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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

Firstly, we increased the two genes, RhlA and RhlB from P. aeruginosa PAO1 strain respectively by PCR. Secondly, in order to make primers for RhlA and RhlB, we ligated former and reverse sequences which had hybridized area by PCR. Primers created ourselves cost less than that was ordered, because its length was big. Using the produced 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 pSB1C3 and transformed E coli. DH5α. We confirmed these sequences. Unfortunately, the objective constructs were not get. BioBricks we submitted are bellows. BBa_K317998: RhlA BBa_K317999: RhlB