Difference between revisions of "Part:BBa K5237017"

 
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<body>
 
<body>
 
   <!-- Part summary -->
 
   <!-- Part summary -->
   <section id="1">
+
   <section>
 
     <h1>FRET-Acceptor: TetR-mScarlet-I</h1>
 
     <h1>FRET-Acceptor: TetR-mScarlet-I</h1>
 
     <p>This composite part is a fusion protein of TetR and the red fluorescent protein mScarlet-I. It was used as a FRET
 
     <p>This composite part is a fusion protein of TetR and the red fluorescent protein mScarlet-I. It was used as a FRET
Line 40: Line 47:
 
       This part was used to measure the proximity of two DNA strands by FRET fluorescence measurements.
 
       This part was used to measure the proximity of two DNA strands by FRET fluorescence measurements.
 
     </p>
 
     </p>
     <p>&nbsp;</p>
+
     <p> </p>
 
   </section>
 
   </section>
   <div id="toc" class="toc">
+
   <div class="toc" id="toc">
 
     <div id="toctitle">
 
     <div id="toctitle">
 
       <h1>Contents</h1>
 
       <h1>Contents</h1>
Line 54: Line 61:
 
         <ul>
 
         <ul>
 
           <li class="toclevel-2 tocsection-2.1"><a href="#2.1"><span class="tocnumber">2.1</span> <span
 
           <li class="toclevel-2 tocsection-2.1"><a href="#2.1"><span class="tocnumber">2.1</span> <span
                 class="toctext">Oct1-DBD</span></a>
+
                 class="toctext">TetR DNA Binding Protein</span></a>
 
           </li>
 
           </li>
 
           <li class="toclevel-2 tocsection-2.2"><a href="#2.2"><span class="tocnumber">2.2</span> <span
 
           <li class="toclevel-2 tocsection-2.2"><a href="#2.2"><span class="tocnumber">2.2</span> <span
                 class="toctext">mNeonGreen</span></a>
+
                 class="toctext">mScarlet-I</span></a>
 
           </li>
 
           </li>
 
           <li class="toclevel-2 tocsection-2.3"><a href="#2.3"><span class="tocnumber">2.3</span> <span
 
           <li class="toclevel-2 tocsection-2.3"><a href="#2.3"><span class="tocnumber">2.3</span> <span
Line 65: Line 72:
 
       </li>
 
       </li>
 
       <li class="toclevel-1 tocsetction-3"><a href="#3"><span class="tocnumber">3</span> <span class="toctext">Assembly
 
       <li class="toclevel-1 tocsetction-3"><a href="#3"><span class="tocnumber">3</span> <span class="toctext">Assembly
             and part evolution</span></a>
+
             and Part Evolution</span></a>
 
       </li>
 
       </li>
 
       <li class="toclevel-1 tocsection-5"><a href="#4"><span class="tocnumber">4</span> <span
 
       <li class="toclevel-1 tocsection-5"><a href="#4"><span class="tocnumber">4</span> <span
Line 74: Line 81:
 
       <li class="toclevel-1 tocsection-8"><a href="#6"><span class="tocnumber">6</span> <span
 
       <li class="toclevel-1 tocsection-8"><a href="#6"><span class="tocnumber">6</span> <span
 
             class="toctext">References</span></a>
 
             class="toctext">References</span></a>
 +
      </li>
 
       </li>
 
       </li>
 
     </ul>
 
     </ul>
 
   </div>
 
   </div>
 
   <section>
 
   <section>
 +
    <p><br /><br /></p>
 
     <font size="5"><b>The PICasSO Toolbox </b> </font>
 
     <font size="5"><b>The PICasSO Toolbox </b> </font>
    <p><br></p>
+
     <div class="thumb" style="margin-top:10px;"></div>
     <div class="thumb"></div>
+
    <div class="thumbinner" style="width:550px"><img alt="" class="thumbimage"
      <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">
+
        src="https://static.igem.wiki/teams/5237/wetlab-results/registry-part-collection-engineering-cycle-example-overview.svg"
        <div class="thumbcaption">
+
        style="width:99%;" />
          <i><b>Figure 1: Example how the part collection can be used to engineer new staples</b></i>
+
      <div class="thumbcaption">
        </div>
+
        <i><b>Figure 1: How our part collection can be used to engineer new staples</b></i>
 
       </div>
 
       </div>
 
     </div>
 
     </div>
   
 
 
 
     <p>
 
     <p>
       <br>
+
       <br />
       The 3D organization of the genome plays a crucial role in regulating gene expression in eukaryotic cells,
+
       While synthetic biology has in the past focused on engineering the genomic sequence of organisms, the <b>3D
       impacting cellular behavior, evolution, and disease. Beyond the linear DNA sequence, the spatial arrangement of
+
        spatial organization</b> of DNA is well-known to be an important layer of information encoding in
      chromatin, influenced by DNA-DNA interactions, shapes pathways of gene regulation. However, the tools to precisely
+
      particular in eukaryotes, playing a crucial role in
       manipulate this genomic architecture remain limited, rendering it challenging to explore the full potential of the
+
      gene regulation and hence
       3D genome in synthetic biology. We - iGEM Team Heidelberg 2024 - have developed PICasSO, a powerful molecular
+
       cell fate, disease development, evolution, and more. However, tools to precisely manipulate and control the
       toolbox based on various DNA-binding proteins to address this issue.
+
       genomic spatial
 
+
      architecture are limited, hampering the exploration of
 +
       3D genome engineering in synthetic biology. We - the iGEM Team Heidelberg 2024 - have developed PICasSO, a
 +
       <b>powerful
 +
        molecular toolbox for rationally engineering genome 3D architectures</b> in living cells, based on
 +
      various DNA-binding proteins.
 
