Team:Paris Bettencourt/Notebook/VitaminB12
Ferment It Yourself
iGEM Paris-Bettencourt 2O15
- Background
- Design
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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
Figure 1: Result of the growth of different strains on different media.- Tested the YEL+agar medium with the following strains:
- P. freudenreichii
- Lactobacillus lactis
- E. coli
- Lactobacillus plantarum
- Saccharomyces cerevisiae
- Result (after 24 hours): figure 1.
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
Figure 2: Gel result of the 16S rRNA PCR.
Lane 10: PCR on the 10 ng/μl of extracted DNA, Lane 5: PCR on the 5 ng/μl of extracted DNA, +: positive control that should have worked.- 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 - Result on Figure 2.
- The Mastermix was probably not working.
September 8th
- Tried again a PCR with the same primers, but a new mastermix.
- Result: figure 3. It worked!
Figure 3: Gel result of the 16S PCR. "-" lane:Negative control, "x" lane:PCR on the 10 ng/μl.
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
