Difference between revisions of "Part:BBa K5237004"

 
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<partinfo>BBa_K5237000</partinfo>
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<partinfo>BBa_K5237004</partinfo>
 
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<body>
  <!-- Part summary -->
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<!-- Part summary -->
  <section id="1">
+
<section>
    <h1>Half-Staple: Oct1-DBD</h1>
+
<h1>Half staple: Oct1-DBD</h1>
    <p>Oct1-DBD is the DNA-binding domain of the human Oct1 transcription factor, binding specifically to the octamer
+
<p>Oct1-DBD is the DNA-binding domain of the human Oct1 transcription factor. It can be readily fused with other
       motif (5'-ATGCAAAT-3') with high affinity and stability.</p>
+
      DNA-binding proteins to form a functional staple to DNA-DNA proximity. We used this part as a component for our
     <p>&nbsp;</p>
+
       Simple
  </section>
+
      staple (<a href="https://parts.igem.org/Part:BBa_K5237006" target="_blank">BBa_K5237006</a>) and also fused to
  <div id="toc" class="toc">
+
      mNeonGreen, as part of a FRET readout system (<a href="https://parts.igem.org/Part:BBa_K5237016" target="_blank">BBa_K5237016</a>).
    <div id="toctitle">
+
     </p>
      <h1>Contents</h1>
+
<p></p>
    </div>
+
</section>
    <ul>
+
<div class="toc" id="toc">
      <li class="toclevel-1 tocsection-1"><a href="#1"><span class="tocnumber">1</span> <span class="toctext">Sequence
+
<div id="toctitle">
             overview</span></a>
+
<h1>Contents</h1>
      </li>
+
</div>
      <li class="toclevel-1 tocsection-2"><a href="#2"><span class="tocnumber">2</span> <span class="toctext">Usage and
+
<ul>
 +
<li class="toclevel-1 tocsection-1"><a href="#1"><span class="tocnumber">1</span> <span class="toctext">Sequence
 +
             Overview</span></a>
 +
</li>
 +
<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-5"><a href="#4"><span class="tocnumber">4</span> <span class="toctext">Results</span></a>
            class="toctext">Results</span></a>
+
<ul>
      </li>
+
<li class="toclevel-2 tocsection-4"><a href="#4.1"><span class="tocnumber">4.1</span> <span class="toctext">Protein Expression and EMSA</span></a>
      <li class="toclevel-1 tocsection-8"><a href="#5"><span class="tocnumber">5</span> <span
+
</li>
            class="toctext">References</span></a>
+
<li class="toclevel-2 tocsection-5"><a href="#4.2"><span class="tocnumber">4.2</span> <span class="toctext"><i>In Silico</i> Characterization Using DaVinci</span></a>
      </li>
+
</li>
    </ul>
+
</ul>
  </div>
+
</li>
  <section id="2">
+
<li class="toclevel-1 tocsection-8"><a href="#5"><span class="tocnumber">5</span> <span class="toctext">References</span></a>
    <font size="5"><b>The PICasSO Toolbox </b> </font>
+
</li>
 
+
</ul>
    <p>The 3D organization of the genome plays a crucial role in regulating gene expression in eukaryotic cells,
+
</div>
       impacting cellular behavior, evolution, and disease. Beyond the linear DNA sequence, the spatial arrangement of
+
<section><p><br/><br/></p>
      chromatin, influenced by DNA-DNA interactions, shapes pathways of gene regulation. However, tools to precisely
+
<font size="5"><b>The PICasSO Toolbox </b> </font>
       manipulate this genomic architecture remain limited, making it challenging to explore the full potential of the 3D
+
<div class="thumb" style="margin-top:10px;"></div>
       genome in synthetic biology. To address this issue, team Heidelberg developed PICasSO.
+
<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%;"/>
 +
<div class="thumbcaption">
 +
<i><b>Figure 1: How our part collection can be used to engineer new staples</b></i>
 +
</div>
 +
</div>
 +
<p>
 +
<br/>
 +
      While synthetic biology has in the past focused on engineering the genomic sequence of organisms, the <b>3D
 +
        spatial organization</b> 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
 +
      <b>powerful
 +
        molecular toolbox for rationally engineering genome 3D architectures</b> in living cells, based on
 +
      various DNA-binding proteins.
 
     </p>
 
     </p>
    <p>The <b>PICasSO part collection</b> offers a comprehensive, modular platform for precise manipulation of DNA-DNA
+
<p>
      proximity in living cells, enabling researchers to recreate natural 3D genomic interactions, such as enhancer
+
      The <b>PICasSO</b> part collection offers a comprehensive, modular platform for precise manipulation and
      hijacking, or design entirely new spatial architectures for gene regulation. Beyond its versatility, PICasSO
+
      <b>re-programming
      includes robust measurement systems to support the engineering, optimization, and testing of new staples, ensuring
+
        of DNA-DNA interactions</b> using engineered "protein staples" in living cells. This enables
       functionality both in vivo and in vitro.
+
      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 <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>With its combination of staple systems, functionalization options, and measurement tools, PICasSO provides a
+
<p>At its heart, the PICasSO part collection consists of three categories. <br/><b>(i)</b> Our <b>DNA-binding
       complete solution for designing, testing, and refining new systems for spatial DNA organization. This toolbox
+
        proteins</b>
       unlocks new possibilities in synthetic biology, from studying fundamental genomic interactions to creating
+
       include our
       innovative gene therapies.</p>
+
      finalized Cas staple experimentally validated using an artificial "enhancer hijacking" system as well as
    <p>
+
      "half staples" that can be combined by scientists to compose entirely
       <font size="4"><b>Our parts collection includes:</b></font><br>
+
       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. <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
 +
      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
 +
      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.
 
