Team:Paris Bettencourt/Notebook/Foot Odor

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June

23-06-2014

Goal: To obtain single colonies of both Wt and mutant B.Subtilis

We received the wt (1A1) and mutant BKE24040 (isovalerate dehydrogenase beta KO), BKE24050 (isovalerate dehydrogenase alpha KO), BKE24080 (Leucine dehydrogenase KO) from BGSC on 23-06-2014. The wt strain (1A1) was on a paper strip. We received 3 strips from BGSC (Bacillus genetic stock centre). Where as the mutant strains arrived on perti-plates.

Procedure

We plated two strips in two different LBA plates. The plates were free from antibiotics. We placed the strips gently on the LBA and added 1 drop of sterile H2O. After few seconds we streaked the paper strip on the LBA and discarded the strip. The LBA plates were incubated at 37° C. We took single colony from the mutant B.subtilis petri-plates and streaked them on LBA+EM petri plates.

Results

Good growth of both the wt and mutant B.subtilis were observed followed by the overnight incubation.

24-06-2014

To obtain single colonies of Wt b.Subtilis from 23-06-14 plate

Procedure

we took a single colony and streaked it on a new LBA with and w/o Erythromycin.

Results

No colonies in LBA + EM. Colonies observed in LBA w/o EM

27-06-2014

To prepare LBA and LB broth for B.Subtilis mutants

Procedure

We added 2.5 ul of the EM (20mg/ml) to 50ml of LB broth. We added 25 ul of EM to 250 ml of LBA and prepared the plates & stored them at 4° C.

30-06-2014

Goal: To prepare 10% glycerol stock of wt and mutant (BKE24040, BKE24050, BKE24080) B.Subtilis

Procedure

We inoculated Single colony of both wt (1A1) and mutant (BKE24040, BKE24050, BKE24080) in 5ml of LB broth supplemented w/o EM for wt and with EM for mutants. Incubated the colonies @ 37°C overnight. After the overnight incubation the tubes were stirred well. 750 ul of the culture is transferred to cryogenic tube and 750 ul of 60% glycerol is added to the culture to obtain 10% glycerol stock.

Entry	Strain	Resistance	Source	Strain	Glycerol Stock Label
sPB.011	BKE24040	EM	BGSC	B.subtilis with 2-oxoisovalerate dehydrogenase beta subunit knockout	G.11
sPB.012	BKE24050	EM	BGSC	B.subtilis with 2-oxoisovalerate dehydrogenase alpha subunit knockout	G.12
sPB.013	BKE24080	EM	BGSC	B.subtilis with leucine dehydrogenase knockout	G.13
sPB.014	1A1	-	BGSC	B.subtilis wt strain	G.14

The cryo tubes were stored at -20°C.

July

15-07-14

To perform colony PCR on mutant & wt B.subtilis to confirm the knock out of gene of our interest and also to confirm the presence of ERM cassette in the loci of the knocked out genes

Work plan:

BKDA alpha
Primer category	F	R	Deletion Strain	WT
Control primer	oPB.023	oPB.025	2944 bp (A)	2920 bp (B)
Deletion primer	oPB.023	oPB.029	2230 bp (1)	No Band (2)
Deletion primer (cassette reversed)	oPB.029	oPB.025	1653 bp (DP1)	No Band (DP2)
BKDA beta
Primer category	F	R	Deletion Strain	WT
Control primer	oPB.026	oPB.028	2869 bp (C)	2836 bp (D)
Deletion primer	oPB.026	oPB.029	1629 bp (3)	No Band (4)
Deletion primer (cassette reversed)	oPB.029	oPB.028	2179 bp (DP3)	No Band (DP4)
BCD
Primer category	F	R	Deletion Strain	WT
Control primer	oPB.020	oPB.022	2636 bp (E)	2714 bp (F)
Deletion primer	oPB.020	oPB.029	1529 bp (5)	No Band (6)
Deletion primer (cassette reversed)	oPB.029	oPB.022	2046 bp (DP5)	No Band (DP6)

Reaction mix:

Reagent	Volume (uL)
	20X
NF         ddH2O	236
5x Phusion HF Buffer	80
10 mM dNTPs	8
DMSO	12
DNA Template	1
Phusion Polymerase	4

PCR NEB Phusion DNA polymerase (thermocycler parameters)

Status	Temperature (°C)	Time (sec)	Process
Start	98	30	Melt
Melt	98	10	x35 Cycles
Anneal	52	30	x35 Cycles
Extend	72	90	x35 Cycles
Finish	72	300	Extend
Store	10	∞	Store

Procedure

After the PCR the reaction mix in the tubes were loaded on individual well. The 1X agarose gel was used for the electrophoresis. The 1kb plus ladder (thermo fisher inc.) was used for comparing the bands.

