Difference between revisions of "Part:BBa K5237007"

 
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    <section id="1">
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    background: none !important;
      <h1>Staple subunit: GCN4</h1>
+
     padding-right: 0px !important;
      <p>GCN4 is a yeast transcription factor belonging to the bZip family of DNA-binding proteins. It consists of a basic
+
  }
        region and a leucine zipper dimerization domain, binding DNA as a homodimer via its N-terminal region</p>
+
      <p>&nbsp;</p>
+
     </section>
+
    <div id="toc" class="toc">
+
      <div id="toctitle">
+
        <h1>Contents</h1>
+
      </div>
+
      <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>
+
        </li>
+
        <li class="toclevel-1 tocsetction-3"><a href="#3"><span class="tocnumber">3</span> <span class="toctext">Assembly
+
              and part evolution</span></a>
+
        </li>
+
        <li class="toclevel-1 tocsection-5"><a href="#4"><span class="tocnumber">4</span> <span
+
              class="toctext">Results</span></a>
+
              <ul>
+
                <li class="toclevel-2 tocsection-6"><a href="#4.1"><span class="tocnumber">4.1</span class="toctext">Protein expression and purification</span></a>
+
                </li>
+
                <li class="toclevel-2 tocsection-7"><a href="#4.2"><span class="tocnumber">4.2</span class="toctext">Electrophoretic Mobility shift assay</span></a></li>
+
              </ul>
+
  
        </li>
+
</style>
        <li class="toclevel-1 tocsection-8"><a href="#5"><span class="tocnumber">5</span> <span
+
<body>
              class="toctext">References</span></a>
+
<!-- Part summary -->
        </li>
+
<section>
      </ul>
+
<h1>Staple Subunit: GCN4</h1>
    </div>
+
<p>GCN4 is a yeast transcription factor belonging to the bZip family of DNA-binding proteins.
    <section>
+
      We used GCN4 to study DNA-binding kinetics in our "Mini staples" that bring two DNA target sites into proximity by
      <font size="5"><b>The PICasSO Toolbox </b> </font>
+
      binding them simultaneously.
      <p><br></p>
+
    </p>
      <div class="thumb"></div>
+
<p> </p>
        <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">
+
</section>
          <div class="thumbcaption">
+
<div class="toc" id="toc">
            <i><b>Figure 1: Example how the part collection can be used to engineer new staples</b></i>
+
<div id="toctitle">
          </div>
+
<h1>Contents</h1>
        </div>
+
</div>
       </div>
+
<ul>
        
+
<li class="toclevel-1 tocsection-1"><a href="#1"><span class="tocnumber">1</span> <span class="toctext">Sequence
 