     </p>
 
     </p>
 
+
<p>
    <table style="width: 65%;">
+
      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
      <td colspan="2" align="left"><b>DNA-binding proteins: </b>
+
        exceptional performance as described on our iGEM wiki and can serve as a reference.</mark> The other
         The building blocks for engineering of custom staples for DNA-DNA interactions with a modular system ensuring
+
      parts in
        easy assembly.</td>
+
      the
      <tbody>
+
      collection are versatile building blocks designed to provide future iGEMers with the flexibility to engineer
        <tr>
+
      their
        <tr>
+
      own custom Cas staples, enabling further optimization and innovation in the new field of 3D genome
          <td><a href="https://parts.igem.org/Part:BBa_K2926000" target="_blank">BBa_K5237000</a></td>
+
      engineering.<br/>
          <td>Half-Staple: Oct1-DBD</td>
+
</p>
        </tr>
+
<p>
        <tr>
+
<font size="4"><b>Our part collection includes:</b></font><br/>
          <td><a href="https://parts.igem.org/Part:BBa_K2926001" target="_blank">BBa_K5237001</a></td>
+
</p>
          <td>Half-Staple: TetR</td>
+
<table style="width: 90%; padding-right:10px;">
        </tr>
+
<td align="left" colspan="3"><b>DNA-Binding Proteins: </b>
        <tr>
+
         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_K2926002" target="_blank">BBa_K5237002</a></td>
+
<tbody>
          <td>Simple-Staple: TetR-Oct1</td>
+
<tr bgcolor="#FFD700">
        </tr>
+
<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>
          <td><a href="https://parts.igem.org/Part:BBa_K2926003" target="_blank">BBa_K5237003</a></td>
+
<td>Entry vector for simple fgRNA cloning via SapI</td>
          <td>Half-Staple: GCN4</td>
+
</tr>
        </tr>
+
<tr bgcolor="#FFD700">
        <tr>
+
<td><a href="https://parts.igem.org/Part:BBa_K5237001" target="_blank">BBa_K5237001</a></td>
          <td><a href="https://parts.igem.org/Part:BBa_K2926004" target="_blank">BBa_K5237004</a></td>
+
<td>Staple Subunit: dMbCas12a-Nucleoplasmin NLS</td>
          <td>Half-Staple: rGCN4</td>
+
<td>Staple subunit that can be combined with crRNA or fgRNA and dSpCas9 to form a functional staple
        </tr>
+
          </td>
        <tr>
+
</tr>
          <td><a href="https://parts.igem.org/Part:BBa_K2926005" target="_blank">BBa_K5237005</a></td>
+
<tr bgcolor="#FFD700">
          <td>Mini-Staple: bGCN4</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>
+
<td>Staple subunit that can be combined with a sgRNA or fgRNA and dMbCas12a to form a functional staple
          <td><a href="https://parts.igem.org/Part:BBa_K2926006" target="_blank">BBa_K5237006</a></td>
+
          </td>
          <td>fgRNA Entryvector: MbCas12a-SpCas9</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_K2926007" target="_blank">BBa_K5237007</a></td>
+
<td>Cas Staple: SV40 NLS-dMbCas12a-dSpCas9-Nucleoplasmin NLS</td>
          <td>Half-Staple: dMbCas12a-NLS</td>
+
<td>Functional Cas staple that can be combined with sgRNA and crRNA or fgRNA to bring two DNA strands into
        </tr>
+
            close
        <tr>
+
            proximity
          <td><a href="https://parts.igem.org/Part:BBa_K2926008" target="_blank">BBa_K5237008</a></td>
+
          </td>
          <td>Half-Staple: NLS-dSpCas9-NLS</td>
+
</tr>
        </tr>
+
<tr>
        <tr bgcolor="#FFD700">
+
<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_K2926009" target="_blank">BBa_K5237009</a></td>
+
<td>Staple Subunit: Oct1-DBD</td>
          <td>Cas-Staple: NLS-dMbCas12a-dSpCas9-NLS</td>
+
<td>Staple subunit that can be combined to form a functional staple, for example with TetR.<br/>
        </tr>
+
            Can also be combined with a fluorescent protein as part of the FRET proximity assay</td>
      </tbody>
+
</tr>
      <td colspan="2" align="left"><b>Functional elements: </b>
+
<tr>
         Protease cleavable peptide linkers and inteins are used to control and modify staples for further optimization
+
<td><a href="https://parts.igem.org/Part:BBa_K5237005" target="_blank">BBa_K5237005</a></td>
         for custom applications.</td>
+
<td>Staple Subunit: TetR</td>
      <tbody>
+
<td>Staple subunit that can be combined to form a functional staple, for example with Oct1.<br/>
        <tr>
+
            Can also be combined with a fluorescent protein as part of the FRET proximity assay</td>
          <td><a href="https://parts.igem.org/Part:BBa_K2926010" target="_blank">BBa_K5237010</a></td>
+
</tr>
          <td>fgRNA processing casette</td>
+
<tr>
        </tr>
+
<td><a href="https://parts.igem.org/Part:BBa_K5237006" target="_blank">BBa_K5237006</a></td>
        <tr bgcolor="#FFD700">
+
<td>Simple Staple: TetR-Oct1</td>
