Difference between revisions of "Team:DTU-Denmark/Proof"

 
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                         <h1>Proof of concept<p class="lead">We have succesfully design and assembled our own plasmid compatible with replication in <i>E. coli, Y. Lipolytica</i> and the BioBrick standard. We demonstrated this by making our own BioBrick device enabeling heterologous protein expression</p></h1>
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                         <h1>Proof of concept<p class="lead">We have succesfully design and assembled our own plasmid compatible with replication in <i>Escherichia coli, Yarrowia lipolytica</i> and the BioBrick standard. We demonstrated this by making our own BioBrick device enabeling heterologous protein expression</p></h1>
 
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         <div><a class="anchor" id="section-1"></a>
 
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         <h2 class="h2">Assembly of BioBricks in Our Own Plasmid</h2>
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         <h2 class="h2">Assembly of BioBricks in pSB1A8YL - The BioBrick Plasmid</h2>
 
              
 
              
 
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                 <small>Jonathan Safran Joer, <cite title="Source Title">Everything is Illuminated</cite></small>
 
                 <small>Jonathan Safran Joer, <cite title="Source Title">Everything is Illuminated</cite></small>
 
             </blockquote>
 
             </blockquote>
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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>
 
  
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                <p>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>.</p>
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<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><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 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>
 
<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>
  
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  <h2 class="h3">Our Proof of Concept</h3><p>
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<div><a class="anchor" id="section-2"></a>
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  <h2 class="h2">Our Proof of Concept</h2><p>
 
<ul>
 
<ul>
 
<li>Design and assembly of a new plasmid (pSB1A8YL)</li>
 
<li>Design and assembly of a new plasmid (pSB1A8YL)</li>
 
<li>Expression of three BioBricks devices with pSB1A8YL in <i>E. coli</i></li>
 
<li>Expression of three BioBricks devices with pSB1A8YL in <i>E. coli</i></li>
 
<li>Transformation in <i>Y. lipolytica</i> and detection of the plasmid with inserts</li>
 
<li>Transformation in <i>Y. lipolytica</i> and detection of the plasmid with inserts</li>
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<li>Express a heterologous protein in <i>Y. lipolytica</i></p>
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<li>Express a heterologous protein in <i>Y. lipolytica</i>
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</ul> </p>
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</ul>  
  
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<h2 class="h3">Design of Plasmid</h3>
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<h2 class="h2">Design of Plasmid</h2>
  
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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 <i>Y. lipolytica<i/>. </p>
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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 <i>E.coli</i> and pSL16-CEN1-1(227) for replication in <i>Y. lipolytica</i>. </p>
  
 
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                 <figcaption class="figure-caption"><strong>Figure 1:</strong> Sequence map of pSB1A8YL. The colored blocks represents the following: <b>Orange</b>: pUC19 part. <b>Blue</b>: modified pSL16-CEN1-1(227) part, <b>pink</b>: BioBrick prefix, <b>purple</b>: BioBrick suffix, <b>red</b>: terminator, <b>green</b>: selection markers, <b>grey</b>: origin of replication.  The fully annotated sequence can be found <a href="http://parts.igem.org/Part:BBa_K2117009">HERE</a>. </figcaption>
 
                 <figcaption class="figure-caption"><strong>Figure 1:</strong> Sequence map of pSB1A8YL. The colored blocks represents the following: <b>Orange</b>: pUC19 part. <b>Blue</b>: modified pSL16-CEN1-1(227) part, <b>pink</b>: BioBrick prefix, <b>purple</b>: BioBrick suffix, <b>red</b>: terminator, <b>green</b>: selection markers, <b>grey</b>: origin of replication.  The fully annotated sequence can be found <a href="http://parts.igem.org/Part:BBa_K2117009">HERE</a>. </figcaption>
 
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<h2 class="h3">Is it Compatible with the BioBrick Standard?</h3>
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<h2 class="h2">Is it Compatible with the BioBrick Standard?</h2>
 