+
            Overview</span></a>
       <p>
+
</li>
         <br>
+
<li class="toclevel-1 tocsection-2"><a href="#2"><span class="tocnumber">2</span> <span class="toctext">Usage and
        The 3D organization of the genome plays a crucial role in regulating gene expression in eukaryotic cells,
+
            Biology</span></a>
         impacting cellular behavior, evolution, and disease. Beyond the linear DNA sequence, the spatial arrangement of
+
</li>
         chromatin, influenced by DNA-DNA interactions, shapes pathways of gene regulation. However, the tools to precisely
+
<li class="toclevel-1 tocsetction-3"><a href="#3"><span class="tocnumber">3</span> <span class="toctext">Assembly
        manipulate this genomic architecture remain limited, rendering it challenging to explore the full potential of the
+
            and Part Evolution</span></a>
        3D genome in synthetic biology. We - iGEM Team Heidelberg 2024 - have developed PICasSO, a powerful molecular
+
</li>
        toolbox based on various DNA-binding proteins to address this issue.
+
<li class="toclevel-1 tocsection-5"><a href="#4"><span class="tocnumber">4</span> <span class="toctext">Results</span></a>
 +
<ul>
 +
<li class="toclevel-2 tocsection-6"><a href="#4.1"><span class="tocnumber">4.1</span> <span class="toctext">Protein Expression and
 +
                Purification</span></a>
 +
</li>
 +
<li class="toclevel-2 tocsection-7"><a href="#4.2"><span class="tocnumber">4.2</span> <span class="toctext">Electrophoretic Mobility
 +
                Shift Assay</span></a>
 +
</li>
 +
<li class="toclevel-2 tocsection-8"><a href="#4.3"><span class="tocnumber">4.3</span> <span class="toctext"><i>In Silico</i>
 +
                Characterization using DaVinci</span></a>
 +
</li>
 +
</ul>
 +
</li>
 +
<li class="toclevel-1 tocsection-8"><a href="#5"><span class="tocnumber">5</span> <span class="toctext">References</span></a>
 +
</li>
 +
</ul>
 +
</div>
 +
<section><p><br/><br/></p>
 +
<font size="5"><b>The PICasSO Toolbox </b> </font>
 +
<div class="thumb" style="margin-top:10px;"></div>
 +
<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>
 +
      The <b>PICasSO</b> part collection offers a comprehensive, modular platform for precise manipulation and
 +
      <b>re-programming
 +
        of DNA-DNA interactions</b> 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 <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>At its heart, the PICasSO part collection consists of three categories. <br/><b>(i)</b> Our <b>DNA-binding
 +
        proteins</b>
 +
      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. <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>
 +
      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.</mark> 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.<br/>
 +
</p>
 +
<p>
 +
<font size="4"><b>Our part collection includes:</b></font><br/>
 +
</p>
 +
<table style="width: 90%; padding-right:10px;">
 +
<td align="left" colspan="3"><b>DNA-Binding Proteins: </b>
 +
         Modular building blocks for engineering of custom staples to mediate defined DNA-DNA interactions <i>in vivo</i></td>
 +
<tbody>
 +
<tr bgcolor="#FFD700">
 +
<td><a href="https://parts.igem.org/Part:BBa_K5237000" target="_blank">BBa_K5237000</a></td>
 +
<td>Fusion Guide RNA Entry Vector MbCas12a-SpCas9</td>
 +
<td>Entry vector for simple fgRNA cloning via SapI</td>
 +
</tr>
 +
<tr bgcolor="#FFD700">
 +
<td><a href="https://parts.igem.org/Part:BBa_K5237001" target="_blank">BBa_K5237001</a></td>
 +
<td>Staple Subunit: dMbCas12a-Nucleoplasmin NLS</td>
 +
<td>Staple Subunit that can be combined with crRNA or fgRNA and dSpCas9 to form a functional staple
 +
          </td>
 +
</tr>
 +
<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>
 +
</tr>
 +
<tr>
 +
<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>Functional Cas staple that can be combined with sgRNA and crRNA or fgRNA to bring two DNA strands into
 +
            close
 +
            proximity
 +
          </td>
 +
</tr>
 +
<tr>
 +
<td><a href="https://parts.igem.org/Part:BBa_K5237004" target="_blank">BBa_K5237004</a></td>
 +
<td>Staple Subunit: Oct1-DBD</td>
 +
<td>Staple subunit that can be combined to form a functional staple, for example with TetR.<br/>
 +
            Can also be combined with a fluorescent protein as part of the FRET proximity assay</td>
 +
</tr>
 +
<tr>
 +
<td><a href="https://parts.igem.org/Part:BBa_K5237005" target="_blank">BBa_K5237005</a></td>
 +
<td>Staple Subunit: TetR</td>
 +
<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>
 +
</tr>
 +
<tr>
 +
<td><a href="https://parts.igem.org/Part:BBa_K5237006" target="_blank">BBa_K5237006</a></td>
 +
<td>Simple Staple: TetR-Oct1</td>
 +
<td>Functional staple that can be used to bring two DNA strands in close proximity</td>
 +
</tr>
 +
<tr>
 +
<td><a href="https://parts.igem.org/Part:BBa_K5237007" target="_blank">BBa_K5237007</a></td>
 +
<td>Staple Subunit: GCN4</td>
 +
<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_K5237008" target="_blank">BBa_K5237008</a></td>
 +
<td>Staple Subunit: rGCN4</td>
 +
<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_K5237009" target="_blank">BBa_K5237009</a></td>
 +
<td>Mini Staple: bGCN4</td>
 +
<td>
 +
            Assembled staple with minimal size that can be further engineered</td>
 +
</tr>
 +
</tbody>
 +
<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
 +
        for custom applications</td>
 +
<tbody>
 +
<tr bgcolor="#FFD700">
 +
<td><a href="https://parts.igem.org/Part:BBa_K5237010" target="_blank">BBa_K5237010</a></td>
 +
<td>Cathepsin B-cleavable Linker: GFLG</td>
 +
<td>Cathepsin B-cleavable peptide linker that can be used to combine two staple subunits to make
 +
            responsive
 +
            staples</td>
 +
</tr>
 +
<tr>
 +
<td><a href="https://parts.igem.org/Part:BBa_K5237011" target="_blank">BBa_K5237011</a></td>
 +