     </p>
 
     </p>
 
     <p>
 
     <p>
 
       The <b>PICasSO</b> part collection offers a comprehensive, modular platform for precise manipulation and
 
       The <b>PICasSO</b> part collection offers a comprehensive, modular platform for precise manipulation and
       re-programming
+
       <b>re-programming
      of DNA-DNA interactions using protein staples in living cells, enabling researchers to recreate natural 3D genomic
+
        of DNA-DNA interactions</b> using engineered "protein staples" in living cells. This enables
       interactions, such as enhancer hijacking, or to design entirely new spatial architectures for gene regulation.
+
      researchers to recreate naturally occurring alterations of 3D genomic
       Beyond its versatility, PICasSO includes robust assay systems to support the engineering, optimization, and
+
       interactions, such as enhancer hijacking in cancer, or to design entirely new spatial architectures for
       testing of new staples, ensuring functionality <i>in vitro</i> and <i>in vivo</i>. We took special care to include
+
      artificial gene regulation and cell function control.
       parts crucial for testing every step of the cycle (design, build, test, learn) when engineering new parts
+
       Specifically, the fusion of two DNA binding proteins enables to artificially bring otherwise distant genomic
 +
      loci into
 +
      spatial proximity.
 +
      To unlock the system's full potential, we introduce versatile <b>chimeric CRISPR/Cas complexes</b>,
 +
      connected either at
 +
      the protein or - in the case of CRISPR/Cas-based DNA binding moieties - the guide RNA level. These complexes are
 +
      referred to as protein- or Cas staples, respectively. Beyond its
 +
      versatility with regard to the staple constructs themselves, PICasSO includes <b>robust assay</b> systems to
 +
      support the engineering, optimization, and
 +
       testing of new staples <i>in vitro</i> and <i>in vivo</i>. Notably, the PICasSO toolbox was developed in a
 +
       design-build-test-learn <b>engineering cycle closely intertwining wet lab experiments and computational
 +
        modeling</b> and iterated several times, yielding a collection of well-functioning and -characterized
 +
      parts.
 
     </p>
 
     </p>
 
+
     <p>At its heart, the PICasSO part collection consists of three categories. <br /><b>(i)</b> Our <b>DNA-binding
     <p>At its heart, the PICasSO part collection consists of three categories. <br><b>(i)</b> Our <b>DNA-binding proteins</b>
+
        proteins</b>
 
       include our
 
       include our
       finalized enhancer hijacking Cas staple as well as half staples that can be used by scientists to compose entirely
+
       finalized Cas staple experimentally validated using an artificial "enhancer hijacking" system as well as
       new Cas staples in the future. We also include our simple staples that serve as controls for successful stapling
+
      "half staples" that can be combined by scientists to compose entirely
       and can be further engineered to create alternative, simpler and more compact staples. <br>
+
       new Cas staples in the future. We also include our Simple staples comprised of particularly small, simple
       <b>(ii)</b> As <b>functional elements</b>, we list additional parts that enhance the functionality of our Cas and Basic staples. These
+
      and robust DNA binding domains well-known to the synthetic biology community, which serve as controls for
       consist of
+
      successful stapling
       protease-cleavable peptide linkers and inteins that allow condition-specific, dynamic stapling <i>in vivo</i>.
+
       and can be further engineered to create alternative, simpler, and more compact staples. <br />
       Besides staple functionality, we also include the parts to enable the efficient delivery of PICasSO's constructs with our
+
       <b>(ii)</b> As <b>functional elements</b>, we list additional parts that enhance and expand the
       interkingdom conjugation system. <br>
+
      functionality of our Cas and
       <b>(iii)</b> As the final component of our collection, we provide parts that support the use of our <b>custom readout
+
      Basic staples. These
         systems</b>. These include components of our established FRET-based proximity assay system, enabling users to
+
       consist of staples dependent on
 +
       cleavable peptide linkers targeted by cancer-specific proteases or inteins that allow condition-specific,
 +
      dynamic stapling <i>in vivo</i>.
 +
       We also include several engineered parts that enable the efficient delivery of PICasSO's constructs into
 +
      target cells, including mammalian cells,
 +
      with our new
 +
       interkingdom conjugation system. <br />
 +
       <b>(iii)</b> As the final category of our collection, we provide parts that underlie our <b>custom
 +
        readout
 +
         systems</b>. These include components of our established FRET-based proximity assay system, enabling
 +
      users to
 
       confirm
 
       confirm
       accurate stapling. Additionally, we offer a complementary, application-oriented testing system for functional
+
       accurate stapling. Additionally, we offer a complementary, application-oriented testing system based on a
       readout via a luciferase reporter, which allows for straightforward experimental simulation of enhancer hijacking.
+
       luciferase reporter, which allows for straightforward experimental assessment of functional enhancer
 +
      hijacking events
 +
      in mammalian cells.
 
     </p>
 
     </p>
 
     <p>
 
     <p>
       The following table gives a complete overview of all parts in our PICasSO toolbox. The highlighted parts showed
+
       The following table gives a comprehensive overview of all parts in our PICasSO toolbox. <mark
      exceptional performance as described on our iGEM wiki and can serve as a reference. The other parts in the
+
        style="background-color: #FFD700; color: black;">The highlighted parts showed
       collection are versatile building blocks designed to provide future iGEMers with the flexibility to engineer their
+
        exceptional performance as described on our iGEM wiki and can serve as a reference.</mark> The other
       own custom Cas staples, enabling further optimization and innovation.<br>
+
      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>
 
     <p>
 
     <p>
       <font size="4"><b>Our part collection includes:</b></font><br>
+
       <font size="4"><b>Our part collection includes:</b></font><br />
 
     </p>
 
     </p>
 
+
     <table style="width: 90%; padding-right:10px;">
     <table style="width: 90%;">
+
       <td align="left" colspan="3"><b>DNA-Binding Proteins: </b>
       <td colspan="3" align="left"><b>DNA-binding proteins: </b>
+
         Modular building blocks for engineering of custom staples to mediate defined DNA-DNA interactions <i>in vivo</i>
         The building blocks for engineering of custom staples for DNA-DNA interactions with a modular system ensuring
+
      </td>
        easy assembly.</td>
+
 
       <tbody>
 
       <tbody>
 
         <tr bgcolor="#FFD700">
 
         <tr bgcolor="#FFD700">
 
           <td><a href="https://parts.igem.org/Part:BBa_K5237000" target="_blank">BBa_K5237000</a></td>
 
           <td><a href="https://parts.igem.org/Part:BBa_K5237000" target="_blank">BBa_K5237000</a></td>
           <td>fgRNA Entryvector MbCas12a-SpCas9</td>
+
           <td>Fusion Guide RNA Entry Vector MbCas12a-SpCas9</td>
           <td>Entryvector for simple fgRNA cloning via SapI</td>
+
           <td>Entry vector for simple fgRNA cloning via SapI</td>
 