          <td><a href="https://parts.igem.org/Part:BBa_K2926011" target="_blank">BBa_K5237011</a></td>
+
<td>Functional staple that can be used to bring two DNA strands in close proximity</td>
          <td>Cathepsin B-Cleavable Linker (GFLG)</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_K29260012" target="_blank">BBa_K5237012</a></td>
+
<td>Staple Subunit: GCN4</td>
          <td>Cathepsin B Expression Cassette</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_K29260013" target="_blank">BBa_K5237013</a></td>
+
<td><a href="https://parts.igem.org/Part:BBa_K5237008" target="_blank">BBa_K5237008</a></td>
          <td>Caged NpuN Intein</td>
+
<td>Staple Subunit: rGCN4</td>
        </tr>
+
<td>Staple subunit that can be combined to form a functional staple, for example with rGCN4</td>
        <tr>
+
</tr>
          <td><a href="https://parts.igem.org/Part:BBa_K29260014" target="_blank">BBa_K5237014</a></td>
+
<tr>
          <td>Caged NpuC Intein</td>
+
<td><a href="https://parts.igem.org/Part:BBa_K5237009" target="_blank">BBa_K5237009</a></td>
        </tr>
+
<td>Mini Staple: bGCN4</td>
        <tr>
+
<td>
          <td><a href="https://parts.igem.org/Part:BBa_K29260015" target="_blank">BBa_K5237015</a></td>
+
            Assembled staple with minimal size that can be further engineered</td>
          <td>DNA delivery: Intimin anti-EGFR Nanobody</td>
+
</tr>
        </tr>
+
</tbody>
      </tbody>
+
<td align="left" colspan="3"><b>Functional Elements: </b>
      <td colspan="2" align="left"><b>Readout Systems: </b>
+
         Protease-cleavable peptide linkers and inteins are used to control and modify staples for further
         FRET and enhancer recruitment to measure proximity of stapled DNA in bacterial and mammalian living cells
+
        optimization
        enabling swift testing and easy development for new systems.</td>
+
         for custom applications</td>
      <tbody>
+
<tbody>
        <tr bgcolor="#FFD700">
+
<tr bgcolor="#FFD700">
          <td><a href="https://parts.igem.org/Part:BBa_K29260016" target="_blank">BBa_K5237016</a></td>
+
<td><a href="https://parts.igem.org/Part:BBa_K5237010" target="_blank">BBa_K5237010</a></td>
          <td>FRET-Donor: mNeonGreen-Oct1</td>
+
<td>Cathepsin B-cleavable Linker: GFLG</td>
        </tr>
+
<td>Cathepsin B-cleavable peptide linker that can be used to combine two staple subunits to make
        <tr bgcolor="#FFD700">
+
            responsive
          <td><a href="https://parts.igem.org/Part:BBa_K2926017" target="_blank">BBa_K5237017</a></td>
+
            staples</td>
          <td>FRET-Acceptor: TetR-mScarlet-I</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_K2926018" target="_blank">BBa_K5237018</a></td>
+
<td>Cathepsin B Expression Cassette</td>
          <td>Oct1 Binding Casette</td>
+
<td>Expression cassette for the overexpression of cathepsin B</td>
        </tr>
+
</tr>
        <tr>
+
<tr>
          <td><a href="https://parts.igem.org/Part:BBa_K2926019" target="_blank">BBa_K5237019</a></td>
+
<td><a href="https://parts.igem.org/Part:BBa_K5237012" target="_blank">BBa_K5237012</a></td>
          <td>TetR Binding Cassette</td>
+
<td>Caged NpuN Intein</td>
        </tr>
+
<td>A caged NpuN split intein fragment that undergoes protein <i>trans</i>-splicing after protease
        <td><a href="https://parts.igem.org/Part:BBa_K2926020" target="_blank">BBa_K5237020</a></td>
+
            activation, which can be used to create functionalized staple
        <td>Cathepsin B-Cleavable Trans-Activator: NLS-Gal4-GFLG-VP64</td>
+
            subunits</td>
         </tr>
+
</tr>
        <tr>
+
<tr>
          <td><a href="https://parts.igem.org/Part:BBa_K2926021" target="_blank">BBa_K5237021</a></td>
+
<td><a href="https://parts.igem.org/Part:BBa_K5237013" target="_blank">BBa_K5237013</a></td>
          <td>NLS-Gal4-VP64</td>
+
<td>Caged NpuC Intein</td>
        </tr>
+
<td>A caged NpuC split intein fragment that undergoes protein <i>trans</i>-splicing after protease
        <td><a href="https://parts.igem.org/Part:BBa_K2926022" target="_blank">BBa_K5237022</a></td>
+
            activation, which can be used to create functionalized staple
        <td>mCherry Expression Cassette: UAS, minimal Promotor, mCherry</td>
+
            subunits</td>
        </tr>
+
</tr>
        <tr>
+
<tr>
          <td><a href="https://parts.igem.org/Part:BBa_K2926023" target="_blank">BBa_K5237023</a></td>
+
<td><a href="https://parts.igem.org/Part:BBa_K5237014" target="_blank">BBa_K5237014</a></td>
          <td>UAS binding Firefly Luciferase Readout System</td>
+
<td>Fusion Guide RNA Processing Casette</td>
        </tr>
+
<td>Processing cassette to produce multiple fgRNAs from one transcript, that can be used for
 
