Difference between revisions of "Part:BBa K5237009"

 
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<body>
  <!-- Part summary -->
+
<!-- Part summary -->
  <section id="0">
+
<section>
    <h1>Mini staple:</h1>
+
<h1>Mini Staple: bGCN4</h1>
    <p>
+
<p>
       The Mini staple is a fusion of GCN4 and rGCN4, engineered to bind two DNA strands simultaneously and induce proximity.
+
       The bGCN4 Mini staple is a fusion of the basic leucine zipper GCN4 and rGCN4, offering a smaller, compact protein
       With the Mini staples, we aimed to engineer a smaller alternative to the simple staples.
+
       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.
 
     </p>
 
     </p>
    <p>&nbsp;</p>
+
<p> </p>
  </section>
+
</section>
  <div id="toc" class="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-5"><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-6">
+
</li>
            <a href="#4.1"><span class="tocnumber">4.1</span> <span class="toctext">Protein Expression and
+
<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>
                Purification</span></a>
+
<ul>
          </li>
+
<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>
          <li class="toclevel-2 tocsection-7">
+
</li>
            <a href="#4.2"><span class="tocnumber">4.2</span> <span class="toctext">Electrophoretic Mobility Shift
+
<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>
                Assay</span></a>
+
</li>
            <ul>
+
</ul>
              <li class="toclevel-3 tocsection-8">
+
</li>
                <a href="#4.2"><span class="tocnumber">4.2.1</span> <span class="toctext">Qualitative DNA binding
+
<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>
                    analysis</span></a>
+
</li>
              </li>
+
</ul>
              <li class="toclevel-3 tocsection-9">
+
</li>
                <a href="#4.2"><span class="tocnumber">4.2.2</span> <span class="toctext">Quantitative DNA binding
+
<li class="toclevel-1 tocsection-8"><a href="#5"><span class="tocnumber">5</span> <span class="toctext">References</span></a>
                    analysis</span></a>
+
</li>
              </li>
+
</ul>
            </ul>
+
</div>
          </li>
+
<section><p><br/><br/></p>
        </ul>
+
<font size="5"><b>The PICasSO Toolbox </b> </font>
      </li>
+
<div class="thumb" style="margin-top:10px;"></div>
      <li class="toclevel-1 tocsection-8"><a href="#5"><span class="tocnumber">5</span> <span
+
<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%;"/>
            class="toctext">References</span></a>
+
<div class="thumbcaption">
      </li>
+
<i><b>Figure 1: How Our Part Collection can be Used to Engineer New Staples</b></i>
    </ul>
+
</div>
  </div>
+
</div>
  <section>
+
<p>
    <font size="5"><b>The PICasSO Toolbox </b> </font>
+
<br/>
    <p><br></p>
+
       While synthetic biology has in the past focused on engineering the genomic sequence of organisms, the <b>3D
    <div class="thumb"></div>
+
        spatial organization</b> of DNA is well-known to be an important layer of information encoding in
      <div class="thumbinner" style="width:550px"><img alt="" src="https://static.igem.wiki/teams/5237/wetlab-results/registry-part-collection-engineering-cycle-example-overview.svg" style="width:99%;" class="thumbimage">
+
      particular in eukaryotes, playing a crucial role in
        <div class="thumbcaption">
+
      gene regulation and hence
          <i><b>Figure 1: Example how the part collection can be used to engineer new staples</b></i>
+
       cell fate, disease development, evolution, and more. However, tools to precisely manipulate and control the
        </div>
+
       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
 
