Difference between revisions of "Part:BBa K5237009"

 
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  <!-- Part summary -->
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<!-- Part summary -->
  <section id="0">
+
<section>
    <h1>Mini staple: bGCN4</h1>
+
<h1>Mini Staple: bGCN4</h1>
    <p>
+
<p>
       The bGCN4 Mini Staple is a fusion of the basic leucine zipper GCN4 and rGCN4, offering a smaller, compact protein
+
       The bGCN4 Mini staple is a fusion of the basic leucine zipper GCN4 and rGCN4, offering a smaller, compact protein
       staple. With its well-characterized subunits and strong in silico and experimental validation, this Mini Staple
+
       staple. With its well-characterized subunits and strong <i>in silico</i> and experimental validation, this Mini staple
 
       serves as a versatile foundation for expanding to similar staples.
 
       serves as a versatile foundation for expanding to similar staples.
 
     </p>
 
     </p>
    <p> </p>
+
<p> </p>
  </section>
+
</section>
  <div class="toc" id="toc">
+
<div class="toc" id="toc">
    <div id="toctitle">
+
<div id="toctitle">
      <h1>Contents</h1>
+
<h1>Contents</h1>
    </div>
+
</div>
    <ul>
+
<ul>
      <li class="toclevel-1 tocsection-1"><a href="#1"><span class="tocnumber">1</span> <span class="toctext">Sequence
+
<li class="toclevel-1 tocsection-1"><a href="#1"><span class="tocnumber">1</span> <span class="toctext">Sequence
 
             overview</span></a>
 
             overview</span></a>
      </li>
+
</li>
      <li class="toclevel-1 tocsection-2"><a href="#2"><span class="tocnumber">2</span> <span class="toctext">Usage and
+
<li class="toclevel-1 tocsection-2"><a href="#2"><span class="tocnumber">2</span> <span class="toctext">Usage and
 
             Biology</span></a>
 
             Biology</span></a>
      </li>
+
</li>
      <li class="toclevel-1 tocsetction-3"><a href="#3"><span class="tocnumber">3</span> <span class="toctext">Assembly
+
<li class="toclevel-1 tocsetction-3"><a href="#3"><span class="tocnumber">3</span> <span class="toctext">Assembly
 
             and part evolution</span></a>
 
             and part evolution</span></a>
      </li>
+
</li>
      <li class="toclevel-1 tocsection-4"><a href="#4"><span class="tocnumber">4</span> <span
+
<li class="toclevel-1 tocsection-4"><a href="#4"><span class="tocnumber">4</span> <span class="toctext">Results</span></a>
            class="toctext">Results</span></a>
+
<ul>
        <ul>
+
<li class="toclevel-2 tocsection-4.1"><a href="#4.1"><span class="tocnumber">4.1</span> <span class="toctext">Protein Expression and Purification</span></a>
          <li class="toclevel-2 tocsection-4.1"><a href="#4.1"><span class="tocnumber">4.1</span> <span
+
</li>
                class="toctext">Protein Expression and Purification</span></a>
+
<li class="toclevel-2 tocsection-4.2"><a href="#4.2"><span class="tocnumber">4.2</span> <span class="toctext">Electrophoretic Mobility Shift Assay</span></a>
          </li>
+
<ul>
          <li class="toclevel-2 tocsection-4.2"><a href="#4.2"><span class="tocnumber">4.2</span> <span
+
<li class="toclevel-3 tocsection-4.2.1"><a href="#4.2.1"><span class="tocnumber">4.2.1</span> <span class="toctext">Qualitative DNA Binding Analysis</span></a>
                class="toctext">Electrophoretic Mobility Shift Assay</span></a>
+
</li>
            <ul>
+
<li class="toclevel-3 tocsection-4.2.2"><a href="#4.2.2"><span class="tocnumber">4.2.2</span> <span class="toctext">Quantitative DNA binding Analysis</span></a>
              <li class="toclevel-3 tocsection-4.2.1"><a href="#4.2.1"><span class="tocnumber">4.2.1</span> <span
+
</li>
                    class="toctext">Qualitative DNA binding analysis</span></a>
+
</ul>
              </li>
+
</li>
              <li class="toclevel-3 tocsection-4.2.2"><a href="#4.2.2"><span class="tocnumber">4.2.2</span> <span
+
<li class="toclevel-2 tocsection-4.3"><a href="#4.3"><span class="tocnumber">4.3</span> <span class="toctext"><i>In Silico</i> Characterization using DaVinci</span></a>
                    class="toctext">Quantitative DNA binding analysis</span></a>
+
</li>
              </li>
+
</ul>
            </ul>
+
</li>
          </li>
+
<li class="toclevel-1 tocsection-8"><a href="#5"><span class="tocnumber">5</span> <span class="toctext">References</span></a>
          <li class="toclevel-2 tocsection-4.3"><a href="#4.3"><span class="tocnumber">4.3</span> <span
+
</li>
                class="toctext">In Silico Characterization using DaVinci</span></a>
+
</ul>
          </li>
+
</div>
        </ul>
+
<section><p><br/><br/></p>
      </li>
+
<font size="5"><b>The PICasSO Toolbox </b> </font>
      <li class="toclevel-1 tocsection-8"><a href="#5"><span class="tocnumber">5</span> <span
+
<div class="thumb" style="margin-top:10px;"></div>
            class="toctext">References</span></a>
+
<div class="thumbinner" style="width:550px"><img alt="" class="thumbimage" src="https://static.igem.wiki/teams/5237/wetlab-results/registry-part-collection-engineering-cycle-example-overview.svg" style="width:99%;"/>
      </li>
+
<div class="thumbcaption">
    </ul>
+
<i><b>Figure 1: How Our Part Collection can be Used to Engineer New Staples</b></i>
  </div>
+
</div>
  <section>
+
</div>
    <p><br /><br /></p>
+
<p>
    <font size="5"><b>The PICasSO Toolbox </b> </font>
+
<br/>
    <div class="thumb" style="margin-top:10px;"></div>
+
       While synthetic biology has in the past focused on engineering the genomic sequence of organisms, the <b>3D
    <div class="thumbinner" style="width:550px"><img alt="" class="thumbimage"
+
        spatial organization</b> of DNA is well-known to be an important layer of information encoding in
        src="https://static.igem.wiki/teams/5237/wetlab-results/registry-part-collection-engineering-cycle-example-overview.svg"
+
      particular in eukaryotes, playing a crucial role in
        style="width:99%;" />
+
       gene regulation and hence
      <div class="thumbcaption">
+
       cell fate, disease development, evolution, and more. However, tools to precisely manipulate and control the
        <i><b>Figure 1: How our part collection can be used to engineer new staples</b></i>
+
      genomic spatial
      </div>
+
       architecture are limited, hampering the exploration of
    </div>
+
       3D genome engineering in synthetic biology. We - the iGEM Team Heidelberg 2024 - have developed PICasSO, a
 
+
      <b>powerful
    <p>
+
         molecular toolbox for rationally engineering genome 3D architectures</b> in living cells, based on
      <br />
+
      various DNA-binding proteins.
       Next to the well-studied linear DNA sequence, the <b>3D spatial organization</b> of DNA plays a crucial role in
+
       gene regulation,
+
       cell fate, disease development and more. However, the tools to precisely manipulate this genomic
+
       architecture remain limited, rendering it challenging to explore the full potential of the
+
       3D genome in synthetic biology. We - iGEM Team Heidelberg 2024 - have developed PICasSO, a <b>powerful
+
         molecular toolbox</b> based on various DNA-binding proteins to address this issue.
+
 
     </p>
 
     </p>
    <p>
+
<p>
 
       The <b>PICasSO</b> part collection offers a comprehensive, modular platform for precise manipulation and
 
       The <b>PICasSO</b> part collection offers a comprehensive, modular platform for precise manipulation and
 
       <b>re-programming
 
       <b>re-programming
         of DNA-DNA interactions</b> using protein staples in living cells, enabling researchers to recreate natural 3D
+
         of DNA-DNA interactions</b> using engineered "protein staples" in living cells. This enables
      genomic
+
      researchers to recreate naturally occurring alterations of 3D genomic
       interactions, such as enhancer hijacking, or to design entirely new spatial architectures for gene regulation.
+
       interactions, such as enhancer hijacking in cancer, or to design entirely new spatial architectures for
       Specifically, the fusion of two DNA binding proteins enables to artifically bring distant genomic loci into
+
      artificial gene regulation and cell function control.
       proximty.
+
       Specifically, the fusion of two DNA binding proteins enables to artificially bring otherwise distant genomic
       To unlock the system's full potential, we introduce versatile <b>chimeric CRISPR/Cas complexes</b>, connected
+
      loci into
       either on
+
       spatial proximity.
       the protein or the guide RNA level. These1 complexes are reffered to as protein- or Cas staples. Beyond its
+
       To unlock the system's full potential, we introduce versatile <b>chimeric CRISPR/Cas complexes</b>,
       versatility, PICasSO includes <b>robust assay</b> systems to support the engineering, optimization, and
+
       connected either at
       testing of new staples, ensuring functionality <i>in vitro</i> and <i>in vivo</i>. We took special care to include
+
       the protein or - in the case of CRISPR/Cas-based DNA binding moieties - the guide RNA level. These complexes are
       parts crucial for testing every step of the cycle (design, build, test, learn) when <b>engineering new parts</b>.
+
      referred to as protein- or Cas staples, respectively. Beyond its
 +
       versatility with regard to the staple constructs themselves, PICasSO includes <b>robust assay</b> systems to
 +
      support the engineering, optimization, and
 +
       testing of new staples <i>in vitro</i> and <i>in vivo</i>. Notably, the PICasSO toolbox was developed in a
 +
       design-build-test-learn <b>engineering cycle closely intertwining wet lab experiments and computational
 +
        modeling</b> and iterated several times, yielding a collection of well-functioning and -characterized
 +
      parts.
 
     </p>
 
     </p>
    <p>At its heart, the PICasSO part collection consists of three categories. <br /><b>(i)</b> Our <b>DNA-binding
+
<p>At its heart, the PICasSO part collection consists of three categories. <br/><b>(i)</b> Our <b>DNA-binding
 
         proteins</b>
 
         proteins</b>
 
       include our
 
       include our
       finalized enhancer hijacking Cas staple as well as half staples that can be used by scientists to compose entirely
+
       finalized Cas staple experimentally validated using an artificial "enhancer hijacking" system as well as
       new Cas staples in the future. We also include our Simple staples that serve as controls for successful stapling
+
      "half staples" that can be combined by scientists to compose entirely
       and can be further engineered to create alternative, simpler and more compact staples. <br />
+
       new Cas staples in the future. We also include our Simple staples comprised of particularly small, simple
      <b>(ii)</b> As <b>functional elements</b>, we list additional parts that enhance the functionality of our Cas and
+
      and robust DNA binding domains well-known to the synthetic biology community, which serve as controls for
 +
      successful stapling
 +
       and can be further engineered to create alternative, simpler, and more compact staples. <br/>
 +
<b>(ii)</b> As <b>functional elements</b>, we list additional parts that enhance and expand the
 +
      functionality of our Cas and
 
       Basic staples. These
 
       Basic staples. These
       consist of
+
       consist of staples dependent on
       protease-cleavable peptide linkers and inteins that allow condition-specific, dynamic stapling <i>in vivo</i>.
+
       cleavable peptide linkers targeted by cancer-specific proteases or inteins that allow condition-specific,
       Besides staple functionality, we also include the parts to enable the efficient delivery of PICasSO's constructs
+
      dynamic stapling <i>in vivo</i>.
       with our
+
       We also include several engineered parts that enable the efficient delivery of PICasSO's constructs into
       interkingdom conjugation system. <br />
+
      target cells, including mammalian cells,
      <b>(iii)</b> As the final category of our collection, we provide parts that support the use of our <b>custom
+
       with our new
 +
       interkingdom conjugation system. <br/>
 +
<b>(iii)</b> As the final category of our collection, we provide parts that underlie our <b>custom
 
         readout
 
         readout
         systems</b>. These include components of our established FRET-based proximity assay system, enabling users to
+
         systems</b>. These include components of our established FRET-based proximity assay system, enabling
 +
      users to
 
       confirm
 
       confirm
       accurate stapling. Additionally, we offer a complementary, application-oriented testing system for functional
+
       accurate stapling. Additionally, we offer a complementary, application-oriented testing system based on a
       readouts via a luciferase reporter, which allows for straightforward experimental simulation of enhancer hijacking
+
       luciferase reporter, which allows for straightforward experimental assessment of functional enhancer
 +
      hijacking events
 
       in mammalian cells.
 
       in mammalian cells.
 