+
            multiplexed 3D
      </tbody>
+
            genome reprogramming</td>
    </table>
+
</tr>
    </p>
+
<tr>
  </section>
+
<td><a href="https://parts.igem.org/Part:BBa_K5237015" target="_blank">BBa_K5237015</a></td>
  <section id="1">
+
<td>Intimin anti-EGFR Nanobody</td>
    <h1>1. Sequence overview</h1>
+
<td>Interkingdom conjugation between bacteria and mammalian cells, as an alternative delivery tool for
  </section>
+
            large
 +
            constructs</td>
 +
</tr>
 +
<tr>
 +
<td><a href="https://parts.igem.org/Part:BBa_K4643003" target="_blank">BBa_K4643003</a></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
 +
            delivery</td>
 +
</tr>
 +
</tbody>
 +
<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>
 +
</tr>
 +
<tr bgcolor="#FFD700">
 +
<td><a href="https://parts.igem.org/Part:BBa_K5237017" target="_blank">BBa_K5237017</a></td>
 +
<td>FRET-Acceptor: TetR-mScarlet-I</td>
 +
<td>Acceptor part for the FRET assay binding the TetR binding cassette, which can be used to visualize
 +
            DNA-DNA
 +
            proximity</td>
 +
</tr>
 +
<tr>
 +
<td><a href="https://parts.igem.org/Part:BBa_K5237018" target="_blank">BBa_K5237018</a></td>
 +
<td>Oct1 Binding Casette</td>
 +
<td>DNA sequence containing 12 Oct1 binding motifs, compatible with various assays such as the FRET
 +
            proximity assay</td>
 +
</tr>
 +
<tr>
 +
<td><a href="https://parts.igem.org/Part:BBa_K5237019" target="_blank">BBa_K5237019</a></td>
 +
<td>TetR Binding Cassette</td>
 +
<td>DNA sequence containing 12 Oct1 binding motifs, can be used for different assays such as the
 +
            FRET
 +
            proximity assay</td>
 +
</tr>
 +
<td><a href="https://parts.igem.org/Part:BBa_K5237020" target="_blank">BBa_K5237020</a></td>
 +
<td>Cathepsin B-Cleavable <i>Trans</i>-Activator: NLS-Gal4-GFLG-VP64</td>
 +
<td>Readout system that responds to protease activity, which was used to test cathepsin B-cleavable linker
 +
         </td>
 +
<tr>
 +
<td><a href="https://parts.igem.org/Part:BBa_K5237021" target="_blank">BBa_K5237021</a></td>
 +
<td>NLS-Gal4-VP64</td>
 +
<td><i>Trans</i>-activating enhancer, that can be used to simulate enhancer hijacking</td>
 +
</tr>
 +
<td><a href="https://parts.igem.org/Part:BBa_K5237022" target="_blank">BBa_K5237022</a></td>
 +
<td>mCherry Expression Cassette: UAS, minimal Promoter, mCherry</td>
 +
<td>Readout system for enhancer binding, which was used to test cathepsin B-cleavable linker</td>
 +
<tr>
 +
<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 and UAS binding cassette, that was used for the simulated enhancer hijacking assay</td>
 +
</tr>
 +
<tr>
 +
<td><a href="https://parts.igem.org/Part:BBa_K5237024" target="_blank">BBa_K5237024</a></td>
 +
<td>TRE-minimal Promoter- Firefly Luciferase</td>
 +
<td>Contains firefly luciferase controlled by a minimal promoter, which was used as a luminescence
 +
            readout for
 +
            simulated enhancer hijacking</td>
 +
</tr>
 +
</tbody>
 +
</table></section>
 +
<section id="1">
 +
<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_K5237000 SequenceAndFeatures</partinfo>
+
<partinfo>BBa_K5237004 SequenceAndFeatures</partinfo>
 
<!--################################-->
 
<!--################################-->
 
 
<html>
 
<html>
 
 
 
<body>
 
<body>
  <section id="2">
+
<section id="2">
    <h1>2. Usage and Biology</h1>
+
<h1>2. Usage and Biology</h1>
    <p>Oct1-DBD is the DNA-binding domain of the human transcription factor Oct1 (POU2F1), which plays a key role in
+
<p>Oct1-DBD is the DNA-binding domain of the human transcription factor Oct1 (POU2F1), which plays a key role in
 
       gene regulation, immune response, and stress adaptation in eukaryotic cells. This domain specifically binds to the
 
       gene regulation, immune response, and stress adaptation in eukaryotic cells. This domain specifically binds to the
       octamer motif (5’-ATGCAAAT-3’) within promoter and enhancer regions, influencing transcriptional activity
+
       octamer motif (5'-ATGCAAAT-3') within promoter and enhancer regions, influencing transcriptional activity
 
       (Lundbäck <i>et al.</i>, 2000). The Oct1-DBD consists of both a POU-specific domain and a POU homeodomain, which
 
       (Lundbäck <i>et al.</i>, 2000). The Oct1-DBD consists of both a POU-specific domain and a POU homeodomain, which
 
       work
 
       work
 
       together to form a stable complex with DNA (Park <i>et al.</i>, 2013, Stepchenko <i>et al.</i> 2021).
 
       together to form a stable complex with DNA (Park <i>et al.</i>, 2013, Stepchenko <i>et al.</i> 2021).
 