+
        molecular toolbox for rationally engineering genome 3D architectures</b> in living cells, based on
    <p>
+
      various DNA-binding proteins.
      <br>
+
       The 3D organization of the genome plays a crucial role in regulating gene expression in eukaryotic cells,
+
       impacting cellular behavior, evolution, and disease. Beyond the linear DNA sequence, the spatial arrangement of
+
      chromatin, influenced by DNA-DNA interactions, shapes pathways of gene regulation. 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 powerful molecular
+
       toolbox 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
       re-programming
+
       <b>re-programming
      of DNA-DNA interactions using protein staples in living cells, enabling researchers to recreate natural 3D genomic
+
        of DNA-DNA interactions</b> using engineered "protein staples" in living cells. This enables
       interactions, such as enhancer hijacking, or to design entirely new spatial architectures for gene regulation.
+
      researchers to recreate naturally occurring alterations of 3D genomic
       Beyond its versatility, PICasSO includes robust assay systems to support the engineering, optimization, and
+
       interactions, such as enhancer hijacking in cancer, or to design entirely new spatial architectures for
       testing of new staples, ensuring functionality <i>in vitro</i> and <i>in vivo</i>. We took special care to include
+
      artificial gene regulation and cell function control.
       parts crucial for testing every step of the cycle (design, build, test, learn) when engineering new parts
+
       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 <b>chimeric CRISPR/Cas complexes</b>,
 +
      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 <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 Basic staples. These
+
      and robust DNA binding domains well-known to the synthetic biology community, which serve as controls for
       consist of
+
      successful stapling
       protease-cleavable peptide linkers and inteins that allow condition-specific, dynamic stapling <i>in vivo</i>.
+
       and can be further engineered to create alternative, simpler, and more compact staples. <br/>
       Besides staple functionality, we also include the parts to enable the efficient delivery of PICasSO's constructs with our
+
<b>(ii)</b> As <b>functional elements</b>, we list additional parts that enhance and expand the
       interkingdom conjugation system. <br>
+
      functionality of our Cas and
      <b>(iii)</b> As the final component of our collection, we provide parts that support the use of our <b>custom readout
+
      Basic staples. These
         systems</b>. These include components of our established FRET-based proximity assay system, enabling users to
+
       consist of staples dependent on
 +
       cleavable peptide linkers targeted by cancer-specific proteases or inteins that allow condition-specific,
 +
      dynamic stapling <i>in vivo</i>.
 +
       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. <br/>
 +
<b>(iii)</b> As the final category of our collection, we provide parts that underlie our <b>custom
 +
        readout
 +
         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
       readout 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.
 
     </p>
 
     </p>
    <p>
+
<p>
       The following table gives a complete overview of all parts in our PICasSO toolbox. The highlighted parts showed
+
       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
      exceptional performance as described on our iGEM wiki and can serve as a reference. The other parts in the
+
        exceptional performance as described on our iGEM wiki and can serve as a reference.</mark> The other
       collection are versatile building blocks designed to provide future iGEMers with the flexibility to engineer their
+
      parts in
       own custom Cas staples, enabling further optimization and innovation.<br>
+
      the
    </p>
+
       collection are versatile building blocks designed to provide future iGEMers with the flexibility to engineer
    <p>
+
      their
      <font size="4"><b>Our part collection includes:</b></font><br>
+
       own custom Cas staples, enabling further optimization and innovation in the new field of 3D genome
    </p>
+
      engineering.<br/>
 
+
</p>
    <table style="width: 90%;">
+
<p>
      <td colspan="3" align="left"><b>DNA-binding proteins: </b>
+
<font size="4"><b>Our part collection includes:</b></font><br/>
         The building blocks for engineering of custom staples for DNA-DNA interactions with a modular system ensuring
+
</p>
        easy assembly.</td>
+
<table style="width: 90%; padding-right:10px;">
      <tbody>
+
<td align="left" colspan="3"><b>DNA-Binding Proteins: </b>
        <tr bgcolor="#FFD700">
+
         Modular building blocks for engineering of custom staples to mediate defined DNA-DNA interactions <i>in vivo</i></td>
          <td><a href="https://parts.igem.org/Part:BBa_K5237000" target="_blank">BBa_K5237000</a></td>
+
<tbody>
          <td>fgRNA Entryvector MbCas12a-SpCas9</td>
+
<tr bgcolor="#FFD700">
          <td>Entryvector for simple fgRNA cloning via SapI</td>
+
<td><a href="https://parts.igem.org/Part:BBa_K5237000" target="_blank">BBa_K5237000</a></td>
        </tr>
+
<td>Fusion Guide RNA Entry Vector MbCas12a-SpCas9</td>
        <tr>
+
<td>Entry vector for simple fgRNA cloning via SapI</td>
          <td><a href="https://parts.igem.org/Part:BBa_K5237001" target="_blank">BBa_K5237001</a></td>
+
</tr>
          <td>Staple subunit: dMbCas12a-Nucleoplasmin NLS</td>
+
<tr bgcolor="#FFD700">
          <td>Staple subunit that can be combined to form a functional staple, for example with fgRNA and dCas9 </td>
+
<td><a href="https://parts.igem.org/Part:BBa_K5237001" target="_blank">BBa_K5237001</a></td>
        </tr>
+
<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
          <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 to form a functional staple, for example with our fgRNA or dCas12a
+
 
