Difference between revisions of "Part:BBa K5237015"

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<body>
  <!-- Part summary -->
+
<!-- Part summary -->
  <section id="1">
+
<section id="1">
    <h1>Intimin anti-EGFR nanobody</h1>
+
<h1>Intimin anti-EGFR nanobody</h1>
    <p>This part contains the N-terminus of intimin harbouring the anti-EGFR nanobody (wild-type 7D12) and can be used for surface display of anti-EGFR nanobodies on <i>E.coli</i>. With this part, we sought to enhance bacterial binding to mammalian cells and potentially improve the chances of DNA delivery via conjugation by the bacterial Type IV secretion system (T4SS). As part of the PICasSO toolbox, we seek to use anti-EGFR adhesins to promote delivery of large DNA constructs encoding the various protein staples (presented in our parts collection) from bacteria to mammalian cells via conjugation to enable controlled modulation of chromatin organization by the delivered staples.</p>
+
<p>This part contains the N-terminus of intimin harbouring the anti-EGFR nanobody (wild-type 7D12) and can be used for surface display of anti-EGFR nanobodies on <i>E.coli</i>. With this part, we sought to enhance bacterial binding to mammalian cells and potentially improve the chances of DNA delivery via conjugation by the bacterial Type IV secretion system (T4SS). As part of the PICasSO toolbox, we seek to use anti-EGFR adhesins to promote delivery of large DNA constructs encoding the various protein staples (presented in our parts collection) from bacteria to mammalian cells via conjugation to enable controlled modulation of chromatin organization by the delivered staples.</p>
 
+
<div class="toc" id="toc">
  <div id="toc" class="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-5"><a href="#4"><span class="tocnumber">4</span> <span class="toctext">Results</span></a>
            class="toctext">Results</span></a>
+
</li>
      </li>
+
<li class="toclevel-1 tocsection-8"><a href="#5"><span class="tocnumber">5</span> <span class="toctext">References</span></a>
      <li class="toclevel-1 tocsection-8"><a href="#5"><span class="tocnumber">5</span> <span
+
</li>
            class="toctext">References</span></a>
+
</ul>
      </li>
+
</div>
    </ul>
+
</section>
  </div>
+
<section><p><br/><br/></p>
  </section>
+
<font size="5"><b>The PICasSO Toolbox </b> </font>
 
+
<div class="thumb" style="margin-top:10px;"></div>
  <section>
+
<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%;"/>
    <font size="5"><b>The PICasSO Toolbox </b> </font>
+
<div class="thumbcaption">
    <p><br></p>
+
<i><b>Figure 1: How our part collection can be used to engineer new staples</b></i>
    <div class="thumb"></div>
+
</div>
      <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">
+
</div>
        <div class="thumbcaption">
+
<p>
          <i><b>Figure 1: Example how the part collection can be used to engineer new staples</b></i>
+
<br/>
        </div>
+
       While synthetic biology has in the past focused on engineering the genomic sequence of organisms, the <b>3D
      </div>
+
        spatial organization</b> of DNA is well-known to be an important layer of information encoding in
    </div>
+
      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
    <p>
+
       genomic spatial
      <br>
+
      architecture are limited, hampering the exploration of
       The 3D organization of the genome plays a crucial role in regulating gene expression in eukaryotic cells,
+
       3D genome engineering in synthetic biology. We - the iGEM Team Heidelberg 2024 - have developed PICasSO, a
       impacting cellular behavior, evolution, and disease. Beyond the linear DNA sequence, the spatial arrangement of
+
       <b>powerful
      chromatin, influenced by DNA-DNA interactions, shapes pathways of gene regulation. However, the tools to precisely
+
        molecular toolbox for rationally engineering genome 3D architectures</b> in living cells, based on
       manipulate this genomic architecture remain limited, rendering it challenging to explore the full potential of the
+
      various DNA-binding proteins.
       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>
          <td>Staple subunit: SV40 NLS-dSpCas9-SV40 NLS</td>
+
</tr>
          <td>Staple subunit that can be combined to form a functional staple, for example with our fgRNA or dCas12a
+
<tr bgcolor="#FFD700">
 +
<td><a href="https://parts.igem.org/Part:BBa_K5237002" target="_blank">BBa_K5237002</a></td>
 +
<td>Staple Subunit: SV40 NLS-dSpCas9-SV40 NLS</td>
 +
<td>Staple subunit that can be combined with a sgRNA or fgRNA and dMbCas12a to form a functional staple
 
