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

 
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{{Paris_Bettencourt/banner|page_id=notebook|page_name=Notebook - Vitamin B12}}
 
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<h3>June</h3>
 
−
Brainstorming and reading literature about vitamin B12. What organism should we take the pathway from, and what organism should we put it in?<br><br>
 
  
−
<b>Overexpress the B12 production pathway in <i>Lactobacillus reuteri</i></b><br>
+
<div id="notebookMenu">
−
The idea is to take an organism already present in idli (<i>Lactobacillus reuteri</i>) and to make it produce even more B12. Its pathway has been studied and we planned to metabolic engineer it as it had already been done in <i>Bacillus megaterium</i> (Biedendieck <i>et al.</i>, 2010).<br>
+
  <ul>
−
The problem is that <i>L. reuteri</i> actually produces pseudo-cobalamin, which has not been proven to be active in mammals (<span style="color: red">Santos reference?</span>)
+
    <li class="stretch"><a href="#july">July</a></li>
 +
    <li><a href="#august">August</a></li>
 +
    <li><a href="#september">September</a></li>
 +
  </ul>
 +
</div>
  
 +
<div id="notebookContent">
 +
<a name="july" class="anchor"><h1>July</h1></a>
 +
<h2 class="date one">July 28th</h2>
 +
<ul>
 +
  <li>Received <i>Propionibacterium freudenreichii subsp. shermanii</i> CIRM BIA1 from the INRA Rennes CIRM collection.</li>
 +
  <li>Made the following media:
 +
  <ul>
 +
    <li><b>MEA (waking lyophilized bacteria up), 1 L</b>
 +
      <table>
 +
        <tr><th>Product</th><th>Quantity</th></tr>
 +
        <tr><td>BHI broth</td><td>37 g</td></tr>
 +
        <tr><td>Soja peptone</td><td>5 g</td></tr>
 +
        <tr><td>Yeast extract</td><td>5 g</td></tr>
 +
        <tr><td>Glucose</td><td>3 g</td></tr>
 +
        <tr><td>Water</td><td>Fill to 1 L</td></tr>
 +
      </table> 
 +
    </li>
 +
    <li><b>YEL (propionibacteria growth medium), 1 L</b>
 +
      <table>
 +
        <tr><th>Product</th><th>Quantity</th></tr>
 +
        <tr><td>Sodium lactate (60% syrup)</td><td>21.4 g</td></tr>
 +
        <tr><td>Tryptone</td><td>10 g</td></tr>
 +
        <tr><td>Yeast extract</td><td>10 g</td></tr>
 +
        <tr><td>K<sub>2</sub>HPO<sub>4</sub>, 3 H<sub>2</sub>O</td><td>328 mg</td></tr>
 +
        <tr><td>MnSO<sub>4</sub>, H<sub>2</sub>O</td><td>56 mg</td></tr>
 +
        <tr><td>Water</td><td>Fill to 1 L</td></tr>
 +
      </table>
 +
    </li>
 +
  </ul>
 +
  </li>
 +
  <li>Successfully grew <i>P. freudenreichii</i> in MEA at 30°C, in aerobic conditions.</li>
 +
  <li>Made a glycerol stock of it: g15.53.</li>
 +
</ul>
  
−
<hr>
+
 
−
<h4>July</h4>
+
 
 +
<h2 class="date one">July 30th</h2>
 +
<div style="float: right; width: 400px;"><img style="float:right" src="https://static.igem.org/mediawiki/2015/f/f7/PB_B12_Result_plates.png" width="400px"><br>
 +
<b>Figure 1:</b> Result of the growth of different strains on different media.</div>
 +
<ul>
 +
  <li>Tested the YEL+agar medium with the following strains:
 +
  <ul>
 +
    <li><i>P. freudenreichii</i></li>
 +
    <li><i>Lactobacillus lactis</i></li>
 +
    <li><i>E. coli</i></li>
 +
    <li><i>Lactobacillus plantarum</i></li>
 +
    <li><i>Saccharomyces cerevisiae</i></li>
 +
  </ul></li>
 +
  <li>Result (after 24 hours): figure 1.</li>
 +
</ul>
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
<a name="august" class="anchor"><h1></h1></a>
 +
 
 +
 
 +
<h2 class="date two">August 3rd</h2>
 +
<ul>
 +
  <li>Plated from the original <i>P. freudenreichii</i> glycerol stock on MEA. Did not grow.</li>
 +
  <li>For now, propioni grows only in MEA liquid (does not grow on MEA+agar nor YEL+agar</li>
 +
</ul>
 +
 
 +
 
 +
.
 +
<h2 class="date two">August 15th</h2>
 +
<ul>
 +
  <li>Successfully grew <i>P. freudenreichii</i> in MEA liquid and YEL liquid</li>
 +
  <li>It seemed to grow much faster. Do I have a contanimation? Need to check.</li>
 +
</ul>
 +
 
 +
 
 +
<h2 class="date two">August 16th</h2>
 +
<ul>
 +
  <li>Made a gram coloration to assess whether the strain I have is gram positive, as <i>P. freudenreichii</i>.</li>
 +
  <li>They seemed purple (as Gram positive bacteria).</li>
 +
  <li>Need further checking.</li>
 +
</ul>
 +
 