           </td>
 
           </td>
        </tr>
+
</tr>
        <tr>
+
<tr bgcolor="#FFD700">
          <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_K5237002" target="_blank">BBa_K5237002</a></td>
          <td>Cas-Staple: SV40 NLS-dMbCas12a-dSpCas9-Nucleoplasmin NLS</td>
+
<td>Staple Subunit: SV40 NLS-dSpCas9-SV40 NLS</td>
          <td>Functional Cas staple that can be combined with sgRNA or fgRNA to bring two DNA strands in close proximity
+
<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_K5237004" target="_blank">BBa_K5237004</a></td>
+
<td><a href="https://parts.igem.org/Part:BBa_K5237003" target="_blank">BBa_K5237003</a></td>
          <td>Staple subunit: Oct1-DBD</td>
+
<td>Cas Staple: SV40 NLS-dMbCas12a-dSpCas9-Nucleoplasmin NLS</td>
          <td>Staple subunit that can be combined to form a functional staple, for example with TetR.<br>
+
<td>Functional Cas staple that can be combined with sgRNA and crRNA or fgRNA to bring two DNA strands into
 +
            close
 +
            proximity
 +
           </td>
 +
</tr>
 +
<tr>
 +
<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 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 taple: 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 colspan="3" align="left"><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
         for custom applications.</td>
+
        optimization
      <tbody>
+
         for custom applications</td>
        <tr bgcolor="#FFD700">
+
<tbody>
          <td><a href="https://parts.igem.org/Part:BBa_K5237010" target="_blank">BBa_K5237010</a></td>
+
<tr bgcolor="#FFD700">
          <td>Cathepsin B-Cleavable Linker (GFLG)</td>
+
<td><a href="https://parts.igem.org/Part:BBa_K5237010" target="_blank">BBa_K5237010</a></td>
          <td>Cathepsin B cleavable peptide linker, that can be used to combine two staple subunits ,to make responsive
+
<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
 
             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>Cathepsin B which can be selectively express to cut the cleavable linker</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>Undergoes protein transsplicing after protease activation, can be used to create functionalized staple
+
<td>A caged NpuN split intein fragment that undergoes protein <i>trans</i>-splicing after protease
             units</td>
+
            activation, which can be used to create functionalized staple
        </tr>
+
             subunits</td>
        <tr>
+
</tr>
          <td><a href="https://parts.igem.org/Part:BBa_K5237013" target="_blank">BBa_K5237013</a></td>
+
<tr>
          <td>Caged NpuC Intein</td>
+
<td><a href="https://parts.igem.org/Part:BBa_K5237013" target="_blank">BBa_K5237013</a></td>
          <td>Undergoes protein transsplicing after protease activation, can be used to create functionalized staple
+
<td>Caged NpuC Intein</td>
             units</td>
+
<td>A caged NpuC split intein fragment that undergoes protein <i>trans</i>-splicing after protease
        </tr>
+
            activation, which can be used to create functionalized staple
        <tr>
+
             subunits</td>
          <td><a href="https://parts.igem.org/Part:BBa_K5237014" target="_blank">BBa_K5237014</a></td>
+
</tr>
          <td>fgRNA processing casette</td>
+
<tr>
          <td>Processing casette to produce multiple fgRNAs from one transcript, can be used for multiplexing</td>
+
<td><a href="https://parts.igem.org/Part:BBa_K5237014" target="_blank">BBa_K5237014</a></td>
        </tr>
+
<td>Fusion Guide RNA Processing Casette</td>
        <tr>
+
<td>Processing cassette to produce multiple fgRNAs from one transcript, that can be used for
          <td><a href="https://parts.igem.org/Part:BBa_K5237015" target="_blank">BBa_K5237015</a></td>
+
            multiplexed 3D
          <td>Intimin anti-EGFR Nanobody</td>
+
            genome reprogramming</td>
          <td>Interkindom conjugation between bacteria and mammalian cells, as alternative delivery tool for large
+
</tr>
 +
<tr>
 +
<td><a href="https://parts.igem.org/Part:BBa_K5237015" target="_blank">BBa_K5237015</a></td>
 +
<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>
      </tbody>
+
<tr>
      <td colspan="3" align="left"><b>Readout Systems: </b>
+
<td><a href="https://parts.igem.org/Part:BBa_K4643003" target="_blank">BBa_K4643003</a></td>
         FRET and enhancer recruitment to measure proximity of stapled DNA in bacterial and mammalian living cells
+
<td>IncP Origin of Transfer</td>
        enabling swift testing and easy development for new systems.</td>
+
<td>Origin of transfer that can be cloned into the plasmid vector and used for conjugation as a
      <tbody>
+
            means of
        <tr bgcolor="#FFD700">
+
            delivery</td>
          <td><a href="https://parts.igem.org/Part:BBa_K5237016" target="_blank">BBa_K5237016</a></td>
+
</tr>
          <td>FRET-Donor: mNeonGreen-Oct1</td>
+
</tbody>
          <td>Donor part for the FRET assay binding the Oct1 binding cassette. Can be used to visualize DNA-DNA
+
<td align="left" colspan="3"><b>Readout Systems: </b>
 +
         FRET and enhancer recruitment readout systems to rapidly assess successful DNA stapling in bacterial and
 +
        mammalian cells
 +
      </td>
 +
<tbody>
 +
<tr bgcolor="#FFD700">
 +
<td><a href="https://parts.igem.org/Part:BBa_K5237016" target="_blank">BBa_K5237016</a></td>
 +
<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
 