<td>Cathepsin B Expression Cassette</td>
 +
<td>Expression cassette for the overexpression of cathepsin B</td>
 +
</tr>
 +
<tr>
 +
<td><a href="https://parts.igem.org/Part:BBa_K5237012" target="_blank">BBa_K5237012</a></td>
 +
<td>Caged NpuN Intein</td>
 +
<td>A caged NpuN split intein fragment that undergoes protein <i>trans</i>-splicing after protease
 +
            activation, which can be used to create functionalized staple
 +
            subunits</td>
 +
</tr>
 +
<tr>
 +
<td><a href="https://parts.igem.org/Part:BBa_K5237013" target="_blank">BBa_K5237013</a></td>
 +
<td>Caged NpuC Intein</td>
 +
<td>A caged NpuC split intein fragment that undergoes protein <i>trans</i>-splicing after protease
 +
            activation, which can be used to create functionalized staple
 +
            subunits</td>
 +
</tr>
 +
<tr>
 +
<td><a href="https://parts.igem.org/Part:BBa_K5237014" target="_blank">BBa_K5237014</a></td>
 +
<td>Fusion Guide RNA Processing Casette</td>
 +
<td>Processing cassette to produce multiple fgRNAs from one transcript, that can be used for
 +
            multiplexed 3D
 +
            genome reprogramming</td>
 +
</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>
 +
</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>
 +
</html>
 +
<!--################################-->
 +
<span class="h3bb">Sequence and Features</span>
 +
<partinfo>BBa_K5237007 SequenceAndFeatures</partinfo>
 +
<!--################################-->
 +
<html>
 +
<section id="2">
 +
<h1>2. Usage and Biology</h1>
 +
<p>
 +
    GCN4 is a yeast transcription factor from the bZip family of DNA-binding proteins, first discovered by McKnight and
 +
    co-workers in 1988.
 +
    The bZip motif features a coiled-coil leucine zipper dimerization domain paired with a highly charged basic region
 +
    that binds to DNA.
 +
    GCN4 binds specifically to the cyclic AMP response element (CRE) DNA sequence (5' ATGACGTCAT 3') in the
 +
    promoter regions of target genes, primarily through its basic residues at the N-terminus.
 +
  </p>
 +
<p>
 +
    In our project we fused GCN4 to rGCN4 (<a href="https://parts.igem.org/Part:BBa_K5237008" target="_blank">BBa_K5237008</a>)
 +
    to create a 150 amino acid long "Mini staple" that can bring two DNA target sites into close proximity.
 +
  </p>
 +
<p>
 +
    The DNA-binding properties of GCN4 were tested using an electrophoretic mobility shift assay (EMSA) to quantify
 +
    binding affinity and calculate kinetics.
 +
    EMSA is a widely adopted method to study DNA-protein interactions. It works on the principle that nucleic acids
 +
    bound to proteins exhibit reduced
 +
    electrophoretic mobility compared to unbound nucleic acids (Hellman &amp; Fried, 2007). EMSA can be employed both
 +
    qualitatively, to assess DNA-binding
 +
    capabilities, and quantitatively, to determine critical parameters such as binding stoichiometry and the apparent
 +
    dissociation constant (K<sub>d</sub>)
 +
    (Fried, 1989).
 +
  </p>
 +
</section>
 +
<section id="3">
 +
<h1>3. Assembly and Part Evolution</h1>
 +
<p>
 +
    The GCN4 amino acid sequence was taken from literature (Hollenbeck et al. 2001) and codon optimized for <i>E.
 +
      coli</i>.
 +
    A FLAG-tag (DYKDDDDK) was added to the N-terminus for protein purification. The FLAG-tag can be cleaved off using an
 +
    enterokinase, if necessary.
 +
    The FLAG-GCN4 sequence was cloned into a T7 expression vector and expressed using <i>E. coli</i> BL21 (DE3) cells.
 +
  </p>
 +
</section>
 +
<section id="4">
 +
<h1>4. Results</h1>
 +
<section id="4.1">
 +
<h2>4.1 Protein Expression and Purification</h2>
 +
<p>The FLAG-GCN4 protein could be readily expressed in <i>E. coli</i>. The protein was purified using an
 +
      anti-FLAG resin. Fractions taken during purification were analyzed by SDS-PAGE and the protein concentration of
 +
      the eluted protein determined with a lowry protein assay.
 +
      A yield of 1.18 mg/mL was obtained, corresponding to 153 µM of monomeric FLAG-GCN4.
 +
    </p>
 +
<div class="thumb">
 +
<div class="thumbinner" style="width:500px">
 +
<img alt="" class="thumbimage" src="https://static.igem.wiki/teams/5237/wetlab-results/mist-sds-page-expression-validation.svg" style="width:99%;"/>
 +
<div class="thumbcaption">
 +
<i><b>Figure 2: SDS-PAGE Analysis of FLAG-GCN4 Purification</b> Fractions analysed for each protein are the
 +
            raw lysate, flow
 +
            through and eluate.
 +
            Depicted is GCN4 (this part), rGCN4 (<a href="https://parts.igem.org/Part:BBa_K5237008" target="_blank">BBa_K5237008</a>), and bGCN4 (<a href="https://parts.igem.org/Part:BBa_K5237009" target="_blank">BBa_K5237009</a>). Protein size is indicated next to construct name and purified band
 +
            with protein of interest highlighted by a red box.</i>
 +
</div>
 +
</div>
 +
</div>
 +
</section>
 +
<section id="4.2">
 +
<h2>4.2 Electrophoretic Mobility Shift Assay</h2>
 +
<div class="thumb tright" style="margin:0;">
 +
<div class="thumbinner" style="width:310px;">
 +
<img alt="" class="thumbimage" src="https://static.igem.wiki/teams/5237/figures-corrected/leucin-zipper-emsa.svg" style="width:99%;"/>
 +
<div class="thumbcaption">
 +
<i>
 +
<b>Figure 3: Overview Image of Electrophoretic Mobility Shift Assay (EMSA)</b>
 +
</i>
 +
</div>
 +
</div>
 +
</div>
 +
<p align="justify"></p>
 +
    The Electrophoretic mobility shift assay (EMSA) is a widely adopted method used to study DNA-protein
 +
    interactions. EMSA functions on the basis that nucleic acids bound to proteins have reduced electrophoretic
 +
    mobility, compared to their unbound counterpart. (Hellman &amp; Fried, 2007). Mobility-shift
 +
    assays can both be used to qualitatively assess DNA binding capabilities or quantitatively to determine binding
 +
    stoichiometry and kinetics such as the apparent dissociation constant (K<sub>d</sub>) (Fried, 1989).
  