         </tr>
 
         </tr>
         <tr>
+
         <tr bgcolor="#FFD700">
 
           <td><a href="https://parts.igem.org/Part:BBa_K5237001" target="_blank">BBa_K5237001</a></td>
 
           <td><a href="https://parts.igem.org/Part:BBa_K5237001" target="_blank">BBa_K5237001</a></td>
           <td>Staple subunit: dMbCas12a-Nucleoplasmin NLS</td>
+
           <td>Staple Subunit: dMbCas12a-Nucleoplasmin NLS</td>
           <td>Staple subunit that can be combined to form a functional staple, for example with fgRNA and dCas9 </td>
+
           <td>Staple subunit that can be combined with crRNA or fgRNA and dSpCas9 to form a functional staple
 +
          </td>
 
         </tr>
 
         </tr>
         <tr>
+
         <tr bgcolor="#FFD700">
 
           <td><a href="https://parts.igem.org/Part:BBa_K5237002" target="_blank">BBa_K5237002</a></td>
 
           <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: SV40 NLS-dSpCas9-SV40 NLS</td>
           <td>Staple subunit that can be combined to form a functional staple, for example with our fgRNA or dCas12a
+
           <td>Staple subunit that can be combined with a sgRNA or fgRNA and dMbCas12a to form a functional staple
 
           </td>
 
           </td>
 
         </tr>
 
         </tr>
 
         <tr>
 
         <tr>
 
           <td><a href="https://parts.igem.org/Part:BBa_K5237003" target="_blank">BBa_K5237003</a></td>
 
           <td><a href="https://parts.igem.org/Part:BBa_K5237003" target="_blank">BBa_K5237003</a></td>
           <td>Cas-Staple: SV40 NLS-dMbCas12a-dSpCas9-Nucleoplasmin NLS</td>
+
           <td>Cas Staple: SV40 NLS-dMbCas12a-dSpCas9-Nucleoplasmin NLS</td>
           <td>Functional Cas staple that can be combined with sgRNA or fgRNA to bring two DNA strands in close proximity
+
           <td>Functional Cas staple that can be combined with sgRNA and crRNA or fgRNA to bring two DNA strands into
 +
            close
 +
            proximity
 
           </td>
 
           </td>
 
         </tr>
 
         </tr>
 
         <tr>
 
         <tr>
 
           <td><a href="https://parts.igem.org/Part:BBa_K5237004" target="_blank">BBa_K5237004</a></td>
 
           <td><a href="https://parts.igem.org/Part:BBa_K5237004" target="_blank">BBa_K5237004</a></td>
           <td>Staple subunit: Oct1-DBD</td>
+
           <td>Staple Subunit: Oct1-DBD</td>
           <td>Staple subunit that can be combined to form a functional staple, for example with TetR.<br>
+
           <td>Staple subunit that can be combined to form a functional staple, for example with TetR.<br />
 
             Can also be combined with a fluorescent protein as part of the FRET proximity assay</td>
 
             Can also be combined with a fluorescent protein as part of the FRET proximity assay</td>
 
         </tr>
 
         </tr>
 
         <tr>
 
         <tr>
 
           <td><a href="https://parts.igem.org/Part:BBa_K5237005" target="_blank">BBa_K5237005</a></td>
 
           <td><a href="https://parts.igem.org/Part:BBa_K5237005" target="_blank">BBa_K5237005</a></td>
           <td>Staple subunit: TetR</td>
+
           <td>Staple Subunit: TetR</td>
           <td>Staple subunit that can be combined to form a functional staple, for example with Oct1.<br>
+
           <td>Staple subunit that can be combined to form a functional staple, for example with Oct1.<br />
 
             Can also be combined with a fluorescent protein as part of the FRET proximity assay</td>
 
             Can also be combined with a fluorescent protein as part of the FRET proximity assay</td>
 
         </tr>
 
         </tr>
 
         <tr>
 
         <tr>
 
           <td><a href="https://parts.igem.org/Part:BBa_K5237006" target="_blank">BBa_K5237006</a></td>
 
           <td><a href="https://parts.igem.org/Part:BBa_K5237006" target="_blank">BBa_K5237006</a></td>
           <td>Simple taple: TetR-Oct1</td>
+
           <td>Simple Staple: TetR-Oct1</td>
 
           <td>Functional staple that can be used to bring two DNA strands in close proximity</td>
 
           <td>Functional staple that can be used to bring two DNA strands in close proximity</td>
 
         </tr>
 
         </tr>
 
         <tr>
 
         <tr>
 
           <td><a href="https://parts.igem.org/Part:BBa_K5237007" target="_blank">BBa_K5237007</a></td>
 
           <td><a href="https://parts.igem.org/Part:BBa_K5237007" target="_blank">BBa_K5237007</a></td>
           <td>Staple subunit: GCN4</td>
+
           <td>Staple Subunit: GCN4</td>
 
           <td>Staple subunit that can be combined to form a functional staple, for example with rGCN4</td>
 
           <td>Staple subunit that can be combined to form a functional staple, for example with rGCN4</td>
 
         </tr>
 
         </tr>
 
         <tr>
 
         <tr>
 
           <td><a href="https://parts.igem.org/Part:BBa_K5237008" target="_blank">BBa_K5237008</a></td>
 
           <td><a href="https://parts.igem.org/Part:BBa_K5237008" target="_blank">BBa_K5237008</a></td>
           <td>Staple subunit: rGCN4</td>
+
           <td>Staple Subunit: rGCN4</td>
 
           <td>Staple subunit that can be combined to form a functional staple, for example with rGCN4</td>
 
           <td>Staple subunit that can be combined to form a functional staple, for example with rGCN4</td>
 
         </tr>
 
         </tr>
 
         <tr>
 
         <tr>
 
           <td><a href="https://parts.igem.org/Part:BBa_K5237009" target="_blank">BBa_K5237009</a></td>
 
           <td><a href="https://parts.igem.org/Part:BBa_K5237009" target="_blank">BBa_K5237009</a></td>
           <td>Mini staple: bGCN4</td>
+
           <td>Mini Staple: bGCN4</td>
 
