Difference between revisions of "Part:BBa K5237024"

 
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TRE-minimal promoter- firefly luciferase
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===Usage and Biology===
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  <!-- Part summary -->
 +
  <section id="1">
 +
    <h1>TRE-minimal promoter- firefly luciferase</h1>
 +
    <p>
 +
      This part contains the tetO binding site (BBa_K5237019) , a minimal promoter and a firefly luciferase gene. With a
 +
      VP64 coming in close proximity to the minimal promoter transcription factors are recruited, initiating expression
 +
      of firefly luciferase. The described mechanism is utilized in our enhancer hijacking assay for prove of Cas
 +
      stapling.
 +
 
 +
    </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>
 +
      </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=""
 +
        src="https://static.igem.wiki/teams/5237/wetlab-results/registry-part-collection-engineering-cycle-example-overview.svg"
 +
        style="width:99%;" class="thumbimage">
 +
      <div class="thumbcaption">
 +
        <i><b>Figure 1: How our part collection can be used to engineer new staples</b></i>
 +
      </div>
 +
    </div>
 +
    </div>
 +
 
 +
 
 +
    <p>
 +
      <br>
 +
      Next to the well-studied linear DNA sequence, the 3D spatial organization of DNA plays a crucial role in gene
 +
      regulation,
 +
      cell fate, disease development and more. However, the tools to precisely manipulate this genomic architecture
 +
      remain limited, rendering it challenging to explore the full potential of the
 +
      3D genome in synthetic biology. We - iGEM Team Heidelberg 2024 - have developed PICasSO, a powerful molecular
 +
      toolbox based on various DNA-binding proteins to address this issue.
 +
    </p>
 +
    <p>
 +
      The <b>PICasSO</b> part collection offers a comprehensive, modular platform for precise manipulation and
 +
      re-programming
 +
      of DNA-DNA interactions using protein staples in living cells, enabling researchers to recreate natural 3D genomic
 +
      interactions, such as enhancer hijacking, or to design entirely new spatial architectures for gene regulation.
 +
      Beyond its versatility, PICasSO includes robust assay systems to support the engineering, optimization, and
 +
      testing of new staples, ensuring functionality <i>in vitro</i> and <i>in vivo</i>. We took special care to include
 +
      parts crucial for testing every step of the cycle (design, build, test, learn) when engineering new 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 enhancer hijacking Cas staple as well as half staples that can be used by scientists to compose entirely
 +
      new Cas staples in the future. We also include our Simple staples that 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 the functionality of our Cas and
 +
      Basic staples. These
 +
      consist of
 +
      protease-cleavable peptide linkers and inteins that allow condition-specific, dynamic stapling <i>in vivo</i>.
 +
      Besides staple functionality, we also include the parts to enable the efficient delivery of PICasSO's constructs
 +
      with our
 +
      interkingdom conjugation system. <br>
 +
      <b>(iii)</b> As the final category of our collection, we provide parts that support the use of 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 for functional
 +
      readouts via a luciferase reporter, which allows for straightforward experimental simulation of enhancer hijacking
 +
      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.<br>
 +
    </p>
 +
    <p>
 +
      <font size="4"><b>Our part collection includes:</b></font><br>
 +
    </p>
 +
 
 +
    <table style="width: 90%; padding-right:10px;">
 +
      <td colspan="3" align="left"><b>DNA-binding proteins: </b>
 +
        The building blocks for engineering of custom staples for DNA-DNA interactions with a modular system ensuring
 +
        easy assembly.</td>
 +
      <tbody>
 +
        <tr bgcolor="#FFD700">
 +
          <td><a href="https://parts.igem.org/Part:BBa_K5237000" target="_blank">BBa_K5237000</a></td>
 +
          <td>fgRNA Entry vector MbCas12a-SpCas9</td>
 +
          <td>Entryvector 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 sgRNA or fgRNA and dCas9 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 witha sgRNA or fgRNA and dCas12avto 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 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 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>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.
 +
            Can be used to create functionalized staples
 +
            units</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.
 +
            Can be used to create functionalized staples
 +
            units</td>
 +
        </tr>
 +
        <tr>
 +
          <td><a href="https://parts.igem.org/Part:BBa_K5237014" target="_blank">BBa_K5237014</a></td>
 +
          <td>fgRNA processing casette</td>
 +
          <td>Processing casette to produce multiple fgRNAs from one transcript, that can be used for multiplexed 3D
 +
            genome reprograming</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>Interkindom conjugation between bacteria and mammalian cells, as 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 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_K5237016" target="_blank">BBa_K5237016</a></td>
 +
          <td>FRET-Donor: mNeonGreen-Oct1</td>
 +
          <td>FRET Donor-Fluorpohore fused to Oct1-DBD that binds to 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, 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 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>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K5237024 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K5237024 SequenceAndFeatures</partinfo>
 +
<!--################################-->
 +
 +
<html>
 +
 +
 +
<section id="2">
 +
  <h1>2. Usage and Biology</h1>
  