Results

30-07-14

To prepare a 100X synthetic sweat master mix

Procedure

Mix the chemicals mentioned in the table below and mix them properly to have a homogeneous mixture

Quantity	unit	Chemical
2.6	g/L	L-Leucine
0.3	g/L	L-Cysteine
30.3	g/L	L-Serine
8.1	g/L	L-Alanine
0.1	g/L	L-Arginine
4.3	g/L	L-Histidine
6.6	g/L	L-Threonine
3.2	g/L	L-Valine
2	g/L	L-Isoleucine
1.19	g/L	L-Lysine
2.1	g/L	L-Phenylalanine
3.4	g/L	L-Tyrosine
2.2	g/L	L-Asparagine
2.3	g/L	L-Glutamic acid
0.7	g/L	L-Methionine
0.3	g/L	L-Glutamine
5.5	g/L	L-Aspartic acid
8.6	g/L	L-Ornithine
7.3	g/L	L-Citrulline
0.1	g/L	L-Ascorbic acid
18	g/L	D-Glucose
0.4	g/L	Creatinine
5.5	g/L	Sodium pyruvate
12	g/L	Potassium hydrogen carbonate
0.2	g/L	NaH2PO4
5.7	g/L	Calcium sulfate

SHAKE THE VESSEL WELL BEFORE USE

Take 1.39 g/l of the above master mix and dissolve it in double distilled water. Add the fatty acids as per the requirement of your experiment. The pH of the synthetic sweat could be adjusted with the aid of NaoH (use the pH meter).

Note: Autoclave the glass vessel with 15g/L of agar to prepare the LBA+ synthetic sweat plates. If not autoclave the vessel without the agar for preparing the broth.

August

11-08-2014

To produce synthetic sweat without Fatty acids

Procedure

we are going to take 0.051 g (since the master mix was 100X) of the synthetic sweat master mix and dissolve it in one liter of distilled water. The PH of the synthetic sweat was adjusted to 6.5 with aid of Hcl and NaoH. The vessel was autoclaved and stored at 4° C for future use.

25-08-14

Smell test of WT and Mutant

Goal: Measure the smell intensity of WT versus mutant
Protocole: from LB Plate of 1A1, BCD, BKDAa, BKDAb: start 5ml liquid culture (times two).
(Media preparation: M9 minimal media, with glucose as carbon source. Add tryptophan to each of the culture.)
Put one colony with a loop in each tube, culture the tubes overnight at 37°c (150 rpm).
Then we do the same, expect that we add leucine to the mdeia (500µl of 1% Leucine into 4500µl of M9) We repeat the experience twice.


Next day, after 20 hours of culture, we take the OD: Exp01
1A1= 0.180
1A1+Leu= 0.264
BCD= 0.047
BCD+Leu = 0.472
BKDAa= 0.084
BKDAa+Leu= 0.128
BKDAb= 0.088
BKDAb+Leu= 0.114

Exp02
1A1= 0.422
1A1+Leu= 0.505
BCD=0.305
BCD+Leu =0.304
BKDAa=0.322
BKDAa+Leu=0.707
BKDAb=0.179
BKDAb+Leu=0.160

We use the tubes from the exp02 and start with three members of the team. We ask the volunteer to smell the samples (choosen randomly) and to rate the intensity between 0 and 5.

Results:
1A1= 4/3/2
1A1+Leu= 1/3/4
BCD=1/3/1
BCD+Leu =2/2/0.5
BKDAa=1/3/1
BKDAa+Leu=1/2/3
BKDAb=Not grown enough
BKDAb+Leu=Not grown enough
At 22 hours of growth, we take again the OD Exp01
1A1= 0.266
1A1+Leu= 0.414
BCD= 0.195
BCD+Leu = 0.129
BKDAa= 0.059
BKDAa+Leu= 0.109
BKDAb= 0.122
BKDAb+Leu= 0.124

Exp02
1A1= 0.464
1A1+Leu= 0.508
BCD=0.388
BCD+Leu =0.332
BKDAa=0.328
BKDAa+Leu=0.711
We do then the smell test again (on exp02 tubes expect BKDAB from exp01 on 5 volunteers. We ask the participants to grade the intensity from 0 to 10.) 1A1= 5/7/2/4/2
1A1+Leu= 5/4/2/7/4
BCD=2/3/4/2/3.5
BCD+Leu =2/3/4/1/6
BKDAa=3/6/2/2/3
BKDAa+Leu=2/4/3/1/0
BKDAb=2/5/2/5/3
BKDAb+Leu=3/4/5/7/1.5

19-08-14

To access the capability of the wt and mutant (BKE24040, BKE24050, BKE24080) B.Subtilis to grow in synthetic sweat

Procedure

We inoculated Single colony of both wt (1A1) and mutant (BKE24040, BKE24050, BKE24080) in 5ml of synthetic sweat supplemented w/o EM for wt and with EM for mutants. Incubated the colonies @ 37°C overnight.

Results

No growth observed for both wt and mutant B.subtilis

Date 1

Goal

Text

Procedure

Text

Results

September

09/10/2014

Odor intensity of wt and mutant

We use 4 strains 1A1, ΔBCD, ΔBKDAa and ΔBKDAb.