           <td>
 
           <td>
 
             Assembled staple with minimal size that can be further engineered</td>
 
             Assembled staple with minimal size that can be further engineered</td>
 
         </tr>
 
         </tr>
 
       </tbody>
 
       </tbody>
       <td colspan="3" align="left"><b>Functional elements: </b>
+
       <td align="left" colspan="3"><b>Functional Elements: </b>
         Protease cleavable peptide linkers and inteins are used to control and modify staples for further optimization
+
         Protease-cleavable peptide linkers and inteins are used to control and modify staples for further
         for custom applications.</td>
+
        optimization
 +
         for custom applications</td>
 
       <tbody>
 
       <tbody>
 
         <tr bgcolor="#FFD700">
 
         <tr bgcolor="#FFD700">
 
           <td><a href="https://parts.igem.org/Part:BBa_K5237010" target="_blank">BBa_K5237010</a></td>
 
           <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 Linker: GFLG</td>
           <td>Cathepsin B cleavable peptide linker, that can be used to combine two staple subunits ,to make responsive
+
           <td>Cathepsin B-cleavable peptide linker that can be used to combine two staple subunits to make
 +
            responsive
 
             staples</td>
 
             staples</td>
 
         </tr>
 
         </tr>
Line 209: Line 253:
 
           <td><a href="https://parts.igem.org/Part:BBa_K5237011" target="_blank">BBa_K5237011</a></td>
 
           <td><a href="https://parts.igem.org/Part:BBa_K5237011" target="_blank">BBa_K5237011</a></td>
 
           <td>Cathepsin B Expression Cassette</td>
 
           <td>Cathepsin B Expression Cassette</td>
           <td>Cathepsin B which can be selectively express to cut the cleavable linker</td>
+
           <td>Expression cassette for the overexpression of cathepsin B</td>
 
         </tr>
 
         </tr>
 
         <tr>
 
         <tr>
           <td><a href="https://parts.igem.org/Part:BBa_K52370012" target="_blank">BBa_K5237012</a></td>
+
           <td><a href="https://parts.igem.org/Part:BBa_K5237012" target="_blank">BBa_K5237012</a></td>
 
           <td>Caged NpuN Intein</td>
 
           <td>Caged NpuN Intein</td>
           <td>Undergoes protein transsplicing after protease activation, can be used to create functionalized staple
+
           <td>A caged NpuN split intein fragment that undergoes protein <i>trans</i>-splicing after protease
             units</td>
+
            activation, which can be used to create functionalized staple
 +
             subunits</td>
 
         </tr>
 
         </tr>
 
         <tr>
 
         <tr>
           <td><a href="https://parts.igem.org/Part:BBa_K52370013" target="_blank">BBa_K5237013</a></td>
+
           <td><a href="https://parts.igem.org/Part:BBa_K5237013" target="_blank">BBa_K5237013</a></td>
 
           <td>Caged NpuC Intein</td>
 
           <td>Caged NpuC Intein</td>
           <td>Undergoes protein transsplicing after protease activation, can be used to create functionalized staple
+
           <td>A caged NpuC split intein fragment that undergoes protein <i>trans</i>-splicing after protease
             units</td>
+
            activation, which can be used to create functionalized staple
 +
             subunits</td>
 
         </tr>
 
         </tr>
 
         <tr>
 
         <tr>
           <td><a href="https://parts.igem.org/Part:BBa_K52370014" target="_blank">BBa_K5237014</a></td>
+
           <td><a href="https://parts.igem.org/Part:BBa_K5237014" target="_blank">BBa_K5237014</a></td>
           <td>fgRNA processing casette</td>
+
           <td>Fusion Guide RNA Processing Casette</td>
           <td>Processing casette to produce multiple fgRNAs from one transcript, can be used for multiplexing</td>
+
           <td>Processing cassette to produce multiple fgRNAs from one transcript, that can be used for
 +
            multiplexed 3D
 +
            genome reprogramming</td>
 
         </tr>
 
         </tr>
 
         <tr>
 
         <tr>
           <td><a href="https://parts.igem.org/Part:BBa_K52370015" target="_blank">BBa_K5237015</a></td>
+
           <td><a href="https://parts.igem.org/Part:BBa_K5237015" target="_blank">BBa_K5237015</a></td>
 
           <td>Intimin anti-EGFR Nanobody</td>
 
           <td>Intimin anti-EGFR Nanobody</td>
           <td>Interkindom conjugation between bacteria and mammalian cells, as alternative delivery tool for large
+
           <td>Interkingdom conjugation between bacteria and mammalian cells, as an alternative delivery tool for
 +
            large
 
             constructs</td>
 
             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>
 
         </tr>
 
       </tbody>
 
       </tbody>
       <td colspan="3" align="left"><b>Readout Systems: </b>
+
       <td align="left" colspan="3"><b>Readout Systems: </b>
         FRET and enhancer recruitment to measure proximity of stapled DNA in bacterial and mammalian living cells
+
         FRET and enhancer recruitment readout systems to rapidly assess successful DNA stapling in bacterial and
        enabling swift testing and easy development for new systems.</td>
+
        mammalian cells
 +
      </td>
 
       <tbody>
 
       <tbody>
 
         <tr bgcolor="#FFD700">
 
         <tr bgcolor="#FFD700">
           <td><a href="https://parts.igem.org/Part:BBa_K52370016" target="_blank">BBa_K5237016</a></td>
+
           <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: mNeonGreen-Oct1</td>
           <td>Donor part for the FRET assay binding the Oct1 binding cassette. Can be used to visualize DNA-DNA
+
           <td>FRET donor-fluorophore fused to Oct1-DBD that binds to the Oct1 binding cassette, which can be used to
 +
            visualize
 +
            DNA-DNA
 
             proximity</td>
 
             proximity</td>
 
         </tr>
 
         </tr>
Line 248: Line 307:
 
           <td><a href="https://parts.igem.org/Part:BBa_K5237017" target="_blank">BBa_K5237017</a></td>
 
           <td><a href="https://parts.igem.org/Part:BBa_K5237017" target="_blank">BBa_K5237017</a></td>
 