     </p>
 
     </p>
    <p>
+
<p>
       The following table gives a comprehensive overview of all parts in our PICasSO toolbox. <mark
+
       The following table gives a comprehensive overview of all parts in our PICasSO toolbox. <mark style="background-color: #FFD700; color: black;">The highlighted parts showed
        style="background-color: #FFD700; color: black;">The highlighted parts showed
+
         exceptional performance as described on our iGEM wiki and can serve as a reference.</mark> The other
         exceptional performance as described on our iGEM wiki and can serve as a reference.</mark> The other parts in
+
      parts in
 
       the
 
       the
       collection are versatile building blocks designed to provide future iGEMers with the flexibility to engineer their
+
       collection are versatile building blocks designed to provide future iGEMers with the flexibility to engineer
       own custom Cas staples, enabling further optimization and innovation.<br />
+
      their
    </p>
+
       own custom Cas staples, enabling further optimization and innovation in the new field of 3D genome
    <p>
+
      engineering.<br/>
      <font size="4"><b>Our part collection includes:</b></font><br />
+
</p>
    </p>
+
<p>
    <table style="width: 90%; padding-right:10px;">
+
<font size="4"><b>Our part collection includes:</b></font><br/>
      <td align="left" colspan="3"><b>DNA-binding proteins: </b>
+
</p>
         The building blocks for engineering of custom staples for DNA-DNA interactions with a modular system ensuring
+
<table style="width: 90%; padding-right:10px;">
        easy assembly.</td>
+
<td align="left" colspan="3"><b>DNA-Binding Proteins: </b>
      <tbody>
+
         Modular building blocks for engineering of custom staples to mediate defined DNA-DNA interactions <i>in vivo</i></td>
        <tr bgcolor="#FFD700">
+
<tbody>
          <td><a href="https://parts.igem.org/Part:BBa_K5237000" target="_blank">BBa_K5237000</a></td>
+
<tr bgcolor="#FFD700">
          <td>fgRNA Entry vector MbCas12a-SpCas9</td>
+
<td><a href="https://parts.igem.org/Part:BBa_K5237000" target="_blank">BBa_K5237000</a></td>
          <td>Entryvector for simple fgRNA cloning via SapI</td>
+
<td>Fusion Guide RNA Entry Vector MbCas12a-SpCas9</td>
        </tr>
+
<td>Entry vector for simple fgRNA cloning via SapI</td>
        <tr bgcolor="#FFD700">
+
</tr>
          <td><a href="https://parts.igem.org/Part:BBa_K5237001" target="_blank">BBa_K5237001</a></td>
+
<tr bgcolor="#FFD700">
          <td>Staple subunit: dMbCas12a-Nucleoplasmin NLS</td>
+
<td><a href="https://parts.igem.org/Part:BBa_K5237001" target="_blank">BBa_K5237001</a></td>
          <td>Staple subunit that can be combined with sgRNA or fgRNA and dCas9 to form a functional staple</td>
+
<td>Staple Subunit: dMbCas12a-Nucleoplasmin NLS</td>
        </tr>
+
<td>Staple subunit that can be combined with crRNA or fgRNA and dSpCas9 to form a functional staple
        <tr bgcolor="#FFD700">
+
          </td>
          <td><a href="https://parts.igem.org/Part:BBa_K5237002" target="_blank">BBa_K5237002</a></td>
+
</tr>
          <td>Staple subunit: SV40 NLS-dSpCas9-SV40 NLS</td>
+
<tr bgcolor="#FFD700">
          <td>Staple subunit that can be combined witha sgRNA or fgRNA and dCas12avto form a functional staple
+
<td><a href="https://parts.igem.org/Part:BBa_K5237002" target="_blank">BBa_K5237002</a></td>
 +
<td>Staple Subunit: SV40 NLS-dSpCas9-SV40 NLS</td>
 +
<td>Staple subunit that can be combined with a sgRNA or fgRNA and dMbCas12a to form a functional staple
 
           </td>
 
           </td>
        </tr>
+
</tr>
        <tr>
+
<tr>
          <td><a href="https://parts.igem.org/Part:BBa_K5237003" target="_blank">BBa_K5237003</a></td>
+
<td><a href="https://parts.igem.org/Part:BBa_K5237003" target="_blank">BBa_K5237003</a></td>
          <td>Cas Staple: SV40 NLS-dMbCas12a-dSpCas9-Nucleoplasmin NLS</td>
+
<td>Cas Staple: SV40 NLS-dMbCas12a-dSpCas9-Nucleoplasmin NLS</td>
          <td>Functional Cas staple that can be combined with sgRNA or fgRNA to bring two DNA strands into close
+
<td>Functional Cas staple that can be combined with sgRNA and crRNA or fgRNA to bring two DNA strands into
 +
            close
 
             proximity
 
             proximity
 
           </td>
 
           </td>
        </tr>
+
</tr>
        <tr>
+
<tr>
          <td><a href="https://parts.igem.org/Part:BBa_K5237004" target="_blank">BBa_K5237004</a></td>
+
<td><a href="https://parts.igem.org/Part:BBa_K5237004" target="_blank">BBa_K5237004</a></td>
          <td>Staple subunit: Oct1-DBD</td>
+
<td>Staple Subunit: Oct1-DBD</td>
          <td>Staple subunit that can be combined to form a functional staple, for example with TetR.<br />
+
<td>Staple subunit that can be combined to form a functional staple, for example with TetR.<br/>
 
             Can also be combined with a fluorescent protein as part of the FRET proximity assay</td>
 
             Can also be combined with a fluorescent protein as part of the FRET proximity assay</td>
        </tr>
+
</tr>
        <tr>
+
<tr>
          <td><a href="https://parts.igem.org/Part:BBa_K5237005" target="_blank">BBa_K5237005</a></td>
+
<td><a href="https://parts.igem.org/Part:BBa_K5237005" target="_blank">BBa_K5237005</a></td>
          <td>Staple subunit: TetR</td>
+
<td>Staple Subunit: TetR</td>
          <td>Staple subunit that can be combined to form a functional staple, for example with Oct1.<br />
+
<td>Staple subunit that can be combined to form a functional staple, for example with Oct1.<br/>
 
             Can also be combined with a fluorescent protein as part of the FRET proximity assay</td>
 
             Can also be combined with a fluorescent protein as part of the FRET proximity assay</td>
        </tr>
+
</tr>
        <tr>
+
<tr>
          <td><a href="https://parts.igem.org/Part:BBa_K5237006" target="_blank">BBa_K5237006</a></td>
+
<td><a href="https://parts.igem.org/Part:BBa_K5237006" target="_blank">BBa_K5237006</a></td>
          <td>Simple staple: TetR-Oct1</td>
+
<td>Simple Staple: TetR-Oct1</td>
          <td>Functional staple that can be used to bring two DNA strands in close proximity</td>
+
<td>Functional staple that can be used to bring two DNA strands in close proximity</td>
        </tr>
+
</tr>
        <tr>
+
<tr>
          <td><a href="https://parts.igem.org/Part:BBa_K5237007" target="_blank">BBa_K5237007</a></td>
+
<td><a href="https://parts.igem.org/Part:BBa_K5237007" target="_blank">BBa_K5237007</a></td>
          <td>Staple subunit: GCN4</td>
+
<td>Staple Subunit: GCN4</td>
          <td>Staple subunit that can be combined to form a functional staple, for example with rGCN4</td>
+
<td>Staple subunit that can be combined to form a functional staple, for example with rGCN4</td>
        </tr>
+
</tr>
        <tr>
+
<tr>
          <td><a href="https://parts.igem.org/Part:BBa_K5237008" target="_blank">BBa_K5237008</a></td>
+
<td><a href="https://parts.igem.org/Part:BBa_K5237008" target="_blank">BBa_K5237008</a></td>
          <td>Staple subunit: rGCN4</td>
+
<td>Staple Subunit: rGCN4</td>
          <td>Staple subunit that can be combined to form a functional staple, for example with rGCN4</td>
+
<td>Staple subunit that can be combined to form a functional staple, for example with rGCN4</td>
        </tr>
+
</tr>
        <tr>
+
<tr>
          <td><a href="https://parts.igem.org/Part:BBa_K5237009" target="_blank">BBa_K5237009</a></td>
+
<td><a href="https://parts.igem.org/Part:BBa_K5237009" target="_blank">BBa_K5237009</a></td>
          <td>Mini staple: bGCN4</td>
+
<td>Mini Staple: bGCN4</td>
          <td>
+
<td>
 
             Assembled staple with minimal size that can be further engineered</td>
 
             Assembled staple with minimal size that can be further engineered</td>
        </tr>
+
</tr>
      </tbody>
+
</tbody>
      <td align="left" colspan="3"><b>Functional elements: </b>
+
<td align="left" colspan="3"><b>Functional Elements: </b>
         Protease-cleavable peptide linkers and inteins are used to control and modify staples for further optimization
+
         Protease-cleavable peptide linkers and inteins are used to control and modify staples for further
 +
        optimization
 
         for custom applications</td>
 
         for custom applications</td>
      <tbody>
+
<tbody>
        <tr bgcolor="#FFD700">
+
<tr bgcolor="#FFD700">
          <td><a href="https://parts.igem.org/Part:BBa_K5237010" target="_blank">BBa_K5237010</a></td>
+
<td><a href="https://parts.igem.org/Part:BBa_K5237010" target="_blank">BBa_K5237010</a></td>
          <td>Cathepsin B-cleavable Linker: GFLG</td>
+
<td>Cathepsin B-cleavable Linker: GFLG</td>
          <td>Cathepsin B-cleavable peptide linker that can be used to combine two staple subunits to make responsive
+
<td>Cathepsin B-cleavable peptide linker that can be used to combine two staple subunits to make
 +
            responsive
 
