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 demonstrated this by using four parts from previous years and combined them as devices in our plasmid
Assembly of BioBricks in our own plasmid
"...and when is enough proof enough?"
Jonathan Safran Joer, Everything is Illuminated
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.
Our proof of concept:
- Design and assembly of a new plasmid (pSB1A8YL)
- Expression of three BioBricks devices with pSB1A8YL in E. coli
- Transformation in Y. lipolytica and detection of the plasmid with inserts
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.
Next step was to show that pSB1A8YL could be transformed into our yeast Y. lipolytica. We used a transformation protocol received from Cory Schwartz, University of California. We designed a composite part containing a TEF promoter and the gene for human proinsulin BBa_K2117002.
This BioBrick was inserted into pSB1A8YL. We wanted to show that we were able to transform, replicate and detect the presence of BBa_K2117002 in Y. lipolytica.
Therefore we performed colony PCR on the transformants.
*Insert picture of gel*We have successfully:
- Designed a functional plasmid for transformation and replication i both E. coli and Y. lipolytica
- Proved that pSB1A8YL is compatible with the BioBrick standard
- Showed that we were able to assemble and express a composite part in E. coli
- Demonstrated that the plasmid is able to be transformed and replicated in Y. lipolytica