           </td>
 
           </td>
        </tr>
+
</tr>
        <tr>
+
<tr>
          <td><a href="https://parts.igem.org/Part:BBa_K5237003" target="_blank">BBa_K5237003</a></td>
+
<td><a href="https://parts.igem.org/Part:BBa_K5237003" target="_blank">BBa_K5237003</a></td>
          <td>Cas-Staple: SV40 NLS-dMbCas12a-dSpCas9-Nucleoplasmin NLS</td>
+
<td>Cas Staple: SV40 NLS-dMbCas12a-dSpCas9-Nucleoplasmin NLS</td>
          <td>Functional Cas staple that can be combined with sgRNA or fgRNA to bring two DNA strands in close proximity
+
<td>Functional Cas staple that can be combined with sgRNA and crRNA or fgRNA to bring two DNA strands into
 +
            close
 +
            proximity
 
           </td>
 
           </td>
        </tr>
+
</tr>
        <tr>
+
<tr>
          <td><a href="https://parts.igem.org/Part:BBa_K5237004" target="_blank">BBa_K5237004</a></td>
+
<td><a href="https://parts.igem.org/Part:BBa_K5237004" target="_blank">BBa_K5237004</a></td>
          <td>Staple subunit: Oct1-DBD</td>
+
<td>Staple Subunit: Oct1-DBD</td>
          <td>Staple subunit that can be combined to form a functional staple, for example with TetR.<br>
+
<td>Staple subunit that can be combined to form a functional staple, for example with TetR.<br/>
 
             Can also be combined with a fluorescent protein as part of the FRET proximity assay</td>
 
             Can also be combined with a fluorescent protein as part of the FRET proximity assay</td>
        </tr>
+
</tr>
        <tr>
+
<tr>
          <td><a href="https://parts.igem.org/Part:BBa_K5237005" target="_blank">BBa_K5237005</a></td>
+
<td><a href="https://parts.igem.org/Part:BBa_K5237005" target="_blank">BBa_K5237005</a></td>
          <td>Staple subunit: TetR</td>
+
<td>Staple Subunit: TetR</td>
          <td>Staple subunit that can be combined to form a functional staple, for example with Oct1.<br>
+
<td>Staple subunit that can be combined to form a functional staple, for example with Oct1.<br/>
 
             Can also be combined with a fluorescent protein as part of the FRET proximity assay</td>
 
             Can also be combined with a fluorescent protein as part of the FRET proximity assay</td>
        </tr>
+
</tr>
        <tr>
+
<tr>
          <td><a href="https://parts.igem.org/Part:BBa_K5237006" target="_blank">BBa_K5237006</a></td>
+
<td><a href="https://parts.igem.org/Part:BBa_K5237006" target="_blank">BBa_K5237006</a></td>
          <td>Simple 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_K52370012" 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_K52370013" 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_K52370014" 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_K52370015" 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_K52370016" 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 Trans-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>Trans-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_K5237015 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K5237015 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>This part contains the N-terminus of intimin harbouring the anti-EGFR (wild-type 7D12) adhesin and can be used for surface display of anti-EGFR nanobodies on <i>E.coli</i>. Salema et al., (2013) showed efficient presentation of nanobodies on the surface of <i>E.coli</i> K-12 cells by fusing them to the β domain of intimin. The β domain of intimin comes from the pNeae2 plasmid (addgene #168300) and encodes an N-terminal signal peptide for Sec-dependent translocation into the periplasm, a LysM domain that interacts with the peptidoglycan and provides anchoring, and a β-barrel that inserts into the outer membrane. The coding sequence of the anti-EGFR nanobody is located in the C-terminus (that is exposed to the extracellular milieu) between the E tag (GAPVPYPDPLEP) and the myc tag (EQKLISEED). These tags can be utilized for purification or detection of the protein.</p>
+
<p>This part contains the N-terminus of intimin harbouring the anti-EGFR (wild-type 7D12) adhesin and can be used for surface display of anti-EGFR nanobodies on <i>E.coli</i>. Salema et al., (2013) showed efficient presentation of nanobodies on the surface of <i>E.coli</i> K-12 cells by fusing them to the β domain of intimin. The β domain of intimin comes from the pNeae2 plasmid (addgene #168300) and encodes an N-terminal signal peptide for Sec-dependent translocation into the periplasm, a LysM domain that interacts with the peptidoglycan and provides anchoring, and a β-barrel that inserts into the outer membrane. The coding sequence of the anti-EGFR nanobody is located in the C-terminus (that is exposed to the extracellular milieu) between the E tag (GAPVPYPDPLEP) and the myc tag (EQKLISEED). These tags can be utilized for purification or detection of the protein.</p>
   