           <td>FRET-Acceptor: TetR-mScarlet-I</td>
 
           <td>FRET-Acceptor: TetR-mScarlet-I</td>
           <td>Acceptor part for the FRET assay binding the TetR binding cassette. Can be used to visualize DNA-DNA
+
           <td>Acceptor part for the FRET assay binding the TetR binding cassette, which can be used to visualize
 +
            DNA-DNA
 
             proximity</td>
 
             proximity</td>
 
         </tr>
 
         </tr>
Line 254: Line 314:
 
           <td><a href="https://parts.igem.org/Part:BBa_K5237018" target="_blank">BBa_K5237018</a></td>
 
           <td><a href="https://parts.igem.org/Part:BBa_K5237018" target="_blank">BBa_K5237018</a></td>
 
           <td>Oct1 Binding Casette</td>
 
           <td>Oct1 Binding Casette</td>
           <td>DNA sequence containing 12 Oct1 binding motifs, can be used for different assays such as the FRET
+
           <td>DNA sequence containing 12 Oct1 binding motifs, compatible with various assays such as the FRET
 
             proximity assay</td>
 
             proximity assay</td>
 
         </tr>
 
         </tr>
Line 260: Line 320:
 
           <td><a href="https://parts.igem.org/Part:BBa_K5237019" target="_blank">BBa_K5237019</a></td>
 
           <td><a href="https://parts.igem.org/Part:BBa_K5237019" target="_blank">BBa_K5237019</a></td>
 
           <td>TetR Binding Cassette</td>
 
           <td>TetR Binding Cassette</td>
           <td>DNA sequence containing 12 Oct1 binding motifs, can be used for different assays such as the FRET
+
           <td>DNA sequence containing 12 Oct1 binding motifs, can be used for different assays such as the
 +
            FRET
 
             proximity assay</td>
 
             proximity assay</td>
 
         </tr>
 
         </tr>
 
         <td><a href="https://parts.igem.org/Part:BBa_K5237020" target="_blank">BBa_K5237020</a></td>
 
         <td><a href="https://parts.igem.org/Part:BBa_K5237020" target="_blank">BBa_K5237020</a></td>
 
         <td>Cathepsin B-Cleavable Trans-Activator: NLS-Gal4-GFLG-VP64</td>
 
         <td>Cathepsin B-Cleavable Trans-Activator: NLS-Gal4-GFLG-VP64</td>
         <td>Readout system that responds to protease activity. It was used to test Cathepsin-B cleavable linker.</td>
+
         <td>Readout system that responds to protease activity, which was used to test cathepsin B-cleavable linker
         </tr>
+
         </td>
 
         <tr>
 
         <tr>
 
           <td><a href="https://parts.igem.org/Part:BBa_K5237021" target="_blank">BBa_K5237021</a></td>
 
           <td><a href="https://parts.igem.org/Part:BBa_K5237021" target="_blank">BBa_K5237021</a></td>
 
           <td>NLS-Gal4-VP64</td>
 
           <td>NLS-Gal4-VP64</td>
           <td>Trans-activating enhancer, that can be used to simulate enhancer hijacking. </td>
+
           <td>Trans-activating enhancer, that can be used to simulate enhancer hijacking</td>
 
         </tr>
 
         </tr>
 
         <td><a href="https://parts.igem.org/Part:BBa_K5237022" target="_blank">BBa_K5237022</a></td>
 
         <td><a href="https://parts.igem.org/Part:BBa_K5237022" target="_blank">BBa_K5237022</a></td>
         <td>mCherry Expression Cassette: UAS, minimal Promotor, mCherry</td>
+
         <td>mCherry Expression Cassette: UAS, minimal Promoter, mCherry</td>
         <td>Readout system for enhancer binding. It was used to test Cathepsin-B cleavable linker.</td>
+
         <td>Readout system for enhancer binding, which was used to test cathepsin B-cleavable linker</td>
        </tr>
+
 
         <tr>
 
         <tr>
 
           <td><a href="https://parts.igem.org/Part:BBa_K5237023" target="_blank">BBa_K5237023</a></td>
 
           <td><a href="https://parts.igem.org/Part:BBa_K5237023" target="_blank">BBa_K5237023</a></td>
           <td>Oct1 - 5x UAS binding casette</td>
+
           <td>Oct1 - 5x UAS Binding Casette</td>
           <td>Oct1 and UAS binding cassette, that was used for the simulated enhancer hijacking assay.</td>
+
           <td>Oct1 and UAS binding cassette, that was used for the simulated enhancer hijacking assay</td>
 
         </tr>
 
         </tr>
 
         <tr>
 
         <tr>
 
           <td><a href="https://parts.igem.org/Part:BBa_K5237024" target="_blank">BBa_K5237024</a></td>
 
           <td><a href="https://parts.igem.org/Part:BBa_K5237024" target="_blank">BBa_K5237024</a></td>
           <td>TRE-minimal promoter- firefly luciferase</td>
+
           <td>TRE-minimal Promoter- Firefly Luciferase</td>
           <td>Contains Firefly luciferase controlled by a minimal promoter. It was used as a luminescence readout for
+
           <td>Contains firefly luciferase controlled by a minimal promoter, which was used as a luminescence
             simulated enhancer hijacking.</td>
+
            readout for
 +
             simulated enhancer hijacking</td>
 
         </tr>
 
         </tr>
 
       </tbody>
 
       </tbody>
 
     </table>
 
     </table>
    </p>
 
 
   </section>
 
   </section>
 
   <section id="1">
 
   <section id="1">
Line 297: Line 357:
  
 
</html>
 
</html>
 
 
<!--################################-->
 
<!--################################-->
<span class='h3bb'>Sequence and Features</span>
+
<span class="h3bb">Sequence and Features</span>
 
<partinfo>BBa_K5237017 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K5237017 SequenceAndFeatures</partinfo>
 
<!--################################-->
 
<!--################################-->
 
 
<html>
 
<html>
 
 
 
<section id="2">
 
<section id="2">
 
   <h1>2. Usage and Biology</h1>
 
   <h1>2. Usage and Biology</h1>
 
   <section id="2.1">
 
   <section id="2.1">
     <h1>2.1 TetR DNA binding protein</h1>
+
     <h1>2.1 TetR DNA Binding Protein</h1>
 
     <p>
 
     <p>
 
       The tetracycline repressor protein (tetR) is naturally present in gram-negative bacteria and is involved in the
 
