Difference between revisions of "Team:Aix-Marseille/Design"
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Latest revision as of 23:56, 18 September 2015
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 X component. 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.
When the cytochrome C will come back to its reduced state, 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.
Our constructions
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.
To test our enzymes, we will mesure the oxygen level with an oxymeter and by spectrophotometry.
We will select enzymes with the best results: one lipoxygenase, one laccase and one cytochrome C.
Using a linker, we will fuse the selected laccase with the selected cytochrome C and test again the activity. We will compared our results.
Production
Once our constructions done, we transform its into BL21 cells.
These E.coli K-12 allow the expression of proteins controlled by the T7 promotor and induced by IPTG.
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.
Purification
We added a His-tag following the sequence of genes coding our proteins.
Using columns containing nickel ions, His-tagged proteins are bound with high specificity and affinity. Other proteins pass through the matrix during the washing step. His-tagged proteins are eluted using imidazole buffer.
