Team:Paris Bettencourt/Notebook/VitaminB12
Ferment It Yourself
iGEM Paris-Bettencourt 2O15
- Background
- Design
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Vitamin A Vitamin B2 Vitamin B12 Phytase Riboswitch Differentiation on E. coli Differentiation on S. cerevisiae Manufacturing Idli and Micro-organisms July
July 28th
- Received Propionibacterium freudenreichii subsp. shermanii CIRM BIA1 from the INRA Rennes CIRM collection.
- Made the following media:
- MEA (waking lyophilized bacteria up), 1 L
Product Quantity BHI broth 37 g Soja peptone 5 g Yeast extract 5 g Glucose 3 g Water Fill to 1 L - YEL (propionibacteria growth medium), 1 L
Product Quantity Sodium lactate (60% syrup) 21.4 g Tryptone 10 g Yeast extract 10 g K2HPO4, 3 H2O 328 mg MnSO4, H2O 56 mg Water Fill to 1 L
- MEA (waking lyophilized bacteria up), 1 L
- Successfully grew P. freudenreichii in MEA at 30°C, in aerobic conditions.
- Made a glycerol stock of it: g15.53.
July 30th
- Tested the YEL+agar medium with the following strains:
- P. freudenreichii
- Lactobacillus lactis
- E. coli
- Lactobacillus plantarum
- Saccharomyces cerevisiae
- Result (after 24 hours) are on the right
August 3rd
- Plated from the original P. freudenreichii glycerol stock on MEA. Did not grow.
- For now, propioni grows only in MEA liquid (does not grow on MEA+agar nor YEL+agar
August 15th
- Successfully grew P. freudenreichii in MEA liquid and YEL liquid
- It seemed to grow much faster. Do I have a contanimation? Need to check.
August 16th
- Made a gram coloration to assess whether the strain I have is gram positive, as P. freudenreichii.
- They seemed purple (as Gram positive bacteria).
- Need further checking.
August 17th
- Tried to extract the genomic DNA to sequence the 16s rRNA.
- Problem: followed only the short sheet in the Qiagen DNeasy Blood & Tissue extraction kit, which was not made for gram-positive bacteria.
- No DNA extracted at all.
August 27th
- Re-used the kit, this time adding lysozyme (10 mg/ml) to degrade the cell wall, but still no DNA out of the extraction.
August 31st
- Made enzymatic lysis buffer for the Qiagen kit:
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Product 20 mM Tris-Cl, pH 8.0 2 mM sodium EDTA 1.2% Triton X-100 Immediately before use, add lysozyme to 20 mg/ml - Unfortunately we did not have Tris (> 20 mM) nor EDTA (> 2 mM). Instead we used a solution of Tris 10 mM and EDTA 1 mM.
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- Followed the rest of the protocol, starting with 1 ml and 0.5 ml of overnight culture of P. freudenreichii.
- Very low (null) results:
Start volume DNA extracted 1 ml 4.6 ng/μl 0.5 ml 2.2 ng/μl
September 1st
- Made a 1 M Tris solution, adjusted the pH to 8.3
- Remade an enzymatic lysis buffer, with the right reagents this time.
- Tried again the DNeasy extraction kit. Got the following yield:
Start volume DNA extracted 1 ml 10.3 ng/μl 0.5 ml 5.2 ng/μl
The yield is better, will try a PCR on it. - Made a PCR following this protocol and using the following primers:
Name Sequence 16s universal primer 27F AGA GTT TGA TCM TGG CTC AG 16s universal primer 1492R CGG TTA CCT TGT TAC GAC TT
References
Biedendieck, R., Malten, M., Barg, H., Bunk, B., Martens, J. H., Deery, E., ... & Jahn, D. (2010). Metabolic engineering of cobalamin (vitamin B12) production in Bacillus megaterium. Microbial biotechnology, 3(1), 24-37.
B12 measurement: http://iioab.org/Vol2%282%292011/Karmi%20et%20al-IIOABJ-2%20%282%29-2011-23-32p.pdf