 +
  <p>
 +
    The tetracycline response element, part of the TetR family of regulators (TFRs), is central to the regulation of
 +
    antibiotic resistance genes, especially through its role in the Tet operon (TetO). In this system, the TetR protein
 +
    acts
 +
    as a repressor by binding to the TetO operator, inhibiting the expression of tetracycline resistance genes. Upon
 +
    binding
 +
    tetracycline or its analogs, TetR undergoes a conformational change, releasing TetO and allowing the transcription
 +
    of
 +
    target genes. This system is widely utilized in molecular biology as a controlled gene expression tool, particularly
 +
    in
 +
    inducible gene expression systems in both prokaryotic and eukaryotic cells (Cuthbertson and Nodwell (2013)).
 +
  </p>
 +
  <p>
 +
    Firefly luciferases are enzymes responsible for the bioluminescence seen in fireflies, catalyzing the oxidation of
 +
    luciferin in the presence of ATP, magnesium ions, and oxygen. This reaction produces light and is highly efficient,
 +
    with
 +
    little heat released, making it a popular tool in molecular biology for reporter assays. The gene encoding firefly
 +
    luciferase is widely used in research to monitor gene expression, quantify cellular ATP levels, and study
 +
    transcriptional activity due to its sensitivity and ease of detection (Xie <i>et al.</i> (2010))
 +
  </p>
 +
  <p>
 +
    We utilize the recognition site for plasmid to plasmid stapling with our Cas staples. A fgRNA targeting Tre and Oct1
 +
    on
 +
    the other plasmid, is the key factor in the Cas staple, bringing them together. When the transactivator, Gal4-VP64,
 +
    binds as well we have transactivation as a readout for functioning staples.
 +
  </p>
 +
</section>
 +
<section id="3">
 +
  <h1>3. Assembly and part evolution</h1>
 +
  <p>
 +
    The cloning strategy designed for TetO allows for the easy assembly of repetitive repeats. It follows the procedure
 +
    outlined by Sladitschek and Neveu (2015). Briefly, the oligos can be inserted into a vector digested with SalI and
 +
    XhoI, yielding a vector with three binding repeats flanked by these restriction sites. The vector can be linearized
 +
    with either SalI or XhoI, as both enzymes create compatible overhangs. The annealed oligos can then be ligated into
 +
    the vector, resulting in six binding repeats, with the middle sequence losing its cleavage site compatibility. This
 +
    process can be repeated to achieve the desired number of repeats by digesting the vector and re-ligating the oligos.
 +
    For the experiments conducted, a folding plasmid with 12 repeats was created. Since the registry has some
 +
    limitations regarding sequence depository, the binding cassette is flanked by SalI and XhoI, and the top and bottom
 +
    oligos with the fitting overhangs are annotated.
 +
  </p>
 +
</section>
 +
<section id="4">
 +
  <h1>4. Results</h1>
 +
  <p>
 +
    We were able to show our enhancer plasmid to work great with the Cas staples and the reporter plasmid. For the whole
 +
    assay, an enhancer plasmid and the reporter plasmid was used. The reporter plasmid has firefly luciferase behind
 +
    several repeats of a Cas9 targeted sequence. The enhancer plasmid has the Oct1 being targeted by Cas12a. By
 +
    introducing a fgRNA staple (<a href="https://parts.igem.org/PArt:BBa_K5237000">BBa_K5237000</a>) and a Gal4-VP64 (<a
 +
      href="https://parts.igem.org/PArt:BBa_K5237021">BBa_K5237021</a>), expression of the luciferase is induced
 +
    (Fig. 2 A).
 +
    Cells were again normalized against ubiquitous renilla expression.
 +
    Using no linker between the two spacers showed similar relative luciferase activity to the baseline control (Fig. 2 B). An extension of the linker from 20 nt up to 40 nt resulted in an increasingly higher
 +
    expression of the reporter gene. These results suggest an extension of the linker might lead to better
 +
    transactivation when hijacking an enhancer/activator.
 +
  </p>
 +
  <div class="thumb">
 +
    <div class="thumbinner" style="width:60%;">
 +
      <img alt="" class="thumbimage" src="https://static.igem.wiki/teams/5237/wetlab-results/results-eh-2.svg"
 +
        style="width:99%;" />
 +
      <div class="thumbcaption">
 +
        <i>
 +
          <b>Figure 2: Applying Fusion Guide RNAs for Cas staples.</b> <b>A</b>, schematic overview of the assay.
 +
          An enhancer
 +
          plasmid and a reporter plasmid are brought into proximity by a fgRNA Cas staple complex binding both
 +
          plasmids. Target
 +
          sequences were included in multiple repeats prior to the functional elements. Firefly luciferase serves as
 +
          the reporter
 +
          gene, the enhancer is constituted by multiple Gal4 repeats that are bound by a Gal4-VP64 fusion.
 +
          <b>B</b>, results of using a fgRNA Cas staple for trans activation of firefly luciferase. Firefly
 +
          luciferase activity was measured 48h after transfection. Normalized against ubiquitously expressed Renilla
 +
          luciferase.
 +
          Statistical significance was calculated with ordinary One-way ANOVA with Dunn's method for multiple
 +
          comparisons (*p &lt;
 +
          0.05; **p &lt; 0.01; ***p &lt; 0.001; mean +/- SD). The assay included sgRNAs and fgRNAs with linker
 +
          lengths from 0 nt
 +
          to 40 nt.
 +
        </i>
 +
      </div>
 +
    </div>
 +
  </div>
 +
</section>
 +
<section id="5">
 +
  <h1>5. References</h1>
 +
  <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. Scientific reports, 5, 10907. <a
 +
      href="https://doi.org/10.1038/srep10907" target="_blank">https://doi.org/10.1038/srep10907</a></p>
 +
</section>
 +
</body>
  