+
<p>The idea behind engineering this part was to use it for increasing cell-cell contact between bacteria and mammalian cells and thereby potentially enhancing the chances of inter-kingdom conjugational DNA transfer. The inspiration to test inter-kingdom conjugation came from the fact that all the events leading to DNA transfer by conjugation are driven by the donor bacterium and the protein components are typically plasmid encoded, making it possible for any type of cell to serve as the recipient (Waters, 2001). Furthermore, as the primary trigger for conjugation remains obscure, we hypothesized that cell-cell contact might be one of the main determinants for conjugation to occur efficiently. Robledo <i>et al.</i>(2022) showed that enhanced cell-cell contact mediated by synthetic adhesins led to a 100-fold increase in conjugation efficiency between bacteria in liquid media, where the RP4 conjugative system is particularly ineffective. This combination of knowledge motivated us to engineer conjugation as a generalized DNA delivery tool for large plasmid constructs (around ~100 kb). Figure 2 below presents an illustration of our idea.</p>
    <p>The idea behind engineering this part was to use it for increasing cell-cell contact between bacteria and mammalian cells and thereby potentially enhancing the chances of inter-kingdom conjugational DNA transfer. The inspiration to test inter-kingdom conjugation came from the fact that all the events leading to DNA transfer by conjugation are driven by the donor bacterium and the protein components are typically plasmid encoded, making it possible for any type of cell to serve as the recipient (Waters, 2001). Furthermore, as the primary trigger for conjugation remains obscure, we hypothesized that cell-cell contact might be one of the main determinants for conjugation to occur efficiently. Robledo <i>et al.</i>(2022) showed that enhanced cell-cell contact mediated by synthetic adhesins led to a 100-fold increase in conjugation efficiency between bacteria in liquid media, where the RP4 conjugative system is particularly ineffective. This combination of knowledge motivated us to engineer conjugation as a generalized DNA delivery tool for large plasmid constructs (around ~100 kb). Figure 2 below presents an illustration of our idea.</p>
+
<div class="thumb">
 
+
<div class="thumbinner" style="width:60%;">
    <div class="thumb">
+
<img alt="Inter-kingdom conjugation" class="thumbimage" src="https://static.igem.wiki/teams/5237/figures-corrected/conjugation-mammalian-bacteria.svg" style="width:99%;"/>
      <div class="thumbinner" style="width:60%;">
+
<div class="thumbcaption">
        <img alt="Inter-kingdom conjugation" src="https://static.igem.wiki/teams/5237/figures-corrected/conjugation-mammalian-bacteria.svg" style="width:99%;" class="thumbimage">
+
<i><b>Figure 2. Illustration of DNA delivery from bacteria to mammalian cells via conjugation.</b>A conjugative helper plasmid catalyzes transfer of an <i><a href="https://parts.igem.org/Part:BBa_K4643003">oriT</a></i> carrying mobilizable plasmid from bacteria to mammalian cells. Anti-EGFR adhesins are shown to stabilize the mating pair.</i>
        <div class="thumbcaption">
+
</div>
          <i><b>Figure 2. Illustration of DNA delivery from bacteria to mammalian cells via conjugation.</b>A conjugative helper plasmid catalyzes transfer of an <i><a href="https://parts.igem.org/Part:BBa_K4643003">oriT</a></i> carrying mobilizable plasmid from bacteria to mammalian cells. Anti-EGFR adhesins are shown to stabilize the mating pair.</i>
+
</div>
      </div>
+
<p>This part is a member of the PICasSO toolbox and can be used to promote adhesion to mammalian cell surfaces as EGFR is a common mammalian surface receptor. This part finds its application as an alternative DNA delivery tool to mammalian cells for parts in the PICasSO Toolbox such as the dCas-based staples (<a href="https://parts.igem.org/Part:BBa_K5237001" target="_blank"> BBa_K5237001</a>, <a href="https://parts.igem.org/Part:BBa_K5237002" target="_blank"> BBa_K5237002</a>, <a href="https://parts.igem.org/Part:BBa_K5237003" target="_blank">BBa_K5237003</a> ) along with fgRNAs, which are encoded by rather large plasmids. It is known that lipofection efficiency decreases with increasing plasmid size (Kreiss <i>et al.</i>, 1999), so conjugation may be employed as an alternative tool to deliver such large plasmids to mammalian cells <i>in vitro</i>, to not only enable engineering of chromatin conformations, but also to allow for its controlled modulation in a stimulus-responsive manner. Moreover, the anti-EGFR nanobody can easily be swapped with other nanobodies to alter the target specificity or binding affinities.</p>
  </div>
+
</div></section>
 