             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, can be used for different 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
         </tr>
+
         </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>
        <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>
    </p>
+
<section id="1">
  </section>
+
<h1>1. Sequence Overview</h1>
  <section id="1">
+
</section>
    <h1>1. Sequence overview</h1>
+
  </section>
+
 
</body>
 
</body>
 
 
</html>
 
</html>
 
 
<!--################################-->
 
<!--################################-->
<span class='h3bb'>Sequence and Features</span>
+
<span class="h3bb">Sequence and Features</span>
 
<partinfo>BBa_K5237009 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K5237009 SequenceAndFeatures</partinfo>
 
<!--################################-->
 
<!--################################-->
 
 
<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 directly
+
     consists of a coiled-coil leucine zipper dimerization domain, and a highly charged basic region that binds to DNA
    contacts and binds to DNA (Hollenbeck & Oakley, 2000). One well characterized example is the General Control Protein
+
    (Hollenbeck &amp; Oakley, 2000). One well characterized example is the General Control
     4 (GCN4), a well-characterized transcriptional activator from yeast (Arndt & Fink, 1986). At its N-terminus, GCN4
+
     Protein 4 (GCN4), a well-characterized transcriptional activator from yeast (Arndt &amp; Fink, 1986).<br/>
    contains basic residues, the so-called bZip domain, through which it binds specifically to the CRE (cyclic AMP
+
    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 <i>et al.</i>, 2002). A variant of GCN4 with the DNA
 
     response element) DNA sequence (5' ATGACGTCAT 3') (Hollenbeck <i>et al.</i>, 2002). A variant of GCN4 with the DNA
     binding bZip-domain
+
     binding bZip-domain at the C-terminus (rGCN4) has been engineered to bind to the inverted CRE sequence, INV2 (5'
    at the C-terminus (rGCN4) has been engineered to bind to the inverted CRE sequence, INV2 (5' GTCAtaTGAC 3', upper
+
    GTCAtaTGAC 3', upper
 
     case letters indicate direct interaction between protein and DNA) with similar affinity
 
     case letters indicate direct interaction between protein and DNA) with similar affinity
     (Hollenbeck <i>et al.</i>, 2001). By genetically fusing GCN4 to rGCN4, we created a small bivalent DNA binding
+
     (Hollenbeck <i>et al.</i>, 2001). By genetically fusing GCN4 to rGCN4, we created a small, bivalent DNA binding
     staple with less than 150 amino acids, which was for its DNA binding and stapling capabilities.</p>
+
     staple with less than 150 amino acids.</p>
 
</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 sequence for GCN4 and rGCN4 was 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.
+
     linker designs, based on our dry lab <a href="https://2024.igem.wiki/heidelberg/model" target="_blank">DaVinci</a>
 +
    model.
 
