Ferment It Yourself

iGEM Paris-Bettencourt 2O15

Vitamin A	Vitamin B2	Vitamin B12
Phytase	Riboswitch	Differentiation on E. coli
Differentiation on S. cerevisiae	Manufacturing	Idli and Micro-organisms

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.

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

Team:Paris Bettencourt/Notebook/VitaminB12