+
    <p>This part is a member of the PICasSO toolbox and can be used to promote adhesion to mammalian cell surfaces as EGFR is a common mammalian surface receptor. This part finds its application as an alternative DNA delivery tool to mammalian cells for parts in the PICasSO Toolbox such as the dCas-based staples (<a href="https://parts.igem.org/Part:BBa_K5237001" target="_blank"> BBa_K5237001</a>, <a href="https://parts.igem.org/Part:BBa_K5237002" target="_blank"> BBa_K5237002</a>, <a href="https://parts.igem.org/Part:BBa_K5237003" target="_blank">BBa_K5237003</a> ) along with fgRNAs, which are encoded by rather large plasmids. It is known that lipofection efficiency decreases with increasing plasmid size (Kreiss <i>et al.</i>, 1999), so conjugation may be employed as an alternative tool to deliver such large plasmids to mammalian cells <i>in vitro</i>, to not only enable engineering of chromatin conformations, but also to allow for its controlled modulation in a stimulus-responsive manner. Moreover, the anti-EGFR nanobody can easily be swapped with other nanobodies to alter the target specificity or binding affinities.</p>
+
</section>
+
 
+
 
<section id="3">
 
<section id="3">
    <h1>3. Assembly and part evolution</h1>
+
<h1>3. Assembly and part evolution</h1>
    <p>The pNeae2 plasmid was obtained from addgene (#168300). Codon optimized DNA sequence of chain L anti-EGFR (7D12) nanobody (Schmitz et al., 2013) for expression in <i>E.coli</i> was procured as a gBlock containing 5' and 3' restriction sites for SfiI and NotI. Attempts at cloning the anti-EGFR nanobody by restriction ligation (using SfiI and NotI) into the coding sequence of intimin in pNeae2 (suggested cloning strategy by Salema et al., (2013)) were unsuccessful as indicated by test digests and Sanger sequencing. To circumvent vector re-ligation issues that we encountered, Gibson assembly shall be attempted in the near future.</p>
+
<p>The pNeae2 plasmid was obtained from addgene (#168300). Codon optimized DNA sequence of chain L anti-EGFR (7D12) nanobody (Schmitz et al., 2013) for expression in <i>E.coli</i> was procured as a gBlock containing 5' and 3' restriction sites for SfiI and NotI. Attempts at cloning the anti-EGFR nanobody by restriction ligation (using SfiI and NotI) into the coding sequence of intimin in pNeae2 (suggested cloning strategy by Salema et al., (2013)) were unsuccessful as indicated by test digests and Sanger sequencing. To circumvent vector re-ligation issues that we encountered, Gibson assembly shall be attempted in the near future.</p>
 