 +
    <p>
 +
      To analyze the binding DNA affinity an EMSA was performed, in which
 +
      GCN4 was incubated in binding buffer with a 20 bp DNA probe containing the <i>CRE</i> GCN4 binding
 +
      sequence (5' ATGACGTCAT 3') until equilibration.
 +
      Subsequently the formed protein-DNA complexes were loaded on a native PAGE. Afterwards the DNA bands were stained
 +
      with SYBR-safe. <br/>
 +
      To further analyze DNA binding, quantitative shift assays were performed for GCN4 and rGCN4 (<a href="https://parts.igem.org/Part:BBa:K5237008">BBa_K5237008</a>).
 +
      0.5 µM DNA were incubated with varying concentrations of protein until equilibration. After
 +
      electrophoresis, bands were stained with SYBR-Safe and quantified based on pixel intensity. The
 +
      obtained values were fitted to equation 1, describing formation of a 2:1 protein-DNA complex:
 +
      <br><br/>
 +
      Θ<sub>app</sub> = Θ<sub>min</sub> + (Θ<sub>max</sub> - Θ<sub>min</sub>) ×
 +
      (K<sub>a</sub><sup>2</sup> [L]<sub>tot</sub><sup>2</sup>) / (1 + K<sub>a</sub><sup>2</sup>
 +
      [L]<sub>tot</sub><sup>2</sup>)
 +
      <span style="float: right;">Equation 1</span>
 +
<br/><br/>
 +
      Here [L]<sub>tot</sub> describes the total protein monomer concentration, K<sub>a</sub>
 +
      corresponds
 +
      to the apparent monomeric equilibration constant. The Θ<sub>min/max</sub> values are the
 +
      experimentally
 +
      determined site saturation values (For this experiment 0 and 1 were chosen for min and max
 +
      respectively).
 +
    </br></p>
 +
<div class="thumb">
 +
<div class="thumbinner" style="width:500px">
 +
<img alt="" class="thumbimage" src="https://static.igem.wiki/teams/5237/wetlab-results/mist-leucine-zipper-kd-plot.svg" style="width:99%;"/>
 +
<div class="thumbcaption">
 +
<i><b>Figure 4: Quantitative Assessment of Binding Affinity for GCN4 and rGCN4.</b> Proteins
 +
            of
 +
            different concentrations were incubated with 0.5 µM DNA until equilibrium and fraction bound analyzed, after
 +
            gel electrophoresis, by dividing pixel intensity of
 +
            bound fraction with pixel intensity of bound and unbound fraction. At least three separate measurements were
 +
            conducted
 +
            for each data point. Values are presented as mean +/- SD.</i>
 +
</div>
 +
</div>
 +
<p>
 +
        GCN4 binds to its optimal DNA binding motif with an apparent dissociation
 +
        constant K<sub>D</sub> of (0.293 ± 0.033) × 10<sup>-6</sup> M, which is almost identical to the
 +
        rGCN4 dissociation constant
 +
        to its target sequence (INVii)  K<sub>D</sub> of (0.298 ± 0.030) × 10<sup>-6</sup> M.
 +
        Comparing them to literature values, our dissociation constants are approximately a factor 10 higher then those
 +
        described in literature ((9±6) × 10<sup>-8</sup> M for
 +
        GCN4 and (2.9±0.8) × 10<sup>-8</sup> M for rGCN4) (Hollenbeck <i class="italic">et al.</i>, 2001). The
 +
        differences could be due to the lower sensitivity of SYBR-Safe staining compared to radio-labeled oligos.
 +
        Most likely, the protein concentration was miscalculated due to the presence of additional (lower intensity)
 +
        bands in
 +
        the SDS-PAGE analysis, indicating co-purification of small amounts of unspecific proteins.
 +
        <br/><br/>
 +
        The FLAG-tag fusion to the N-terminus of proteins could potentially decrease binding affinity, likely due
 +
        to steric hindrance affecting the interaction with DNA. Interestingly, the differences in binding affinity
 +
        between GCN4 and rGCN4 appear negligible. Since GCN4 binds to DNA via its N-terminus and rGCN4 binds
 +
        C-terminally, the FLAG-tag likely does not directly influence DNA binding. However, it may influence the
 +
        dimerization of the proteins, which is necessary for DNA binding. To further investigate this, the
 +
        FLAG-tag can be cleaved using an enterokinase and potential changes in binding affinity analyzed.
 +
        Furthermore, coiled coil formation, and the amount of dimeric and monomeric proteins could be further analyzed
 +
        with circular dichroism spectroscopy (Greenfield, 2006).
 