             staples</td>
 
             staples</td>
        </tr>
+
</tr>
        <tr>
+
<tr>
          <td><a href="https://parts.igem.org/Part:BBa_K5237011" target="_blank">BBa_K5237011</a></td>
+
<td><a href="https://parts.igem.org/Part:BBa_K5237011" target="_blank">BBa_K5237011</a></td>
          <td>Cathepsin B Expression Cassette</td>
+
<td>Cathepsin B Expression Cassette</td>
          <td>Expression Cassette for the overexpression of cathepsin B</td>
+
<td>Expression cassette for the overexpression of cathepsin B</td>
        </tr>
+
</tr>
        <tr>
+
<tr>
          <td><a href="https://parts.igem.org/Part:BBa_K5237012" target="_blank">BBa_K5237012</a></td>
+
<td><a href="https://parts.igem.org/Part:BBa_K5237012" target="_blank">BBa_K5237012</a></td>
          <td>Caged NpuN Intein</td>
+
<td>Caged NpuN Intein</td>
          <td>A caged NpuN split intein fragment that undergoes protein <i>trans</i>-splicing after protease activation.
+
<td>A caged NpuN split intein fragment that undergoes protein <i>trans</i>-splicing after protease
             Can be used to create functionalized staples
+
             activation, which can be used to create functionalized staple
             units</td>
+
             subunits</td>
        </tr>
+
</tr>
        <tr>
+
<tr>
          <td><a href="https://parts.igem.org/Part:BBa_K5237013" target="_blank">BBa_K5237013</a></td>
+
<td><a href="https://parts.igem.org/Part:BBa_K5237013" target="_blank">BBa_K5237013</a></td>
          <td>Caged NpuC Intein</td>
+
<td>Caged NpuC Intein</td>
          <td>A caged NpuC split intein fragment that undergoes protein <i>trans</i>-splicing after protease activation.
+
<td>A caged NpuC split intein fragment that undergoes protein <i>trans</i>-splicing after protease
             Can be used to create functionalized staples
+
             activation, which can be used to create functionalized staple
             units</td>
+
             subunits</td>
        </tr>
+
</tr>
        <tr>
+
<tr>
          <td><a href="https://parts.igem.org/Part:BBa_K5237014" target="_blank">BBa_K5237014</a></td>
+
<td><a href="https://parts.igem.org/Part:BBa_K5237014" target="_blank">BBa_K5237014</a></td>
          <td>fgRNA processing casette</td>
+
<td>Fusion Guide RNA Processing Casette</td>
          <td>Processing casette to produce multiple fgRNAs from one transcript, that can be used for multiplexed 3D
+
<td>Processing cassette to produce multiple fgRNAs from one transcript, that can be used for
             genome reprograming</td>
+
            multiplexed 3D
        </tr>
+
             genome reprogramming</td>
        <tr>
+
</tr>
          <td><a href="https://parts.igem.org/Part:BBa_K5237015" target="_blank">BBa_K5237015</a></td>
+
<tr>
          <td>Intimin anti-EGFR Nanobody</td>
+
<td><a href="https://parts.igem.org/Part:BBa_K5237015" target="_blank">BBa_K5237015</a></td>
          <td>Interkindom conjugation between bacteria and mammalian cells, as alternative delivery tool for large
+
<td>Intimin anti-EGFR Nanobody</td>
 +
<td>Interkingdom conjugation between bacteria and mammalian cells, as an alternative delivery tool for
 +
            large
 
             constructs</td>
 
             constructs</td>
        </tr>
+
</tr>
        <tr>
+
<tr>
          <td><a href="https://parts.igem.org/Part:BBa_K4643003" target="_blank">BBa_K4643003</a></td>
+
<td><a href="https://parts.igem.org/Part:BBa_K4643003" target="_blank">BBa_K4643003</a></td>
          <td>incP origin of transfer</td>
+
<td>IncP Origin of Transfer</td>
          <td>Origin of transfer that can be cloned into the plasmid vector and used for conjugation as a means of
+
<td>Origin of transfer that can be cloned into the plasmid vector and used for conjugation as a
 +
            means of
 
             delivery</td>
 
             delivery</td>
        </tr>
+
</tr>
      </tbody>
+
</tbody>
      <td align="left" colspan="3"><b>Readout Systems: </b>
+
<td align="left" colspan="3"><b>Readout Systems: </b>
         FRET and enhancer recruitment to measure proximity of stapled DNA in bacterial and mammalian living cells
+
         FRET and enhancer recruitment readout systems to rapidly assess successful DNA stapling in bacterial and
        enabling swift testing and easy development for new systems</td>
+
        mammalian cells
      <tbody>
+
      </td>
        <tr bgcolor="#FFD700">
+
<tbody>
          <td><a href="https://parts.igem.org/Part:BBa_K5237016" target="_blank">BBa_K5237016</a></td>
+
<tr bgcolor="#FFD700">
          <td>FRET-Donor: mNeonGreen-Oct1</td>
+
<td><a href="https://parts.igem.org/Part:BBa_K5237016" target="_blank">BBa_K5237016</a></td>
          <td>FRET Donor-Fluorpohore fused to Oct1-DBD that binds to the Oct1 binding cassette. Can be used to visualize
+
<td>FRET-Donor: mNeonGreen-Oct1</td>
 +
<td>FRET donor-fluorophore fused to Oct1-DBD that binds to the Oct1 binding cassette, which can be used to
 +
            visualize
 
             DNA-DNA
 
             DNA-DNA
 
             proximity</td>
 
             proximity</td>
        </tr>
+
</tr>
        <tr bgcolor="#FFD700">
+
<tr bgcolor="#FFD700">
          <td><a href="https://parts.igem.org/Part:BBa_K5237017" target="_blank">BBa_K5237017</a></td>
+
<td><a href="https://parts.igem.org/Part:BBa_K5237017" target="_blank">BBa_K5237017</a></td>
          <td>FRET-Acceptor: TetR-mScarlet-I</td>
+
<td>FRET-Acceptor: TetR-mScarlet-I</td>
          <td>Acceptor part for the FRET assay binding the TetR binding cassette. Can be used to visualize DNA-DNA
+
<td>Acceptor part for the FRET assay binding the TetR binding cassette, which can be used to visualize
 +
            DNA-DNA
 
             proximity</td>
 
             proximity</td>
        </tr>
+
</tr>
        <tr>
+
<tr>
          <td><a href="https://parts.igem.org/Part:BBa_K5237018" target="_blank">BBa_K5237018</a></td>
+
<td><a href="https://parts.igem.org/Part:BBa_K5237018" target="_blank">BBa_K5237018</a></td>
          <td>Oct1 Binding Casette</td>
+
<td>Oct1 Binding Casette</td>
          <td>DNA sequence containing 12 Oct1 binding motifs, compatible with various assays such as the FRET
+
<td>DNA sequence containing 12 Oct1 binding motifs, compatible with various assays such as the FRET
 
             proximity assay</td>
 
             proximity assay</td>
        </tr>
+
</tr>
        <tr>
+
<tr>
          <td><a href="https://parts.igem.org/Part:BBa_K5237019" target="_blank">BBa_K5237019</a></td>
+
<td><a href="https://parts.igem.org/Part:BBa_K5237019" target="_blank">BBa_K5237019</a></td>
          <td>TetR Binding Cassette</td>
+
<td>TetR Binding Cassette</td>
          <td>DNA sequence containing 12 Oct1 binding motifs, can be used for different assays such as the FRET
+
<td>DNA sequence containing 12 Oct1 binding motifs, can be used for different assays such as the
 +
            FRET
 
             proximity assay</td>
 
             proximity assay</td>
        </tr>
+
</tr>
        <td><a href="https://parts.igem.org/Part:BBa_K5237020" target="_blank">BBa_K5237020</a></td>
+
<td><a href="https://parts.igem.org/Part:BBa_K5237020" target="_blank">BBa_K5237020</a></td>
        <td>Cathepsin B-Cleavable Trans-Activator: NLS-Gal4-GFLG-VP64</td>
+
<td>Cathepsin B-Cleavable <i>Trans</i>-Activator: NLS-Gal4-GFLG-VP64</td>
        <td>Readout system that responds to protease activity. It was used to test cathepsin B-cleavable linker</td>
+
<td>Readout system that responds to protease activity, which was used to test cathepsin B-cleavable linker
 
+
        </td>
        <tr>
+
<tr>
          <td><a href="https://parts.igem.org/Part:BBa_K5237021" target="_blank">BBa_K5237021</a></td>
+
<td><a href="https://parts.igem.org/Part:BBa_K5237021" target="_blank">BBa_K5237021</a></td>
          <td>NLS-Gal4-VP64</td>
+
<td>NLS-Gal4-VP64</td>
          <td>Trans-activating enhancer, that can be used to simulate enhancer hijacking</td>
+
<td><i>Trans</i>-activating enhancer, that can be used to simulate enhancer hijacking</td>
        </tr>
+
</tr>
        <td><a href="https://parts.igem.org/Part:BBa_K5237022" target="_blank">BBa_K5237022</a></td>
+
<td><a href="https://parts.igem.org/Part:BBa_K5237022" target="_blank">BBa_K5237022</a></td>
        <td>mCherry Expression Cassette: UAS, minimal Promotor, mCherry</td>
+
<td>mCherry Expression Cassette: UAS, minimal Promoter, mCherry</td>
        <td>Readout system for enhancer binding. It was used to test cathepsin B-cleavable linker</td>
+
<td>Readout system for enhancer binding, which was used to test cathepsin B-cleavable linker</td>
 
+
<tr>
        <tr>
+
<td><a href="https://parts.igem.org/Part:BBa_K5237023" target="_blank">BBa_K5237023</a></td>
          <td><a href="https://parts.igem.org/Part:BBa_K5237023" target="_blank">BBa_K5237023</a></td>
+
<td>Oct1 - 5x UAS Binding Casette</td>
          <td>Oct1 - 5x UAS binding casette</td>
+
<td>Oct1 and UAS binding cassette, that was used for the simulated enhancer hijacking assay</td>
          <td>Oct1 and UAS binding cassette, that was used for the simulated enhancer hijacking assay</td>
+
</tr>
        </tr>
+
<tr>
        <tr>
+
<td><a href="https://parts.igem.org/Part:BBa_K5237024" target="_blank">BBa_K5237024</a></td>
          <td><a href="https://parts.igem.org/Part:BBa_K5237024" target="_blank">BBa_K5237024</a></td>
+
<td>TRE-minimal Promoter- Firefly Luciferase</td>
          <td>TRE-minimal promoter- firefly luciferase</td>
+
<td>Contains firefly luciferase controlled by a minimal promoter, which was used as a luminescence
          <td>Contains Firefly luciferase controlled by a minimal promoter. It was used as a luminescence readout for
+
            readout for
 
             simulated enhancer hijacking</td>
 
             simulated enhancer hijacking</td>
        </tr>
+
</tr>
      </tbody>
+
</tbody>
    </table>
+
</table></section>
  </section>
+
<section id="1">
  <section id="1">
+
<h1>1. Sequence Overview</h1>
    <h1>1. Sequence overview</h1>
+
</section>
  </section>
+
 
</body>
 
</body>
 
 
</html>
 
</html>
 
<!--################################-->
 
<!--################################-->
Line 337: Line 355:
 
<html>
 
<html>
 
<section id="2">
 
<section id="2">
  <h1>2. Usage and Biology</h1>
+
<h1>2. Usage and Biology</h1>
  <p>Small basic-region leucine zipper (bZip) proteins are characterized by their DNA-binding. The bZip motif
+
<p>Small basic-region leucine zipper (bZip) proteins are characterized by their DNA-binding. The bZip motif
 
     consists of a coiled-coil leucine zipper dimerization domain, and a highly charged basic region that binds to DNA
 
     consists of a coiled-coil leucine zipper dimerization domain, and a highly charged basic region that binds to DNA
 
     (Hollenbeck &amp; Oakley, 2000). One well characterized example is the General Control
 
     (Hollenbeck &amp; Oakley, 2000). One well characterized example is the General Control
     Protein 4 (GCN4), a well-characterized transcriptional activator from yeast (Arndt &amp; Fink, 1986).<br>
+
     Protein 4 (GCN4), a well-characterized transcriptional activator from yeast (Arndt &amp; Fink, 1986).<br/>
 
