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Team:Paris Bettencourt/Notebook/Old People Smell
From 2014.igem.org
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 3 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 after 5 days
| 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 - 30min (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 1 | iGEM medium 2 | iGEM medium 3 | iGEM medium 4 | |
| Nose | x | x | x | x |
| Forehead | x | x | x | x |
| NEB (control) | x | x | x | x |
August 13th
Results of M9 plates after 7 days
| M9 100% glucose | M9 50%-50% | M9 100% 2-nonenal | |
| Arm | Many very small colonies | 17 including 6 big ones | 6 very small? |
| Armpit | Many very small colonies | About 20 very small | 1 or 2 really small? |
| Forehead | 32 | 8 | 1 |
| NEB (control) | Many | many | 0 |
I plated on LBA the colony developped on 100% 2-nonenal from the Urszula's forehead to developp it.
August 14th
The colony developped on M9 100% 2-nonenal developped perfectly on LBA plates. It seems to be Staphylococcus aureus. I transfered colonies on 3 M9 100% 2-nonenal plates and I started 2 liquid cultures: one in LB and one in M9 100% 2-nonenal.
August 17th
We started new approach: Directed evolution of Corynebacterium striatum in the liquid medium Corynebacterium striatum from the glycerol stock was incubated in 37°C in medium 95% (4.75mL) LB and 5% M9 Minimal Media 100% 2-nonenal (0.25 mL)
August 18th
1) After four days of culture, I measured the absorbance of the liquid cultures of Staphylococcus aureus in LB and in M9 100% 2-nonenal medium.
| A | |
| LB | 1.647 |
| M9 100% 2-nonenal medium | 0.109 |
S.aureus still develops on 2-nonenal but its fitness is much lower than on LB. I let it grow a few days more.
2) M9 plates from August 6th
It seems like we got small colonies on every 100% 2-nonenal plate (NEB, Forehead, Arm and Armpit). For each one, I prepared 2 liquid cultures: 1 LB and 1 M9 100% 2-nonenal.
3)Corynebacterium striatum directed evolution
Absorbance has been measured for 2 tubes. A = 0 in both tubes. I will give it one more day
4)Directed evolution of the body samples
I proceeded similarly as started yesterday: incubated in 37°C in medium 95% (4.75mL) LB and 5% M9 Minimal Media 100% 2-nonenal (0.25 mL) with a sample from my forehead and another sample from my nose skin.
August 19th
1) Directed evolution - gradual approach
Corynebacterium striatum (Samples 1 and 2) in 95%LB:5% M9 2-nonenal medium grew well and the nose and forehead samples as well
| Absorbance [OD600] | |||||
| date | Sample 1 | Sample 2 | Forehead | Nose | medium |
| 18.08 10h25 | 0 | 0 | x | x | 95% LB:5% M9 2-nonenal |
| 19.08 12h35 | 2.474 | 2.338 | 2.370 | 2.983 | 95% LB:5% M9 2-nonenal |
I centrifuged all 4 samples, discarded supernatant and changed the medium to 90% LB and 10% M9 2-nonenal
2) Staphylococcus aureus
I plated on LBA the forehead colonies from August 15th which had developped on 2-nonenal. Next week we will make a olfactive test to estimate the decrease of 2-nonenal using S.aureus. This strain will be called (N+)-S.aureus.
August 20th
1) Directed evolution - gradual approach
Corynebacterium striatum and nose and forehead samples density in 90%LB:10% M9 2-nonenal medium
| Absorbance [OD600] | |||||
| date | Sample 1 | Sample 2 | Forehead | Nose | medium |
| 18.08 10h25 | 0 | 0 | x | x | 95% LB:5% M9 2-nonenal |
| 19.08 12h35 | 2.474 | 2.338 | 2.370 | 2.983 | 95% LB:5% M9 2-nonenal |
| 20.08 10h35 | 2.31 | 1.54 | 2.806 | 2.76 | 90% LB:10% M9 2-nonenal |
I centrifuged all 4 samples, discharged supernatant and changed the medium to 85% LB and 15% M9 2-nonenal
2) Plates (Samples 1 and 2)
The LB plate of (N+)-S.aureus has many colonies. I made a liquid culture to get a glycerol stock.3) New M9
I prepared 2x50mL of each media:
- M9 - 2-nonenal (120uL mixed for 3uL of Tween80 added after autoclave for 50mL of medium)
- M9 - 2-nonenal (120uL mixed for 3uL of Tween80 added after autoclave for 50mL of medium) + 0.5g of peptone for 50mL of medium
I want to try to make M9 a faster medium. I started a liquid culture of (N+)-S.aureus and measured the initial density of each medium.
| OD600 | |
| M9 - 2-nonenal | 0.885 |
| M9 - 2-nonenal + peptone | 0.994 |
August 21th
1) Glycerol stock
I made a glycerol stock using (N+)-S.aureus liquid culture in LB. The strain is now called sPB.050.
2) M9 overnight liquid cultures
After 24 hours, I measured the OD600 of the M9 liquid cultures with or without 2-nonenal.
| OD600 before culture | OD600 after 24H culture | |
| M9 - 2-nonenal | 0.885 | 0.424 |
| M9 - 2-nonenal + peptone | 0.994 | 0.511 |
The result can be surprising because OD600 has decreased. But we figured out thant the main element which sets OD600 is the concentration of 2-nonenal because our medium is an emulsion. That is why OD600 is really variable according to the mixture. There is here a balance between the bacterian growth which increases OD600, correlated to a decrease of 2-nonenal concentration which lowers OD600 because it is consumed by the cells. It would be interesting to plot OD600 as a function of 2-nonenal concentration and CFU.
To plot OD600 as a function of CFU, I used the (N+)-S.aureus liquid culture in LB, putting different volumes in each tube. Then I centrifuged the tubes (1min-5000tr/min), discarded the supernatant, added 1mL of PBS and resuspended the cell by vortexing. I measured OD600 (with 500uL of solution) and then plated the solutions (100uL in each plate).
| Tube | Volume of LB culture (uL) | OD600 |
| 1 | 1000 | 2.581 |
| 2 | 500 | 1.758 |
| 3 | 250 | 1.104 |
| 4 | 200 | 0.927 |
| 5 | 150 | 0.819 |
| 6 | 100 | 0.555 |
To plot OD600 as a function of 2-nonenal concentration, I mixed 1mL of 2-nonenal with 5% of Tween80 (50uL). Then I prepared these solutions with a total volume of 1mL, and I measured their OD600 after 30min:
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