Team:DTU-Denmark/Proof
Proof of concept
We have succesfully design and assembled our own plasmid compatible with replication in E. coli, Y. Lipolytica and the BioBrick standard. We demonstrate this by using 4 part from previous years and combine them as devices in our plasmid
Assembly of BioBricks in our own plasmid
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This page is intended to give brief overview of how we fulfilled gold medal requirement #3. For much more information please visit (Molecular Tools).
Yarrowia Lipolytica has a great potential to be a very versatile cell factory due to its ability to grow on a wide range of substrates. However, working with this unconventional yeast is troublesome due to the lack of molecular tools that can be used for genetic engineering.
We wanted to open up for the possibility of using Y. lipolytica in the future to produce any desired product while growing on any kind of substrate. Our aim was to develop a plasmid able to replicate in E. coli for easy cloning and propagation. The plasmid should also be compatible with Y. lipolytica along with being compatible with the BioBrick standard.
First step was to design the plasmid. We designed a plasmid (pSB1A8YL) based on the high copy plasmid Puc19 for replication in E.coli and pSL16-CEN1-1(227) for replication in Y. lipolytica.
Our next step was to prove that our plasmid was compatible with the BioBrick standard. We made three BioBricks by combining BioBricks already in the registry: the Anderson promoter (BBa_K880005) and paired this with the chromoproteins: amilCP (BBa_K592009), amilGFP (BBa_K592010) or mRFP(E1010). We assembles these BioBricks in our plasmid and transformed them into chemically competent DH5alpha cells.
The coloured colonies show that we were able to assemble a BioBrick device in our own designed plasmid, transform it into E. coli and express a chromoprotein proving that the BioBrick devices work.
