Difference between revisions of "Team:Paris Bettencourt/Notebook/Manufacturing"

Line 23: Line 23:
 
Basically, we are going to try different ways to distribute the strains and see how well they survived for each process.<br>
 
Basically, we are going to try different ways to distribute the strains and see how well they survived for each process.<br>
 
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.
 
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.
 +
 
<h2>Material</h2>
 
<h2>Material</h2>
 +
<li>Glycerol stock of Sc. mcherry</li>
 
<li>YPD broth</li>
 
<li>YPD broth</li>
 
<li>Agar</li>
 
<li>Agar</li>
 
<li>Geneticin, 100mg/ml</li>
 
<li>Geneticin, 100mg/ml</li>
 
<li>Osmosed water</li>
 
<li>Osmosed water</li>
−
<li>Glycerol stock of Sc. mcherry</li>
 
 
<li>Sterile eppendorf tubes</li>
 
<li>Sterile eppendorf tubes</li>
 
<li>Sterile plates and beads</li>
 
<li>Sterile plates and beads</li>
 +
 
<h2>Procedure</h2>
 
<h2>Procedure</h2>
 
<img src="https://static.igem.org/mediawiki/2015/5/5f/PBmanufacturingdilutions.jpg" width="400px" style="display:block; margin-right: 20px; float:left;">
 
<img src="https://static.igem.org/mediawiki/2015/5/5f/PBmanufacturingdilutions.jpg" width="400px" style="display:block; margin-right: 20px; float:left;">
 
<li>Make a culture of Sc. mcherry in 10ml of YPD media adding 20µL of geneticin</li>
 
<li>Make a culture of Sc. mcherry in 10ml of YPD media adding 20µL of geneticin</li>
−
(We did the culture on the 25th of july)
+
(We did cultures on the 25th of july)
 
<li>Two days later, centrifuge the culture tube</li>
 
<li>Two days later, centrifuge the culture tube</li>
 
<li>Throw the media away and replace it by 10ml of osmosed water</li>
 
<li>Throw the media away and replace it by 10ml of osmosed water</li>
Line 42: Line 44:
 
<br>
 
<br>
 
That is what we did today, and we plated the different dilutions.
 
That is what we did today, and we plated the different dilutions.
 +
 +
  
 
<br><br>
 
<br><br>
 
<h1 class="date one">July 28th</h1>
 
<h1 class="date one">July 28th</h1>
 
<h2>Goal</h2>
 
<h2>Goal</h2>
−
Our first idea to distribute the yeast is to simply dry them and collect the dry cells.<br>
+
Our first idea to distribute the yeast was to simply dry them and collect the dry cells, without adding anything.<br>
 +
Today is a first try, to have an idea of how to do it.<br>
 +
 
 +
<h2>Material</h2>
 +
<li>YPD broth</li>
 +
<li>Agar</li>
 +
<li>Geneticin, 100mg/ml</li>
 +
<li>Osmosed water</li>
 +
<li>Glycerol stock of Sc. mcherry</li>
 +
<li>Sterile eppendorf tubes</li>
 +
<li>Sterile plates and beads</li>
 +
 
 
<h2>Procedure</h2>
 
<h2>Procedure</h2>
 
<ul>
 
<ul>
−
<li>Centrifuge the culture tube</li>
+
<li>Centrifuge a culture tube</li>
 
<li>Throw the media away</li>
 
<li>Throw the media away</li>
−
<li>Add a very small amount of water to detach the yeasts from the tube easily</li>
+
<li>Add 300µL of water to detach the yeasts from the tube easily</li>
 
<li>Spread on aluminium</li>
 
<li>Spread on aluminium</li>
−
<li>Let it dry for 2 hours</li>
+
<li>Let it dry for 2 hours in the open air or in the incubator (2 different tests)</li>
 
<li>Scratch the aluminium to detach the dried yeasts</li>
 
<li>Scratch the aluminium to detach the dried yeasts</li>
−
<li>Plate the powder obtained to see if Sc. M Cherry survived </li>
+
<li>Put the powder in 10ml of osmosed water</li>
 +
<li>Plate the solution to see if Sc. M Cherry survived</li>
 