     A FLAG-tag (DYKDDDDK) was added to the N-terminus for protein purification. The FLAG-tag can be cleaved off using an
 
     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.
 
     Enterokinase, if necessary.
     The FLAG-GCN4 sequence was cloned into a T7 expression vector and expressed using <i>E. coli</i> BL21 (DE3) cells.
+
     Expression of FLAG-GCN4 was done under an IPTG inducible T7 promoter in <i>E. coli</i> BL21 (DE3) cells.
 
   </p>
 
   </p>
 
</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
+
     predict improved dimerization and DNA binding.
    improved dimerization and DNA binding.
+
 
   </p>
 
   </p>
  <section id="4.1">
+
<section id="4.1">
    <h1>4.1 Protein Expression and Purification</h1>
+
<h2>4.1 Protein Expression and Purification</h2>
    <p>
+
<p>
       The bZip proteins GCN4, rGCN4 and their fusion bGCN4 were fused N-terminally to a FLAG-tag (DYKDDDDK). All
+
       The bZip proteins GCN4, rGCN4 and the fusion thereof, bGCN4, were fused N-terminally to a FLAG-tag (DYKDDDDK).
       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=""
+
<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%;" class="thumbimage">
+
<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>
    </div>
+
</section>
  </section>
+
<section id="4.2">
  <section>
+
<h2>4.2 Electrophoretic Mobility Shift Assay</h2>
    <section id="4.2">
+
<div class="thumb tright">
      <h1>4.2 Electrophoretic Mobility Shift Assay</h1>
+
<div class="thumbinner" style="width:310px;">
      <p>
+
<img alt="" class="thumbimage" src="https://static.igem.wiki/teams/5237/figures-corrected/leucin-zipper-emsa.svg" style="width:99%;"/>
        The Electrophoretic mobility shift assay (EMSA) is a widely adopted method used to study DNA-protein
+
<div class="thumbcaption">
        interactions. EMSa functions on the basis that nucleic acids bound to proteins have reduced electrophoretic
+
<i>
        mobility, compared to their counterpart. (Hellman & Fried, 2007). Mobility-shift
+
<b>Figure 3: Overview Image of Electrophoretic Mobility Shift Assay (EMSA)</b>
        assays can both be used to qualitatively assess DNA binding capabilities or quantitatively to determine binding
+
</i>
        stoichiometry and kinetics such as the apparent dissociation constant (K<sub>d</sub>) (Fried, 1989).
+
</div>
 +
</div>
 +
</div>
 +
<p align="justify">
 +
    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 &amp; 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 (K<sub>d</sub>) (Fried, 1989).
 +
    </p>
 +
<section id="4.2.1" style="clear:both;">
 +
<h2>4.2.1 Qualitative DNA Binding Analysis</h2>
 +
<p>
 +
        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 <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
 +
        (Fig. 4).
 
