Difference between revisions of "Team:Paris Bettencourt/Notebook/VitaminB12"

 
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     <img src="https://static.igem.org/mediawiki/2015/1/1a/PB_Ewen_gelJason.png" width="100px"><br>
 
     <b>Figure 3:</b> Gel result of the 16S PCR. <b>"-" lane:</b>Negative control, <b>"x" lane:</b>PCR on the 10 ng/μl.
 
     <b>Figure 3:</b> Gel result of the 16S PCR. <b>"-" lane:</b>Negative control, <b>"x" lane:</b>PCR on the 10 ng/μl.
 
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   <li>Result: figure 3. It worked!</li>
 
   <li>Result: figure 3. It worked!</li>
 
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Latest revision as of 22:14, 18 September 2015

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
      ProductQuantity
      BHI broth37 g
      Soja peptone5 g
      Yeast extract5 g
      Glucose3 g
      WaterFill to 1 L
    • YEL (propionibacteria growth medium), 1 L
      ProductQuantity
      Sodium lactate (60% syrup)21.4 g
      Tryptone10 g
      Yeast extract10 g
      K2HPO4, 3 H2O328 mg
      MnSO4, H2O56 mg
      WaterFill 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


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:
    • 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 volumeDNA extracted
    1 ml4.6 ng/μl
    0.5 ml2.2 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 volumeDNA extracted
    1 ml10.3 ng/μl
    0.5 ml5.2 ng/μl

    The yield is better, will try a PCR on it.
  • Made a PCR following this protocol and using the following primers:
    NameSequence
    16s universal primer 27FAGA GTT TGA TCM TGG CTC AG
    16s universal primer 1492RCGG TTA CCT TGT TAC GAC TT
  • Result on Figure 2.
  • The Mastermix was probably not working.

September 8th


    Figure 3: Gel result of the 16S PCR. "-" lane:Negative control, "x" lane:PCR on the 10 ng/μl.
  • Tried again a PCR with the same primers, but a new mastermix.
  • Result: figure 3. It worked!

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