       The tetracycline repressor protein (tetR) is naturally present in gram-negative bacteria and is involved in the
 
       resistance mechanism against tetracycline (and derivatives). It does so by tightly controlling the gene expression
 
       resistance mechanism against tetracycline (and derivatives). It does so by tightly controlling the gene expression
 
       of <i>tetA</i>, which encodes an efflux pump responsible for removing tetracycline from the cell.
 
       of <i>tetA</i>, which encodes an efflux pump responsible for removing tetracycline from the cell.
       TetR binds selectively to two plaindromic recognition sequences (<i>tetO</i>>1,2) with high affinity. For DNA
+
       TetR binds selectively to two plaindromic recognition sequences (<i>tetO</i>&gt;1,2) with high affinity. For DNA
       binding to occur tetR adopts a homodimeric structure and binds with two &#945;-helix-turn- &#945;-helix motifs
+
       binding to occur tetR adopts a homodimeric structure and binds with two α-helix-turn- α-helix motifs
 
       (HTH) to two tandemly oriented tetO sequences. In the presence of tetracycline or its analogs, tetR undergoes a
 
       (HTH) to two tandemly oriented tetO sequences. In the presence of tetracycline or its analogs, tetR undergoes a
 
       conformational change, which prevents it from binding to DNA, therby allowing gene expression(Orth <i>et al.</i>
 
       conformational change, which prevents it from binding to DNA, therby allowing gene expression(Orth <i>et al.</i>
 
       2000; Kisker <i>et al.</i> 1995).
 
       2000; Kisker <i>et al.</i> 1995).
       <br>
+
       <br />
 
       Due to its robust and highly regulatable DNA-binding properties, tetR has become a widely adopted tool in
 
       Due to its robust and highly regulatable DNA-binding properties, tetR has become a widely adopted tool in
 
       synthetic
 
       synthetic
 
       biology. Its ease of modification and ability to function in both prokaryotic and eukaryotic systems have made it
 
       biology. Its ease of modification and ability to function in both prokaryotic and eukaryotic systems have made it
       an essential element in the development of gene regulation systems (Berens & Hillen, 2004).
+
       an essential element in the development of gene regulation systems (Berens &amp; Hillen, 2004).
       <br>
+
       <br />
 
       In our project, tetR was integrated into the design of a modular DNA-stapling system because of its
 
       In our project, tetR was integrated into the design of a modular DNA-stapling system because of its
 
       well-characterized behavior, ensuring reliable DNA interactions.
 
       well-characterized behavior, ensuring reliable DNA interactions.
Line 330: Line 386:
 
   </section>
 
   </section>
 
   <section id="2.2">
 
   <section id="2.2">
     <h1>2.2mScarlet-I</h1>
+
     <h1>2.2 mScarlet-I</h1>
 
     <p>
 
     <p>
 
       mScarlet-I is a rapidly-maturing monomeric red fluorescent protein designed by Bindels <i>et al.</i> in 2017. It
 
       mScarlet-I is a rapidly-maturing monomeric red fluorescent protein designed by Bindels <i>et al.</i> in 2017. It
Line 343: Line 399:
 
       intensity and make it an ideal candidate for Förster Resonance Energy Transfer assays (McCullock <i>et al.</i>
 
       intensity and make it an ideal candidate for Förster Resonance Energy Transfer assays (McCullock <i>et al.</i>
 
       2020). When paired with mNeonGreen, mScarlet-I
 
       2020). When paired with mNeonGreen, mScarlet-I
 +
    </p>
 
     </p>
 
     </p>
 
   </section>
 
   </section>
Line 355: Line 412:
 
       sensitivity is governed by the Förster radius (R₀), which is the distance at which 50% energy transfer occurs.
 
       sensitivity is governed by the Förster radius (R₀), which is the distance at which 50% energy transfer occurs.
 
       Factors affecting FRET efficiency include the overlap of the donor's emission spectrum with the acceptor's
 
       Factors affecting FRET efficiency include the overlap of the donor's emission spectrum with the acceptor's
       absorption spectrum, the quantum yield of the donor, and the relative orientation of the fluorophores (Wu &
+
       absorption spectrum, the quantum yield of the donor, and the relative orientation of the fluorophores (Wu &amp;
 
       Brand,
 
       Brand,
 
       1994). These characteristics allow FRET to detect interactions such as protein-DNA binding or DNA proximity in
 
       1994). These characteristics allow FRET to detect interactions such as protein-DNA binding or DNA proximity in
Line 363: Line 420:
 
       <i>et al.</i>, 2017; Shaner <i>et al.</i>, 2013). FRET's sensitivity to small changes in distance makes it
 
       <i>et al.</i>, 2017; Shaner <i>et al.</i>, 2013). FRET's sensitivity to small changes in distance makes it
 
       especially powerful
 
       especially powerful
       for analyzing molecular interactions in living cells (Okamoto & Sako, 2017).
+
       for analyzing molecular interactions in living cells (Okamoto &amp; Sako, 2017).
 
     </p>
 
     </p>
 
     <div class="thumb">
 
     <div class="thumb">
       <div class="thumbinner" style="width:550px"><img alt=""
+
       <div class="thumbinner" style="width:550px"><img alt="" class="thumbimage"
           src="https://static.igem.wiki/teams/5237/wetlab-results/fpbase-fret-mng-msci.svg" style="width:99%;"
+
           src="https://static.igem.wiki/teams/5237/wetlab-results/fpbase-fret-mng-msci.svg" style="width:99%;" />
          class="thumbimage">
+
 
         <div class="thumbcaption">
 
         <div class="thumbcaption">
           <i><b>Figure 2: Overview of excitation and emission spectrum of mNeonGreen and m-Scarlet and it's properties
+
           <i><b>Figure 2: Overview of Excitation and Emission Spectrum of mNeonGreen and m-Scarlet and it's Properties
               as a FRET pair</b></i>
+
               as a FRET Pair</b></i>
 
         </div>
 
         </div>
 
       </div>
 
       </div>
Line 378: Line 434:
 
</section>
 
</section>
 
<section id="3">
 
<section id="3">
   <h1>3. Assembly and part evolution</h1>
+
   <h1>3. Assembly and Part Evolution</h1>
 