       </p>
 
       </p>
      <dive class="thumb">
+
<p>
         <div class="thumbinner">
+
         The EMSA is a widely adopted method used to study DNA-protein
          <img alt="" src="https://static.igem.wiki/teams/5237/figures-corrected/leucin-zipper-emsa.svg"
+
         interactions. EMSA functions on the basis that nucleic acids, bound to proteins, have reduced electrophoretic
          style="width:99%;" class="thumbimage">
+
        mobility, compared to their counterpart. (Hellman &amp; Fried, 2007). Mobility-shift
          <div class="thumbcaption">
+
         assays can be used both to qualitatively assess DNA binding capabilities or quantitatively to determine binding
            <i>
+
         stoichiometry and kinetics such as the apparent dissociation constant (K<sub>d</sub>) (Fried, 1989).<br/><br/>
              <b>Figure 3: Overview Image of Electrophoretic Mobility Shift Assay (EMSA)</b>
+
            </i>
+
          </div>
+
        </div>
+
      </div>
+
      <section>
+
        <h1>4.2.1 Qualitative DNA binding analysis</h1>
+
        <p>
+
          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).
+
          DNA binding could only be shown for GCN4 and rGCN4, no visible band was observed for the bGCN4 fusion protein
+
          (Figure 4).
+
         </p>
+
        <div class="thumb">
+
          <div class="thumbinner" style="width:550px"><img alt=""
+
              src="https://static.igem.wiki/teams/5237/wetlab-results/mist-emsa-quali.svg" style="width:99%;"
+
              class="thumbimage">
+
            <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 200
+
                µM
+
                of protein
+
                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
+
                KH<sub>2</sub>HPO<sub>4</sub>, 0.1 % (v/v) IGEPAL&#174; CA-360, 1 mM EDTA). Gel electorphoresis 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.</i>
+
            </div>
+
          </div>
+
         </div>
+
      </section>
+
      <section>
+
        <h1>4.2.2 Quantitative DNA binding analysis</h1>
+
         <p>
+
          To further analyze DNA binding, 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:
+
          <br><br>
+
          Θ<sub>app</sub> = Θ<sub>min</sub> + (Θ<sub>max</sub> - Θ<sub>min</sub>) &#215;
+
          (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>)
+
          <span style="float: right;">Equation 1</span>
+
          <br><br>
+
          Here [L]<sub>tot</sub> describes the total protein monomer concentration, K<sub>a</sub>
+
          corresponds
+
          to the apparent monomeric equilibration constant. The Θ<sub>min/max</sub> values are the
+
          experimentally
+
          determined site saturation values (For this experiment 0 and 1 were chosen for min and max
+
          respectively). GCN4 binds to its optimal DNA binding motif with an apparent dissociation
+
          constant K<sub>k</sub> of (0.2930.033)&#215;10<sup>-6</sup> M, which is almost identical to the
+
          rGCN4 binding
+
          affinity to INVii a <sub>d</sub> of (0.2980.030)&#215;10<sup>-6</sup> M.
+
        </p>
+
  