</section>
 
</section>
 
 
<section id="4">
 
<section id="4">
    <h1>4. Results</h1>
+
<h1>4. Results</h1>
    <h2><b>Successful expression of myc-tagged intimin by <i>E.coli</i> 10-beta</b></h2>
+
<h2><b>Successful expression of myc-tagged intimin by <i>E.coli</i> 10-beta</b></h2>
    <p>Despite unsuccessful cloning of anti-EGFR adhesin into the coding sequence of intimin, protein expression was tested in <i>E.coli</i> 10-beta since they will be utilized as the donor strain in our upcoming conjugation tests - due to their ability to stably maintain and tolerate large plasmid constructs. Since <i>E.coli</i> 10-beta is not a typical strain used for protein expression, it was important to test their ability to overexpress outer membrane proteins and its potential toxicity to the cells.  
+
<p>Despite unsuccessful cloning of anti-EGFR adhesin into the coding sequence of intimin, protein expression was tested in <i>E.coli</i> 10-beta since they will be utilized as the donor strain in our upcoming conjugation tests - due to their ability to stably maintain and tolerate large plasmid constructs. Since <i>E.coli</i> 10-beta is not a typical strain used for protein expression, it was important to test their ability to overexpress outer membrane proteins and its potential toxicity to the cells.  
 
      
 
      
     <p><i>E.coli</i> 10-beta were transformed with pNeae2, and protein expression was induced with 50 µM IPTG. A Western Blot against the myc epitope on the C-terminus of intimin revealed expression of myc-tagged intimin of the expected size (~76 kDa) after induction (Figure 3). However, a smear of several bands was noted which could be a consequence of several factors included but not limited to protein degradation, expression of truncated proteins or high SDS-PAGE protein load. Nevertheless, this result still suggests that <i>E.coli</i> 10-beta could be used as the donor strain for our future conjugation assays owing to their ability to express full-length intimin.</p>
+
     <p><i>E.coli</i> 10-beta were transformed with pNeae2, and protein expression was induced with 50 µM IPTG. A Western Blot against the myc epitope on the C-terminus of intimin revealed expression of myc-tagged intimin of the expected size (~76 kDa) after induction (Figure 3). However, a smear of several bands was noted which could be a consequence of several factors included but not limited to protein degradation, expression of truncated proteins or high SDS-PAGE protein load. Nevertheless, this result still suggests that <i>E.coli</i> 10-beta could be used as the donor strain for our future conjugation assays owing to their ability to express full-length intimin.</p>
   
+
<div class="thumb">
    <div class="thumb">
+
<div class="thumbinner" style="width:30%;">
          <div class="thumbinner" style="width:30%;">
+
<img alt="Anti-myc Western Blot" class="thumbimage" src="https://static.igem.wiki/teams/5237/wetlab-results/anti-myc-wb-intimin-myc2.png" style="width:99%;"/>
            <img alt="Anti-myc Western Blot" src="https://static.igem.wiki/teams/5237/wetlab-results/anti-myc-wb-intimin-myc2.png" style="width:99%;" class="thumbimage">
+
<div class="thumbcaption">
            <div class="thumbcaption">
+
<i><b>Figure 3: Fluorescence western blot scan showing expression of myc-tagged intimin by <i>E.coli</i> 10-beta after IPTG induction.</b> <i>E.coli</i> 10-beta were transformed with pNeae2, induced with 50 µM IPTG and lysed. Lane 1 was loaded with 30 µg of total protein from the <i>E.coli</i> lysate after IPTG induction and Lane 2 was loaded with 30 µg of total protein from the uninduced <i>E.coli</i> lysate. The blue arrow indicates the position of the myc-tagged intimin (~76 kDa)</i>
              <i><b>Figure 3: Fluorescence western blot scan showing expression of myc-tagged intimin by <i>E.coli</i> 10-beta after IPTG induction.</b> <i>E.coli</i> 10-beta were transformed with pNeae2, induced with 50 µM IPTG and lysed. Lane 1 was loaded with 30 µg of total protein from the <i>E.coli</i> lysate after IPTG induction and Lane 2 was loaded with 30 µg of total protein from the uninduced <i>E.coli</i> lysate. The blue arrow indicates the position of the myc-tagged intimin (~76 kDa)</i>
+
</div>
            </div>
+
</div>
          </div>
+
</div>
    </div>
+
<h2><b>Outlook</b></h2>
    <h2><b>Outlook</b></h2>
+
<p>Upcoming experiments shall address the interaction between the anti-EGFR adhesin and EGFR <i>in vitro</i> and also examine the increased localization of adhesin-expressing bacteria on the surface of mammalian cells. Ultimately, this part shall be used in conjugation assays between bacteria and mammalian cells to delineate the role of cell-cell contact in promoting inter-kingdom conjugation.</p>
    <p>Upcoming experiments shall address the interaction between the anti-EGFR adhesin and EGFR <i>in vitro</i> and also examine the increased localization of adhesin-expressing bacteria on the surface of mammalian cells. Ultimately, this part shall be used in conjugation assays between bacteria and mammalian cells to delineate the role of cell-cell contact in promoting inter-kingdom conjugation.</p>
+
</p></section>
</section>
+
 