     At its N-terminus, GCN4 contains basic residues, the so-called bZip domain, through which it binds specifically to
 
     At its N-terminus, GCN4 contains basic residues, the so-called bZip domain, through which it binds specifically to
 
     the CRE (cyclic AMP
 
     the CRE (cyclic AMP
Line 352: Line 370:
 
</section>
 
</section>
 
<section id="3">
 
<section id="3">
  <h1>3. Assembly and part evolution</h1>
+
<h1>3. Assembly and Part Evolution</h1>
  <p>
+
<p>
     The amino acid sequences for GCN4 and rGCN4 were obtained from literature (Hollenbeck et al. 2001), and
+
     The amino acid sequences for GCN4 and rGCN4 were obtained from literature (Hollenbeck <i>et al.</i> 2001), and
 
     codon-optimized for <i>Escherichia coli</i>.
 
     codon-optimized for <i>Escherichia coli</i>.
     The two leucine zipper were combined with a GSG linker harbouring a BamHI site to adapt the construct with different
+
     The two leucine zippers were combined with a GSG linker harbouring a BamHI site to adapt the construct with different
 
     linker designs, based on our dry lab <a href="https://2024.igem.wiki/heidelberg/model" target="_blank">DaVinci</a>
 
     linker designs, based on our dry lab <a href="https://2024.igem.wiki/heidelberg/model" target="_blank">DaVinci</a>
 
     model.
 
     model.
Line 365: Line 383:
 
</section>
 
</section>
 
<section id="4">
 
<section id="4">
  <h1>4. Results</h1>
+
<h1>4. Results</h1>
  <p>
+
<p>
 
     The current version of the part with GCN4-rGCN4 fusion, linked by a GSG peptide linker has not demonstrated any DNA
 
     The current version of the part with GCN4-rGCN4 fusion, linked by a GSG peptide linker has not demonstrated any DNA
 
     binding in the tests conducted thus far.
 
     binding in the tests conducted thus far.
     Nevertheless, we belive the part to still be a valuable addition, as it can be further engineered with different
+
     Nevertheless, we believe the part to still be a valuable addition, as it can be further engineered with different
 
     linker types to
 
     linker types to
 
     create a functioning staple. Through dry lab modelling, various linker designs have already been simulated to
 
     create a functioning staple. Through dry lab modelling, various linker designs have already been simulated to
 
     predict improved dimerization and DNA binding.
 
     predict improved dimerization and DNA binding.
 
   </p>
 
   </p>
  <section id="4.1">
+
<section id="4.1">
    <h2>4.1 Protein Expression and Purification</h2>
+
<h2>4.1 Protein Expression and Purification</h2>
    <p>
+
<p>
 
       The bZip proteins GCN4, rGCN4 and the fusion thereof, bGCN4, were fused N-terminally to a FLAG-tag (DYKDDDDK).
 
       The bZip proteins GCN4, rGCN4 and the fusion thereof, bGCN4, were fused N-terminally to a FLAG-tag (DYKDDDDK).
 
       All proteins could be readily
 
       All proteins could be readily
 
       expressed under the T7 promoter in <i class="”italic”">E. coli</i> BL21 DE3 and purified with Anti-FLAG affinity
 
       expressed under the T7 promoter in <i class="”italic”">E. coli</i> BL21 DE3 and purified with Anti-FLAG affinity
       columns. The purity of the proteins was confirmed by SDS-PAGE (Figure 2).
+
       columns. The purity of the proteins was confirmed by SDS-PAGE (Fig. 2).
 
     </p>
 
     </p>
    <div class="thumb"></div>
+
<div class="thumb"></div>
    <div class="thumbinner" style="width:550px"><img alt="" class="thumbimage"
+
<div class="thumbinner" style="width:550px"><img alt="" class="thumbimage" src="https://static.igem.wiki/teams/5237/wetlab-results/mist-sds-page-expression-validation.svg" style="width:99%;"/>
        src="https://static.igem.wiki/teams/5237/wetlab-results/mist-sds-page-expression-validation.svg"
+
<div class="thumbcaption">
        style="width:99%;" />
+
<i><b>Figure 2: SDS-PAGE Analysis of Protein Purification.</b> Analysis of fractions eluate of purified protein
      <div class="thumbcaption">
+
        <i><b>Figure 2: SDS-PAGE analysis of protein purification.</b>Analysis of fractions eluate of purified protein
+
 
           taken during Anti-FLAG affinity chromatography
 
           taken during Anti-FLAG affinity chromatography
           1 µL of each sample was prepared with Leammli buffer and loaded on 4-15% TGX-Gel. Correct bands of interest
+
           1 µL of each sample was prepared with Laemmli buffer and loaded on 4-15% TGX-Gel. Correct bands of interest
 
           are highlighted by red</i>
 
           are highlighted by red</i>
      </div>
+
</div>
    </div>
+
</div>
  </section>
+
</section>
  <section id="4.2">
+
<section id="4.2">
    <h2>4.2 Electrophoretic Mobility Shift Assay</h2>
+
<h2>4.2 Electrophoretic Mobility Shift Assay</h2>
    <div class="thumb tright">
+
<div class="thumb tright">
      <div class="thumbinner" style="width:310px;">
+
<div class="thumbinner" style="width:310px;">
        <img alt="" class="thumbimage"
+
<img alt="" class="thumbimage" src="https://static.igem.wiki/teams/5237/figures-corrected/leucin-zipper-emsa.svg" style="width:99%;"/>
          src="https://static.igem.wiki/teams/5237/figures-corrected/leucin-zipper-emsa.svg" style="width:99%;" />
+
<div class="thumbcaption">
        <div class="thumbcaption">
+
<i>
          <i>
+
<b>Figure 3: Overview Image of Electrophoretic Mobility Shift Assay (EMSA)</b>
            <b>Figure 3: Overview Image of Electrophoretic Mobility Shift Assay (EMSA)</b>
+
</i>
          </i>
+
</div>
        </div>
+
</div>
      </div>
+
</div>
    </div>
+
<p align="justify">
    <p align="justify">
+
 
     The Electrophoretic mobility shift assay (EMSA) is a widely adopted method used to study DNA-protein
 
     The Electrophoretic mobility shift assay (EMSA) is a widely adopted method used to study DNA-protein
 
     interactions. EMSA functions on the basis that nucleic acids, bound to proteins, have reduced electrophoretic
 
     interactions. EMSA functions on the basis that nucleic acids, bound to proteins, have reduced electrophoretic
Line 414: Line 429:
 
     stoichiometry and kinetics such as the apparent dissociation constant (K<sub>d</sub>) (Fried, 1989).
 
     stoichiometry and kinetics such as the apparent dissociation constant (K<sub>d</sub>) (Fried, 1989).
 
     </p>
 
     </p>
 
+
<section id="4.2.1" style="clear:both;">
    <section id="4.2.1" style="clear:both;">
+
<h2>4.2.1 Qualitative DNA Binding Analysis</h2>
      <h2>4.2.1 Qualitative DNA binding analysis</h2>
+
<p>
      <p>
+
 
         To asses possible DNA binding, a qualitative EMSA was performed with the purified proteins. The same binding
 
         To asses possible DNA binding, a qualitative EMSA was performed with the purified proteins. The same binding
         buffer conditions were used, as previously desribed for GCN4 and rGCN4 (Hollenbeck <i>et al.</i> 2001).
+
         buffer conditions were used, as previously described for GCN4 and rGCN4 (Hollenbeck <i>et al.</i> 2001).
 
         DNA binding could only be shown for GCN4 and rGCN4, no visible band was observed for the bGCN4 fusion protein
 
         DNA binding could only be shown for GCN4 and rGCN4, no visible band was observed for the bGCN4 fusion protein
         (Figure 4).
+
         (Fig. 4).
 
       </p>
 
       </p>
 
+
<p>
      <p>
+
         The EMSA is a widely adopted method used to study DNA-protein
         The Electrophoretic mobility shift assay (EMSA) is a widely adopted method used to study DNA-protein
+
 
         interactions. EMSA functions on the basis that nucleic acids, bound to proteins, have reduced electrophoretic
 
         interactions. EMSA functions on the basis that nucleic acids, bound to proteins, have reduced electrophoretic
 
         mobility, compared to their counterpart. (Hellman &amp; Fried, 2007). Mobility-shift
 
         mobility, compared to their counterpart. (Hellman &amp; Fried, 2007). Mobility-shift
 
         assays can be used both to qualitatively assess DNA binding capabilities or quantitatively to determine binding
 
         assays can be used both to qualitatively assess DNA binding capabilities or quantitatively to determine binding
         stoichiometry and kinetics such as the apparent dissociation constant (K<sub>d</sub>) (Fried, 1989).<br><br>
+
         stoichiometry and kinetics such as the apparent dissociation constant (K<sub>d</sub>) (Fried, 1989).<br/><br/>
  
 
         To analyze the binding DNA affinity an EMSA was performed, in which
 
         To analyze the binding DNA affinity an EMSA was performed, in which
Line 435: Line 448:
 
         sequence (5' ATGACGTCAT 3') or the <i>INVii</i> (rGCN4 binding) sequence (5' GTCAtaTGAC 3') until equilibration.
 
         sequence (5' ATGACGTCAT 3') or the <i>INVii</i> (rGCN4 binding) sequence (5' GTCAtaTGAC 3') until equilibration.
 
         Subsequently the formed protein-DNA complexes were loaded on a native PAGE. Afterwards the DNA bands were
 
         Subsequently the formed protein-DNA complexes were loaded on a native PAGE. Afterwards the DNA bands were
         stained with SYBR-safe. <br>
+
         stained with SYBR-safe. <br/>
 
         The bGCN4 fusion protein did not show any DNA binding for both target sites.  
 
         The bGCN4 fusion protein did not show any DNA binding for both target sites.  
 
       </p>
 
       </p>
 
+
<div class="thumb">
      <div class="thumb">
+
<div class="thumbinner" style="width:550px"><img alt="" class="thumbimage" src="https://static.igem.wiki/teams/5237/wetlab-results/mist-emsa-quali.svg" style="width:99%;"/>
        <div class="thumbinner" style="width:550px"><img alt="" class="thumbimage"
+
<div class="thumbcaption">
            src="https://static.igem.wiki/teams/5237/wetlab-results/mist-emsa-quali.svg" style="width:99%;" />
+
<i><b>Figure 4: Qualitative EMSA DNA Binding</b>
          <div class="thumbcaption">
+
            <i><b>Figure 4: Qualitative EMSA DNA binding</b>
+
 
               0.5 µM of annealed oligos (20 bp) containing either one CRE or INVii binding site were incubated with
 
               0.5 µM of annealed oligos (20 bp) containing either one CRE or INVii binding site were incubated with
 
               200
 
               200
Line 450: Line 461:
 
               and equilibrated in binding buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na <sub>2</sub>HPO<sub>4</sub>, 1.8
 
               and equilibrated in binding buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na <sub>2</sub>HPO<sub>4</sub>, 1.8
 
               mM
 
               mM
               KH<sub>2</sub>HPO<sub>4</sub>, 0.1 % (v/v) IGEPAL® CA-360, 1 mM EDTA). Gel electorphoresis was
+
               KH<sub>2</sub>HPO<sub>4</sub>, 0.1 % (v/v) IGEPAL® CA-360, 1 mM EDTA). Gel electrophoresis was
 
               performed with a pre-equilibrated TGX-Gel in TBE running buffer.
 
               performed with a pre-equilibrated TGX-Gel in TBE running buffer.
 