       </p>
 
       </p>
      <p>
+
</div>
        The <b>PICasSO</b> part collection offers a comprehensive, modular platform for precise manipulation and
+
</section>
        re-programming
+
<section id="4.3">
        of DNA-DNA interactions using protein staples in living cells, enabling researchers to recreate natural 3D genomic
+
<h2>4.3 <i>In Silico</i> Characterization using DaVinci</h2>
        interactions, such as enhancer hijacking, or to design entirely new spatial architectures for gene regulation.
+
<div class="thumb tright" style="margin:0;">
        Beyond its versatility, PICasSO includes robust assay systems to support the engineering, optimization, and
+
<div class="thumbinner" style="width:300px;">
        testing of new staples, ensuring functionality <i>in vitro</i> and <i>in vivo</i>. We took special care to include
+
<iframe allowfullscreen="" class="thumbimage" frameborder="0" height="315" sandbox="allow-same-origin allow-scripts allow-popups allow-forms" src="https://video.igem.org/videos/embed/36edba03-5fef-4b19-9b2f-2c802e126660?loop=1&amp;title=0&amp;warningTitle=0" style="width:99%;" title="Heidelberg: GCN4-MD (2024)" width="560"></iframe>
        parts crucial for testing every step of the cycle (design, build, test, learn) when engineering new parts
+
<div class="thumbcaption">
      </p>
+
<i><b>Figure 5: Molecular Dynamics Simulation of GCN4</b>
 
+
</i>
      <p>At its heart, the PICasSO part collection consists of three categories. (i) Our <b>DNA-binding proteins</b>
+
</div>
        include our
+
</div>
        finalized enhancer hijacking Cas staple as well as half staples that can be used by scientists to compose entirely
+
</div>
        new Cas staples in the future. We also include our simple staples that serve as controls for successful stapling
+
<p>
        and can be further engineered to create alternative, simpler and more compact staples. (ii) As <b>functional
+
      We developed DaVinci, an <i>in silico</i> model, for rapid engineering and optimization of our PICasSO system. DaVinci
          elements</b>, we list additional parts that enhance the functionality of our Cas and Basic staples. These
+
      serves as a digital twin to PICasSO, analyzing the forces acting on the system, refining experimental parameters,
        consist of
+
      and identifying optimal interactions between protein staples and target DNA. The model was calibrated using
        protease-cleavable peptide linkers and inteins that allow condition-specific, dynamic stapling <i>in vivo</i>.
+
      literature data and experimental affinity results from rGCN4 EMSA assays with purified proteins.
        Besides staple functionality, we also include the parts to enable the efficient delivery of PICasSO's with our
+
      <br/>
        interkingdom conjugation system.
+
      DaVinci operates in three phases: static structure prediction, all-atom dynamics simulation, and long-range DNA
      </p>
+
      dynamics simulation. We applied the first two phases to our components, allowing us to characterize the structure
      <p>
+
      and dynamics of the DNA-binding interactions.
        (iii) As the final component of our collection, we provide parts that support the use of our <b>custom readout
+
      <br/>
          systems</b>. These include components of our established FRET-based proximity assay system, enabling users to
+
      For our bivalent DNA-binding Mini staple (<a href="https://parts.igem.org/Part:BBa_K5237009">BBa_K5237009</a>),
        confirm
+
      consisting of GCN4 fused via a GSG-linker to rGCN4
        accurate stapling. Additionally, we offer a complementary, application-oriented testing system for functional
+
      (<a href="https://parts.igem.org/Part:BBa_K5237008">BBa_K5237008</a>), we predicted the structure and binding
        readout via a luciferase reporter, which allows for straightforward experimental simulation of enhancer hijacking.
+
       affinity and tested various linker options. We evaluated
      </p>
+
       the flexibility and rigidity of the constructs using pLDDT values from the predictions. Flexible linkers, like
      <p>
+
       ('GGGGS')n, and rigid linkers, like ('EAAAK')n, were assessed (Arai et al., 2001). Predictions were colored by
        The following table gives a complete overview of all parts in our PICasSO toolbox. The highlighted parts showed
+
       pLDDT scores, providing insights into chain rigidity (Akdel et al., 2022; Guo et al., 2022). Construct C (Fig. 5)
        exceptional performance as described on our iGEM wiki and can serve as a reference. The other parts in the
+
       was tested in the wet lab as part of BBa_K5237009, but it failed to bind DNA due to excessive rigidity, which
        collection are versatile building blocks designed to provide future iGEMers with the flexibility to engineer their
+
      inhibited subunit dimerization.
        own custom Cas staples, enabling further optimization and innovation
+
    </p>
      </p>
+
<div class="thumb">
      <p>
+
<div class="thumbinner" style="width:80%;">
        <font size="4"><b>Our parts collection includes:</b></font><br>
+
<img alt="" class="thumbimage" src="https://static.igem.wiki/teams/5237/model/structure-figure-3.svg" style="width:99%;"/>
      </p>
+
<div class="thumbcaption">
 