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===Functional Parameters===
+
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<partinfo>BBa_K5237024 parameters</partinfo>
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Revision as of 04:54, 1 October 2024

BBa_K5237024

TRE-minimal promoter- firefly luciferase

This part contains the tetO binding site (BBa_K5237019) , a minimal promoter and a firefly luciferase gene. With a VP64 coming in close proximity to the minimal promoter transcription factors are recruited, initiating expression of firefly luciferase. The described mechanism is utilized in our enhancer hijacking assay for prove of Cas stapling.

 



The PICasSO Toolbox
Figure 1: How our part collection can be used to engineer new staples


Next to the well-studied linear DNA sequence, the 3D spatial organization of DNA plays a crucial role in gene regulation, cell fate, disease development and more. However, the tools to precisely manipulate this genomic architecture remain limited, rendering it challenging to explore the full potential of the 3D genome in synthetic biology. We - iGEM Team Heidelberg 2024 - have developed PICasSO, a powerful molecular toolbox based on various DNA-binding proteins to address this issue.

The PICasSO part collection offers a comprehensive, modular platform for precise manipulation and re-programming of DNA-DNA interactions using protein staples in living cells, enabling researchers to recreate natural 3D genomic interactions, such as enhancer hijacking, or to design entirely new spatial architectures for gene regulation. Beyond its versatility, PICasSO includes robust assay systems to support the engineering, optimization, and testing of new staples, ensuring functionality in vitro and in vivo. We took special care to include parts crucial for testing every step of the cycle (design, build, test, learn) when engineering new parts.

At its heart, the PICasSO part collection consists of three categories.
(i) Our DNA-binding proteins include our finalized enhancer hijacking Cas staple as well as half staples that can be used by scientists to compose entirely new Cas staples in the future. We also include our Simple staples that 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 the functionality of our Cas and Basic staples. These consist of protease-cleavable peptide linkers and inteins that allow condition-specific, dynamic stapling in vivo. Besides staple functionality, we also include the parts to enable the efficient delivery of PICasSO's constructs with our interkingdom conjugation system.
(iii) As the final category of our collection, we provide parts that support the use of 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 for functional readouts via a luciferase reporter, which allows for straightforward experimental simulation of enhancer hijacking 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.

Our part collection includes:

DNA-binding proteins: The building blocks for engineering of custom staples for DNA-DNA interactions with a modular system ensuring easy assembly.
BBa_K5237000 fgRNA Entry vector MbCas12a-SpCas9 Entryvector for simple fgRNA cloning via SapI
BBa_K5237001 Staple subunit: dMbCas12a-Nucleoplasmin NLS Staple subunit that can be combined with sgRNA or fgRNA and dCas9 to form a functional staple
BBa_K5237002 Staple subunit: SV40 NLS-dSpCas9-SV40 NLS Staple subunit that can be combined witha sgRNA or fgRNA and dCas12avto form a functional staple
BBa_K5237003 Cas Staple: SV40 NLS-dMbCas12a-dSpCas9-Nucleoplasmin NLS Functional Cas staple that can be combined with sgRNA 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. Can be used to create functionalized staples units
BBa_K5237013 Caged NpuC Intein A caged NpuC split intein fragment that undergoes protein trans-splicing after protease activation. Can be used to create functionalized staples units
BBa_K5237014 fgRNA processing casette Processing casette to produce multiple fgRNAs from one transcript, that can be used for multiplexed 3D genome reprograming
BBa_K5237015 Intimin anti-EGFR Nanobody Interkindom conjugation between bacteria and mammalian cells, as 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 to measure proximity of stapled DNA in bacterial and mammalian living cells enabling swift testing and easy development for new systems
BBa_K5237016 FRET-Donor: mNeonGreen-Oct1 FRET Donor-Fluorpohore fused to Oct1-DBD that binds to the Oct1 binding cassette. 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. 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. It 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 Promotor, mCherry Readout system for enhancer binding. It 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. It 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 99
    Illegal XbaI site found at 61
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 99
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 99
    Illegal BamHI site found at 78
    Illegal XhoI site found at 48
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 99
    Illegal XbaI site found at 61
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 99
    Illegal XbaI site found at 61
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 952