               Gel-bands were visualized by staining with SYBR Safe and imaged with an UV transilluminator.</i>
 
               Gel-bands were visualized by staining with SYBR Safe and imaged with an UV transilluminator.</i>
          </div>
+
</div>
        </div>
+
</div>
      </div>
+
</div>
    </section>
+
</section>
    <section id="4.2.2">
+
<section id="4.2.2">
      <h2>4.2.2 Quantitative DNA binding analysis</h2>
+
<h2>4.2.2 Quantitative DNA Binding Analysis</h2>
      <p>
+
<p>
 
         To further analyze DNA binding of the staple subunits, quantitative shift assays were performed for GCN4 and rGCN4. Here
 
         To further analyze DNA binding of the staple subunits, quantitative shift assays were performed for GCN4 and rGCN4. Here
 
         0.5 µM DNA were incubated with varying concentrations of protein until equilibration. After
 
         0.5 µM DNA were incubated with varying concentrations of protein until equilibration. After
 
         electrophoresis, bands were stained with SYBR-Safe and quantified based on pixel intensity. The
 
         electrophoresis, bands were stained with SYBR-Safe and quantified based on pixel intensity. The
 
         obtained values were fitted to equation 1, describing formation of a 2:1 protein-DNA complex:
 
         obtained values were fitted to equation 1, describing formation of a 2:1 protein-DNA complex:
         <br /><br />
+
         <br><br>
 
         Θ<sub>app</sub> = Θ<sub>min</sub> + (Θ<sub>max</sub> - Θ<sub>min</sub>) ×
 
         Θ<sub>app</sub> = Θ<sub>min</sub> + (Θ<sub>max</sub> - Θ<sub>min</sub>) ×
 
         (K<sub>a</sub><sup>2</sup> [L]<sub>tot</sub><sup>2</sup>) / (1 + K<sub>a</sub><sup>2</sup>
 
         (K<sub>a</sub><sup>2</sup> [L]<sub>tot</sub><sup>2</sup>) / (1 + K<sub>a</sub><sup>2</sup>
 
         [L]<sub>tot</sub><sup>2</sup>)
 
         [L]<sub>tot</sub><sup>2</sup>)
 
         <span style="float: right;">Equation 1</span>
 
         <span style="float: right;">Equation 1</span>
        <br /><br />
+
<br><br>
 
         Here [L]<sub>tot</sub> describes the total protein monomer concentration, K<sub>a</sub>
 
         Here [L]<sub>tot</sub> describes the total protein monomer concentration, K<sub>a</sub>
 
         corresponds
 
         corresponds
Line 476: Line 487:
 
         determined site saturation values (For this experiment 0 and 1 were chosen for min and max
 
         determined site saturation values (For this experiment 0 and 1 were chosen for min and max
 
         respectively).  
 
         respectively).  
       </p>
+
       </br></br></br></br></p>
      <div class="thumb">
+
<div class="thumb">
        <div class="thumbinner" style="width:550px"><img alt="" class="thumbimage"
+
<div class="thumbinner" style="width:550px"><img alt="" class="thumbimage" src="https://static.igem.wiki/teams/5237/wetlab-results/mist-leucine-zipper-kd-plot.svg" style="width:99%;"/>
            src="https://static.igem.wiki/teams/5237/wetlab-results/mist-leucine-zipper-kd-plot.svg"
+
<div class="thumbcaption">
            style="width:99%;" />
+
<i><b>Figure 5: K<sub>d</sub> Calculation of GCN4 and rGCN4</b>
          <div class="thumbcaption">
+
            <i><b>Figure 5: K<sub>d</sub> Calculation of GCN4 and rGCN4</b>
+
 
               Quantitative assessment of binding affinity for GCN4 and rGCN4. Proteins of varying
 
               Quantitative assessment of binding affinity for GCN4 and rGCN4. Proteins of varying
 
               concentrations were incubated with 0.5 µM DNA (20 bp, containing one binding site for CRE or INVii) in
 
               concentrations were incubated with 0.5 µM DNA (20 bp, containing one binding site for CRE or INVii) in
Line 491: Line 500:
 
               least three separate measurements were conducted for each data point. Values are presented as mean +/-
 
               least three separate measurements were conducted for each data point. Values are presented as mean +/-
 
               SD</i>
 
               SD</i>
          </div>
+
</div>
        </div>
+
</div>
      </div>
+
</div>
    <p>
+
<p>
 
       GCN4 binds to its optimal DNA binding motif with an apparent dissociation
 
       GCN4 binds to its optimal DNA binding motif with an apparent dissociation
       constant K<sub>D</sub> of (0.293 &#177; 0.033) × 10<sup>-6</sup> M, which is almost identical to the
+
       constant K<sub>D</sub> of (0.293 ± 0.033) × 10<sup>-6</sup> M, which is almost identical to the
 
       rGCN4 dissociation constant
 
       rGCN4 dissociation constant
       to INVii a K<sub>D</sub> of (0.298 &#177; 0.030) × 10<sup>-6</sup> M.
+
       to INVii a K<sub>D</sub> of (0.298 ± 0.030) × 10<sup>-6</sup> M.
 
       Comparing them to literature values, our dissociation constants are approximately a factor 10 higher then those
 
       Comparing them to literature values, our dissociation constants are approximately a factor 10 higher then those
       described in literature ((96) × 10<sup>-8</sup> M for
+
       described in literature ((9±6) × 10<sup>-8</sup> M for
       GCN4 and (2.90.8) × 10<sup>-8</sup> M for rGCN4) (Hollenbeck <i class="italic">et al.</i>, 2001). The
+
       GCN4 and (2.9 ± 0.8) × 10<sup>-8</sup> M for rGCN4) (Hollenbeck <i class="italic">et al.</i>, 2001). The
 
       differences could be due to the lower sensitivity of SYBR-Safe staining compared to radio-labeled oligos.
 
       differences could be due to the lower sensitivity of SYBR-Safe staining compared to radio-labeled oligos.
 
       Most likely, the protein concentration was miscalculated due to the presence of additional (lower intensity)
 
       Most likely, the protein concentration was miscalculated due to the presence of additional (lower intensity)
 
       bands in
 
       bands in
 
       the SDS-PAGE analysis, indicating co-purification of small amounts of unspecific proteins.
 
       the SDS-PAGE analysis, indicating co-purification of small amounts of unspecific proteins.
       <br /><br />
+
       <br/><br/>
 
       The FLAG-tag fusion to the N-terminus of proteins could potentially decrease binding affinity, likely due
 
       The FLAG-tag fusion to the N-terminus of proteins could potentially decrease binding affinity, likely due
 
       to steric hindrance affecting the interaction with DNA. Interestingly, the differences in binding affinity
 
       to steric hindrance affecting the interaction with DNA. Interestingly, the differences in binding affinity
Line 516: Line 525:
 
       with circular dichroism spectroscopy (Greenfield, 2006).  
 
       with circular dichroism spectroscopy (Greenfield, 2006).  
 
     </p>
 
     </p>
    </section>
+
</section>
    <section id="4.3">
+
<section id="4.3">
      <h2>4.3 <i>In Silico</i> Characterization using DaVinci</h2>
+
<h2>4.3 <i>In Silico</i> Characterization Using DaVinci</h2>
      <p>
+
<div class="thumb tright" style="margin:0;">
        We developed the in silico model <a href="https://2024.igem.wiki/heidelberg/model" target="_blank">DaVinci</a>
+
<div class="thumbinner" style="width:300px;">
         for rapid engineering
+
<iframe allowfullscreen="" class="thumbimage" frameborder="0" height="315" sandbox="allow-same-origin allow-scripts allow-popups allow-forms" src="https://video.igem.org/videos/embed/a00b62a2-3330-4a5a-85ee-f6ed8fc361d4?loop=1&amp;title=0&amp;warningTitle=0" style="width:99%;" title="Heidelberg: bGCN4-MD (2024)" width="560"></iframe>
        and development of our PiCasSO system.
+
<div class="thumbcaption">
         DaVinci acts as a digital twin to PiCasSO, designed to understand the forces acting on our system,
+
<i><b>Figure 6: Molecular Dynamics Simulation of GCN4</b>
        refine experimental parameters, and find optimal connections between protein staples and target DNA.
+
</i></div>
         We calibrated DaVinci with literature and our own experimental affinity data obtained via EMSA assays and
+
</div>
        purified proteins.<br>
+
</div>
         DaVinci is divided into three phases: static structure prediction, all-atom dynamics simulation, and long-ranged
+
<p>
         dna dynamics simulation. We applied the first two to our parts, characterizing structure and dynamics of the
+
         We developed DaVinci, an <i>in silico</i> model, for rapid engineering and optimization of our PICasSO system. DaVinci
        dna-binding
+
         serves as a digital twin to PICasSO, analyzing the forces acting on the system, refining experimental parameters,
        interaction.
+
        and identifying optimal interactions between protein staples and target DNA. The model was calibrated using
      </p>
+
         literature data and experimental affinity results from rGCN4 EMSA assays with purified proteins.
      <p>
+
        <br/>
         In our efforts to create a bivalent DNA binding protein with minimal size, we created a Mini staple (<a
+
         DaVinci operates in three phases: static structure prediction, all-atom dynamics simulation, and long-range DNA
          href="https://parts.igem.org/Part:BBa_K5237009" target="_blank">BBa_K5237009</a>)
+
         dynamics simulation. We applied the first two phases to our components, allowing us to characterize the structure
         consisting of
+
        and dynamics of the DNA-binding interactions.
        GCN4 fused with an GSG-linker to rGCN4 (<a href="https://parts.igem.org/Part:BBa_K5237008"
+
        <br/>
          target="_blank">BBa_K5237008</a>). The structure and binding affinity of GCN4 were predicted and calculated.
+
         For our bivalent DNA-binding Mini staple (<a href="https://parts.igem.org/Part:BBa_K5237009">BBa_K5237009</a>),
         Furthermore different possible linkers were tested.<br>
+
         consisting of GCN4 fused via a GSG-linker to rGCN4
         We assessed the flexibility and rigidity of our constructs through the pLDDT values assigned
+
        (<a href="https://parts.igem.org/Part:BBa_K5237008">BBa_K5237008</a>), we predicted the structure and binding
        during the predictions. Figure 2 illustrates the variation in linkers using the ('GGGGS')<sub>n</sub>
+
         affinity and tested various linker options. We evaluated
        sequence for flexible linkers and the ('EAAAK')<sub>n</sub> sequence for rigid linkers (Arai <i>et al.</i>, 2001).
+
         the flexibility and rigidity of the constructs using pLDDT values from the predictions. Flexible linkers, like
         The predictions are colored by their pLDDT scores, which act as a surrogate measure of
+
        ('GGGGS')n, and rigid linkers, like ('EAAAK')n, were assessed (Arai <i>et al.</i>, 2001). Predictions were colored by
        chain rigidity (Akdel <i>et al.</i>, 2022; Guo <i>et al.</i>, 2022). The construct C (Fig. 5) was tested as part
+
         pLDDT scores, providing insights into chain rigidity (Akdel <i>et al.</i>, 2022; Guo <i>et al.</i>, 2022). Construct C (Fig. 5)
        BBa_K5237009 in the Wetlab - it did not bind DNA on any side as the structure is to rigid
+
        was tested in the wet lab as part of BBa_K5237009, but it failed to bind DNA due to excessive rigidity, which
         which hinders dimerisation of the two subunits
+
         inhibited subunit dimerization.
 