   <p>
 
   <p>
     As part of our engineering efforts an engineered tetR-mScarlet was tested, that binds DNA as a monomer. This scTetR is a fusion of
+
     As part of our engineering efforts an engineered tetR-mScarlet was tested, that binds DNA as a monomer. This scTetR
     two tetR proteins with a flexible linker which was described to maintain the same DNA-binding affinity and specificity as wild-type tetR (Kaminoka <i>et al</i>. 2006; Krueger <i>et al.</i>2003)</p>
+
    is a fusion of
 +
     two tetR proteins with a flexible linker which was described to maintain the same DNA-binding affinity and
 +
    specificity as wild-type tetR (Kaminoka <i>et al</i>. 2006; Krueger <i>et al.</i>2003)</p>
 
   <p>
 
   <p>
     Our experiments with the scTetR-mScarlet-I fusion protein showed no significant expression. Since the scTetR-mScarlet-I fusion protein was expressed as part of a polycistronic transcript, the results should be taken with caution.
+
     Our experiments with the scTetR-mScarlet-I fusion protein showed no significant expression. Since the
     Nonetheless we belive that qualitative assesements can still be drawn, because the same construct with tetR-mScarlet-I showed a good expression (Figure 2, 3).
+
    scTetR-mScarlet-I fusion protein was expressed as part of a polycistronic transcript, the results should be taken
 +
    with caution.
 +
     Nonetheless we belive that qualitative assesements can still be drawn, because the same construct with
 +
    tetR-mScarlet-I showed a good expression (Figure 2, 3).
 
   </p>
 
   </p>
 
   <div class="thumb"></div>
 
   <div class="thumb"></div>
   <div class="thumbinner" style="width:300px"><img alt="pic"
+
   <div class="thumbinner" style="width:300px"><img alt="pic" class="thumbimage"
       src="https://static.igem.wiki/teams/5237/engineering/sist-mut-t7-mscarlet-i.svg"
+
       src="https://static.igem.wiki/teams/5237/engineering/sist-mut-t7-mscarlet-i.svg" style="width:99%;" />
      style="width:99%;" class="thumbimage">
+
 
     <div class="thumbcaption">
 
     <div class="thumbcaption">
       <i><b>Figure 2: Fluoresence measurement of scTetR with mutant T7 polymerase.</b> Fluorescence intensity of mScarlet-I (ex. 560 nm, em. 600 nm) was measured 18 h after induction with IPTG and normalized to cell count (OD<sub>600</sub>).  
+
       <i><b>Figure 3: Fluoresence Measurement of scTetR with Mutant T7 Polymerase.</b> Fluorescence intensity of
      Positive control consists of a tetR-mNeonGreen fusion protein.</i>
+
        mScarlet-I (ex. 560 nm, em. 600 nm) was measured 18 h after induction with IPTG and normalized to cell count
 +
        (OD<sub>600</sub>).
 +
        Positive control consists of a tetR-mNeonGreen fusion protein.</i>
 
     </div>
 
     </div>
  </div>
 
 
   </div>
 
   </div>
 
</section>
 
</section>
Line 409: Line 470:
 
     an Oct1 binding site (<a href="https://parts.igem.org/Part:BBa_K5237018">BBa_K5237018</a>) for the staple and FRET
 
     an Oct1 binding site (<a href="https://parts.igem.org/Part:BBa_K5237018">BBa_K5237018</a>) for the staple and FRET
 
     donor binding.
 
     donor binding.
     <br><br>
+
     <br /><br />
 
     When tetR-Oct1 binds its respective sites on both plasmids, it brings mNeonGreen and mScarlet-I
 
     When tetR-Oct1 binds its respective sites on both plasmids, it brings mNeonGreen and mScarlet-I
 
     into close proximity, facilitating FRET between the two fluorophores. Successful stapling of the
 
     into close proximity, facilitating FRET between the two fluorophores. Successful stapling of the
Line 416: Line 477:
 
     consisting of a direct fusion of mNeonGreen and mScarlet-I, ensures maximal FRET efficiency and
 
     consisting of a direct fusion of mNeonGreen and mScarlet-I, ensures maximal FRET efficiency and
 
     serves as a benchmark for the assay.
 
     serves as a benchmark for the assay.
     <br><br>
+
  </p>
 +
  <div class="thumb">
 +
     <div class="thumbinner" style="width:60%;">
 +
      <img alt="" class="thumbimage" src="https://static.igem.wiki/teams/5237/figures-corrected/basic-staple-fret.svg"
 +
        style="width:99%;" />
 +
      <div class="thumbcaption">
 +
        <i>
 +
          <b>Figure 4: Overview FRET Assay</b>
 +
        </i>
 +
      </div>
 +
    </div>
 +
  </div>
 +
  <p>
 
     Fluorescence intensity, normalized to cell count, of mNeonGreen and mScarlet-I was measured 18 h
 
     Fluorescence intensity, normalized to cell count, of mNeonGreen and mScarlet-I was measured 18 h
     after induction with varying IPTG concentration (Figure 3). An increasing
+
     after induction with varying IPTG concentration (Fig. 5). An increasing
 
     expression strength
 
     expression strength
 
     is visible for decreasing IPTG concentrations. Fluorescence intensity of the positive control was
 
     is visible for decreasing IPTG concentrations. Fluorescence intensity of the positive control was
Line 428: Line 501:
 
     measurement of the donor and acceptor showed similar intensities, with only a small significant
 
     measurement of the donor and acceptor showed similar intensities, with only a small significant
 
     difference for mNeonGreen. A large difference could be measured between the staple and negative
 
     difference for mNeonGreen. A large difference could be measured between the staple and negative
     control, indicating proximity induced FRET. (Figure 3)
+
     control, indicating proximity induced FRET. (Fig. 5)
 
   </p>
 
   </p>
 
   <div class="thumb">
 
   <div class="thumb">
     <div class="thumbinner" style="width:700px"><img alt="pic"
+
     <div class="thumbinner" style="width:700px"><img alt="pic" class="thumbimage"
         src="https://static.igem.wiki/teams/5237/wetlab-results/sist-results-panel-fret.svg" style="width:99%;"
+
         src="https://static.igem.wiki/teams/5237/wetlab-results/sist-results-panel-fret.svg" style="width:99%;" />
        class="thumbimage">
+
 
       <div class="thumbcaption">
 
       <div class="thumbcaption">
         <i><b>Figure 3: Fluorescence of mNeonGreen, mScarlet-I and FRET.</b>
+
         <i><b>Figure 5: Fluorescence of mNeonGreen, mScarlet-I and FRET.</b>
 
           Fluorescence intensity of mNeonGreen (ex. 490 nm, em. 530 nm),
 
           Fluorescence intensity of mNeonGreen (ex. 490 nm, em. 530 nm),
 
           mScarlet-I (ex. 560 nm, em. 600 nm), and FRET (ex. 490 nm, em. 600 nm) was measured 18 h after induction with
 
           mScarlet-I (ex. 560 nm, em. 600 nm), and FRET (ex. 490 nm, em. 600 nm) was measured 18 h after induction with
 
           IPTG and normalized to cell count (OD<sub>600</sub>).
 