2. Usage and Biology

The tetracycline response element, part of the TetR family of regulators (TFRs), is central to the regulation of antibiotic resistance genes, especially through its role in the Tet operon (TetO). In this system, the TetR protein acts as a repressor by binding to the TetO operator, inhibiting the expression of tetracycline resistance genes. Upon binding tetracycline or its analogs, TetR undergoes a conformational change, releasing TetO and allowing the transcription of target genes. This system is widely utilized in molecular biology as a controlled gene expression tool, particularly in inducible gene expression systems in both prokaryotic and eukaryotic cells (Cuthbertson and Nodwell (2013)).

Firefly luciferases are enzymes responsible for the bioluminescence seen in fireflies, catalyzing the oxidation of luciferin in the presence of ATP, magnesium ions, and oxygen. This reaction produces light and is highly efficient, with little heat released, making it a popular tool in molecular biology for reporter assays. The gene encoding firefly luciferase is widely used in research to monitor gene expression, quantify cellular ATP levels, and study transcriptional activity due to its sensitivity and ease of detection (Xie et al. (2010))

We utilize the recognition site for plasmid to plasmid stapling with our Cas staples. A fgRNA targeting Tre and Oct1 on the other plasmid, is the key factor in the Cas staple, bringing them together. When the transactivator, Gal4-VP64, binds as well we have transactivation as a readout for functioning staples.

3. Assembly and part evolution

The cloning strategy designed for TetO allows for the easy assembly of repetitive repeats. It follows the procedure outlined by Sladitschek and Neveu (2015). Briefly, the oligos can be inserted into a vector digested with SalI and XhoI, yielding a vector with three binding repeats flanked by these restriction sites. The vector can be linearized with either SalI or XhoI, as both enzymes create compatible overhangs. The annealed oligos can then be ligated into the vector, resulting in six binding repeats, with the middle sequence losing its cleavage site compatibility. This process can be repeated to achieve the desired number of repeats by digesting the vector and re-ligating the oligos. For the experiments conducted, a folding plasmid with 12 repeats was created. Since the registry has some limitations regarding sequence depository, the binding cassette is flanked by SalI and XhoI, and the top and bottom oligos with the fitting overhangs are annotated.

4. Results

We were able to show our enhancer plasmid to work great with the Cas staples and the reporter plasmid. For the whole assay, an enhancer plasmid and the reporter plasmid was used. The reporter plasmid has firefly luciferase behind several repeats of a Cas9 targeted sequence. The enhancer plasmid has the Oct1 being targeted by Cas12a. By introducing a fgRNA staple (BBa_K5237000) and a Gal4-VP64 (BBa_K5237021), expression of the luciferase is induced (Fig. 2 A). Cells were again normalized against ubiquitous renilla expression. Using no linker between the two spacers showed similar relative luciferase activity to the baseline control (Fig. 2 B). An extension of the linker from 20 nt up to 40 nt resulted in an increasingly higher expression of the reporter gene. These results suggest an extension of the linker might lead to better transactivation when hijacking an enhancer/activator.

Figure 2: Applying Fusion Guide RNAs for Cas staples. A, schematic overview of the assay. An enhancer plasmid and a reporter plasmid are brought into proximity by a fgRNA Cas staple complex binding both plasmids. Target sequences were included in multiple repeats prior to the functional elements. Firefly luciferase serves as the reporter gene, the enhancer is constituted by multiple Gal4 repeats that are bound by a Gal4-VP64 fusion. B, results of using a fgRNA Cas staple for trans activation of firefly luciferase. Firefly luciferase activity was measured 48h after transfection. Normalized against ubiquitously expressed Renilla luciferase. Statistical significance was calculated with ordinary One-way ANOVA with Dunn's method for multiple comparisons (*p < 0.05; **p < 0.01; ***p < 0.001; mean +/- SD). The assay included sgRNAs and fgRNAs with linker lengths from 0 nt to 40 nt.

5. References

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. Scientific reports, 5, 10907. https://doi.org/10.1038/srep10907