+
 
       </p>
 
       </p>
      <div class="thumb">
+
<div class="thumb">
        <div class="thumbinner" style="width:80%;">
+
<div class="thumbinner" style="width:80%;">
          <img alt="" class="thumbimage"
+
<img alt="" class="thumbimage" src="https://static.igem.wiki/teams/5237/model/structure-figure-3.svg" style="width:99%;"/>
            src="https://static.igem.wiki/teams/5237/model/structure-figure-3.svg" style="width:99%;" />
+
<div class="thumbcaption">
          <div class="thumbcaption">
+
<i><b>Figure 6: Variation of Linkers Connecting Our Mini Staples.</b>
            <i><b>Figure 5: Variation of linkers connecting our mini staples.</b>
+
 
               Panels A (BBa_K5237007) and B (BBa_K5237008) show
 
               Panels A (BBa_K5237007) and B (BBa_K5237008) show
               orientations of the leucine zipper, each bound to DNA. Panels C to I display linker variations colored by their pLDDT
+
               orientations of the leucine zipper, each bound to DNA. Panels C to I display linker variations colored by
               confidence score, which serves as a surrogate for chain flexibility (Akdel et al., 2022). Note that panels H and I are
+
              their pLDDT
               not bound to the second DNA strand. All structures were predicted using the AlphaFold server (Google DeepMind,
+
               confidence score, which serves as a surrogate for chain flexibility (Akdel <i>et al.</i>, 2022). Note that panels H
 +
              and I are
 +
               not bound to the second DNA strand. All structures were predicted using the AlphaFold server (Google
 +
              DeepMind,
 
               2024).
 
               2024).
 
             </i>
 
             </i>
          </div>
+
</div>
        </div>
+
</div>
      </div>
+
</div>
 
+
</section>
    </section>
+
</section>
  </section>
+
<section id="5">
  <section id="5">
+
<h1>5. References</h1>
    <h1>5. References</h1>
+
<p>Akdel, M., Pires, D. E. V., Pardo, E. P., Janes, J., Zalevsky, A. O., Meszaros, B., Bryant, P., Good, L. L.,
    <p>Akdel, M., Pires, D. E. V., Pardo, E. P., Janes, J., Zalevsky, A. O., Meszaros, B., Bryant, P., Good, L. L.,
+
 
       Laskowski, R. A., Pozzati, G., Shenoy, A., Zhu, W., Kundrotas, P., Serra, V. R., Rodrigues, C. H. M., Dunham, A.
 
       Laskowski, R. A., Pozzati, G., Shenoy, A., Zhu, W., Kundrotas, P., Serra, V. R., Rodrigues, C. H. M., Dunham, A.
 
       S., Burke, D., Borkakoti, N., Velankar, S., … Beltrao, P. (2022). A structural biology community assessment of
 
       S., Burke, D., Borkakoti, N., Velankar, S., … Beltrao, P. (2022). A structural biology community assessment of
       AlphaFold2 applications. <i>Nat Struct Mol Biol</i>, 29(11), 1056–1067. <a
+
       AlphaFold2 applications. <i>Nat Struct Mol Biol</i>, 29(11), 1056–1067. <a href="https://doi.org/10.1038/s41594-022-00849-w" target="_blank">https://doi.org/10.1038/s41594-022-00849-w</a>
        href="https://doi.org/10.1038/s41594-022-00849-w" target="_blank">https://doi.org/10.1038/s41594-022-00849-w</a>
+
</p>
    </p>
+
<p>Arai, R., Ueda, H., Kitayama, A., Kamiya, N., &amp; Nagamune, T. (2001). Design of the linkers which effectively
    <p>Arai, R., Ueda, H., Kitayama, A., Kamiya, N., &amp; Nagamune, T. (2001). Design of the linkers which effectively
+
 
       separate domains of a bifunctional fusion protein. <i>Protein Engineering, Design and Selection</i>, 14(8),
 
       separate domains of a bifunctional fusion protein. <i>Protein Engineering, Design and Selection</i>, 14(8),
       529–532. <a href="https://doi.org/10.1093/protein/14.8.529"
+
       529–532. <a href="https://doi.org/10.1093/protein/14.8.529" target="_blank">https://doi.org/10.1093/protein/14.8.529</a></p>
        target="_blank">https://doi.org/10.1093/protein/14.8.529</a></p>
+
<p>Arndt, K., &amp; Fink, G. R. (1986). GCN4 protein, a positive transcription factor in yeast, binds general
    <p>Arndt, K., &amp; Fink, G. R. (1986). GCN4 protein, a positive transcription factor in yeast, binds general
+
 
       control promoters at all 5’ TGACTC 3’ sequences. <em>Proceedings of the National Academy of Sciences,
 
       control promoters at all 5’ TGACTC 3’ sequences. <em>Proceedings of the National Academy of Sciences,
 
         83</em>(22),
 
         83</em>(22),
       8516–8520. <a href="https://doi.org/10.1073/pnas.83.22.8516"
+
       8516–8520. <a href="https://doi.org/10.1073/pnas.83.22.8516" target="_blank">https://doi.org/10.1073/pnas.83.22.8516</a></p>
        target="_blank">https://doi.org/10.1073/pnas.83.22.8516</a></p>
+
<p>Chen, X., Zaro, J. L., &amp; Shen, W.-C. (2013). Fusion protein linkers: Property, design and functionality.
    <p>Chen, X., Zaro, J. L., &amp; Shen, W.-C. (2013). Fusion protein linkers: Property, design and functionality.
+
       <i>Advanced Drug Delivery Reviews</i>, 65(10), 1357–1369. <a href="https://doi.org/10.1016/j.addr.2012.09.039" target="_blank">https://doi.org/10.1016/j.addr.2012.09.039</a>
       <i>Advanced Drug Delivery Reviews</i>, 65(10), 1357–1369. <a href="https://doi.org/10.1016/j.addr.2012.09.039"
+
</p>
        target="_blank">https://doi.org/10.1016/j.addr.2012.09.039</a>
+
<p>Google DeepMind. (2024). AlphaFold Server. <a href="https://alphafoldserver.com/terms" target="_blank">https://alphafoldserver.com/terms</a></p>
    </p>
+
<p>Guo, H.-B., Perminov, A., Bekele, S., Kedziora, G., Farajollahi, S., Varaljay, V., Hinkle, K., Molinero, V.,
    <p>Google DeepMind. (2024). AlphaFold Server. <a href="https://alphafoldserver.com/terms"
+
        target="_blank">https://alphafoldserver.com/terms</a></p>
+
    <p>Guo, H.-B., Perminov, A., Bekele, S., Kedziora, G., Farajollahi, S., Varaljay, V., Hinkle, K., Molinero, V.,
+
 
       Meister, K., Hung, C., Dennis, P., Kelley-Loughnane, N., &amp; Berry, R. (2022). AlphaFold2 models indicate that
 
       Meister, K., Hung, C., Dennis, P., Kelley-Loughnane, N., &amp; Berry, R. (2022). AlphaFold2 models indicate that
       protein sequence determines both structure and dynamics. <i>Scientific Reports</i>, 12(1), 10696. <a
+
       protein sequence determines both structure and dynamics. <i>Scientific Reports</i>, 12(1), 10696. <a href="https://doi.org/10.1038/s41598-022-14382-9" target="_blank">https://doi.org/10.1038/s41598-022-14382-9</a>
        href="https://doi.org/10.1038/s41598-022-14382-9" target="_blank">https://doi.org/10.1038/s41598-022-14382-9</a>
+
</p>
    </p>
+
<p>Ellenberger, T. E., Brandl, C. J., Struhl, K., &amp; Harrison, S. C. (1992). The GCN4 basic region leucine
    <p>Ellenberger, T. E., Brandl, C. J., Struhl, K., &amp; Harrison, S. C. (1992). The GCN4 basic region leucine
+
 
       zipper
 
       zipper
 
       binds DNA as a dimer of uninterrupted alpha helices: Crystal structure of the protein-DNA complex. <em>Cell,
 
       binds DNA as a dimer of uninterrupted alpha helices: Crystal structure of the protein-DNA complex. <em>Cell,
         71</em>(7), 1223–1237. <a href="https://doi.org/10.1016/s0092-8674(05)80070-4"
+
         71</em>(7), 1223–1237. <a href="https://doi.org/10.1016/s0092-8674(05)80070-4" target="_blank">https://doi.org/10.1016/s0092-8674(05)80070-4</a></p>
        target="_blank">https://doi.org/10.1016/s0092-8674(05)80070-4</a></p>
+
<p>Hollenbeck, J. J., Gurnon, D. G., Fazio, G. C., Carlson, J. J., &amp; Oakley, M. G. (2001). A GCN4 Variant with
    <p>Hollenbeck, J. J., Gurnon, D. G., Fazio, G. C., Carlson, J. J., &amp; Oakley, M. G. (2001). A GCN4 Variant with
+
 
       a
 
       a
 
       C-Terminal Basic Region Binds to DNA with Wild-Type Affinity. <em>Biochemistry, 40</em>(46), 13833–13839.</p>
 
       C-Terminal Basic Region Binds to DNA with Wild-Type Affinity. <em>Biochemistry, 40</em>(46), 13833–13839.</p>
    <p>Hollenbeck, J. J., McClain, D. L., &amp; Oakley, M. G. (2002). The role of helix stabilizing residues in GCN4
+
<p>Hollenbeck, J. J., McClain, D. L., &amp; Oakley, M. G. (2002). The role of helix stabilizing residues in GCN4
       basic region folding and DNA binding. <em>Protein Science, 11</em>(11), 2740–2747. <a
+
       basic region folding and DNA binding. <em>Protein Science, 11</em>(11), 2740–2747. <a href="https://doi.org/10.1110/ps.0211102" target="_blank">https://doi.org/10.1110/ps.0211102</a></p>
        href="https://doi.org/10.1110/ps.0211102" target="_blank">https://doi.org/10.1110/ps.0211102</a></p>
+
<p>Hollenbeck, J. J., &amp; Oakley, M. G. (2000). GCN4 Binds with High Affinity to DNA Sequences Containing a
    <p>Hollenbeck, J. J., &amp; Oakley, M. G. (2000). GCN4 Binds with High Affinity to DNA Sequences Containing a
+
 
