• July
• August
• September

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

• 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:

  • 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.
• 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

• Result:

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.
• The Mastermix was probably not working.

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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