         <div class="thumb">
+
         To analyze the binding DNA affinity an EMSA was performed, in which
          <div class="thumbinner" style="width:550px"><img alt=""
+
        bGCN4 was incubated in binding buffer with a 20 bp DNA probe containing the either the <i>CRE</i> (GCN4 binding)
              src="https://static.igem.wiki/teams/5237/wetlab-results/mist-leucine-zipper-kd-plot.svg" style="width:99%;"
+
        sequence (5' ATGACGTCAT 3') or the <i>INVii</i> (rGCN4 binding) sequence (5' GTCAtaTGAC 3') until equilibration.
              class="thumbimage">
+
        Subsequently the formed protein-DNA complexes were loaded on a native PAGE. Afterwards the DNA bands were
            <div class="thumbcaption">
+
        stained with SYBR-safe. <br/>
              <i><b>Figure 5: K<sub>d</sub> Calculation of GCN4 and rGCN4</b>
+
        The bGCN4 fusion protein did not show any DNA binding for both target sites.
                Quantitative assessment of binding affinity for GCN4 and rGCN4. Proteins of varying
+
      </p>
                concentrations were incubated with 0.5 µM DNA (20 bp, containing one binding site for CRE or INVii) in
+
<div class="thumb">
                Binding buffer 1, and the bound fraction
+
<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%;"/>
                analyzed by dividing pixel intensity of bound fraction with pixel intensity of bound and unbound fraction
+
<div class="thumbcaption">
                using ImageJ. At
+
<i><b>Figure 4: Qualitative EMSA DNA Binding</b>
                least three separate measurements were conducted for each data point. Values are presented as mean +/-
+
              0.5 µM of annealed oligos (20 bp) containing either one CRE or INVii binding site were incubated with
                SD</i>
+
              200
            </div>
+
              µM
          </div>
+
              of protein
        </div>
+
              and equilibrated in binding buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na <sub>2</sub>HPO<sub>4</sub>, 1.8
      <p>
+
              mM
        To better understand the proteins, quantitative analysis was done to determine the apparent dissociation constant
+
              KH<sub>2</sub>HPO<sub>4</sub>, 0.1 % (v/v) IGEPAL® CA-360, 1 mM EDTA). Gel electrophoresis was
        for GCN4 and rGCN4. <br>
+
              performed with a pre-equilibrated TGX-Gel in TBE running buffer.
        For the purified proteins GCN4, rGCN4 and the fusion bGCN4 a qualitative gel was run with high protein concentration (Figure 3).
+
              Gel-bands were visualized by staining with SYBR Safe and imaged with an UV transilluminator.</i>
        Bands for GCN4 and rGCN4 were visible but no band for bGCN4 could be detected, indicating a
+
</div>
        lack of DNA binding. This suggests that the dimerization, necessary for DNA binding, is disrupted by the
+
</div>
        GSG-linker (Ellenberger <i class="italic">et al.</i>, 1992; Liu <i class="italic">et al.</i>, 2006; Lupas
+
</div>
        <i class="italic">et al.</i>, 2017; Woolfson, 2023). To better understand possible problems in dimerization circular dichroism can be used to analyze secondary structure
+
</section>
        and proper coiled coil formation (Greenfield, 2006). Further engineering can be done by testing out
+
<section id="4.2.2">
        various linkers with specific properties to ensure correct folding and dimerization (Chen <i class="italic">et
+
<h2>4.2.2 Quantitative DNA Binding Analysis</h2>
          al.</i>, 2013). The apparent binding kinetics calculated for GCN4 ((0.2930.033) &#215; 10<sup>-6</sup> M) and rGCN4
+
<p>
        ((0.2980.030) &#215; 10<sup>-6</sup> M) are
+
        To further analyze DNA binding of the staple subunits, quantitative shift assays were performed for GCN4 and rGCN4. Here
        approximately a factor 10 higher then those described in literature ((96) &#215; 10<sup>-8</sup> M for
+
         0.5 µM DNA were incubated with varying concentrations of protein until equilibration. After
        GCN4 and (2.90.8) &#215; 10<sup>-8</sup> M for rGCN4) (Hollenbeck <i class="italic">et al.</i>, 2001). The
+
        electrophoresis, bands were stained with SYBR-Safe and quantified based on pixel intensity. The
         differences could be due to the lower sensitivity of SYBR-Safe
+
         obtained values were fitted to equation 1, describing formation of a 2:1 protein-DNA complex:
         staining compared to radio-labeled oligos.
+
 
         <br><br>
 
         <br><br>
         The FLAG-tag fusion to the N-terminus of proteins could potentially decrease binding affinity, likely due
+
         Θ<sub>app</sub> = Θ<sub>min</sub> + (Θ<sub>max</sub> - Θ<sub>min</sub>) ×
        to steric hindrance affecting the interaction with DNA. Interestingly, the differences in binding affinity
+
        (K<sub>a</sub><sup>2</sup> [L]<sub>tot</sub><sup>2</sup>) / (1 + K<sub>a</sub><sup>2</sup>
        between GCN4 and rGCN4 appear negligible. Since GCN4 binds to DNA via its N-terminus and rGCN4 binds
+
        [L]<sub>tot</sub><sup>2</sup>)
        C-terminally, the FLAG-tag likely does not directly influence DNA binding. However, it may influence the
+
        <span style="float: right;">Equation 1</span>
        dimerization of the proteins, which is necessary for DNA binding. To further investigate this, the
+
<br><br>
        FLAG-tag can be cleaved using an enterokinase and potential changes in binding affinity analyzed
+
        Here [L]<sub>tot</sub> describes the total protein monomer concentration, K<sub>a</sub>
 +
        corresponds
 +
        to the apparent monomeric equilibration constant. The Θ<sub>min/max</sub> values are the
 +
        experimentally
 +
        determined site saturation values (For this experiment 0 and 1 were chosen for min and max
 +
        respectively).
 +
      </br></br></br></br></p>
 +
<div class="thumb">
 +
<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%;"/>
 +
<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
 +
              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</i>
 +
</div>
 +
</div>
 +
</div>
 +
<p>
 +
      GCN4 binds to its optimal DNA binding motif with an apparent dissociation
 +
      constant K<sub>D</sub> of (0.293 ± 0.033) × 10<sup>-6</sup> M, which is almost identical to the
 +
      rGCN4 dissociation constant
 +
      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
 +
      described in literature ((9±6) × 10<sup>-8</sup> M for
 +
      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.
 +
      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.
 +
      <br/><br/>
 +
      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).
 +
    </p>
 +
</section>
 +
<section id="4.3">
 +
<h2>4.3 <i>In Silico</i> Characterization Using DaVinci</h2>
 +
<div class="thumb tright" style="margin:0;">
 +
<div class="thumbinner" style="width:300px;">
 +
<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>
 +
<div class="thumbcaption">
 +
<i><b>Figure 6: Molecular Dynamics Simulation of GCN4</b>
 +
</i></div>
 +
</div>
 +
</div>
 +
<p>
 +
        We developed DaVinci, an <i>in silico</i> 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.
 +
        <br/>
 +
        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.
 +
        <br/>
 +
        For our bivalent DNA-binding Mini staple (<a href="https://parts.igem.org/Part:BBa_K5237009">BBa_K5237009</a>),
 +
        consisting of GCN4 fused via a GSG-linker to rGCN4
 +
        (<a href="https://parts.igem.org/Part:BBa_K5237008">BBa_K5237008</a>), 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 <i>et al.</i>, 2001). Predictions were colored by
 +
        pLDDT scores, providing insights into chain rigidity (Akdel <i>et al.</i>, 2022; Guo <i>et al.</i>, 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.
 