     </p>
 
     </p>
    <p>In synthetic biology, Oct1-DBD was previously used for plasmid display technology due to its strong binding
+
<p>In synthetic biology, Oct1-DBD has been previously used for plasmid display technology due to its strong binding
       affinity (K<sub>D</sub> = 9 &times 10<sup>-11</sup> M). Proteins fused with Oct1-DBD showed increased expression
+
       affinity (K<sub>D</sub> = 9 × 10<sup>-11</sup> M), additionally proteins fused with Oct1-DBD showed increased
       and protein solubility
+
      expression
      (Parker <i>et al.</i> 2020).
+
       and protein solubility (Parker <i>et al.</i> 2020).
 
     </p>
 
     </p>
    <p>
+
<p>
 
       This part was further used in <a href="https:parts.igem.org/Part:BBa_K5237002" target="_blank">BBa_K5237002</a> as
 
       This part was further used in <a href="https:parts.igem.org/Part:BBa_K5237002" target="_blank">BBa_K5237002</a> as
 
       a fusion with tetR, resulting in a bivalent DNA binding staple, and
 
       a fusion with tetR, resulting in a bivalent DNA binding staple, and
       also fused to mNeonGree, as part of a FRET readout system (<a href="https:parts.igem.org/Part:BBa_K5237016"
+
       also fused to mNeonGreen, as part of a FRET readout system (<a href="https:parts.igem.org/Part:BBa_K5237016" target="_blank">BBa_K5237016</a>).
        target="_blank">BBa_K5237016</a>).
+
 
     </p>
 
     </p>
 
+
</section>
 
+
<section id="3">
  </section>
+
<h1>3. Assembly and Part Evolution</h1>
  <section id="3">
+
<p>The Oct1-DBD amino acid sequence was obtained from UniProt (<a href="https://www.uniprot.org/uniprot/P14859" target="_blank">P14859</a>, POU domain, class 2, transcription factor 1)
    <h1>3. Assembly and part evolution</h1>
+
       and the DNA binding domain was extracted based on information given from Park <i>et al.</i> 2013 &amp; 2020.
    <p>The Oct1-DBD amino acid sequence was obtained from <a href="https://www.uniprot.org/uniprot/P14859"
+
        target="_blank">UniProt (P14859, POU domain, class 2, transcription factor 1)</a>
+
       and DNA binding domain extracted based on information given from Park <i>et al.</i> 2013 & 2020.
+
 
       An <i>E. coli</i> codon optimized DNA sequence was obtained through gene synthesis and used to clone further
 
       An <i>E. coli</i> codon optimized DNA sequence was obtained through gene synthesis and used to clone further
       constructs
+
       constructs.
 
     </p>
 
     </p>
  </section>
+
</section>
  <section id="4">
+
<section id="4">
    <h1>4. Results</h1>
+
<h1>4. Results</h1>
    <p>Oct1 was N-terminally fused to the His6-mNeonGreen, and expressed under a T7 expression protein and subsequently
+
<section id="4.1">
      purified using metal affinity chromatography with Ni-NTA beads.
+
<h2>4.1 Protein Expression and EMSA</h2>
      [!!Picture of SDS-Page sds-page-mng-oct1-expression.svg]
+
<p>Oct1 was N-terminally fused to His6-mNeonGreen. This fusion protein was expressed under a T7 promoter and
      DNA binding affinity was estimated with an electrophoretic mobility shift assay (EMSA). For this, three different
+
        subsequently
      buffer conditions were tested (Binding buffer 1: 137 mM NaCl, 2.7 mM KCl, 10 mM Na<sub>2</sub>HPO<sub>4</sub>, 1.8
+
        purified using metal affinity chromatography with Ni-NTA beads (Fig. 2, left).
      mM KH<sub>2</sub>HPO<sub>4</sub>,
+
        DNA binding affinity was estimated with an electrophoretic mobility shift assay (EMSA). For this, three
      0.1 % (v/v) IGEPAL&#174; CA-360, 1 mM EDTA; Binding buffer 2: 10 mM Tris, 50 mM KCl; NaP250:
+
        different
      Na<sub>2</sub>HPO<sub>4</sub>, 150 mM
+
        buffer conditions were tested (Binding buffer 1: 137 mM NaCl, 2.7 mM KCl, 10 mM Na<sub>2</sub>HPO<sub>4</sub>,
      NaCl, 250 mM Imidazol). DNA binding could only be detected for Binding buffer 1.
+
        1.8
    </p>
+
        mM KH<sub>2</sub>HPO<sub>4</sub>,
    <div class="thumb"></div>
+
        0.1 % (v/v) IGEPAL® CA-360, 1 mM EDTA; Binding buffer 2: 10 mM Tris, 50 mM KCl; NaP250:
    <div class="thumbinner" style="width:452px;"><a href="placeholder" class="image"><img alt=""
+
        Na<sub>2</sub>HPO<sub>4</sub>, 150 mM
          src="https://static.igem.wiki/teams/5237/wetlab-results/sds-page-mng-oct1-expression.svg" width="450"
+
        NaCl, 250 mM Imidazol). DNA binding could only be detected for Binding buffer 1. (Fig. 2, right)
          height="547" class="thumbimage"></a>
+
      </p>
      <div class="thumbcaption">
+
<div class="thumb">
        <i><b>Figure 1</b> SDS-PAGE analysis of His<sub>6</sub>-mNeonGreen-Oct1-DBD</i><br>
+
<div class="thumbinner" style="width:90%;">
        <i>Lane 1: raw lysate of E. coli expression culture after steril-filtration; Lane 2: Flow through of first wash (10 bed volumes of NaP10 (Na<sub>2</sub>HPO<sub>4</sub>, 150 mM NaCl, 10 mM
+
<div style="display: flex; justify-content: center; border:none;">
          Imidazol)); Lane 3: Flow through of second wash (10 bed volumes of NaP20 (Na<sub>2</sub>HPO<sub>4</sub>, 150 mM NaCl, 20 mM
+
<div>
          Imidazol)) Lane 4: Elution of purified protein.<br>
+
<img alt="" class="thumbimage" src="https://static.igem.wiki/teams/5237/wetlab-results/sds-page-mng-oct1-expression.svg" style="height: 300px; width: auto; border:none;"/>
        1 µL of each fraction was loaded after mixing and heating with 4x Laeemli buffer, on a 4-15 % TGX-Gel. The expected band size of the protein is 56 840.23 Da, which is highlited in red on the gel.</i>
+
</div>
       </div>
+
<div>
    </div>
+
<a href="Fig2_right">
    </div>
+
<img alt="" class="thumbimage" src="https://static.igem.wiki/teams/5237/wetlab-results/emsa-oct1-binding-buffer-optimization.svg" style="height: 300px; width: auto; border:none;"/>
 
