Difference between revisions of "Team:Aix-Marseille/Design"

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    <h2 class="title wow bounce in up"><span style="color:#8E3B8C"><span style="font-family:Armalite Rifle">Our constructions</h2></span></span>
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    <div class="space30"></div>
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    <p class="space20"><div align="justify"><span style="font-family: Courier New">First, we will produce our enzymes independantly (lippoxygenase, laccase and cytochrome C). Usually, cytochrome C uses the Sec-pathway and folds into the periplasm. Howerver, we could not produce proteins that help the periplasmic folding. So we added CXXCH and CCHL sequences that will help the cytochrome C to use the Tat-pathway and allowed its cytoplasmic folding. Then we will test all the enzymes and different combinations on polymers.
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<p class="space10"><div align="justify"><span style="font-family: Courier New">To test our enzymes, we will mesure the oxygen level with an oxymeter and by spectrophotometry.</div></p></span>
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<p class="space50"><div align="justify"><span style="font-family: Courier New">We will select enzymes with the best results: one lipoxygenase, one laccase and one cytochrome C.
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Using a linker, we will fuse the selected laccase with the selected cytochrome C and test again the activity. We will compared our results. </div></p></span>
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    <h2 class="title wow bounce in up"><span style ="color:#000000"><span style="font-family:Armalite Rifle">Production</span></span></h2>
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    <p class="space20"><div align="justify"><span style ="color:#000000"><span style="font-family:Courier New">Once our constructions done, we transform its into BL21 cells.
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These E.coli K-12 allow the expression of proteins controlled by the T7 promotor and induced by IPTG.
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Bacteria cells were resuspended into a lysis buffer and were sonicated. Sonication of bacteria is used to break cells that contain our proteins to be purified. After, a centrifugation separates broken/unbroken cells and allows us to collect soluble proteins into the supernatant.
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Revision as of 10:47, 12 September 2015

Chew fight

Project design

Our project this year is to work on the chewing gum degradation. Our research on this topic reveals that chewing gum is composed of different compounds. They are hydrophilic or hydrophobic. When you chew a chewing gum, the hydrophilic part is solubilized by your saliva. Once done, you throw away the hydrophobic part called the gum base. The gum base is composed of polymers : polyisoprene and butadiene.

So we searched for enzymes which are able to degrade polymers. We found two enzymes: lipoxygenases and laccases.

At the beginning, our main problem was that laccase is able to oxidize the ruthenium. But we did not want to use it in our project because of its scarcity, its toxicity and impact on the environment. We found that the cytochrome C which uses iron could replace it and be oxidized by laccases. We decided to try and asked if it could help to degrade our polymers. We selected several enzymes that we believed could be great to use in E.coli strain.

Our strategy

Our strategy is to use a reduced laccase which will be oxidized by the oxygen present in the air.

To return at a reduced state, laccase will oxidize the cytochrom C previously excited by light. A superoxide anion radical will be formed.

When the cytochrome C will come back to its reduced state and the superoxide anion radical to oxygen, the polymers will be in a radical state.

Thanks to oxygen, it will form an epoxide that will be hydrolyzed and become a diol.

So we think that the polymer degradation will be more efficient by this method.