       </p>
 
       </p>
    </section>
+
<div class="thumb">
  </section>
+
<div class="thumbinner" style="width:80%;">
 +
<img alt="" class="thumbimage" src="https://static.igem.wiki/teams/5237/model/structure-figure-3.svg" style="width:99%;"/>
 +
<div class="thumbcaption">
 +
<i><b>Figure 6: Variation of Linkers Connecting Our Mini Staples.</b>
 +
              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 <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).
 +
            </i>
 +
</div>
 +
</div>
 +
</div>
 +
</section>
 
</section>
 
</section>
 
<section id="5">
 
<section id="5">
  <h1>5. References</h1>
+
<h1>5. References</h1>
  <p>Arndt, K., & 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, 83</em>(22), 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>
+
<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.
  <p>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. <em>Cell, 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>
+
      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 href="https://doi.org/10.1038/s41594-022-00849-w" target="_blank">https://doi.org/10.1038/s41594-022-00849-w</a>
  <p>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. <em>Biochemistry, 40</em>(46), 13833–13839.</p>
+
</p>
 
+
<p>Arai, R., Ueda, H., Kitayama, A., Kamiya, N., &amp; Nagamune, T. (2001). Design of the linkers which effectively
  <p>Hollenbeck, J. J., McClain, D. L., & 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 href="https://doi.org/10.1110/ps.0211102" target="_blank">https://doi.org/10.1110/ps.0211102</a></p>
+
      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" target="_blank">https://doi.org/10.1093/protein/14.8.529</a></p>
  <p>Hollenbeck, J. J., & Oakley, M. G. (2000). GCN4 Binds with High Affinity to DNA Sequences Containing a Single 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>
+
<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,
  <p>Liu, J., Zheng, Q., Deng, Y., Cheng, C.-S., Kallenbach, N. R., & 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></p>
+
        83</em>(22),
 
+
      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>
  <p>Lupas, A. N., Bassler, J., & Dunin-Horkawicz, S. (2017). The Structure and Topology of α-Helical Coiled 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>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>
  <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></p>
+
</p>
 
+
<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
 +
      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>
 +
</p>
 +
<p>Ellenberger, T. E., Brandl, C. J., Struhl, K., &amp; 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. <em>Cell,
 +
        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>
 +
<p>Hollenbeck, J. J., Gurnon, D. G., Fazio, G. C., Carlson, J. J., &amp; Oakley, M. G. (2001). A GCN4 Variant with
 +
      a
 +
      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
 +
      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>
 +
<p>Hollenbeck, J. J., &amp; Oakley, M. G. (2000). GCN4 Binds with High Affinity to DNA Sequences Containing a
 +
      Single
 +
      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>
 +
<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>
 +
</p>
 +
<p>Lupas, A. N., Bassler, J., &amp; Dunin-Horkawicz, S. (2017). The Structure and Topology of α-Helical Coiled
 +
      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>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