+
 
<section id="5">
 
<section id="5">
    <h1>5. References</h1>
+
<h1>5. References</h1>
    <p>Kreiss, P., Cameron, B., Rangara, R., Mailhe, P., Aguerre-Charriol, O., Airiau, M., Scherman, D., Crouzet, J., & Pitard, B. (1999). Plasmid DNA size does not affect the physicochemical properties of lipoplexes but modulates gene transfer efficiency. Nucleic Acids Research, 27(19). https://doi.org/10.1093/nar/27.19.3792</p>
+
<p>Kreiss, P., Cameron, B., Rangara, R., Mailhe, P., Aguerre-Charriol, O., Airiau, M., Scherman, D., Crouzet, J., &amp; Pitard, B. (1999). Plasmid DNA size does not affect the physicochemical properties of lipoplexes but modulates gene transfer efficiency. Nucleic Acids Research, 27(19). https://doi.org/10.1093/nar/27.19.3792</p>
    <p>Robledo, M., Álvarez, B., Cuevas, A., González, S., Ruano-Gallego, D., Fernández, L. Á., & De La Cruz, F. (2022). Targeted bacterial conjugation mediated by synthetic cell-to-cell adhesions. Nucleic Acids Research, 50(22). https://doi.org/10.1093/nar/gkac1164</p>
+
<p>Robledo, M., Álvarez, B., Cuevas, A., González, S., Ruano-Gallego, D., Fernández, L. Á., &amp; De La Cruz, F. (2022). Targeted bacterial conjugation mediated by synthetic cell-to-cell adhesions. Nucleic Acids Research, 50(22). https://doi.org/10.1093/nar/gkac1164</p>
    <p>Salema, V., Marín, E., Martínez-Arteaga, R., Ruano-Gallego, D., Fraile, S., Margolles, Y., Teira, X., Gutierrez, C., Bodelón, G., & Fernández, L. Á. (2013). Selection of Single Domain Antibodies from Immune Libraries Displayed on the Surface of E. coli Cells with Two β-Domains of Opposite Topologies. PLoS ONE, 8(9). https://doi.org/10.1371/journal.pone.0075126</p>
+
<p>Salema, V., Marín, E., Martínez-Arteaga, R., Ruano-Gallego, D., Fraile, S., Margolles, Y., Teira, X., Gutierrez, C., Bodelón, G., &amp; Fernández, L. Á. (2013). Selection of Single Domain Antibodies from Immune Libraries Displayed on the Surface of E. coli Cells with Two β-Domains of Opposite Topologies. PLoS ONE, 8(9). https://doi.org/10.1371/journal.pone.0075126</p>
    <p>Schmitz, K. R., Bagchi, A., Roovers, R. C., Van Bergen En Henegouwen, P. M. P., & Ferguson, K. M. (2013). Structural evaluation of EGFR inhibition mechanisms for nanobodies/VHH domains. Structure, 21(7).https://doi.org/10.1016/j.str.2013.05.008</p>
+
<p>Schmitz, K. R., Bagchi, A., Roovers, R. C., Van Bergen En Henegouwen, P. M. P., &amp; Ferguson, K. M. (2013). Structural evaluation of EGFR inhibition mechanisms for nanobodies/VHH domains. Structure, 21(7).https://doi.org/10.1016/j.str.2013.05.008</p>
    <p>Waters, V. L. (2001). Conjugation between bacterial and mammalian cells. Nature Genetics, 29(4). https://doi.org/10.1038/ng779</p>
+
<p>Waters, V. L. (2001). Conjugation between bacterial and mammalian cells. Nature Genetics, 29(4). https://doi.org/10.1038/ng779</p>
 
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Revision as of 07:27, 2 October 2024

