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 according to this protocol. 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:

Tube	2-nonenal + Tween80 (uL)	Water (uL)	2-nonenal concentration (%tot. volume)	OD600(blank with Nuclease Free water)
1	40	960	4%	1.957
2	30	970	3%	1.318
3	20	980	2%	1.265
4	15	985	1.5%	0.667
5	10	990	1%	0.504
6	5	995	0.5%	0.344

3)Directed evolution

	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
21.08 9h35	2.857	1.358	2.858	++++	85% LB:15% M9 2-nonenal

We started to have doubts about accuracy of the OD600 measurments because just by adding moe 2-nonenal to the media, we increase the density. However, with the measurments we did (2-nonenal + TWIN concentration vs. OD) we hope to estimate the cell content correctly.

We modified the protocol. We measure OD600. We take 10um from of the overnight colony and we put it into the new medium with 5% more 2-nonenal and 5% less LB. This will allow us to see better growth of bacteria.

August 22th

Directed evolution

I measured OD600

	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
21.08 9h35	2.857	1.358	2.858	++++	85% LB:15% M9 2-nonenal
22.08 11h30	2.23	2.43	2.099	1.907	80% LB:20% M9 2-nonenal

Then I took 10um from each sample and I put it into the medium 75%LB : 25% M9nonenal and put it into incubator 37C for overnight.

August 23th

Directed evolution

I measured OD600

	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
21.08 9h35	2.857	1.358	2.858	++++	85% LB:15% M9 2-nonenal
22.08 11h30	2.23	2.43	2.099	1.907	80% LB:20% M9 2-nonenal
23.08 14h10	1.492	1.035	1.107	1.735	75% LB:25% M9 2-nonenal

Then I took 10um from each sample and I put it into the medium 70%LB : 30% M9nonenal and put it into incubator 37C for overnight.

August 24th

Directed evolution

I measured OD600

	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
21.08 9h35	2.857	1.358	2.858	++++	85% LB:15% M9 2-nonenal
22.08 11h30	2.23	2.43	2.099	1.907	80% LB:20% M9 2-nonenal
23.08 14h10	1.492	1.035	1.107	1.735	75% LB:25% M9 2-nonenal
24.08 16h10	1.53	1.876	1.437	2.385	70% LB:30% M9 2-nonenal

Then I took 10um from each sample and I put it into the medium 65%LB : 35% M9nonenal and put it into incubator 37C for overnight.

August 25th

1) Olfactive test experiment

This week we are leading our olfactive test experiment using sPB.050 and M9-2-nonenal medium. Each day of the week we start a liquid culture of sPB.050 in M9-2-nonenal: on Friday we will have 5 cultures, each one 0, 1, 2, 3 or 4 days old. We will plate each of this culture on LB to determine the CFU and have all the tubes smelled and rated by the team according to the intensity with a blind test.

2)Plating the team's forehead samples

I plated on LB and on M9-2-nonenal Alexandre's, Sylvia's, Cristina's, Naresh's, Henry's and my forehead samples in order to find other bacteria developping on 2-nonenal.

3) Directed evolution

I measured OD600

	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
21.08 9h35	2.857	1.358	2.858	++++	85% LB:15% M9 2-nonenal
22.08 11h30	2.23	2.43	2.099	1.907	80% LB:20% M9 2-nonenal
23.08 14h10	1.492	1.035	1.107	1.735	75% LB:25% M9 2-nonenal
24.08 16h10	1.53	1.867	1.437	2.385	70% LB:30% M9 2-nonenal
25.08 11h30	1.604	1.873	1.329	2.143	65% LB:35% M9 2-nonenal

Then I took 10um from each sample and I put it into the medium 60%LB : 40% M9nonenal and put it into incubator 37C for overnight.

August 26th

Directed evolution

I measured OD600

	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
21.08 9h35	2.857	1.358	2.858	++++	85% LB:15% M9 2-nonenal
22.08 11h30	2.23	2.43	2.099	1.907	80% LB:20% M9 2-nonenal
23.08 14h10	1.492	1.035	1.107	1.735	75% LB:25% M9 2-nonenal
24.08 16h10	1.53	1.867	1.437	2.385	70% LB:30% M9 2-nonenal
25.08 11h30	1.604	1.873	1.329	2.143	65% LB:35% M9 2-nonenal
26.08 14h00	0.609	1.283	0.501	0.487	60% LB:40% M9 2-nonenal

Then I took 10um from each sample and I put it into the medium 55%LB : 45% M9nonenal and put it into incubator 37C for overnight.

August 27th

1) Directed evolution

I measured OD600

	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
21.08 9h35	2.857	1.358	2.858	++++	85% LB:15% M9 2-nonenal
22.08 11h30	2.23	2.43	2.099	1.907	80% LB:20% M9 2-nonenal
23.08 14h10	1.492	1.035	1.107	1.735	75% LB:25% M9 2-nonenal
24.08 16h10	1.53	1.867	1.437	2.385	70% LB:30% M9 2-nonenal
25.08 11h30	1.604	1.873	1.329	2.143	65% LB:35% M9 2-nonenal
26.08 14h00	0.609	1.283	0.501	0.487	60% LB:40% M9 2-nonenal
27.08 15h50	0.898	0.847	0.942	0.874	55% LB:35% M9 2-nonenal

Then I took 10um from each sample and I put it into the medium 50%LB : 50% M9nonenal and put it into incubator 37C for overnight.

2) I got a suggestion from other member of the team that bacteria from the body samples growing on the LB plates in the experiment they may differ depending on the way I put them on the plate. So far, we have been taking samples by scratching body surface with a loop. This time we took a cotton pad and inoculated it in LB in 37C overnight in order to plate them on 2-nonenal plates next day

3) Gram colouration. I performed the gram colouration in order identify the pSB.050. According to the observation the bacteria are gram negative (fuchsia colour).

August 28th

Directed evolution

I measured OD600

	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
21.08 9h35	2.857	1.358	2.858	++++	85% LB:15% M9 2-nonenal
22.08 11h30	2.23	2.43	2.099	1.907	80% LB:20% M9 2-nonenal
23.08 14h10	1.492	1.035	1.107	1.735	75% LB:25% M9 2-nonenal
24.08 16h10	1.53	1.867	1.437	2.385	70% LB:30% M9 2-nonenal
25.08 11h30	1.604	1.873	1.329	2.143	65% LB:35% M9 2-nonenal
26.08 14h00	0.609	1.283	0.501	0.487	60% LB:40% M9 2-nonenal
27.08 15h50	0.898	0.847	0.942	0.874	55% LB:45% M9 2-nonenal
28.08 11h40	-0.316	-0.038	-0.651	-0.011	50% LB:50% M9 2-nonenal

Then I took 10um from each sample and I put it into the medium 45%LB : 55% M9nonenal and put it into incubator 37C for overnight.

The negative numbers may be surprising but taking into account that 2-nonenal with time get degraded (hopefully but bacteria in culture or by oxidation).

I have also plated all 4 samples on LB, 50% M9 2-nonenal:50% LB, 100% M9 2-nonenal plates.

September

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October

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