Team:Paris Bettencourt/Notebook/Old People Smell

From 2014.igem.org

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                                         <p>Then, we made liquid culture of C.striatum in media <b>1)</b> to try if the minima media works.</p>
                                         <p>Then, we made liquid culture of C.striatum in media <b>1)</b> to try if the minima media works.</p>
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                                         <p>* Concentration of the 2-nonenal solution:
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                                         <p><i>* Concentration of the 2-nonenal solution:
In M9 Media, we use 20mL of Glucose 20% (200g/L). The concentration of glucose is C=200/M=1.09 mol/L.
In M9 Media, we use 20mL of Glucose 20% (200g/L). The concentration of glucose is C=200/M=1.09 mol/L.
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Glucose has 6 carbons while 2-nonenal has 9, so we need 2/3 of the glucose volume to have the same amount of carbon atoms. C=2/3*1.09=0.74 mol/L and then 0.74*M=103.7g/L. That's why the concentration of the 2-nonenal solution is 10.73%. We also added Tween80.</p>
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Glucose has 6 carbons while 2-nonenal has 9, so we need 2/3 of the glucose volume to have the same amount of carbon atoms. C=2/3*1.09=0.74 mol/L and then 0.74*M=103.7g/L. That's why the concentration of the 2-nonenal solution is 10.73%. We also added Tween80.</i></p>

Revision as of 15:15, 14 August 2014

Paris Bettencourt 2014



Notebook

Old Person odor

August

August 5th

The goal is to select a bacteria able to develop on 2-nonenal. We made 2 new media:


1) M9 Minimal Media 100% Glucose

  • 200mL M9 Salts
  • 0.1mL CaCl2 1M
  • 2mL MgSO4 1M
  • 20mL Glucose 20%
  • Water: bring to 1L

2) M9 Minimal Media 50% Glucose - 50% 2-Nonenal (according to the number of carbon atoms)

  • 200mL M9 Salts
  • 0.1mL CaCl2 1M
  • 2mL MgSO4 1M
  • 10mL Glucose 20%
  • 10mL 2-nonenal 10,7%*
  • Water: bring to 1L

3) M9 Minimal Media 100% 2-nonenal

  • 200mL M9 Salts
  • 0.1mL CaCl2 1M
  • 2mL MgSO4 1M
  • 20mL 2-nonenal 10,7%*
  • Water: bring to 1L

We autoclaved 6 bottles (2 of 500mL for each)

Then, we made liquid culture of C.striatum in media 1) to try if the minima media works.


* Concentration of the 2-nonenal solution: In M9 Media, we use 20mL of Glucose 20% (200g/L). The concentration of glucose is C=200/M=1.09 mol/L. Glucose has 6 carbons while 2-nonenal has 9, so we need 2/3 of the glucose volume to have the same amount of carbon atoms. C=2/3*1.09=0.74 mol/L and then 0.74*M=103.7g/L. That's why the concentration of the 2-nonenal solution is 10.73%. We also added Tween80.

August 6th

We poured 20 plates of each of the 3 media with 350mL of an agar solution and 100mL of medium.


Then we used 3 plates of each type to culture skin bacteria from 3 spots: arm, armpit and forehead. Ursczula sampled bacteria from her skin with a loop and for each spot she diluted the bacteria in 300uL of NF Water. Then we plated 100uL of each solution in each of the 3 types of plates and incubated them at 37°C.

M9 100% glucoseM9 50%-50%M9 100% 2-nonenal
Armxxx
Armpitxxx
Foreheadxxx
NEB (control)xxx

August 8th

After 2 days, nothind has developped on the M9 plates. M9 media is actually really slow for the development of any strain. That is why we created a new media which would be like the real skin environment: synthetic sweat according to this recipe

According to the recipe, fatty acids seem to be the main source of carbon in synthetic sweat so we replaced them by 2-nonenal.
We made 2 media:
1) without fatty acids
2) without fatty acids and with 2-nonenal mixed with 10% of Tween 80.

August 11th

Results of M9 plates

M9 100% glucoseM9 50%-50%M9 100% 2-nonenal
ArmMany very small colonies60
ArmpitMany very small colonies00
Forehead2750
NEB (control)Manymany0

Analysis:
The fitness of bacteria is better on 100% Glucose than on 50-50%. No colony develops on 2-nonenal, not even NEB. Then we can suggest that 2-nonenal is not used as a carbon source and that the difference of fitness between 100% glucose and 50-50% is only due to the lower amount of glucose in 50-50%. We should have made plates with only 50% of glucose to compare.


We also plated samples on synthetic sweat:

NEBForeheadNose
1) Without fatty acidsxxx
2) Without fatty acids and with 2-nonenalxxx

On August 10th, Jake tried a new medium with peptone added to M9. He made 2 media (20 plates of each):

  • iGEM medium 1 (100% glucose)
    • pancreatic digest of casein: 15 g
    • glucose: 5 g
    • NaCl: l5 g
    • Agar: 15 g
    • Water: 1 L
  • iGEM medium 2 (100% 2-nonenal):
    • pancreatic digest of casein: 15 g
    • 2-nonenal: 10 g
    • Tween 80: 250uL (premix with 2-nonenal before adding to the medium)
    • NaCl: l5 g
    • Agar: 15 g
    • Water: 1 L

I prepared 2 new media:


  • iGEM medium 3 (50% glucose)
    • pancreatic digest of casein: 7.5 g
    • glucose: 1.25 g
    • NaCl: 7.5 g
    • Agar: 7.5 g
    • Water: 0.5 L
  • iGEM medium 4 (50% glucose - 50% 2-nonenal):
    • pancreatic digest of casein: 7.5 g
    • glucose: 1.25 g
    • 2-nonenal: 2.5 g
    • Tween 80: 125uL (premix with 2-nonenal before adding to the autoclaved medium)
    • NaCl: 7.5 g
    • Agar: 7.5 g
    • Water: 0.5 L

I autoclaved them at 111°C - 3min (programm 5).

Then, I added the 2-nonenal to iGEM medium 4: density of nonenal is 0.846g/mL so I put 2.5/0.846 = 2.96 mL of 2-nonenal mixed with 125uL of Tween 80. I poured 20 plates of each medium and then plated before incubating at 37°C:

iGEM medium 1iGEM medium 2iGEM medium 3iGEM medium 4
Nosexxxx
Foreheadxxxx
NEB (control)xxxx

September

Date 1

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Date 2

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October

Date 1

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Date 2

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