so we made for different tests :

• Test 1, original recipe: wheat flour, sugar and ginger
Since the project is designed for rice fermentation and rice is cheaper than wheat, we wanted to try with rice flour.
• Test 2: rice flour, sugar and ginger
Ginger can be expensive so maybe we can avoid using it...
• Test 3: rice flour, sugar
And maybe we can also avoid using sugar
• Test 4: only rice flour

Material

• Culture of Sc. mcherry
• Potatoes
• Flour (wheat and rice)
• Cornmeal
• Sugar
• Ginger
• Plate of YPDagar+Gen(200)
• Osmosed water
• Sterile eppendorf tubes
• Sterile beads

Procedure

• Cook some potatoes
• Save the cooking water, wait till it's not too hot and mix 20ml of it with the yeasts
• Add 20ml of flour (rice or wheat depending on the recipe), 40ml of mashed potatoes
• Depending on the recipe add 20ml of sugar and a teaspoon of ginger
• Add 80ml of cornmeal
• Let it dry for 1 day
• Mill the paste to obtain a powder
• Plate the powder obtained to see how many Sc. mcherry survived

August 3rd

Continuing the powder recipe we began on the 1st of August

After almost two days of drying, we obtained a very hard paste that we milled.
After milling, we had this nice yellow powder you can see on the picture.

We put 100mg of each powder in 10ml of osmosed water, made dilutions,
and plated the solutions in YPDagar+GEN(200) plates.

August 4th

Discussion and New Goal

While doing the recipe with the potatoes (from the 1st to the 3rd of August), we realised it wasn't easy to do and it took lots of time and ingredients.
We decided to reduce the number of ingredients, using only water and rice flour.

Procedure

• Mix the yeasts with 20ml of osmosed water
• Add 40ml 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

The result is a small solid cube, very easy to give away.

Several cubes can be packed together.

We tought it could be a good media of distribution.

We dissolved one cube in 10ml of osmosed water, after weighed it.
Then we made dilutions (10^-1 to 10^-5) and plated 100µL of each on YPDagar+GEN(200)

August 5th

Results of the survival of Sc. mcherry in the potato powder made on the 3rd of August:

Survival rate:

• Wheat flour, sugar and ginger: 14%
• Rice flour, sugar and ginger: 20%
• Rice flour, sugar: 12%
• Rice flour: 4%

Discussion:

We can see that the more ingredients we put, the more the yeasts survived, so each ingredient plays a role in the cells survival.
But as we said before, we don't want to put too much ingredients in the recipe so that it's cheap and easy.
We can also notice that rice flour seems more efficient than wheat flour (but we made only one repetition so nothing is statistically proven).

August 6th

Results of the survival of Sc. mcherry in the cubes made on the 4th of August:

We obtained a survival rate of 69%.

After staying 5hours in the water, the survival rate reached 737%.

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.

Material

• Glycerol stock of G1513
• M17 broth
• Solution of D-Glucose
• Agar
• Erythromycin, 20mg/ml
• Osmosed water
• Sterile eppendorf tubes
• Sterile plates and beads

Procedure

• Make a culture of G1513 in 10ml of M17+1% glucose media adding 5µL of erythromycin
(We did cultures on the 6th of August)
• One day 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
• In the eppendorf tubes, make 10^-1 to 10^-5 dilutions of the G1513/water solution to plate 100µL of each on M17Glucose+agar+Ery(10)

August 10th

Results of the OD test for G1513 made on the 7th of August:

The 10ml solution of G1513/water had an OD of 1,296.
Here is a table of what 100µL of each dilutions grew:

Dilution	Number of colonies
10-1	381
10-2	84
10-3	11

We have, in average, 7,74x10³ cells per ml of the 10^-1 dilution.
so: 1 A = 6,0x10⁴ cells/ml

Procedure

Today, we made new cubes containing Sc. mcherry 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.

Material

• Culture of Sc. mcherry
• 3 potatoes
• Rice flour
• Geneticin, 100mg/ml
• Osmosed water
• Sterile eppendorf tubes and culture tubes
• Sterile plates and beads
• One glass bottle

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 potato water in a culture tube
• Add 20μL of geneticin (100mg/ml)
• Inoculate with Sc. mcherry
• Put in the incubator at 30°C for 2 days

For the mix potato juice with rice flour:

• Put 5ml of the potato water made previously in a culture tube
• Inoculate with Sc. mcherry
• 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 made on the 10th of August

The yeasts survived well: 42%.
(Seven days later, on the 19th of August, we plated cubes from this same test, and obtained a survival rate of 21%.)
But about the G1513, strangely, the number of cells in the plates of M17agar, 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. mcherry produces RFP but not G1513.
Unfortunately, we came to the conclusion that the cells in the M17 plates were yeasts. Actually, Sc. mcherry 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. mcherry.
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 made on the 11th of August

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. mcherry alone, G1513 alone, and a mix of both strains.
Measuring the OD and plating to count the cells

Results of the media test made on the 13th of August

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 potato and sugar concentration.

