Team:Paris Bettencourt/Notebook/Foot Odor
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Revision as of 03:12, 18 October 2014
![](https://static.igem.org/mediawiki/2014/3/3a/Goodytwoshoes.png)
Notebook
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=3/4/5/7/1.519-08-14
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.