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. 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 Date 1 Goal Text Procedure Text Results Date 1 Goal Text Procedure Text Results October Date 1 Goal Text Procedure Text Results Date 1 Goal Text Procedure Text Results Retrieved from "http://2014.igem.org/Team:Paris_Bettencourt/Notebook/Foot_Odor" Recent changes What links here Related changes Special pages My preferences Printable version Permanent link Privacy policy Disclaimers