 +
 
 +
<h2 class="date two">August 17th</h2>
 +
<ul>
 +
  <li>Tried to extract the genomic DNA to sequence the 16s rRNA.</li>
 +
  <li>Problem: followed only the short sheet in the <a href="https://2015.igem.org/Team:Paris_Bettencourt/Protocols#QiagenDNeasy">Qiagen DNeasy Blood & Tissue extraction kit</a>, which was not made for gram-positive bacteria.</li>
 +
  <li>No DNA extracted at all.</li>
 +
</ul>
 +
 
 +
 
 +
<h2 class="date two">August 27th</h2>
 +
<ul>
 +
  <li>Re-used the <a href="https://2015.igem.org/Team:Paris_Bettencourt/Protocols#QiagenDNeasy">kit</a>, this time adding lysozyme (10 mg/ml) to degrade the cell wall, but still no DNA out of the extraction.</li>
 +
</ul>
 +
 
 +
 
 +
<h2 class="date two">August 31st</h2>
 +
<ul>
 +
  <li>Made enzymatic lysis buffer for the <a href="https://2015.igem.org/Team:Paris_Bettencourt/Protocols#QiagenDNeasy">Qiagen kit</a>:
 +
  <ul>
 +
      <li>
 +
      <table>
 +
        <tr><th>Product</th></tr>
 +
        <tr><td>20 mM Tris-Cl, pH 8.0</td></tr>
 +
        <tr><td>2 mM sodium EDTA</td></tr>
 +
        <tr><td>1.2% Triton X-100</td></tr>
 +
        <tr><td>Immediately before use, add lysozyme to 20 mg/ml</td></tr>
 +
      </table></li>
 +
      <li>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.</li>
 +
  </ul>
 +
  </li>
 +
  <li>Followed the rest of the <a href="https://2015.igem.org/Team:Paris_Bettencourt/Protocols#QiagenDNeasy">protocol</a>, starting with 1 ml and 0.5 ml of overnight culture of <i>P. freudenreichii</i>.</li>
 +
  <li>Very low (null) results:
 +
      <table>
 +
        <tr><th>Start volume</th><th>DNA extracted</th></tr>
 +
        <tr><td>1 ml</td><td>4.6 ng/μl</td></tr>
 +
        <tr><td>0.5 ml</td><td>2.2 ng/μl</td></tr>
 +
      </table>
 +
  </li>
 +
</ul>
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
<a name="september" class="anchor"><h1></h1></a>
 +
 
 +
 
 +
<h2 class="date three">September 1st</h2>
 +
  <div style="width: 300px; float: right"><img src="https://static.igem.org/mediawiki/2015/b/b6/PB_Ewen_16PCRGel01.png" width="160px"><br>
 +
  <b>Figure 2: </b> Gel result of the 16S rRNA PCR.<br><b>Lane 10:</b> PCR on the 10 ng/μl of extracted DNA, <b>Lane 5:</b> PCR on the 5 ng/μl of extracted DNA, <b>+:</b> positive control that should have worked.</div>
 +
<ul>
 +
  <li>Made a 1 M Tris solution, adjusted the pH to 8.3</li>
 +
  <li>Remade an enzymatic lysis buffer, with the right reagents this time.</li>
 +
  <li>Tried again the <a href="https://2015.igem.org/Team:Paris_Bettencourt/Protocols#QiagenDNeasy">DNeasy extraction kit</a>. Got the following yield:
 +
      <table>
 +
        <tr><th>Start volume</th><th>DNA extracted</th></tr>
 +
        <tr><td>1 ml</td><td>10.3 ng/μl</td></tr>
 +
        <tr><td>0.5 ml</td><td>5.2 ng/μl</td></tr>
 +
      </table><br>
 +
      The yield is better, will try a PCR on it.
 +
  </li>
 +
  <li>Made a PCR following this <a href="https://2015.igem.org/Team:Paris_Bettencourt/Protocols#FSPCR">protocol</a> and using the following primers:
 +
      <table>
 +
        <tr><th>Name</th><th>Sequence</th></tr>
 +
        <tr><td>16s universal primer <b>27F</b></td><td>AGA GTT TGA TCM TGG CTC AG</td></tr>
 +
        <tr><td>16s universal primer <b>1492R</b></td><td>CGG TTA CCT TGT TAC GAC TT</td></tr>
 +
      </table>
 +
  </li>
 +
  <li>Result on Figure 2.</li>
 +
  <li>The Mastermix was probably not working.</li>
 +
</ul>
 +
<div style="clear:both"></div>
 +
 
 +
 
 +
<h2 class="date three">September 8th</h2>
 +
<ul>
 +
  <div style="float:right; width: 300px;">
 +
    <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.
 +
  </div>
 +
  <li>Tried again a <a href="https://2015.igem.org/Team:Paris_Bettencourt/Protocols#FSPCR">PCR</a> with the same primers, but a new mastermix.</li>
 +
  <li>Result: figure 3. It worked!</li>
 +
</ul>
 +
<div style="clear:both"></div>
  
  
 
<hr>
 
<hr>
 
<h2>References</h2>
 
<h2>References</h2>
−
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.
+
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.<br>
 +
B12 measurement: http://iioab.org/Vol2%282%292011/Karmi%20et%20al-IIOABJ-2%20%282%29-2011-23-32p.pdf<br>
  
 +
</div>
 
</html>
 
</html>
  
 
{{Paris_Bettencourt/footer}}
 
{{Paris_Bettencourt/footer}}

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