+
<i><b>Figure 6: Variation of Linkers Connecting Our Mini Staples.</b>
      <table style="width: 90%;">
+
            Panels A (BBa_K5237007) and B (BBa_K5237008) show
        <td colspan="3" align="left"><b>DNA-binding proteins: </b>
+
            orientations of the leucine zipper, each bound to DNA. Panels C to I display linker variations colored by
          The building blocks for engineering of custom staples for DNA-DNA interactions with a modular system ensuring
+
            their pLDDT
          easy assembly.</td>
+
            confidence score, which serves as a surrogate for chain flexibility (Akdel et al., 2022). Note that panels H
        <tbody>
+
            and I are
          <tr bgcolor="#FFD700">
+
            not bound to the second DNA strand. All structures were predicted using the AlphaFold server (Google
            <td><a href="https://parts.igem.org/Part:BBa_K5237000" target="_blank">BBa_K5237000</a></td>
+
            DeepMind,
            <td>fgRNA Entryvector MbCas12a-SpCas9</td>
+
            2024).
            <td>Entryvector for simple fgRNA cloning via SapI</td>
+
          </i>
          </tr>
+
</div>
          <tr>
+
</div>
            <td><a href="https://parts.igem.org/Part:BBa_K5237001" target="_blank">BBa_K5237001</a></td>
+
</div>
            <td>Staple subunit: dMbCas12a-Nucleoplasmin NLS</td>
+
</section>
            <td>Staple subunit that can be combined to form a functional staple, for example with fgRNA and dCas9 </td>
+
</section>
          </tr>
+
<section id="5">
          <tr>
+
<h1>5. References</h1>
            <td><a href="https://parts.igem.org/Part:BBa_K5237002" target="_blank">BBa_K5237002</a></td>
+
<p>Fried, M. G. (1989). Measurement of protein-DNA interaction parameters by electrophoresis mobility shift assay.
            <td>Staple subunit: SV40 NLS-dSpCas9-SV40 NLS</td>
+
    <em>ELECTROPHORESIS, 10</em>(5-6), 366-376. <a href="https://doi.org/10.1002/elps.1150100515" target="_blank">https://doi.org/10.1002/elps.1150100515</a>
            <td>Staple subunit that can be combined to form a functional staple, for example with our fgRNA or dCas12a
+
</p>
            </td>
+
<p>Akdel, M., Pires, D. E. V., Pardo, E. P., Janes, J., Zalevsky, A. O., Meszaros, B., Bryant, P., Good, L. L.,
          </tr>
+
    Laskowski, R. A., Pozzati, G., Shenoy, A., Zhu, W., Kundrotas, P., Serra, V. R., Rodrigues, C. H. M., Dunham, A. S.,
          <tr>
+
    Burke, D., Borkakoti, N., Velankar, S., … Beltrao, P. (2022). A structural biology community assessment of
            <td><a href="https://parts.igem.org/Part:BBa_K5237003" target="_blank">BBa_K5237003</a></td>
+
    AlphaFold2 applications. <i>Nat Struct Mol Biol, 29</i>(11), 1056–1067. <a href="https://doi.org/10.1038/s41594-022-00849-w" target="_blank">https://doi.org/10.1038/s41594-022-00849-w</a>
            <td>Cas-Staple: SV40 NLS-dMbCas12a-dSpCas9-Nucleoplasmin NLS</td>
+
</p>
            <td>Functional Cas staple that can be combined with sgRNA or fgRNA to bring two DNA strands in close proximity
+
<p>Arai, R., Ueda, H., Kitayama, A., Kamiya, N., &amp; Nagamune, T. (2001). Design of the linkers which effectively
            </td>
+
    separate domains of a bifunctional fusion protein. <i>Protein Engineering, Design and Selection, 14</i>(8), 529–532.
          </tr>
+
    <a href="https://doi.org/10.1093/protein/14.8.529" target="_blank">https://doi.org/10.1093/protein/14.8.529</a>
          <tr>
+
</p>
            <td><a href="https://parts.igem.org/Part:BBa_K5237004" target="_blank">BBa_K5237004</a></td>
+
<p>Chen, X., Zaro, J. L., &amp; Shen, W.-C. (2013). Fusion protein linkers: Property, design and functionality.
            <td>Staple subunit: Oct1-DBD</td>
+
    <i>Advanced Drug Delivery Reviews, 65</i>(10), 1357–1369. <a href="https://doi.org/10.1016/j.addr.2012.09.039" target="_blank">https://doi.org/10.1016/j.addr.2012.09.039</a>
            <td>Staple subunit that can be combined to form a functional staple, for example with TetR.<br>
+
</p>
              Can also be combined with a fluorescent protein as part of the FRET proximity assay</td>
+
<p>Google DeepMind. (2024). AlphaFold Server. <a href="https://alphafoldserver.com/terms" target="_blank">https://alphafoldserver.com/terms</a></p>
          </tr>
+
<p>Greenfield, N. J. (2006). Using circular dichroism spectra to estimate protein secondary structure. Nature
          <tr>
+
    Protocols, 1(6), 2876–2890. <a href="https://doi.org/10.1038/nprot.2006.202">https://doi.org/10.1038/nprot.2006.202</a></p>
            <td><a href="https://parts.igem.org/Part:BBa_K5237005" target="_blank">BBa_K5237005</a></td>
+
<p>Guo, H.-B., Perminov, A., Bekele, S., Kedziora, G., Farajollahi, S., Varaljay, V., Hinkle, K., Molinero, V.,
            <td>Staple subunit: TetR</td>
+
    Meister, K., Hung, C., Dennis, P., Kelley-Loughnane, N., &amp; Berry, R. (2022). AlphaFold2 models indicate that protein
            <td>Staple subunit that can be combined to form a functional staple, for example with Oct1.<br>
+
    sequence determines both structure and dynamics. <i>Scientific Reports, 12</i>(1), 10696. <a href="https://doi.org/10.1038/s41598-022-14382-9" target="_blank">https://doi.org/10.1038/s41598-022-14382-9</a>
              Can also be combined with a fluorescent protein as part of the FRET proximity assay</td>
+
</p>
          </tr>
+
<p>Hellman, L. M., &amp; Fried, M. G. (2007). Electrophoretic mobility shift assay (EMSA) for detecting
          <tr>
+
    protein-nucleic acid interactions. <em>Nature Protocols, 2</em>(8), 1849-1861. <a href="https://doi.org/10.1038/nprot.2007.249" target="_blank">https://doi.org/10.1038/nprot.2007.249</a></p>
            <td><a href="https://parts.igem.org/Part:BBa_K5237006" target="_blank">BBa_K5237006</a></td>
+
<p>Hollenbeck, J. J., Gurnon, D. G., Fazio, G. C., Carlson, J. J., &amp; Oakley, M. G. (2001). A GCN4 Variant with a
            <td>Simple taple: TetR-Oct1</td>
+
    C-Terminal Basic Region Binds to DNA with Wild-Type Affinity. <em>Biochemistry, 40</em>(46), 13833-13839.</p>
            <td>Functional staple that can be used to bring two DNA strands in close proximity</td>
+
<p>Hollenbeck, J. J., &amp; Oakley, M. G. (2000). GCN4 Binds with High Affinity to DNA Sequences Containing a Single
          </tr>
+
    Consensus Half-Site. <em>Biochemistry, 39</em>(21), 6380-6389. <a href="https://doi.org/10.1021/bi992705n" target="_blank">https://doi.org/10.1021/bi992705n</a></p>
          <tr>
+
</section>
            <td><a href="https://parts.igem.org/Part:BBa_K5237007" target="_blank">BBa_K5237007</a></td>
+
</html>
            <td>Staple subunit: GCN4</td>
+
            <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_K5237008" target="_blank">BBa_K5237008</a></td>
+
            <td>Staple subunit: rGCN4</td>
+
            <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_K5237009" target="_blank">BBa_K5237009</a></td>
+
            <td>Mini staple: bGCN4</td>
+
            <td>
+
              Assembled staple with minimal size that can be further engineered</td>
+
          </tr>
+
        </tbody>
+
        <td colspan="3" align="left"><b>Functional elements: </b>
+
          Protease cleavable peptide linkers and inteins are used to control and modify staples for further optimization
+
          for custom applications.</td>
+
        <tbody>
+
          <tr bgcolor="#FFD700">
+
            <td><a href="https://parts.igem.org/Part:BBa_K5237010" target="_blank">BBa_K5237010</a></td>
+
            <td>Cathepsin B-Cleavable Linker (GFLG)</td>
+
            <td>Cathepsin B cleavable peptide linker, that can be used to combine two staple subunits ,to make responsive
+
              staples</td>
+
          </tr>
+
          <tr>
+
            <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 which can be selectively express to cut the cleavable linker</td>
+
          </tr>
+
          <tr>
+
            <td><a href="https://parts.igem.org/Part:BBa_K52370012" target="_blank">BBa_K5237012</a></td>
+
            <td>Caged NpuN Intein</td>
+
            <td>Undergoes protein transsplicing after protease activation, can be used to create functionalized staple
+
              units</td>
+
          </tr>
+
          <tr>
+
            <td><a href="https://parts.igem.org/Part:BBa_K52370013" target="_blank">BBa_K5237013</a></td>
+
            <td>Caged NpuC Intein</td>
+
            <td>Undergoes protein transsplicing after protease activation, can be used to create functionalized staple
+
              units</td>
+
          </tr>
+
          <tr>
+
            <td><a href="https://parts.igem.org/Part:BBa_K52370014" target="_blank">BBa_K5237014</a></td>
+
            <td>fgRNA processing casette</td>
+
            <td>Processing casette to produce multiple fgRNAs from one transcript, can be used for multiplexing</td>
+
          </tr>
+
          <tr>
+
            <td><a href="https://parts.igem.org/Part:BBa_K52370015" target="_blank">BBa_K5237015</a></td>
+
            <td>Intimin anti-EGFR Nanobody</td>
+
            <td>Interkindom conjugation between bacteria and mammalian cells, as alternative delivery tool for large
+
              constructs</td>
+
          </tr>
+
        </tbody>
+
        <td colspan="3" align="left"><b>Readout Systems: </b>
+
          FRET and enhancer recruitment to measure proximity of stapled DNA in bacterial and mammalian living cells
+
          enabling swift testing and easy development for new systems.</td>
+
        <tbody>
+
          <tr bgcolor="#FFD700">
+
            <td><a href="https://parts.igem.org/Part:BBa_K52370016" target="_blank">BBa_K5237016</a></td>
+
            <td>FRET-Donor: mNeonGreen-Oct1</td>
+
            <td>Donor part for the FRET assay binding the Oct1 binding cassette. 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. 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, can be used for different 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 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>
+
          </tr>
+
          <tr>
+
            <td><a href="https://parts.igem.org/Part:BBa_K5237021" target="_blank">BBa_K5237021</a></td>
+
            <td>NLS-Gal4-VP64</td>
+
            <td>Trans-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 Promotor, mCherry</td>
+
          <td>Readout system for enhancer binding. It was used to test Cathepsin-B cleavable linker.</td>
+
          </tr>
+
          <tr>
+
            <td><a href="https://parts.igem.org/Part:BBa_K5237023" target="_blank">BBa_K5237023</a></td>
+
            <td>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. It was used as a luminescence readout for
+
              simulated enhancer hijacking.</td>
+
          </tr>
+
        </tbody>
+
       </table>
+
      </p>
+
    </section>
+
    <section id="1">
+
      <h1>1. Sequence overview</h1>
+
    </section>
+
  </body>
+
 
