Team:Paris Bettencourt/Notebook/Old People Smell

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@iGEM_Paris

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).

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

September

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October

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