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Team:Tokyo-NoKoGen/Project/solubilization
From 2010.igem.org
Objectives:Solubilization
Suppose that you would like to get a object from nature. Can you say it is always easy to pick it up? Such as a gemstone in a mine, you encounter the difficulty that a fascinating or valuable target is in a mixture.
We solve this problem by making rhamnolipids , which is a biosurfactant naturally secreted by Pseudomonas Aeruginosa.
Why is this device needed?
For the first of all, we have to make the target be easily taken up from an mixture. Oil sand is a good example. It is a naturally occurring mixture of sand, clay and several minerals, and bitumen. This bitumen is a material for a usable fuels such as gasoline and diesel, though it is too viscous to produce fuels. One of the solutions is to solubilize a object from a mixture. This is because solubilization system is the first necessary step in Eco Tanker.
What is this device compose inside it?
This device can produce two enzymes. One is transacylase, the other is rhamnosyltransferase. Each protein is expressed by the gene, RhlA and RhlB respectively.
Fig.1 shows Rhamnolipids Biosynthetic Pathway, and you can see where two genes work for.
Transacylase:
It catalyzes the transfer of β-hydroxydecanoyl moieties from acyl carrier protein (ACP) to coenzyme A (CoA). With this reaction, 3-(3-hydroxyalkanoyloxy)alkanoic acids (HAAs), which Fig.2 shows, are produced.
Rhamnosyltransferase:
It catalyzes the reaction, which uses dTDP-L-rhamnose and an HAA as precursors, yielding mono-rhamnolipids(rhamnolipids contains both mono-rhamnolipids and di-rhamnolipid naturally(Fig.3).).
How does this device work in EcoTanker>
Our objective construct is as above. We ligate it to pSB1C3 and transform E. coli DH5α.
progress
At first, we cultured P. aeruginosa on arger plates including nutrient broth and we did cloning of the two genes, RhlA and RhlB from the colonies of P. aeruginosa respectively by colony PCR. Secondly, in order to add promoter, RBS and terminator to RhlA or RhlB respectively, we made mega-primer by PCR. Using the primers, we amplified the genes, promoter-RBS-RhlA-terminator and promoter-RBS-RhlB-terminator. After digested by EcoRI and PstI, we ligated the PCR products to
