Difference between revisions of "Team:DTU-Denmark/Proof"
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<p><i> This page is intended to give brief overview of how we fulfilled gold medal requirement #3. For much more information please visit <a href="https://2016.igem.org/Team:DTU-Denmark/molecular_toolbox">(Molecular Tools)</a>. </i></p> | <p><i> This page is intended to give brief overview of how we fulfilled gold medal requirement #3. For much more information please visit <a href="https://2016.igem.org/Team:DTU-Denmark/molecular_toolbox">(Molecular Tools)</a>. </i></p> | ||
| − | <p><i>Yarrowia lipolytica</i> 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 | + | <p><i>Yarrowia lipolytica</i> 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. </p> |
| − | <p>We | + | <p>We want to open up for the possibility of using <i>Y. lipolytica</i> 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 <i>Escherichia coli</i> for easy cloning and propagation. In addition, the plasmid should also be compatible with both <i>Y. lipolytica</i> along with the BioBrick standard.</p> |
<h2 class="h3">Our Proof of Concept</h3><p> | <h2 class="h3">Our Proof of Concept</h3><p> | ||
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<figure class="figure"> | <figure class="figure"> | ||
<img width=400 id="img2" class="enlarge img-responsive figure-img" src="https://static.igem.org/mediawiki/2016/thumb/f/f0/T--DTU-Denmark--pSB1A8YL_Sequence_map.png/603px-T--DTU-Denmark--pSB1A8YL_Sequence_map.png" alt="DESCRIPTION"> | <img width=400 id="img2" class="enlarge img-responsive figure-img" src="https://static.igem.org/mediawiki/2016/thumb/f/f0/T--DTU-Denmark--pSB1A8YL_Sequence_map.png/603px-T--DTU-Denmark--pSB1A8YL_Sequence_map.png" alt="DESCRIPTION"> | ||
| − | <figcaption class="figure-caption"><strong>Figure 1:</strong> Sequence map of pSB1A8YL. The colored blocks represents the following: Orange: pUC19 part, Blue modified pSL16-CEN1-1(227) part, pink: BioBrick prefix, purple: BioBrick suffix, red: terminator, green selection markers, grey: origin of replication. The | + | <figcaption class="figure-caption"><strong>Figure 1:</strong> Sequence map of pSB1A8YL. The colored blocks represents the following: Orange: pUC19 part, Blue modified pSL16-CEN1-1(227) part, pink: BioBrick prefix, purple: BioBrick suffix, red: terminator, green selection markers, grey: origin of replication. The fully annotated sequence can be found <a href="http://parts.igem.org/Part:BBa_K2117009">HERE</a>. </figcaption> |
</figure> | </figure> | ||
<h2 class="h3">Is it Compatible with the BioBrick Standard?</h3> | <h2 class="h3">Is it Compatible with the BioBrick Standard?</h3> | ||
<p>Our next step was to prove that our plasmid was compatible with the BioBrick standard. | <p>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) | + | 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.</p> | We assembles these BioBricks in our plasmid and transformed them into chemically competent DH5Α cells.</p> | ||
<figure class="figure"> | <figure class="figure"> | ||
<img id="img7" class="enlarge img-responsive figure-img" src="https://static.igem.org/mediawiki/2016/thumb/8/85/T--DTU-Denmark--Colored_colonies.png/799px-T--DTU-Denmark--Colored_colonies.png" alt="DESCRIPTION"> | <img id="img7" class="enlarge img-responsive figure-img" src="https://static.igem.org/mediawiki/2016/thumb/8/85/T--DTU-Denmark--Colored_colonies.png/799px-T--DTU-Denmark--Colored_colonies.png" alt="DESCRIPTION"> | ||
| + | <figcaption class="figure-caption"><strong>Figure 2:</strong> The coloured colonies show that we were able to assemble a BioBrick device in our own designed plasmid, transform it into <i>E. coli</i> and express a chromoprotein proving that the BioBrick devices work. </figcaption> | ||
</figure> | </figure> | ||
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<span class="close img7">×</span> | <span class="close img7">×</span> | ||
<img class="modal-content" id="img7Img"> | <img class="modal-content" id="img7Img"> | ||
| − | <div class="caption"> | + | <div class="caption">The coloured colonies show that we were able to assemble a BioBrick device in our own designed plasmid, transform it into <i>E. coli</i> and express a chromoprotein proving that the BioBrick devices work.</div> |
</div> | </div> | ||
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| − | |||
| − | |||
<h2 class="h3">Expression of Heterologous Proteins</h3> | <h2 class="h3">Expression of Heterologous Proteins</h3> | ||
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<figure class="figure"> | <figure class="figure"> | ||
<img id="img3" class="enlarge img-responsive figure-img" src="https://static.igem.org/mediawiki/2016/4/40/T--DTU-Denmark--GFP_control.png" alt="DESCRIPTION" width="478"> | <img id="img3" class="enlarge img-responsive figure-img" src="https://static.igem.org/mediawiki/2016/4/40/T--DTU-Denmark--GFP_control.png" alt="DESCRIPTION" width="478"> | ||
| − | <figcaption class="figure-caption"><strong>Figure | + | <figcaption class="figure-caption"><strong>Figure 3:</strong> Fluorescence microscopy conducted by a confocal laser microscope with 100x magnification. A and D are taken using standard brightfield, B and E are taken using the GFP filter and with the excitation laser on and C and F are overlays of the two photos where the black bagground has been removed (C is an overlay of A and B, and F is an overlay of D and E). A, B and C are <i>Y. lipolytica</i> PO1f cells with our GFP expressing device (<a href="http://parts.igem.org/Part:BBa_K2117005">BBa_K2117005</a>) shuttled by our plasmid pSB1A8YL. D, E and F are <i>Y. lipolytica</i> PO1f cells with the empty pSB1A8YL plasmid, which serves as a control for the GFP signal. Notice that even though the empty vector control shows trace amounts of auto-fluoresence the strain with the GFP expressing device clearly exhibits higher levels of fluorescence, which proves that our expression system works as intended.</figcaption> |
</figure> | </figure> | ||
Revision as of 21:38, 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.
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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.
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.
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DESCRIPTION
Conclusion
We managed to:
- Design a functional plasmid for transformation and replication i 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
- Demonstrate that the plasmid is able to be transformed and replicated in Y. lipolytica
- Prove heterologous protein expression in Y. lipolytica