</ul>
 
</ul>
−
<h2>Results</h2>
+
 
 +
<h2>Results and discussion</h2>
 
<div class="column-left"><p>
 
<div class="column-left"><p>
 
<br>
 
<br>
 
We obtained this powder:<br><br>
 
We obtained this powder:<br><br>
−
The aluminium is not a good idea:<br>
+
We realised the aluminium was not a good idea:<br>
 
The yeasts stick to it and it gets destroyed when we scratch.<br>
 
The yeasts stick to it and it gets destroyed when we scratch.<br>
 
We have to find another drying paper, more resistant.<br>
 
We have to find another drying paper, more resistant.<br>
Line 69: Line 86:
 
</p></div>
 
</p></div>
 
<div style="clear: both"></div>
 
<div style="clear: both"></div>
 +
  
 
<br>
 
<br>
 
<h1 class="date one">July 29th</h1>
 
<h1 class="date one">July 29th</h1>
−
<H2>Result of the first OD test:</h2>
+
 
−
The 10ml solution of yeasts/water had an OD of 2,557 and 100µL of the 10^-4 dilution grew 359 colonies, so:<br>
+
<H2>Result of OD test made on the 27th of July:</h2>
 +
The 10ml solution of Sc. mcherry/water had an OD of 2,557.<br>
 +
100µL of the 10^-4 dilution grew 359 colonies, so:<br>
 
<b>1 OD = 1,4x10^7 cells/ml</b><br>
 
<b>1 OD = 1,4x10^7 cells/ml</b><br>
−
We launched a similar experience for more repetitions.
+
We repeated the very same experiment today, for more repetitions.
 +
 
  
 
<br><br>
 
<br><br>
 
<h1 class="date one">July 30th</h1>
 
<h1 class="date one">July 30th</h1>
−
<h2>Results of the drying test:</h2>
+
 
 +
<h2>Results of the drying test made on the 28th of July:</h2>
 
Yeasts grew in the plate. We can check that they produce RFP, it's Sc. M Cherry who survived.<br>
 
Yeasts grew in the plate. We can check that they produce RFP, it's Sc. M Cherry who survived.<br>
−
Let's repeat the experience but drying the yeasts on a plastic dish.<br>
+
 
 +
<h2>Procedure</h2>
 +
Today, we are repeating the drying experiment (see July 28th) but drying the yeasts on a plastic dish.<br>
 
This time, we took the OD and plated a known quantity of the obtained powder to be able to calculate the survival rate.
 
This time, we took the OD and plated a known quantity of the obtained powder to be able to calculate the survival rate.
  

Revision as of 11:47, 11 September 2015

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

  • Glycerol stock of Sc. mcherry
  • YPD broth
  • Agar
  • Geneticin, 100mg/ml
  • Osmosed water
  • 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
  • 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 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

  • YPD broth
  • Agar
  • Geneticin, 100mg/ml
  • Osmosed water
  • Glycerol stock of Sc. mcherry
  • 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. M Cherry 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 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 OD = 1,4x10^7 cells/ml
    We repeated the very same 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. M Cherry who survived.

    Procedure

    Today, we are repeating the drying experiment (see July 28th) 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
    • Let it dry for 1 day
    • Mill the paste to obtain a powder
    • Plate the powder obtained to see how many Sc. M Cherry survived

    We decided to try with wheat and rice flour to see if there is a difference. Since the product is designed for rice fermentation, using rice flour would be better!

    We also tried with sugar and ginger, then with only sugar and then without any sugar nor ginger.



    August 3rd

    Results of the second drying test:

    The survival rate is about 70%.

    Procedure


    We milled the dry paste and obtained this nice yellow powder:


    We put a known quantity of powder in 10ml of osmosed water,
    made dilutions, and plated the powder in YPD+gen plates.



    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


    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 plated small amount of the cubes dissolved in water.



    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:


    The survival rate is about 7%.

    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.

    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

    ???????????????

    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

    ????????????????????????????

    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