August 18th

Results of the OD test made on the 17th of August

For Sc. mcherry:
The 10ml solution of Sc. mcherry/water had an OD of 1,820.
100µL of the 10^-4 dilution grew 70 colonies, so:
1 A = 3,8x10⁶ cells/ml
For G1513:
The 10ml solution of G1513/water had an OD of 1,521.
100µL of the dilutions grew:

Dilution	Number of colonies
10-5	131
10-6	21

So 1 A = 1,1x10⁸ cells/ml

August 19th

Results of the cube test made on the 17th of August

• Sc. mcherry alone: 39%
• Sc. mcherry in the mix: 26%
• G1513 in the mix: not plated because we are waiting for the cycloheximide
• 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

We made more cubes with Sc. mcherry alone, G1513 alone, and a mix of both.
We made more OD correlations.

August 24th

Procedure

We made more cubes...

Results of the OD test made on the 20th of August

For Sc. mcherry:
The 10ml solution of Sc. mcherry/water had an OD of 1,828.
100µL of the 10^-5 dilution grew 56 colonies, so:
1 A = 3,1x10⁶ 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 made on the 20th of August

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

August 25th

Results of the OD tests made on the 24th of August

For Sc. mcherry:
The first 10ml solution of Sc. mcherry/water had an OD of 1,921.
100µL of the dilutions grew:

Dilution	Number of colonies
10-4	505
10-5	39

1 A = 2,3x10⁶ cells/mL
The second 10ml solution of Sc. mcherry/water had an OD of 2,523.
100µL of the 10^-5 dilution grew 95 colonies.
so 1 A = 3,8x10⁶ 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 made on the 24th of August

Sc. mcherry alone: 122% et 162%

Sc. mcherry 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.

Material

• Potatoes
• Sugar
• Water
• Culture of Sc. mcherry
• Culture of G1513
• Erythromycin, Geneticin
• Tecan and Tecan plate
• Mineral oil

Procedure

• Make 3 different potato juice solutions(low, medium and high concentration of potatoes) following this protocol
• For each one of these solutions, make samples with different concentration of sugar: 0g/ml; 0,1g/ml; 0,2g/ml; 0,4g/ml.
• Inoculate the strains in these media, with the appropriate antibiotics (do not mix G1513 and Sc. mcherry)
• Put 150μL of the solutions in Tecan plate wells 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 idli.

Procedure

• Following the idli recipe protocol, make idli.
• Weigh the cube
• Dilute the cube in 5ml of osmosed water
• Add this solution to 1 bottle of idli before the fermentation
• After the fermentation, plate 100μL of a 10^-2 dilution of the Idli water

August 31th

Results of the media test made on the 27th of August

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

Results of the idli test

	Number of cells in the top part of the idli, 10-2 dilution	Number of cells in the middle part of the idli, 10-2 dilution	Number of cells in the bottom part of the idli, 10-2 dilution
X	38 / 23	324	71 / 111
Y	0	39 / 27	64 / 47

September 2nd

Results of the second media test made on the 31st of August

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 our advisors, because obviously, there was a misunderstanding.

September 4th

Procedure

We tried 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 6th

Procedure

We made fresh new cultures of G1513 and Sc. mcherry.

September 7th

Results of the Tecan test made on the 4th of September

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

Today we used the fresh cultures we made on the 6th of August to make lots of new cubes and OD test.
After centrifugation, throwing the media and replacing it by osmosed water, the cultures gave us two solutions:

• Solution X: 8,8x10⁷ cells of Sc. mcherry per ml
• Solution Y: 5,2x10⁹ cells of G1513 per ml

We made 3 different types of cubes:

• Cubes Q: 15ml of rice flour, 6ml of osmosed water and 1ml of solution X
• Cubes R: 15ml of rice flour, 5ml of osmosed water, 900μL of solution X and 1ml of solution Y
• Cubes S: 15ml of rice flour, 6ml of osmosed water and 1ml of solution Y

Strains and cubes	Total weight of the cubes (g)	Number of cells per g of cube
Sc. mcherry in Q cubes	13,30	6,6x106
Sc. mcherry in R cubes	13,39	5,9x106
G1513 in R cubes	13,39	3,9x108
Sc. mcherry in S cubes	10,70	4,8x108

We are going to plate one cube of each kind each day and follow the survival rate during a week of drying. For the OD, here are the results we obtained (we obtained them on the 10th of August).