+
  </html>
+
 
+
  <!--################################-->
+
  <span class='h3bb'>Sequence and Features</span>
+
  <partinfo>BBa_K5237007 SequenceAndFeatures</partinfo>
+
  <!--################################-->
+
 
+
  <html>
+
 
+
 
+
  <section id="2">
+
      <h1>2. Usage and Biology</h1>
+
       <p>
+
        GCN4 is a yeast transcription factor from the bZip family of DNA-binding proteins, first discovered by McKnight and co-workers in 1988.
+
        The bZip motif features a coiled-coil leucine zipper dimerization domain paired with a highly charged basic region, which directly interacts
+
        with DNA. GCN4 binds specifically to the cyclic AMP response element (CRE) in the promoter regions of target genes, primarily through its basic
+
        residues at the N-terminus.
+
      </p>
+
      <p>
+
        In our project, GCN4 was employed to study DNA-binding kinetics and develop a minimal "Mini staple" that brings two DNA target sites into proximity
+
        by binding them simultaneously. This "Mini staple" was designed as a versatile tool for precise DNA manipulation in synthetic biology applications.
+
       </p>
+
      <p>
+
        The DNA-binding properties of GCN4 were tested using an electrophoretic mobility shift assay (EMSA) to quantify binding affinity and kinetics.
+
        EMSA is a widely adopted method to study DNA-protein interactions. It works on the principle that nucleic acids bound to proteins exhibit reduced
+
        electrophoretic mobility compared to unbound nucleic acids (Hellman & Fried, 2007). EMSA can be employed both qualitatively, to assess DNA-binding
+
        capabilities, and quantitatively, to determine critical parameters such as binding stoichiometry and the apparent dissociation constant (K<sub>d</sub>)  
+
        (Fried, 1989).
+
       </p>
+
    </section>
+
    <section id="3">
+
      <h1>3. Assembly and part evolution</h1>
+
      <p>
+
        The GCN4 amino acid sequence was taken from literature (Hollenbeck & Oakley 1999) and codon optimized for <i>E. coli</i>.  
+
        A FLAG-tag (DYKDDDDK) was added to the N-terminus for protein purification. The FLAG-tag can be cleaved off using an Enterokinase, if necessary.
+
        The FLAG-GCN4 sequence was cloned into a T7 expression vector and expressed using <i>E. coli</i> BL21 (DE3) cells.
+
       </p>
+
    </section>
+
    <section id="4">
+
      <h1>4. Results</h1>
+
      <section id="4.1">
+
        <h2>4.1 Protein expression and purification</h2>
+
        <p>The FLAG-GCN4 protein could be readily expressed in <i>E. coli</i> BL21 (DE3). The protein was purified using an anti-FLAG resin.
+
          Fractions taken during purification were analyzed by SDS-PAGE. Purified protein was quantified using a Lowry assay, 1.18 mg/mL were obtained, resulting in 153 µM of monomeric FLAG-GCN4.
+
        </p>
+
        <div class="thumb">
+
          <div class="thumbinner" style="width:500px">
+
            <img alt="" src="https://static.igem.wiki/teams/5237/wetlab-results/mist-sds-page-expression-validation.svg" style="width:99%;" class="thumbimage">
+
            <div class="thumbcaption">
+
              <i><b>Figure 2: SDS-PAGE analysis of FLAG-GCN4 purification</b> Fractions analysed are the raw lysate, flow through and eluate.
+
              Depicted is GCN4 (this part), rGCN4 (<a href="https://parts.igem.org/Part:BBa_K5237008"
+
                target="_blank">BBa_K5237008</a>), and bGCN4 (<a href="https://parts.igem.org/Part:BBa_K5237009"
+
                target="_blank">BBa_K5237009</a>)</i>. Protein size is indicated next to construct name and purified band with protein of interest highlighted by a red box.
+
                </i>
+
            </div>
+
          </div>
+
        </div>
+
      </section>
+
      <section id="4.2">
+
        <h2>4.2 Electrophoretic Mobility shift assay</h2>
+
        <p>
+
          GCN4 was tested for its DNA-binding capabilities using an electrophoretic mobility shift assay (EMSA). The protein was incubated with a DNA probe containing the GCN4 binding site. The formation of a protein-DNA complex was analyzed by native PAGE.
+
          To further analyze DNA binding, quantitative shift assays were performed for GCN4 and rGCN4 (<a href="https://parts.igem.org/Part:BBa:K5237008">BBa_K5237008</a>).
+
          0.5 µM DNA were incubated with varying concentrations of protein until equilibration. After
+
          electrophoresis, bands were stained with SYBR-Safe and quantified based on pixel intensity. The
+
          obtained values were fitted to equation 1, describing formation of a 2:1 protein-DNA complex:
+
          <br><br>
+
          Θ<sub>app</sub> = Θ<sub>min</sub> + (Θ<sub>max</sub> - Θ<sub>min</sub>) &#215;
+
          (K<sub>a</sub><sup>2</sup> [L]<sub>tot</sub><sup>2</sup>) / (1 + K<sub>a</sub><sup>2</sup>
+
          [L]<sub>tot</sub><sup>2</sup>)
+
          <span style="float: right;">Equation 1</span>
+
          <br><br>
+
          Here [L]<sub>tot</sub> describes the total protein monomer concentration, K<sub>a</sub>
+
          corresponds
+
          to the apparent monomeric equilibration constant. The Θ<sub>min/max</sub> values are the
+
          experimentally
+
          determined site saturation values (For this experiment 0 and 1 were chosen for min and max
+
          respectively). GCN4 binds to its optimal DNA binding motif with an apparent dissociation
+
          constant K<sub>k</sub> of (0.2930.033)&#215;10<sup>-6</sup> M, which is almost identical to the
+
          rGCN4 binding
+
          affinity to INVii a <sub>d</sub> of (0.2980.030)&#215;10<sup>-6</sup> M.
+
        </p>
+
        <div class="thumb">
+
          <div class="thumbinner" style="width:500px">
+
            <img alt="" src="https://static.igem.wiki/teams/5237/wetlab-results/mist-leucine-zipper-kd-plot.svg" style="width:99%;" class="thumbimage">
+
            <div class="thumbcaption">
+
              <i><b>Figure 3: Quantitative EMSA</b>Quantitative assessment of binding affinity for GCN4 and rGCN4. Proteins of
+
                different concentrations were incubated with 0.5 µM DNA until equilibrium and fraction bound analyzed, after gel electrophoresis, by dividing pixel intensity of
+
                bound fraction with pixel intensity of bound and unbound fraction. At least three separate measurements were conducted
+
                for each data point. Values are presented as mean +/- SD.</i>
+
            </div>
+
        </div>
+
        <p>
+
          The apparent binding kinetics calculated for GCN4 ((0.2930.033) &#215; 10<sup>-6</sup> M) and rGCN4
+
          ((0.2980.030) &#215; 10<sup>-6</sup> M) are
+
          approximately a factor 10 higher then those described in literature ((96) &#215; 10<sup>-8</sup> M for
+
          GCN4 and (2.90.8) &#215; 10<sup>-8</sup> M for rGCN4) (Hollenbeck <i class="italic">et al.</i>, 2001). The
+
          differences could be due to the lower sensitivity of SYBR-Safe staining compared to radio-labeled oligos.
+
          Most likely, the protein concentration was miscalculated due to the presence of additional (lower intensity) bands in
+
          the SDS-PAGE analysis, indicating the co-purification of small amounts of unspecific proteins.
+
          <br><br>
+
          The FLAG-tag fusion to the N-terminus of proteins could potentially decrease binding affinity, likely due
+
          to steric hindrance affecting the interaction with DNA. Interestingly, the differences in binding affinity
+
          between GCN4 and rGCN4 appear negligible. Since GCN4 binds to DNA via its N-terminus and rGCN4 binds
+
          C-terminally, the FLAG-tag likely does not directly influence DNA binding. However, it may influence the
+
          dimerization of the proteins, which is necessary for DNA binding. To further investigate this, the
+
          FLAG-tag can be cleaved using an enterokinase and potential changes in binding affinity analyzed
+
        </p>
+
    </section>
+
    <section id="5">
+
      <h1>5. References</h1>
+
      <p>
+
        Fried, M. G. (1989). Measurement of protein-DNA interaction parameters by electrophoresis mobility shift assay.  
+
        <i>Electrophoresis, 10</i>(5–6), 366–376.  
+
        <a href="https://doi.org/10.1002/elps.1150100515" target="_blank">https://doi.org/10.1002/elps.1150100515</a>
+
      </p>
+
      <p>
+
        Hellman, L. M., & Fried, M. G. (2007). Electrophoretic mobility shift assay (EMSA) for detecting protein-nucleic acid interactions.  
+
        <i>Nature Protocols, 2</i>(8), 1849–1861.  
+
        <a href="https://doi.org/10.1038/nprot.2007.249" target="_blank">https://doi.org/10.1038/nprot.2007.249</a>
+
      </p>
+
      <p>
+
        Hollenbeck, J. J., & Oakley, M. G. (2000). GCN4 binds with high affinity to DNA sequences containing a single consensus half-site.  
+
        <i>Biochemistry, 39</i>(21), 6380–6389.  
+
        <a href="https://doi.org/10.1021/bi992705n" target="_blank">https://doi.org/10.1021/bi992705n</a>
+
      </p>
+
      <p>
+
        Hollenbeck, J. J., Gurnon, D. G., Fazio, G. C., Carlson, J. J., & Oakley, M. G. (2001). A GCN4 variant with a C-terminal basic region binds to DNA with wild-type affinity.
+
        <i>Biochemistry, 40</i>(46), 13833–13839.  
+
        <a href="https://doi.org/10.1021/bi0106916" target="_blank">https://doi.org/10.1021/bi0106916</a>
+
      </p>
+
      <p>
+
        McKnight, S. L., & Tjian, R. (1988). Analysis of transcriptional regulatory proteins of the human genome.
+
        <i>Science, 241</i>(4870), 1306–1313.  
+
        <a href="https://doi.org/10.1126/science.2847199" target="_blank">https://doi.org/10.1126/science.2847199</a>
+
      </p>
+
      <p>
+
        Wu, W., Zhang, L., Yao, L., Tan, X., Liu, X., & Lu, X. (2015). Genetically assembled fluorescent biosensor for in situ detection of bio-synthesized alkanes.  
+
        <i>Scientific Reports, 5</i>, 10907.  
+
        <a href="https://doi.org/10.1038/srep10907" target="_blank">https://doi.org/10.1038/srep10907</a>
+
      </p>
+
    </section>
+
   