<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.  
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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).  
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We made three BioBricks by combining BioBricks already present in the registry: the Anderson promoter <a href="http://parts.igem.org/Part:BBa_K880005">(BBa_K880005)</a>    paired with the chromoproteins: amilCP <a href="http://parts.igem.org/Part:BBa_K592009">(BBa_K592009)</a>, amilGFP <a href="http://parts.igem.org/Part:BBa_K592010">(BBa_K592010)</a>  or mRFP <a href="http://parts.igem.org/Part:BBa_E1010">(E1010)</a>.  
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We assembles these BioBricks in our plasmid and transformed them into chemically competent DH5&#913; cells.</p>  
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We assembled these BioBricks in our plasmid and transformed them into chemically competent DH5&#913; cells.</p>  
  
 
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<h2 class="h2">Expression of Heterologous Proteins</h2>
 
<p>Next step was to show that pSB1A8YL could be transformed into our yeast <i>Y. lipolytica</i> and enable protein expression.  
 
<p>Next step was to show that pSB1A8YL could be transformed into our yeast <i>Y. lipolytica</i> 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 <i>Y. lipolytica</i> (<a href="http://parts.igem.org/Part:BBa_K2117005">(BBa_K2117005)</a>). </p>
 
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 <i>Y. lipolytica</i> (<a href="http://parts.igem.org/Part:BBa_K2117005">(BBa_K2117005)</a>). </p>
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             <figcaption class="figure-caption"><strong>Figure 3:</strong> Fluorescence microscopy conducted by a confocal laser microscope with 100x magnification. <b>A</b> and <b>D</b> are taken using a standard brightfield filter. <b>B</b> and <b>E</b> are taken using the GFP filter and with the excitation laser swithced on. <b>C</b> and <b>F</b> are overlays of the two photos where the black bagground has been removed (<b>C</b> is an overlay of <b>A</b> and <b>B</b>, and <b>F</b> is an overlay of <b>D</b> and <b>E</b>). <b>A</b>, <b>B</b> and <b>C</b> 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. <b>D</b>, <b>E</b> and <b>F</b> 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. This proves that our expression system works as intended.</figcaption>
 
             <figcaption class="figure-caption"><strong>Figure 3:</strong> Fluorescence microscopy conducted by a confocal laser microscope with 100x magnification. <b>A</b> and <b>D</b> are taken using a standard brightfield filter. <b>B</b> and <b>E</b> are taken using the GFP filter and with the excitation laser swithced on. <b>C</b> and <b>F</b> are overlays of the two photos where the black bagground has been removed (<b>C</b> is an overlay of <b>A</b> and <b>B</b>, and <b>F</b> is an overlay of <b>D</b> and <b>E</b>). <b>A</b>, <b>B</b> and <b>C</b> 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. <b>D</b>, <b>E</b> and <b>F</b> 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. This proves that our expression system works as intended.</figcaption>
 
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<h2 class="h3">Conclusion</h3>
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<h2 class="h2">Conclusion</h2>
 
<p>We managed to:  
 
<p>We managed to:  
 
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             <li><a href="#section-1">Assembly of BioBricks</a></li>
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<li><a href="#section-2">Proof of concept</a></li>
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<li><a href="#section-3">Design</a></li>
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Latest revision as of 02:02, 20 October 2016

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Proof of concept

We have succesfully design and assembled our own plasmid compatible with replication in Escherichia coli, Yarrowia lipolytica and the BioBrick standard. We demonstrated this by making our own BioBrick device enabeling heterologous protein expression


Assembly of BioBricks in pSB1A8YL - The BioBrick 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.

DESCRIPTION
Figure 1: 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 HERE.

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 assembled these BioBricks in our plasmid and transformed them into chemically competent DH5Α cells.

DESCRIPTION
Figure 2: 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.

DESCRIPTION
DESCRIPTION
Figure 3: Fluorescence microscopy conducted by a confocal laser microscope with 100x magnification. A and D are taken using a standard brightfield filter. B and E are taken using the GFP filter and with the excitation laser swithced on. 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 Y. lipolytica PO1f cells with our GFP expressing device (BBa_K2117005) shuttled by our plasmid pSB1A8YL. D, E and F are Y. lipolytica 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. This proves that our expression system works as intended.

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

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