Difference between revisions of "Team:DTU-Denmark/Proof"
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| − | <p>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. </p> | + | <p>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 <i>Y. lipolytica<i/>. </p> |
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Revision as of 22:59, 19 October 2016
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 making our own BioBrick device enabeling heterologous protein expression
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 available for genetic engineering.
We want 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 Escherichia coli for easy cloning and propagation. In addition, the plasmid should also be compatible with both Y. lipolytica along 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
- Express a heterologous protein in Y. lipolytica
Design of Plasmid
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.
Is it Compatible with the BioBrick Standard?
Our next step was to prove that our plasmid was compatible with the BioBrick standard. We made three BioBricks by combining BioBricks already present in the registry: the Anderson promoter (BBa_K880005) paired 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 DH5Α cells.
Expression of Heterologous Proteins
Next step was to show that pSB1A8YL could be transformed into our yeast Y. lipolytica and enable protein expression. We used a transformation protocol received from Cory M. Schwartz, University of California. We designed a composite part containing a TEF promoter and hrGFP codon-optimized for Y. lipolytica ((BBa_K2117005)).
This BioBrick was inserted into pSB1A8YL. We wanted to show that we were able to transform, replicate and detect expression of a heterologous protein from ((BBa_K2117005)) in Y. lipolytica.
Conclusion
We managed to:
- Design a functional plasmid for transformation and replication in both E. coli and Y. lipolytica
- Prove that pSB1A8YL is compatible with the BioBrick standard
- Show that we were able to assemble and express a composite part in E. coli
- Prove that the plasmid is able to be transformed and replicated in Y. lipolytica
- Demonstrate heterologous protein expression in Y. lipolytica