       Single
 
       Single
       Consensus Half-Site. <em>Biochemistry, 39</em>(21), 6380–6389. <a href="https://doi.org/10.1021/bi992705n"
+
       Consensus Half-Site. <em>Biochemistry, 39</em>(21), 6380–6389. <a href="https://doi.org/10.1021/bi992705n" target="_blank">https://doi.org/10.1021/bi992705n</a></p>
        target="_blank">https://doi.org/10.1021/bi992705n</a></p>
+
<p>Liu, J., Zheng, Q., Deng, Y., Cheng, C.-S., Kallenbach, N. R., &amp; Lu, M. (2006). A seven-helix coiled coil.
    <p>Liu, J., Zheng, Q., Deng, Y., Cheng, C.-S., Kallenbach, N. R., &amp; Lu, M. (2006). A seven-helix coiled coil.
+
       <em>Proceedings of the National Academy of Sciences, 103</em>(42), 15457–15462. <a href="https://doi.org/10.1073/pnas.0604871103" target="_blank">https://doi.org/10.1073/pnas.0604871103</a>
       <em>Proceedings of the National Academy of Sciences, 103</em>(42), 15457–15462. <a
+
</p>
        href="https://doi.org/10.1073/pnas.0604871103" target="_blank">https://doi.org/10.1073/pnas.0604871103</a>
+
<p>Lupas, A. N., Bassler, J., &amp; Dunin-Horkawicz, S. (2017). The Structure and Topology of α-Helical Coiled
    </p>
+
       Coils. <em>Fibrous Proteins: Structures and Mechanisms, 82</em>, 95–129. <a href="https://doi.org/10.1007/978-3-319-49674-0_4" target="_blank">https://doi.org/10.1007/978-3-319-49674-0_4</a></p>
    <p>Lupas, A. N., Bassler, J., &amp; Dunin-Horkawicz, S. (2017). The Structure and Topology of α-Helical Coiled
+
<p>Woolfson, D. N. (2023). Understanding a protein fold: The physics, chemistry, and biology of α-helical coiled
       Coils. <em>Fibrous Proteins: Structures and Mechanisms, 82</em>, 95–129. <a
+
       coils. <em>Journal of Biological Chemistry, 299</em>(4), 104579. <a href="https://doi.org/10.1016/j.jbc.2023.104579" target="_blank">https://doi.org/10.1016/j.jbc.2023.104579</a>
        href="https://doi.org/10.1007/978-3-319-49674-0_4"
+
</p>
        target="_blank">https://doi.org/10.1007/978-3-319-49674-0_4</a></p>
+
</section>
    <p>Woolfson, D. N. (2023). Understanding a protein fold: The physics, chemistry, and biology of α-helical coiled
+
       coils. <em>Journal of Biological Chemistry, 299</em>(4), 104579. <a
+
        href="https://doi.org/10.1016/j.jbc.2023.104579" target="_blank">https://doi.org/10.1016/j.jbc.2023.104579</a>
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Latest revision as of 12:35, 2 October 2024

BBa_K5237009

Mini Staple: bGCN4

The bGCN4 Mini staple is a fusion of the basic leucine zipper GCN4 and rGCN4, offering a smaller, compact protein staple. With its well-characterized subunits and strong in silico and experimental validation, this Mini staple serves as a versatile foundation for expanding to similar staples.



The PICasSO Toolbox
Figure 1: How Our Part Collection can be Used to Engineer New Staples


While synthetic biology has in the past focused on engineering the genomic sequence of organisms, the 3D spatial organization of DNA is well-known to be an important layer of information encoding in particular in eukaryotes, playing a crucial role in gene regulation and hence cell fate, disease development, evolution, and more. However, tools to precisely manipulate and control the genomic spatial architecture are limited, hampering the exploration of 3D genome engineering in synthetic biology. We - the iGEM Team Heidelberg 2024 - have developed PICasSO, a powerful molecular toolbox for rationally engineering genome 3D architectures in living cells, based on various DNA-binding proteins.

The PICasSO part collection offers a comprehensive, modular platform for precise manipulation and re-programming of DNA-DNA interactions using engineered "protein staples" in living cells. This enables researchers to recreate naturally occurring alterations of 3D genomic interactions, such as enhancer hijacking in cancer, or to design entirely new spatial architectures for artificial gene regulation and cell function control. Specifically, the fusion of two DNA binding proteins enables to artificially bring otherwise distant genomic loci into spatial proximity. To unlock the system's full potential, we introduce versatile chimeric CRISPR/Cas complexes, connected either at the protein or - in the case of CRISPR/Cas-based DNA binding moieties - the guide RNA level. These complexes are referred to as protein- or Cas staples, respectively. Beyond its versatility with regard to the staple constructs themselves, PICasSO includes robust assay systems to support the engineering, optimization, and testing of new staples in vitro and in vivo. Notably, the PICasSO toolbox was developed in a design-build-test-learn engineering cycle closely intertwining wet lab experiments and computational modeling and iterated several times, yielding a collection of well-functioning and -characterized parts.

At its heart, the PICasSO part collection consists of three categories.
(i) Our DNA-binding proteins include our finalized Cas staple experimentally validated using an artificial "enhancer hijacking" system as well as "half staples" that can be combined by scientists to compose entirely new Cas staples in the future. We also include our Simple staples comprised of particularly small, simple and robust DNA binding domains well-known to the synthetic biology community, which serve as controls for successful stapling and can be further engineered to create alternative, simpler, and more compact staples.
(ii) As functional elements, we list additional parts that enhance and expand the functionality of our Cas and Basic staples. These consist of staples dependent on cleavable peptide linkers targeted by cancer-specific proteases or inteins that allow condition-specific, dynamic stapling in vivo. We also include several engineered parts that enable the efficient delivery of PICasSO's constructs into target cells, including mammalian cells, with our new interkingdom conjugation system.
(iii) As the final category of our collection, we provide parts that underlie our custom readout systems. These include components of our established FRET-based proximity assay system, enabling users to confirm accurate stapling. Additionally, we offer a complementary, application-oriented testing system based on a luciferase reporter, which allows for straightforward experimental assessment of functional enhancer hijacking events in mammalian cells.

The following table gives a comprehensive overview of all parts in our PICasSO toolbox. The highlighted parts showed exceptional performance as described on our iGEM wiki and can serve as a reference. The other parts in the collection are versatile building blocks designed to provide future iGEMers with the flexibility to engineer their own custom Cas staples, enabling further optimization and innovation in the new field of 3D genome engineering.

Our part collection includes:

DNA-Binding Proteins: Modular building blocks for engineering of custom staples to mediate defined DNA-DNA interactions in vivo
BBa_K5237000 Fusion Guide RNA Entry Vector MbCas12a-SpCas9 Entry vector for simple fgRNA cloning via SapI
BBa_K5237001 Staple Subunit: dMbCas12a-Nucleoplasmin NLS Staple subunit that can be combined with crRNA or fgRNA and dSpCas9 to form a functional staple
BBa_K5237002 Staple Subunit: SV40 NLS-dSpCas9-SV40 NLS Staple subunit that can be combined with a sgRNA or fgRNA and dMbCas12a to form a functional staple
BBa_K5237003 Cas Staple: SV40 NLS-dMbCas12a-dSpCas9-Nucleoplasmin NLS Functional Cas staple that can be combined with sgRNA and crRNA or fgRNA to bring two DNA strands into close proximity
BBa_K5237004 Staple Subunit: Oct1-DBD Staple subunit that can be combined to form a functional staple, for example with TetR.
Can also be combined with a fluorescent protein as part of the FRET proximity assay
BBa_K5237005 Staple Subunit: TetR Staple subunit that can be combined to form a functional staple, for example with Oct1.
Can also be combined with a fluorescent protein as part of the FRET proximity assay
BBa_K5237006 Simple Staple: TetR-Oct1 Functional staple that can be used to bring two DNA strands in close proximity
BBa_K5237007 Staple Subunit: GCN4 Staple subunit that can be combined to form a functional staple, for example with rGCN4
BBa_K5237008 Staple Subunit: rGCN4 Staple subunit that can be combined to form a functional staple, for example with rGCN4
BBa_K5237009 Mini Staple: bGCN4 Assembled staple with minimal size that can be further engineered
Functional Elements: Protease-cleavable peptide linkers and inteins are used to control and modify staples for further optimization for custom applications
BBa_K5237010 Cathepsin B-cleavable Linker: GFLG Cathepsin B-cleavable peptide linker that can be used to combine two staple subunits to make responsive staples
BBa_K5237011 Cathepsin B Expression Cassette Expression cassette for the overexpression of cathepsin B
BBa_K5237012 Caged NpuN Intein A caged NpuN split intein fragment that undergoes protein trans-splicing after protease activation, which can be used to create functionalized staple subunits
BBa_K5237013 Caged NpuC Intein A caged NpuC split intein fragment that undergoes protein trans-splicing after protease activation, which can be used to create functionalized staple subunits
BBa_K5237014 Fusion Guide RNA Processing Casette Processing cassette to produce multiple fgRNAs from one transcript, that can be used for multiplexed 3D genome reprogramming
BBa_K5237015 Intimin anti-EGFR Nanobody Interkingdom conjugation between bacteria and mammalian cells, as an alternative delivery tool for large constructs
BBa_K4643003 IncP Origin of Transfer Origin of transfer that can be cloned into the plasmid vector and used for conjugation as a means of delivery
Readout Systems: FRET and enhancer recruitment readout systems to rapidly assess successful DNA stapling in bacterial and mammalian cells
BBa_K5237016 FRET-Donor: mNeonGreen-Oct1 FRET donor-fluorophore fused to Oct1-DBD that binds to the Oct1 binding cassette, which can be used to visualize DNA-DNA proximity
BBa_K5237017 FRET-Acceptor: TetR-mScarlet-I Acceptor part for the FRET assay binding the TetR binding cassette, which can be used to visualize DNA-DNA proximity
BBa_K5237018 Oct1 Binding Casette DNA sequence containing 12 Oct1 binding motifs, compatible with various assays such as the FRET proximity assay
BBa_K5237019 TetR Binding Cassette DNA sequence containing 12 Oct1 binding motifs, can be used for different assays such as the FRET proximity assay
BBa_K5237020 Cathepsin B-Cleavable Trans-Activator: NLS-Gal4-GFLG-VP64 Readout system that responds to protease activity, which was used to test cathepsin B-cleavable linker
BBa_K5237021 NLS-Gal4-VP64 Trans-activating enhancer, that can be used to simulate enhancer hijacking
BBa_K5237022 mCherry Expression Cassette: UAS, minimal Promoter, mCherry Readout system for enhancer binding, which was used to test cathepsin B-cleavable linker
BBa_K5237023 Oct1 - 5x UAS Binding Casette Oct1 and UAS binding cassette, that was used for the simulated enhancer hijacking assay
BBa_K5237024 TRE-minimal Promoter- Firefly Luciferase Contains firefly luciferase controlled by a minimal promoter, which was used as a luminescence readout for simulated enhancer hijacking

1. Sequence Overview

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 175
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

2. Usage and Biology

Small basic-region leucine zipper (bZip) proteins are characterized by their DNA-binding. The bZip motif consists of a coiled-coil leucine zipper dimerization domain, and a highly charged basic region that binds to DNA (Hollenbeck & Oakley, 2000). One well characterized example is the General Control Protein 4 (GCN4), a well-characterized transcriptional activator from yeast (Arndt & Fink, 1986).
At its N-terminus, GCN4 contains basic residues, the so-called bZip domain, through which it binds specifically to the CRE (cyclic AMP response element) DNA sequence (5' ATGACGTCAT 3') (Hollenbeck et al., 2002). A variant of GCN4 with the DNA binding bZip-domain at the C-terminus (rGCN4) has been engineered to bind to the inverted CRE sequence, INV2 (5' GTCAtaTGAC 3', upper case letters indicate direct interaction between protein and DNA) with similar affinity (Hollenbeck et al., 2001). By genetically fusing GCN4 to rGCN4, we created a small, bivalent DNA binding staple with less than 150 amino acids.

3. Assembly and Part Evolution

The amino acid sequences for GCN4 and rGCN4 were obtained from literature (Hollenbeck et al. 2001), and codon-optimized for Escherichia coli. The two leucine zippers were combined with a GSG linker harbouring a BamHI site to adapt the construct with different linker designs, based on our dry lab DaVinci model. A FLAG-tag (DYKDDDDK) was added to the N-terminus for protein purification. The FLAG-tag can be cleaved off using an Enterokinase, if necessary. Expression of FLAG-GCN4 was done under an IPTG inducible T7 promoter in E. coli BL21 (DE3) cells.

4. Results

The current version of the part with GCN4-rGCN4 fusion, linked by a GSG peptide linker has not demonstrated any DNA binding in the tests conducted thus far. Nevertheless, we believe the part to still be a valuable addition, as it can be further engineered with different linker types to create a functioning staple. Through dry lab modelling, various linker designs have already been simulated to predict improved dimerization and DNA binding.