+
</a>
  </section>
+
</div>
  <section id="5">
+
</div>
    <h1>5. References</h1>
+
<div class="thumbcaption" style="text-align: justify;">
    <p>
+
<i><b>Figure 2: Expression and DNA Binding Analysis of His<sub>6</sub>-mNeonGreen-Oct1-DBD.</b></i><br/>
      Lundbäck, T., Chang, J.-F., Phillips, K., Luisi, B., & Ladbury, J. E. (2000). Characterization of
+
<i><b>Left image:</b>Lane 1: raw lysate of E. coli expression culture after sterile filtration; Lane 2: Flow
       Sequence-Specific DNA Binding by the Transcription Factor Oct-1. Biochemistry, 39(25), 7570–7579.
+
              through of first wash
      <a href="https://doi.org/10.1021/bi000377h" target="_blank">https://doi.org/10.1021/bi000377h</a>
+
              (10 bed volumes of NaP10 (Na<sub>2</sub>HPO<sub>4</sub>, 150 mM NaCl, 10 mM Imidazol)); Lane 3: Flow
    </p>
+
              through
    <p>
+
              of
      Park, J. H., Kwon, H. W., & Jeong, K. J. (2013). Development of a plasmid display system with an Oct-1 DNA-binding
+
              second wash (10 bed volumes of NaP20 (Na<sub>2</sub>HPO<sub>4</sub>, 150 mM NaCl, 20 mM Imidazol)); Lane
      domain suitable for in vitro screening of engineered proteins. Journal of Bioscience and Bioengineering, 116(2),
+
              4:
      246-252.
+
              Elution of purified protein.<br/>
      <a href="https://doi.org/10.1016/j.jbiosc.2013.02.005"
+
              1 µL of each fraction was loaded after mixing and heating with 4x Laemmli buffer, on a 4-15% TGX-Gel. The
        target="_blank">https://doi.org/10.1016/j.jbiosc.2013.02.005</a>.
+
              expected band size of the protein is 56 840.23 Da, highlighted in red on the gel.<br/>
    </p>
+
<b>Right image</b> Purified mNeonGreen-Oct1 fusion-protein (1000 nM, 100 nM or 10 nM) were equilibrated
    <p>
+
              with 0.5 µM DNA,
      Park, Y., Shin, J., Yang, J., Kim, H., Jung, Y., Oh, H., Kim, Y., Hwang, J., Park, M., Ban, C., Jeong, K. J., Kim,
+
              containing three Oct1 binding sites, in different buffer compositions.
       S.-K., & Kweon, D.-H. (2020). Plasmid Display for Stabilization of Enzymes Inside the Cell to Improve Whole-Cell
+
              (Binding buffer 1: 137 mM NaCl, 2.7 mM KCl, 10 mM Na 2HPO4, 1.8 mM KH2HPO4, 0.1 % (v/v) IGEPAL® CA-360, 1
      Biotransformation Efficiency. Frontiers in Bioengineering and Biotechnology, 7.
+
              mM
      <a href="https://doi.org/10.3389/fbioe.2019.00444" target="_blank">https://doi.org/10.3389/fbioe.2019.00444</a>
+
              EDTA; Binding buffer 2: 10 mM Tris, 50 mM KCl; NaP250: 50 mM NaH2PO4, 150 mM NaCl, 250 mM Imidazol) Bands
    </p>
+
              were visualized with SYBR-Safe staining.</i>
    <p>
+
</div>
      Stepchenko, A. G., Portseva, T. N., Glukhov, I. A., Kotnova, A. P., Lyanova, B. M., Georgieva, S. G., &
+
</div>
 +
</div>
 +
</section>
 +
<section id="4.2">
 +
<h2>4.2 <i>In Silico</i> Characterization using DaVinci</h2>
 +
<p>
 +
        We developed the <i>in silico</i> model <a href="https://2024.igem.wiki/heidelberg/model" target="_blank">DaVinci</a>
 +
        for rapid engineering
 +
        and development of our PICasSO system.
 +
        DaVinci acts as a digital twin to PICasSO, designed to understand the forces acting on our system,
 +
        refine experimental parameters, and find optimal connections between protein staples and target DNA.
 +
        We calibrated DaVinci with literature and our own experimental affinity data calculated from EMSA assays with
 +
        purified proteins
 +
        DaVinci is divided into three phases: static structure prediction, all-atom dynamics simulation, and long-ranged
 +
        DNA
 +
        dynamics simulation. We applied the first two to our parts, characterizing structure and dynamics of the
 +
        DNA-binding interaction.<br/>
 +
        The structures shown in Figure 4 were predicted using the AlphaFold server and the protein-DNA interaction
 +
        further
 +
        analyzed with all atom dynamics simulations. The depicted structures show favorable DNA binding, and no apparent
 +
        problems with the fusion protein and DNA binding were detected.
 +
       </p>
 +
<div class="thumb">
 +
<div class="thumbinner" style="width:80%;">
 +
<img alt="" class="thumbimage" src="https://static.igem.wiki/teams/5237/model/structure-figure-2-png.svg" style="width: 99%;"/>
 +
<div class="thumbcaption">
 +
<i><b>Figure 4: Representations of the Simple Staple Constructs</b>
 +
              Proteins are shown in full color (top row) and by their predicted structural accuracy during DNA
 +
              interaction.
 +
              The linkers were selected based on their structural property providing maximal flexibility. All structures
 +
              were predicted using the AlphaFold server (Google DeepMind, 2024).</i>
 +
</div>
 +
</div>
 +
</div></section>
 +
</section>
 +
<section id="5">
 +
<h1>5. References</h1>
 +
<p>Lundbäck, T., Chang, J.-F., Phillips, K., Luisi, B., &amp; Ladbury, J. E. (2000). Characterization of
 +
       Sequence-Specific DNA Binding by the Transcription Factor Oct-1. <em>Biochemistry, 39</em>(25), 7570–7579. <a href="https://doi.org/10.1021/bi000377h" target="_blank">https://doi.org/10.1021/bi000377h</a></p>
 +
<p>Park, J. H., Kwon, H. W., &amp; Jeong, K. J. (2013). Development of a plasmid display system with an Oct-1
 +
      DNA-binding domain suitable for in vitro screening of engineered proteins. <em>Journal of Bioscience and
 +
        Bioengineering, 116</em>(2), 246–252. <a href="https://doi.org/10.1016/j.jbiosc.2013.02.005" target="_blank">https://doi.org/10.1016/j.jbiosc.2013.02.005</a></p>
 +
<p>Park, Y., Shin, J., Yang, J., Kim, H., Jung, Y., Oh, H., Kim, Y., Hwang, J., Park, M., Ban, C., Jeong, K. J.,
 +
       Kim, S.-K., &amp; Kweon, D.-H. (2020). Plasmid Display for Stabilization of Enzymes Inside the Cell to Improve
 +
      Whole-Cell Biotransformation Efficiency. <em>Frontiers in Bioengineering and Biotechnology, 7</em>. <a href="https://doi.org/10.3389/fbioe.2019.00444" target="_blank">https://doi.org/10.3389/fbioe.2019.00444</a></p>
 +
<p>Stepchenko, A. G., Portseva, T. N., Glukhov, I. A., Kotnova, A. P., Lyanova, B. M., Georgieva, S. G., &amp;
 