BBa_K5237015

Intimin anti-EGFR nanobody

This part contains the N-terminus of intimin harbouring the anti-EGFR nanobody (wild-type 7D12) and can be used for surface display of anti-EGFR nanobodies on E.coli. With this part, we sought to enhance bacterial binding to mammalian cells and potentially improve the chances of DNA delivery via conjugation by the bacterial Type IV secretion system (T4SS). As part of the PICasSO toolbox, we seek to use anti-EGFR adhesins to promote delivery of large DNA constructs encoding the various protein staples (presented in our parts collection) from bacteria to mammalian cells via conjugation to enable controlled modulation of chromatin organization by the delivered 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
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 105
    Illegal EcoRI site found at 2259
    Illegal PstI site found at 401
    Illegal PstI site found at 649
    Illegal PstI site found at 1096
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 105
    Illegal EcoRI site found at 2259
    Illegal PstI site found at 401
    Illegal PstI site found at 649
    Illegal PstI site found at 1096
    Illegal NotI site found at 2410
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 105
    Illegal EcoRI site found at 2259
    Illegal BamHI site found at 1983
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 105
    Illegal EcoRI site found at 2259
    Illegal PstI site found at 401
    Illegal PstI site found at 649
    Illegal PstI site found at 1096
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 105
    Illegal EcoRI site found at 2259
    Illegal PstI site found at 401
    Illegal PstI site found at 649
    Illegal PstI site found at 1096
    Illegal NgoMIV site found at 2004
  • 1000
    COMPATIBLE WITH RFC[1000]

2. Usage and Biology

This part contains the N-terminus of intimin harbouring the anti-EGFR (wild-type 7D12) adhesin and can be used for surface display of anti-EGFR nanobodies on E.coli. Salema et al., (2013) showed efficient presentation of nanobodies on the surface of E.coli K-12 cells by fusing them to the β domain of intimin. The β domain of intimin comes from the pNeae2 plasmid (addgene #168300) and encodes an N-terminal signal peptide for Sec-dependent translocation into the periplasm, a LysM domain that interacts with the peptidoglycan and provides anchoring, and a β-barrel that inserts into the outer membrane. The coding sequence of the anti-EGFR nanobody is located in the C-terminus (that is exposed to the extracellular milieu) between the E tag (GAPVPYPDPLEP) and the myc tag (EQKLISEED). These tags can be utilized for purification or detection of the protein.

The idea behind engineering this part was to use it for increasing cell-cell contact between bacteria and mammalian cells and thereby potentially enhancing the chances of inter-kingdom conjugational DNA transfer. The inspiration to test inter-kingdom conjugation came from the fact that all the events leading to DNA transfer by conjugation are driven by the donor bacterium and the protein components are typically plasmid encoded, making it possible for any type of cell to serve as the recipient (Waters, 2001). Furthermore, as the primary trigger for conjugation remains obscure, we hypothesized that cell-cell contact might be one of the main determinants for conjugation to occur efficiently. Robledo et al.(2022) showed that enhanced cell-cell contact mediated by synthetic adhesins led to a 100-fold increase in conjugation efficiency between bacteria in liquid media, where the RP4 conjugative system is particularly ineffective. This combination of knowledge motivated us to engineer conjugation as a generalized DNA delivery tool for large plasmid constructs (around ~100 kb). Figure 2 below presents an illustration of our idea.

Inter-kingdom conjugation
Figure 2. Illustration of DNA delivery from bacteria to mammalian cells via conjugation.A conjugative helper plasmid catalyzes transfer of an oriT carrying mobilizable plasmid from bacteria to mammalian cells. Anti-EGFR adhesins are shown to stabilize the mating pair.

This part is a member of the PICasSO toolbox and can be used to promote adhesion to mammalian cell surfaces as EGFR is a common mammalian surface receptor. This part finds its application as an alternative DNA delivery tool to mammalian cells for parts in the PICasSO Toolbox such as the dCas-based staples ( BBa_K5237001, BBa_K5237002, BBa_K5237003 ) along with fgRNAs, which are encoded by rather large plasmids. It is known that lipofection efficiency decreases with increasing plasmid size (Kreiss et al., 1999), so conjugation may be employed as an alternative tool to deliver such large plasmids to mammalian cells in vitro, to not only enable engineering of chromatin conformations, but also to allow for its controlled modulation in a stimulus-responsive manner. Moreover, the anti-EGFR nanobody can easily be swapped with other nanobodies to alter the target specificity or binding affinities.

