Team:Paris Bettencourt/Notebook/Old People Smell

From 2014.igem.org

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<TR><TD>19.08 12h35</TD><TD>2.474</TD><TD>2.338</TD><TD>2.370</TD><TD>2.983</TD><TD>95% LB:5% M9 2-nonenal</TD></TR>
<TR><TD>19.08 12h35</TD><TD>2.474</TD><TD>2.338</TD><TD>2.370</TD><TD>2.983</TD><TD>95% LB:5% M9 2-nonenal</TD></TR>
<TR><TD>20.08 10h35</TD><TD>2.31</TD><TD>1.54</TD><TD>2.806</TD><TD>2.76</TD><TD>90% LB:10% M9 2-nonenal</TD></TR>
<TR><TD>20.08 10h35</TD><TD>2.31</TD><TD>1.54</TD><TD>2.806</TD><TD>2.76</TD><TD>90% LB:10% M9 2-nonenal</TD></TR>
 +
<TR><TD>21.08 9h35</TD><TD>2.857</TD><TD>1.358</TD><TD>2.858</TD><TD>++++</TD><TD>85% LB:15% M9 2-nonenal</TD></TR>
</TABLE>
</TABLE>

Revision as of 15:20, 21 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 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% 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 after 5 days

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


August 13th

Results of M9 plates after 7 days

M9 100% glucoseM9 50%-50%M9 100% 2-nonenal
ArmMany very small colonies17 including 6 big ones6 very small?
ArmpitMany very small coloniesAbout 20 very small1 or 2 really small?
Forehead3281
NEB (control)Manymany0

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
LB1.647
M9 100% 2-nonenal medium0.109

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]
dateSample 1Sample 2ForeheadNosemedium
18.08 10h2500xx95% LB:5% M9 2-nonenal
19.08 12h352.4742.3382.3702.98395% 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]
dateSample 1Sample 2ForeheadNosemedium
18.08 10h2500xx95% LB:5% M9 2-nonenal
19.08 12h352.4742.3382.3702.98395% LB:5% M9 2-nonenal
20.08 10h352.311.542.8062.7690% 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-nonenal0.885
M9 - 2-nonenal + peptone0.994


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 cultureOD600 after 24H culture
M9 - 2-nonenal0.8850.424
M9 - 2-nonenal + peptone0.9940.511

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).

TubeVolume of LB culture (uL)OD600
110002.581
25001.758
32501.104
42000.927
51500.819
61000.555

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: Tube2-nonenal + Tween80 (uL)Water2-nonenal concentration (%tot. volume)OD600(blank with Nuclease Free water) 1409604%1.957 2309703%1.318 3209802%1.265 4159851.5%0.667 5109901%0.504 659950.5%0.344

3)Directed evolution

Absorbance [OD600]
dateSample 1Sample 2ForeheadNosemedium
18.08 10h2500xx95% LB:5% M9 2-nonenal
19.08 12h352.4742.3382.3702.98395% LB:5% M9 2-nonenal
20.08 10h352.311.542.8062.7690% LB:10% M9 2-nonenal
21.08 9h352.8571.3582.858++++85% LB:15% M9 2-nonenal

September

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October

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