We culture the cells in M9 minimal media (Final volume= 5ml).
The minimal media was prepared by diluting 1ml of 50% glucose 1.1 ml of mgso4.3H20 2ml of 1% w/v casmino acids 2ml of 10mg/ml of tryptophan in 100 ml of distilled water The media is then filter sterilized under a laminar air flow cabinet.
The cells were grown overnight at 37°c.


The presence of isovaleric acid in the bacterial culture was sensed with double blind smell test, where neither the subject nor the experimenter is aware of the tube labels.
Results:
0% Leucine
1A1=1,5/2,3/2,0/1,5/2,0/2,0
BCD=1,5/2,7/2,/3,3/2,0/2,4
BKDAa=1,0/2,0/0,4/1,2/1,0/1,2
BKDAb=2,0/2,8/3,1/2,4/2,6
0.1% Leucine
1A1=3,5/3,5/5/4/4/3,5
BCD=2/2,5/4/5/3,5/3,2
BKDAa=1/1/2/3/2/1,7
BKDAb=1,5/2/2/3/3/2
0.2% Leucine
1A1=4/4/5/5/4/4
BCD=3/3/4/5/3/4
BKDAa=2/2,5/2,5/3/3/2,4
BKDAb=3/2,5/3,5/2,9/2

Procedure

Text

Results

Goal

Text

Procedure

Text

Results

October

12/10/14

Growth curve of B. subtilis 1A1, ΔBCD, ΔBKDAa and ΔBKDAb. We put one colony of b. subtilis (from a LB plate) in an eppendorf containing 500µl of media (either synthetic sweat, LB or M9 complemented with tryptophane). We then put 100µl in a 96 plates for automatic plate reader. We had 30*l of oil on each holes. The experiment have been done in triplicates.

We used

Procedure

Text

Results

11-17/10/14

Socks experiment: survival of b. subtilis on socks

The bacterial cell culture is diluted in synthetic sweat untill it reaches 0.1 OD. The sock was soaked in the synthetic sweat and hanged till it dry. After this step 2 cm2 of sock was cut and soaked in 5 ml of 1% PBS EACH DAY. The tubes were stirred gently and supernetant is diluted in PBS to prepare 1X and 10X concentration of B. subtilis. The bacteria were then plated on LB agar. The day after we measure the number of colony

RESULTS: (in bacteria per cm²) 1A1:
Day1: 8,125000e+003 1,062500e+004 8,750000e+003
Day2: 4,750000e+003 8,125000e+003 6,250000e+003
Day3: 5,250000e+003 6,062500e+003 6,187500e+003
Day4:3,500000e+003 5,625000e+003 4,187500e+003
Day 5:2,937500e+003 4,062500e+003 3,250000e+003


BCD:
Day1: 2,312500e+003 2,750000e+003 2,875000e+003
Day 2: 2,750000e+003 2,750000e+003 3,125000e+003
Day 3: 1,687500e+003 1,687500e+003 2,375000e+003
Day 4: 2,937500e+003 1,437500e+003
Day 5: 3,312500e+003 1,812500e+003


BKDAa
Day1: 5,125000e+003 3,750000e+003
Day 2:6,250000e+003 3,062500e+003
Day 3:2,750000e+003 2,187500e+003
Day 4: 4,875000e+003 2,875000e+003
Day 5: 3,062500e+003 2,625000e+003


BKDAb Day1: 3,812500e+003 4,812500e+003
Day2: 3,750000e+003 3,750000e+003
Day3: 2,437500e+003 2,500000e+003
Day4: 2,750000e+003 3,812500e+003
Day 5: 2,250000e+003 3,625000e+003

Procedure

Text

Results

13-10-2014

To To perform GC-MS analysis of the wild type and mutant B. subtilis

Procedure

The overnight culture of the strains grown on M9 minimal media was filter sterilized and transferred to sterile 1 ml amber glass tube and sent for analysis.
For the GC-MS analysis, 1µl of the sample was injected into the instrument in split mode. A Rtx5MS- 30m column with 0.25-mm ID and 0.25µm df were used. The GC-MS run uses the following standardized parameters:
- Injection temperature: 300°C
- Interface temperature: 300°C
- Ion Source: 250°C
- Carrier Gas: Helium (flow rate of 1 ml min-1)
The temperature program used was 3 minutes of isothermal heating at 50⁰C followed by heating at 350⁰C for 10 minutes. Mass spectra were recorded at 2 scan sec-1 with a scanning range of 40 to 850 m/z.

Results

X Caliber software is used to process and analyze the data from a GC-MS run. Quantify each component based on peak areas and normalization based on the internal standard.
Mass spectroscopy analysis of the mutant and the wild type strains. url(”https://static.igem.org/mediawiki/2014/2/22/MS-FOOT.pdf”);
Gas chromatography analysis of the mutant and the wild type strains. url(”https://static.igem.org/mediawiki/2014/4/49/GC-FOOT.pdf”)