Difference between revisions of "Team:Paris Bettencourt/Notebook/VitaminB12"
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− | < | + | <div id="notebookMenu"> |
− | < | + | <ul> |
− | + | <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> | |
− | <li> | + | <tr><td>Soja peptone</td><td>5 g</td></tr> |
− | + | <tr><td>Yeast extract</td><td>5 g</td></tr> | |
− | <li> | + | <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> | ||
+ | |||
+ | |||
+ | |||
+ | <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> | ||
+ | <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.<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}} |
Latest revision as of 22:14, 18 September 2015