       Pankratova, E. V. (2021). Primate-specific stress-induced transcription factor POU2F1Z protects human neuronal
 
       Pankratova, E. V. (2021). Primate-specific stress-induced transcription factor POU2F1Z protects human neuronal
       cells from stress. Scientific Reports, 11(1), 18808.
+
       cells from stress. <em>Scientific Reports, 11</em>(1), 18808. <a href="https://doi.org/10.1038/s41598-021-98323-y" target="_blank">https://doi.org/10.1038/s41598-021-98323-y</a></p>
      <a href="https://doi.org/10.1038/s41598-021-98323-y"
+
</section>
        target="_blank">https://doi.org/10.1038/s41598-021-98323-y</a>
+
    </p>
+
  </section>
+
 
</body>
 
</body>
 
 
</html>
 
</html>

Latest revision as of 12:43, 2 October 2024


BBa_K5237004

Half staple: Oct1-DBD

Oct1-DBD is the DNA-binding domain of the human Oct1 transcription factor. It can be readily fused with other DNA-binding proteins to form a functional staple to DNA-DNA proximity. We used this part as a component for our Simple staple (BBa_K5237006) and also fused to mNeonGreen, as part of a FRET readout system (BBa_K5237016).



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
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

2. Usage and Biology

Oct1-DBD is the DNA-binding domain of the human transcription factor Oct1 (POU2F1), which plays a key role in gene regulation, immune response, and stress adaptation in eukaryotic cells. This domain specifically binds to the octamer motif (5'-ATGCAAAT-3') within promoter and enhancer regions, influencing transcriptional activity (Lundbäck et al., 2000). The Oct1-DBD consists of both a POU-specific domain and a POU homeodomain, which work together to form a stable complex with DNA (Park et al., 2013, Stepchenko et al. 2021).