Studied strains	OD of the sample	Number of colonies in the 10-3 dilution of the sample	Number of colonies in the 10-4 dilution of the sample	Number of colonies in the 10-5 dilution of the sample
Sc. mcherry	1,156	+++	347	23
G1513	0,392	+++	439	/

So, for Sc. mcherry: 1A=2,5x10⁷ cells/ml
For G1513: 1A=1,1x10⁸ cells/ml

September 9th

We plated the first cubes today.
Everyday, we will follow the same protocol:

• Weigh the cube
• Dilute the cube in 10ml of osmosed water during 5min
• Dilute the solution obtained 10^-1, 10^-2 and 10^-3 times
• Plate the dilutions

This should only take 20min in total, doing it for all the cubes in the same time.
For more clarity, we will write the results for each cubes on the day we plated it (even if the colonies took 1 or 2 days to grow depending on the strains, so we only add the results 2 days after the plating).

Studied strains	Weight (g)	Number of cells initially in the cube	Number of colonies in the 10-3 dilution	Number of colonies in the 10-4 dilution	Number of cells in the cube after the drying period	Survival rate
Sc. mcherry in Q	0,96	6,3x106	72	10	8,6x106	136,5%
Sc. mcherry in R	0,96	5,7x106	80	11	9,5x106	166,7%
G1513 in R	0,96	3,7x108	79	10	9,0x106	2,4%
G1513 in S	0,72	5,4x108	357	23	2,9x107	5,4%

September 10th

Studied strains	Weight (g)	Number of cells initially in the cube	Number of colonies in the 10-3 dilution	Number of colonies in the 10-4 dilution	Number of cells in the cube after the drying period	Survival rate
Sc. mcherry in Q	0,97	6,4x106	104	5	7,7x106	120,3
Sc. mcherry in R	1,40	8,3x106	150	11	1,3x107	156,6
G1513 in R	1,40	5,5x108	126	/	1,3x107	2,4
G1513 in S	1,19	5,7x108	63	/	6,3x106	1,1%

September 11th

Studied strains	Weight (g)	Number of cells initially in the cube	Number of colonies in the 10-2 dilution	Number of colonies in the 10-3 dilution	Number of cells in the cube after the drying period	Survival rate
Sc. mcherry in Q	1,04	6,9x106		77	7,7x106	111,5%
Sc. mcherry in R	1,28	8,5x106		149	1,5x107	176,5
G1513 in R	1,28	5,0x108	164	/	1,6x106	0,3%
G1513 in S	1,49	5,8x108	299	/	3,0x106	0,5%

September 12th

Studied strains	Weight (g)	Number of cells initially in the cube	Number of colonies in the 10-2 dilution	Number of colonies in the 10-3 dilution	Number of cells in the cube after the drying period	Survival rate
Sc. mcherry in Q	1,50	9,9x106	877	88	1,3x107	131,3%
Sc. mcherry in R	1,12	6,6x106	513	52	7,2x106	109,1%
G1513 in R	1,12	4,4x108	331	/	3,3x106	0,8%
G1513 in S	0,93	4,5x108	368	/	3,7x106	0,8%

September 13th

Studied strains	Weight (g)	Number of cells initially in the cube	Number of colonies in the 10-2 dilution	Number of colonies in the 10-3 dilution	Number of cells in the cube after the drying period	Survival rate
Sc. mcherry in Q	1,17	7,7x106	/	123	1,2x107	155,8%
Sc. mcherry in R	1,50	8,9x106	/	117	1,2x107	134,8%
G1513 in R	1,50	5,9x108	28	/	2,8x105	0,05%
G1513 in S	0,96	4,6x108	30	/	3,0x105	0,07%

September 14th

Studied strains	Weight (g)	Number of cells initially in the cube	Number of colonies in the 10-2 dilution	Number of colonies in the 10-3 dilution	Number of cells in the cube after the drying period	Survival rate
Sc. mcherry in Q	1,18	7,8x106	/	103	1,0x107	128,2%
Sc. mcherry in R	1,33	7,9x106	/	83	8,3x106	105,1%
G1513 in R	1,33	5,2x108	12	/	1,2x105	0,02%
G1513 in S	0,82	3,9x108	49	/	4,9x105	0,1%

September 15th

Studied strains	Weight (g)	Number of cells initially in the cube	Number of colonies in the 10-2 dilution	Number of colonies in the 10-3 dilution	Number of cells in the cube after the drying period	Survival rate
Sc. mcherry in Q	1,08	7,1x106	/	91	9,1x106	128,2
Sc. mcherry in R	1,44	8,5x106	/	84	8,4x106	98,8
G1513 in R	1,44	5,6x108	4	/	4,0x104	0,0%
G1513 in S	1,01	4,9x108	10	/	1,0x105	0,0%