Difference between revisions of "Team:Paris Bettencourt/Project/VitaminB12"
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− | <td class="noTop">Vitamin B12 is already widely naturally synthesized by some natural producers such as <i> | + | <td class="noTop">Vitamin B12 is already widely naturally synthesized by some natural producers such as <i>Propionibacterium freudenrichii</i> which is GRAS (Generally Recognized As Safe) organism. It is however quite impossible to quickly and cheaply quantify it. |
</td> | </td> | ||
<td class="noTop">Introducing <i>Propionibacterium</i> in our idli batter would allow us to highly increase its quantity of vitamin B12. Quantifying vitamin B12 in fermented food in a cheap way was aimed by designing a biosensor based on a vitamin B12 riboswitch associated with a fluorescent protein. | <td class="noTop">Introducing <i>Propionibacterium</i> in our idli batter would allow us to highly increase its quantity of vitamin B12. Quantifying vitamin B12 in fermented food in a cheap way was aimed by designing a biosensor based on a vitamin B12 riboswitch associated with a fluorescent protein. | ||
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<li><b>However</b> this comes with a cost as water soluble vitamins are not stored in the body but <b>excreted</b> through urine.</li> | <li><b>However</b> this comes with a cost as water soluble vitamins are not stored in the body but <b>excreted</b> through urine.</li> | ||
</ul> | </ul> | ||
+ | |||
+ | <h2><i>Propionibacterium freudenreichii</i> grows in idli</h2> | ||
+ | <div class="column-left"> | ||
+ | <p> | ||
+ | <i>Propionibacterium freudenreichii</i> is a bacterium that has the Generally Recognized As Safe (GRAS) status, and is commonly found is cheese such as Emmental. It is a natural big producer of vitamin B12, conferring high amounts of this vitamin on cheese. | ||
+ | Since the biosynthesis of vitamin B12 is a complex process, we decided to engineer the microbiome of idli by directly adding this bacterium to the batter, in the hope of increasing its vitamin B12 content.</p> | ||
+ | <p>As a matter of fact, <i>P. freudenreichii</i> was found to grow well in idli (see figure on the right).</p> | ||
+ | </div> | ||
+ | |||
+ | <div class="column-right"> | ||
+ | <img src="https://static.igem.org/mediawiki/2015/e/e6/ParisBettencourt_propioniGrowthIdli.png" style="width: 100%"><br> | ||
+ | <span class="caption">Comparison of growth of <i>P. freudenreichii</i> in YEL (regular growth medium) and 100 times diluted idli batter.</span> | ||
+ | </div> | ||
+ | <div style="clear: both"></div> | ||
− | <h1 class="date two">Cobalamin Biosensor</h1> | + | <h1 class="date two" id="biosensor">Cobalamin Biosensor</h1> |
<div class="column-left"> | <div class="column-left"> | ||
<h2>Background</h2> | <h2>Background</h2> | ||
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<div style="align: center"> | <div style="align: center"> | ||
<img src="https://static.igem.org/mediawiki/2015/e/ef/Ribomescouilles.png" style="width: 100%"> | <img src="https://static.igem.org/mediawiki/2015/e/ef/Ribomescouilles.png" style="width: 100%"> | ||
− | + | Map of the riboswitch/RFP cassette. | |
</div> | </div> | ||
<br> | <br> | ||
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<div style="clear:both"></div> | <div style="clear:both"></div> | ||
− | <div style="align: center"> | + | <div style="text-align: center"> |
− | <img src="https://static.igem.org/mediawiki/2015/6/6d/Graphs.png" | + | <img src="https://static.igem.org/mediawiki/2015/6/6d/Graphs.png" style="text-align: center"><br> |
− | + | Cobalamin detection with our biosensor. <b>Left:</b> mRFP1/OD = f([AdoCbl]); Exc=585nm and Em=635nm. <b>Right:</b> Venus/OD = f([AdoCbl]) = Positive control; Exc=480nm and Em=520nm. OD was mesured at 600nm. | |
</div> | </div> | ||
+ | <br><br> | ||
− | + | <div class="column-left"> | |
+ | <p> | ||
+ | Normalizing the mRFP1 expression to the Venus expression gives you the power to get rid of any noise coming from the bacterial metabolism, or it’s environment. | ||
+ | </p> | ||
− | + | <p> | |
+ | We were waiting our solution containing the E. coli sensor to reach an OD of 0.1 and dilute it 1:20 with M9-Glucose. Then this diluted solution containing our bacterial sensor was inoculated (75uL) with increasing concentrations of AdoCbl diluted in M9-Glucose (75uL) in plate reader wells covered by 50uL mineral oil. M9 was used because it is free of B12 and thus their is no interference in our measure.</p> | ||
+ | </div> | ||
− | + | <div class="column-right"> | |
+ | <p> | ||
+ | Our riboswitch has been demonstrated to be sensitive to different forms of cobalamin: | ||
+ | <ul> | ||
+ | <li>Natural forms: AdenosylCbl (AKA AdoCbl or Coenzyme B12), MethylCbl, and HydroxyCbl</li> | ||
+ | <li>Hemisynthetic form: CyanoCbl</li> | ||
+ | </ul> | ||
+ | </p> | ||
− | + | <p> | |
− | + | Some other experience would have to be done to determine if our riboswitch construction is sensitive to those Cobalamin forms. | |
− | + | </p> | |
− | + | <p> | |
− | + | All the new results experiences will be updated in the biobrick page. (<a href="http://parts.igem.org/Part:BBa_K1678007">BBa_K1678007</a>). Check for updates if you’re interested in. | |
− | All the new results experiences will be updated in the biobrick page. ( | + | </p> |
− | http://parts.igem.org/Part:BBa_K1678007. Check for updates if you’re interested in. < | + | </div> |
+ | <div style="clear: both"></div> | ||
<h2> Use the construction with a food sample </h2><br> | <h2> Use the construction with a food sample </h2><br> | ||
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+ | <div style="text-align: center"> | ||
+ | <img src="https://static.igem.org/mediawiki/2015/5/5a/Ribosensor.png"><br> | ||
+ | General scheme for Riboswitch/Fluorescent protein based biosensor.<br> | ||
+ | </div> | ||
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Riboswitch is a complex mRNA system where all the element listed above are intricate in a fine manner, disturbing this trying to shorter the sequence could be fatal in the operation of the biosensing construct.<br><br> | Riboswitch is a complex mRNA system where all the element listed above are intricate in a fine manner, disturbing this trying to shorter the sequence could be fatal in the operation of the biosensing construct.<br><br> |
Latest revision as of 15:36, 12 December 2015