4.1 Protein Expression and Purification

The bZip proteins GCN4, rGCN4 and the fusion thereof, bGCN4, were fused N-terminally to a FLAG-tag (DYKDDDDK). All proteins could be readily expressed under the T7 promoter in E. coli BL21 DE3 and purified with Anti-FLAG affinity columns. The purity of the proteins was confirmed by SDS-PAGE (Fig. 2).

Figure 2: SDS-PAGE Analysis of Protein Purification. Analysis of fractions eluate of purified protein taken during Anti-FLAG affinity chromatography 1 µL of each sample was prepared with Laemmli buffer and loaded on 4-15% TGX-Gel. Correct bands of interest are highlighted by red

4.2 Electrophoretic Mobility Shift Assay

Figure 3: Overview Image of Electrophoretic Mobility Shift Assay (EMSA)

The Electrophoretic mobility shift assay (EMSA) is a widely adopted method used to study DNA-protein interactions. EMSA functions on the basis that nucleic acids, bound to proteins, have reduced electrophoretic mobility, compared to their counterpart. (Hellman & Fried, 2007). Mobility-shift assays can both be used to qualitatively assess DNA binding capabilities or quantitatively to determine binding stoichiometry and kinetics such as the apparent dissociation constant (Kd) (Fried, 1989).

4.2.1 Qualitative DNA Binding Analysis

To asses possible DNA binding, a qualitative EMSA was performed with the purified proteins. The same binding buffer conditions were used, as previously described for GCN4 and rGCN4 (Hollenbeck et al. 2001). DNA binding could only be shown for GCN4 and rGCN4, no visible band was observed for the bGCN4 fusion protein (Fig. 4).

The EMSA is a widely adopted method used to study DNA-protein interactions. EMSA functions on the basis that nucleic acids, bound to proteins, have reduced electrophoretic mobility, compared to their counterpart. (Hellman & Fried, 2007). Mobility-shift assays can be used both to qualitatively assess DNA binding capabilities or quantitatively to determine binding stoichiometry and kinetics such as the apparent dissociation constant (Kd) (Fried, 1989).

To analyze the binding DNA affinity an EMSA was performed, in which bGCN4 was incubated in binding buffer with a 20 bp DNA probe containing the either the CRE (GCN4 binding) sequence (5' ATGACGTCAT 3') or the INVii (rGCN4 binding) sequence (5' GTCAtaTGAC 3') until equilibration. Subsequently the formed protein-DNA complexes were loaded on a native PAGE. Afterwards the DNA bands were stained with SYBR-safe.
The bGCN4 fusion protein did not show any DNA binding for both target sites.

Figure 4: Qualitative EMSA DNA Binding 0.5 µM of annealed oligos (20 bp) containing either one CRE or INVii binding site were incubated with 200 µM of protein and equilibrated in binding buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na 2HPO4, 1.8 mM KH2HPO4, 0.1 % (v/v) IGEPAL® CA-360, 1 mM EDTA). Gel electrophoresis was performed with a pre-equilibrated TGX-Gel in TBE running buffer. Gel-bands were visualized by staining with SYBR Safe and imaged with an UV transilluminator.

4.2.2 Quantitative DNA Binding Analysis

To further analyze DNA binding of the staple subunits, quantitative shift assays were performed for GCN4 and rGCN4. Here 0.5 µM DNA were incubated with varying concentrations of protein until equilibration. After electrophoresis, bands were stained with SYBR-Safe and quantified based on pixel intensity. The obtained values were fitted to equation 1, describing formation of a 2:1 protein-DNA complex:

Θapp = Θmin + (Θmax - Θmin) × (Ka2 [L]tot2) / (1 + Ka2 [L]tot2) Equation 1

Here [L]tot describes the total protein monomer concentration, Ka corresponds to the apparent monomeric equilibration constant. The Θmin/max values are the experimentally determined site saturation values (For this experiment 0 and 1 were chosen for min and max respectively).



Figure 5: Kd Calculation of GCN4 and rGCN4 Quantitative assessment of binding affinity for GCN4 and rGCN4. Proteins of varying concentrations were incubated with 0.5 µM DNA (20 bp, containing one binding site for CRE or INVii) in Binding buffer 1, and the bound fraction analyzed by dividing pixel intensity of bound fraction with pixel intensity of bound and unbound fraction using ImageJ. At least three separate measurements were conducted for each data point. Values are presented as mean +/- SD

GCN4 binds to its optimal DNA binding motif with an apparent dissociation constant KD of (0.293 ± 0.033) × 10-6 M, which is almost identical to the rGCN4 dissociation constant to INVii a KD of (0.298 ± 0.030) × 10-6 M. Comparing them to literature values, our dissociation constants are approximately a factor 10 higher then those described in literature ((9±6) × 10-8 M for GCN4 and (2.9 ± 0.8) × 10-8 M for rGCN4) (Hollenbeck et al., 2001). The differences could be due to the lower sensitivity of SYBR-Safe staining compared to radio-labeled oligos. Most likely, the protein concentration was miscalculated due to the presence of additional (lower intensity) bands in the SDS-PAGE analysis, indicating co-purification of small amounts of unspecific proteins.

The FLAG-tag fusion to the N-terminus of proteins could potentially decrease binding affinity, likely due to steric hindrance affecting the interaction with DNA. Interestingly, the differences in binding affinity between GCN4 and rGCN4 appear negligible. Since GCN4 binds to DNA via its N-terminus and rGCN4 binds C-terminally, the FLAG-tag likely does not directly influence DNA binding. However, it may influence the dimerization of the proteins, which is necessary for DNA binding. To further investigate this, the FLAG-tag can be cleaved using an enterokinase and potential changes in binding affinity analyzed. Furthermore, coiled coil formation, and the amount of dimeric and monomeric proteins could be further analyzed with circular dichroism spectroscopy (Greenfield, 2006).

4.3 In Silico Characterization Using DaVinci

Figure 6: Molecular Dynamics Simulation of GCN4

We developed DaVinci, an in silico model, for rapid engineering and optimization of our PICasSO system. DaVinci serves as a digital twin to PICasSO, analyzing the forces acting on the system, refining experimental parameters, and identifying optimal interactions between protein staples and target DNA. The model was calibrated using literature data and experimental affinity results from rGCN4 EMSA assays with purified proteins.
DaVinci operates in three phases: static structure prediction, all-atom dynamics simulation, and long-range DNA dynamics simulation. We applied the first two phases to our components, allowing us to characterize the structure and dynamics of the DNA-binding interactions.
For our bivalent DNA-binding Mini staple (BBa_K5237009), consisting of GCN4 fused via a GSG-linker to rGCN4 (BBa_K5237008), we predicted the structure and binding affinity and tested various linker options. We evaluated the flexibility and rigidity of the constructs using pLDDT values from the predictions. Flexible linkers, like ('GGGGS')n, and rigid linkers, like ('EAAAK')n, were assessed (Arai et al., 2001). Predictions were colored by pLDDT scores, providing insights into chain rigidity (Akdel et al., 2022; Guo et al., 2022). Construct C (Fig. 5) was tested in the wet lab as part of BBa_K5237009, but it failed to bind DNA due to excessive rigidity, which inhibited subunit dimerization.

Figure 6: Variation of Linkers Connecting Our Mini Staples. Panels A (BBa_K5237007) and B (BBa_K5237008) show orientations of the leucine zipper, each bound to DNA. Panels C to I display linker variations colored by their pLDDT confidence score, which serves as a surrogate for chain flexibility (Akdel et al., 2022). Note that panels H and I are not bound to the second DNA strand. All structures were predicted using the AlphaFold server (Google DeepMind, 2024).

5. References

Akdel, M., Pires, D. E. V., Pardo, E. P., Janes, J., Zalevsky, A. O., Meszaros, B., Bryant, P., Good, L. L., Laskowski, R. A., Pozzati, G., Shenoy, A., Zhu, W., Kundrotas, P., Serra, V. R., Rodrigues, C. H. M., Dunham, A. S., Burke, D., Borkakoti, N., Velankar, S., … Beltrao, P. (2022). A structural biology community assessment of AlphaFold2 applications. Nat Struct Mol Biol, 29(11), 1056–1067. https://doi.org/10.1038/s41594-022-00849-w

Arai, R., Ueda, H., Kitayama, A., Kamiya, N., & Nagamune, T. (2001). Design of the linkers which effectively separate domains of a bifunctional fusion protein. Protein Engineering, Design and Selection, 14(8), 529–532. https://doi.org/10.1093/protein/14.8.529

Arndt, K., & Fink, G. R. (1986). GCN4 protein, a positive transcription factor in yeast, binds general control promoters at all 5’ TGACTC 3’ sequences. Proceedings of the National Academy of Sciences, 83(22), 8516–8520. https://doi.org/10.1073/pnas.83.22.8516

Chen, X., Zaro, J. L., & Shen, W.-C. (2013). Fusion protein linkers: Property, design and functionality. Advanced Drug Delivery Reviews, 65(10), 1357–1369. https://doi.org/10.1016/j.addr.2012.09.039

Google DeepMind. (2024). AlphaFold Server. https://alphafoldserver.com/terms

Guo, H.-B., Perminov, A., Bekele, S., Kedziora, G., Farajollahi, S., Varaljay, V., Hinkle, K., Molinero, V., Meister, K., Hung, C., Dennis, P., Kelley-Loughnane, N., & Berry, R. (2022). AlphaFold2 models indicate that protein sequence determines both structure and dynamics. Scientific Reports, 12(1), 10696. https://doi.org/10.1038/s41598-022-14382-9

Ellenberger, T. E., Brandl, C. J., Struhl, K., & Harrison, S. C. (1992). The GCN4 basic region leucine zipper binds DNA as a dimer of uninterrupted alpha helices: Crystal structure of the protein-DNA complex. Cell, 71(7), 1223–1237. https://doi.org/10.1016/s0092-8674(05)80070-4

Hollenbeck, J. J., Gurnon, D. G., Fazio, G. C., Carlson, J. J., & Oakley, M. G. (2001). A GCN4 Variant with a C-Terminal Basic Region Binds to DNA with Wild-Type Affinity. Biochemistry, 40(46), 13833–13839.

Hollenbeck, J. J., McClain, D. L., & Oakley, M. G. (2002). The role of helix stabilizing residues in GCN4 basic region folding and DNA binding. Protein Science, 11(11), 2740–2747. https://doi.org/10.1110/ps.0211102

Hollenbeck, J. J., & Oakley, M. G. (2000). GCN4 Binds with High Affinity to DNA Sequences Containing a Single Consensus Half-Site. Biochemistry, 39(21), 6380–6389. https://doi.org/10.1021/bi992705n

Liu, J., Zheng, Q., Deng, Y., Cheng, C.-S., Kallenbach, N. R., & Lu, M. (2006). A seven-helix coiled coil. Proceedings of the National Academy of Sciences, 103(42), 15457–15462. https://doi.org/10.1073/pnas.0604871103

Lupas, A. N., Bassler, J., & Dunin-Horkawicz, S. (2017). The Structure and Topology of α-Helical Coiled Coils. Fibrous Proteins: Structures and Mechanisms, 82, 95–129. https://doi.org/10.1007/978-3-319-49674-0_4

Woolfson, D. N. (2023). Understanding a protein fold: The physics, chemistry, and biology of α-helical coiled coils. Journal of Biological Chemistry, 299(4), 104579. https://doi.org/10.1016/j.jbc.2023.104579