3. Assembly and part evolution

The pNeae2 plasmid was obtained from addgene (#168300). Codon optimized DNA sequence of chain L anti-EGFR (7D12) nanobody (Schmitz et al., 2013) for expression in E.coli was procured as a gBlock containing 5' and 3' restriction sites for SfiI and NotI. Attempts at cloning the anti-EGFR nanobody by restriction ligation (using SfiI and NotI) into the coding sequence of intimin in pNeae2 (suggested cloning strategy by Salema et al., (2013)) were unsuccessful as indicated by test digests and Sanger sequencing. To circumvent vector re-ligation issues that we encountered, Gibson assembly shall be attempted in the near future.

4. Results

Successful expression of myc-tagged intimin by E.coli 10-beta

Despite unsuccessful cloning of anti-EGFR adhesin into the coding sequence of intimin, protein expression was tested in E.coli 10-beta since they will be utilized as the donor strain in our upcoming conjugation tests - due to their ability to stably maintain and tolerate large plasmid constructs. Since E.coli 10-beta is not a typical strain used for protein expression, it was important to test their ability to overexpress outer membrane proteins and its potential toxicity to the cells.

E.coli 10-beta were transformed with pNeae2, and protein expression was induced with 50 µM IPTG. A Western Blot against the myc epitope on the C-terminus of intimin revealed expression of myc-tagged intimin of the expected size (~76 kDa) after induction (Figure 3). However, a smear of several bands was noted which could be a consequence of several factors included but not limited to protein degradation, expression of truncated proteins or high SDS-PAGE protein load. Nevertheless, this result still suggests that E.coli 10-beta could be used as the donor strain for our future conjugation assays owing to their ability to express full-length intimin.

Anti-myc Western Blot
Figure 3: Fluorescence western blot scan showing expression of myc-tagged intimin by E.coli 10-beta after IPTG induction. E.coli 10-beta were transformed with pNeae2, induced with 50 µM IPTG and lysed. Lane 1 was loaded with 30 µg of total protein from the E.coli lysate after IPTG induction and Lane 2 was loaded with 30 µg of total protein from the uninduced E.coli lysate. The blue arrow indicates the position of the myc-tagged intimin (~76 kDa)

Outlook

Upcoming experiments shall address the interaction between the anti-EGFR adhesin and EGFR in vitro and also examine the increased localization of adhesin-expressing bacteria on the surface of mammalian cells. Ultimately, this part shall be used in conjugation assays between bacteria and mammalian cells to delineate the role of cell-cell contact in promoting inter-kingdom conjugation.

5. References

Kreiss, P., Cameron, B., Rangara, R., Mailhe, P., Aguerre-Charriol, O., Airiau, M., Scherman, D., Crouzet, J., & Pitard, B. (1999). Plasmid DNA size does not affect the physicochemical properties of lipoplexes but modulates gene transfer efficiency. Nucleic Acids Research, 27(19). https://doi.org/10.1093/nar/27.19.3792

Robledo, M., Álvarez, B., Cuevas, A., González, S., Ruano-Gallego, D., Fernández, L. Á., & De La Cruz, F. (2022). Targeted bacterial conjugation mediated by synthetic cell-to-cell adhesions. Nucleic Acids Research, 50(22). https://doi.org/10.1093/nar/gkac1164

Salema, V., Marín, E., Martínez-Arteaga, R., Ruano-Gallego, D., Fraile, S., Margolles, Y., Teira, X., Gutierrez, C., Bodelón, G., & Fernández, L. Á. (2013). Selection of Single Domain Antibodies from Immune Libraries Displayed on the Surface of E. coli Cells with Two β-Domains of Opposite Topologies. PLoS ONE, 8(9). https://doi.org/10.1371/journal.pone.0075126

Schmitz, K. R., Bagchi, A., Roovers, R. C., Van Bergen En Henegouwen, P. M. P., & Ferguson, K. M. (2013). Structural evaluation of EGFR inhibition mechanisms for nanobodies/VHH domains. Structure, 21(7).https://doi.org/10.1016/j.str.2013.05.008

Waters, V. L. (2001). Conjugation between bacterial and mammalian cells. Nature Genetics, 29(4). https://doi.org/10.1038/ng779