Team:Paris Bettencourt/Notebook/Old People Smell
From 2014.igem.org
Home | Team | Official Team Profile | Project | Parts | Modeling | Notebook | Safety | Attributions |
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% glucose | M9 50%-50% | M9 100% 2-nonenal | |
Arm | x | x | x |
Armpit | x | x | x |
Forehead | x | x | x |
NEB (control) | x | x | x |
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% glucose | M9 50%-50% | M9 100% 2-nonenal | |
Arm | Many very small colonies | 6 | 0 |
Armpit | Many very small colonies | 0 | 0 |
Forehead | 27 | 5 | 0 |
NEB (control) | Many | many | 0 |
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:
NEB | Forehead | Nose | |
1) Without fatty acids | x | x | x |
2) Without fatty acids and with 2-nonenal | x | x | x |
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:
September
Date 1
Text
Text
Date 2
Text
Text
October
Date 1
Text
Text
Date 2
Text
Text