In synthetic biology, Oct1-DBD has been previously used for plasmid display technology due to its strong binding affinity (KD = 9 × 10-11 M), additionally proteins fused with Oct1-DBD showed increased expression and protein solubility (Parker et al. 2020).

This part was further used in BBa_K5237002 as a fusion with tetR, resulting in a bivalent DNA binding staple, and also fused to mNeonGreen, as part of a FRET readout system (BBa_K5237016).

3. Assembly and Part Evolution

The Oct1-DBD amino acid sequence was obtained from UniProt (P14859, POU domain, class 2, transcription factor 1) and the DNA binding domain was extracted based on information given from Park et al. 2013 & 2020. An E. coli codon optimized DNA sequence was obtained through gene synthesis and used to clone further constructs.

4. Results

4.1 Protein Expression and EMSA

Oct1 was N-terminally fused to His6-mNeonGreen. This fusion protein was expressed under a T7 promoter and subsequently purified using metal affinity chromatography with Ni-NTA beads (Fig. 2, left). DNA binding affinity was estimated with an electrophoretic mobility shift assay (EMSA). For this, three different buffer conditions were tested (Binding buffer 1: 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2HPO4, 0.1 % (v/v) IGEPAL® CA-360, 1 mM EDTA; Binding buffer 2: 10 mM Tris, 50 mM KCl; NaP250: Na2HPO4, 150 mM NaCl, 250 mM Imidazol). DNA binding could only be detected for Binding buffer 1. (Fig. 2, right)

Figure 2: Expression and DNA Binding Analysis of His6-mNeonGreen-Oct1-DBD.
Left image:Lane 1: raw lysate of E. coli expression culture after sterile filtration; Lane 2: Flow through of first wash (10 bed volumes of NaP10 (Na2HPO4, 150 mM NaCl, 10 mM Imidazol)); Lane 3: Flow through of second wash (10 bed volumes of NaP20 (Na2HPO4, 150 mM NaCl, 20 mM Imidazol)); Lane 4: Elution of purified protein.
1 µL of each fraction was loaded after mixing and heating with 4x Laemmli buffer, on a 4-15% TGX-Gel. The expected band size of the protein is 56 840.23 Da, highlighted in red on the gel.
Right image Purified mNeonGreen-Oct1 fusion-protein (1000 nM, 100 nM or 10 nM) were equilibrated with 0.5 µM DNA, containing three Oct1 binding sites, in different buffer compositions. (Binding buffer 1: 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; Binding buffer 2: 10 mM Tris, 50 mM KCl; NaP250: 50 mM NaH2PO4, 150 mM NaCl, 250 mM Imidazol) Bands were visualized with SYBR-Safe staining.

4.2 In Silico Characterization using DaVinci

We developed the in silico model DaVinci for rapid engineering and development of our PICasSO system. DaVinci acts as a digital twin to PICasSO, designed to understand the forces acting on our system, refine experimental parameters, and find optimal connections between protein staples and target DNA. We calibrated DaVinci with literature and our own experimental affinity data calculated from EMSA assays with purified proteins DaVinci is divided into three phases: static structure prediction, all-atom dynamics simulation, and long-ranged DNA dynamics simulation. We applied the first two to our parts, characterizing structure and dynamics of the DNA-binding interaction.
The structures shown in Figure 4 were predicted using the AlphaFold server and the protein-DNA interaction further analyzed with all atom dynamics simulations. The depicted structures show favorable DNA binding, and no apparent problems with the fusion protein and DNA binding were detected.

Figure 4: Representations of the Simple Staple Constructs Proteins are shown in full color (top row) and by their predicted structural accuracy during DNA interaction. The linkers were selected based on their structural property providing maximal flexibility. All structures were predicted using the AlphaFold server (Google DeepMind, 2024).

5. References

Lundbäck, T., Chang, J.-F., Phillips, K., Luisi, B., & Ladbury, J. E. (2000). Characterization of Sequence-Specific DNA Binding by the Transcription Factor Oct-1. Biochemistry, 39(25), 7570–7579. https://doi.org/10.1021/bi000377h

Park, J. H., Kwon, H. W., & Jeong, K. J. (2013). Development of a plasmid display system with an Oct-1 DNA-binding domain suitable for in vitro screening of engineered proteins. Journal of Bioscience and Bioengineering, 116(2), 246–252. https://doi.org/10.1016/j.jbiosc.2013.02.005

Park, Y., Shin, J., Yang, J., Kim, H., Jung, Y., Oh, H., Kim, Y., Hwang, J., Park, M., Ban, C., Jeong, K. J., Kim, S.-K., & Kweon, D.-H. (2020). Plasmid Display for Stabilization of Enzymes Inside the Cell to Improve Whole-Cell Biotransformation Efficiency. Frontiers in Bioengineering and Biotechnology, 7. https://doi.org/10.3389/fbioe.2019.00444

Stepchenko, A. G., Portseva, T. N., Glukhov, I. A., Kotnova, A. P., Lyanova, B. M., Georgieva, S. G., & Pankratova, E. V. (2021). Primate-specific stress-induced transcription factor POU2F1Z protects human neuronal cells from stress. Scientific Reports, 11(1), 18808. https://doi.org/10.1038/s41598-021-98323-y