+
  </body>
+
 
+
  </html>
+

Latest revision as of 12:39, 2 October 2024

BBa_K5237007

Staple Subunit: GCN4

GCN4 is a yeast transcription factor belonging to the bZip family of DNA-binding proteins. We used GCN4 to study DNA-binding kinetics in our "Mini staples" that bring two DNA target sites into proximity by binding them simultaneously.



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

GCN4 is a yeast transcription factor from the bZip family of DNA-binding proteins, first discovered by McKnight and co-workers in 1988. The bZip motif features a coiled-coil leucine zipper dimerization domain paired with a highly charged basic region that binds to DNA. GCN4 binds specifically to the cyclic AMP response element (CRE) DNA sequence (5' ATGACGTCAT 3') in the promoter regions of target genes, primarily through its basic residues at the N-terminus.

In our project we fused GCN4 to rGCN4 (BBa_K5237008) to create a 150 amino acid long "Mini staple" that can bring two DNA target sites into close proximity.

The DNA-binding properties of GCN4 were tested using an electrophoretic mobility shift assay (EMSA) to quantify binding affinity and calculate kinetics. EMSA is a widely adopted method to study DNA-protein interactions. It works on the principle that nucleic acids bound to proteins exhibit reduced electrophoretic mobility compared to unbound nucleic acids (Hellman & Fried, 2007). EMSA can be employed both qualitatively, to assess DNA-binding capabilities, and quantitatively, to determine critical parameters such as binding stoichiometry and the apparent dissociation constant (Kd) (Fried, 1989).

3. Assembly and Part Evolution

The GCN4 amino acid sequence was taken from literature (Hollenbeck et al. 2001) and codon optimized for E. coli. A FLAG-tag (DYKDDDDK) was added to the N-terminus for protein purification. The FLAG-tag can be cleaved off using an enterokinase, if necessary. The FLAG-GCN4 sequence was cloned into a T7 expression vector and expressed using E. coli BL21 (DE3) cells.

4. Results

4.1 Protein Expression and Purification

The FLAG-GCN4 protein could be readily expressed in E. coli. The protein was purified using an anti-FLAG resin. Fractions taken during purification were analyzed by SDS-PAGE and the protein concentration of the eluted protein determined with a lowry protein assay. A yield of 1.18 mg/mL was obtained, corresponding to 153 µM of monomeric FLAG-GCN4.

Figure 2: SDS-PAGE Analysis of FLAG-GCN4 Purification Fractions analysed for each protein are the raw lysate, flow through and eluate. Depicted is GCN4 (this part), rGCN4 (BBa_K5237008), and bGCN4 (BBa_K5237009). Protein size is indicated next to construct name and purified band with protein of interest highlighted by a red box.

4.2 Electrophoretic Mobility Shift Assay

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

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

To analyze the binding DNA affinity an EMSA was performed, in which GCN4 was incubated in binding buffer with a 20 bp DNA probe containing the CRE GCN4 binding sequence (5' ATGACGTCAT 3') until equilibration. Subsequently the formed protein-DNA complexes were loaded on a native PAGE. Afterwards the DNA bands were stained with SYBR-safe.
To further analyze DNA binding, quantitative shift assays were performed for GCN4 and rGCN4 (BBa_K5237008). 0.5 µM DNA were incubated with varying concentrations of protein until equilibration. After electrophoresis, bands were stained with SYBR-Safe and quantified based on pixel intensity. The obtained values were fitted to equation 1, describing formation of a 2:1 protein-DNA complex:

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

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

Figure 4: Quantitative Assessment of Binding Affinity for GCN4 and rGCN4. Proteins of different concentrations were incubated with 0.5 µM DNA until equilibrium and fraction bound analyzed, after gel electrophoresis, by dividing pixel intensity of bound fraction with pixel intensity of bound and unbound fraction. At least three separate measurements were conducted for each data point. Values are presented as mean +/- SD.

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

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

4.3 In Silico Characterization using DaVinci

Figure 5: Molecular Dynamics Simulation of GCN4

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

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

5. References

Fried, M. G. (1989). Measurement of protein-DNA interaction parameters by electrophoresis mobility shift assay. ELECTROPHORESIS, 10(5-6), 366-376. https://doi.org/10.1002/elps.1150100515

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

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

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

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

Greenfield, N. J. (2006). Using circular dichroism spectra to estimate protein secondary structure. Nature Protocols, 1(6), 2876–2890. https://doi.org/10.1038/nprot.2006.202

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

Hellman, L. M., & Fried, M. G. (2007). Electrophoretic mobility shift assay (EMSA) for detecting protein-nucleic acid interactions. Nature Protocols, 2(8), 1849-1861. https://doi.org/10.1038/nprot.2007.249

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

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