           IPTG and normalized to cell count (OD<sub>600</sub>).
           <b>A&#41;, B&#41;</b> Fluorescence intensity of mNeonGreen and mScarlet-I after induction with different IPTG
+
           <b>A), B)</b> Fluorescence intensity of mNeonGreen and mScarlet-I after induction with different IPTG
 
           concentrations.
 
           concentrations.
           <b>C&#41;</b> Fluorescence intensity of FRET pair induced with 0.025 mM IPTG. Statistical significance was
+
           <b>C)</b> Fluorescence intensity of FRET pair induced with 0.025 mM IPTG. Statistical significance was
 
           tested for with Ordinary two-way ANOVA with Šidák's multiple comparison test, with a single
 
           tested for with Ordinary two-way ANOVA with Šidák's multiple comparison test, with a single
 
           pooled variance. *p &lt; 0.05, ****p &lt; 0.001. Only significant results, within groups are shown. Data is
 
           pooled variance. *p &lt; 0.05, ****p &lt; 0.001. Only significant results, within groups are shown. Data is
           depcited as mean &#177; SD.
+
           depcited as mean ± SD.
 
         </i>
 
         </i>
 
       </div>
 
       </div>
Line 456: Line 528:
 
     mScarlet-I as a donor-acceptor pair. The assay was optimized for maximal FRET efficiency and validated with a
 
     mScarlet-I as a donor-acceptor pair. The assay was optimized for maximal FRET efficiency and validated with a
 
     positive control. The results showed a significant difference in fluorescence intensity between the staple and
 
     positive control. The results showed a significant difference in fluorescence intensity between the staple and
     negative control, indicating successful DNA stapling and FRET. This assay provides a powerful tool to engineer and test out novel Staples.
+
     negative control, indicating successful DNA stapling and FRET. This assay provides a powerful tool to engineer and
 +
    test out novel Staples.
 
     Future work will focus on further optimizing the assay in mammalian cells and quantifying interactions.
 
     Future work will focus on further optimizing the assay in mammalian cells and quantifying interactions.
 
   </p>
 
   </p>
Line 462: Line 535:
 
<section id="6">
 
<section id="6">
 
   <h1>6. References</h1>
 
   <h1>6. References</h1>
   <p>Berens, C., & Hillen, W. (2004). Gene Regulation By Tetracyclines. In J. K. Setlow (Ed.), <em>Genetic Engineering: Principles and Methods</em> (pp. 255–277). Springer US. <a href="https://doi.org/10.1007/978-0-306-48573-2_13" target="_blank">https://doi.org/10.1007/978-0-306-48573-2_13</a></p>
+
   <p>Berens, C., &amp; Hillen, W. (2004). Gene Regulation By Tetracyclines. In J. K. Setlow (Ed.), <em>Genetic
 +
      Engineering: Principles and Methods</em> (pp. 255–277). Springer US. <a
 +
      href="https://doi.org/10.1007/978-0-306-48573-2_13"
 +
      target="_blank">https://doi.org/10.1007/978-0-306-48573-2_13</a></p>
 +
  <p>Bindels, D. S., Haarbosch, L., van Weeren, L., Postma, M., Wiese, K. E., Mastop, M., Aumonier, S., Gotthard, G.,
 +
    Royant, A., Hink, M. A., &amp; Gadella, T. W. J. (2017). mScarlet: A bright monomeric red fluorescent protein for
 +
    cellular imaging. <em>Nature Methods, 14</em>(1), 53–56. <a href="https://doi.org/10.1038/nmeth.4074"
 +
      target="_blank">https://doi.org/10.1038/nmeth.4074</a></p>
 +
  <p>Hochreiter, B., Kunze, M., Moser, B., &amp; Schmid, J. A. (2019). Advanced FRET normalization allows quantitative
 +
    analysis of protein interactions including stoichiometries and relative affinities in living cells. <em>Scientific
 +
      Reports, 9</em>(1), 8233. <a href="https://doi.org/10.1038/s41598-019-44650-0"
 +
      target="_blank">https://doi.org/10.1038/s41598-019-44650-0</a></p>
 +
  <p>Kisker, C., Hinrichs, W., Tovar, K., Hillen, W., &amp; Saenger, W. (1995). The Complex Formed Between Tet Repressor
 +
    and Tetracycline-Mg<sup>2+</sup> Reveals Mechanism of Antibiotic Resistance. <em>Journal of Molecular Biology,
 +
      247</em>(2), 260–280. <a href="https://doi.org/10.1006/jmbi.1994.0138"
 +
      target="_blank">https://doi.org/10.1006/jmbi.1994.0138</a></p>
 +
  <p>McCullock, T. W., MacLean, D. M., &amp; Kammermeier, P. J. (2020). Comparing the performance of mScarlet-I, mRuby3,
 +
    and mCherry as FRET acceptors for mNeonGreen. <em>PLOS ONE, 15</em>(2), e0219886. <a
 +
      href="https://doi.org/10.1371/journal.pone.0219886"
 +
      target="_blank">https://doi.org/10.1371/journal.pone.0219886</a></p>
 +
  <p>Okamoto, K., &amp; Sako, Y. (2017). Recent advances in FRET for the study of protein interactions and dynamics.
 +
    <em>Current Opinion in Structural Biology, 46</em>, 16–23. <a href="https://doi.org/10.1016/j.sbi.2017.03.010"
 +
      target="_blank">https://doi.org/10.1016/j.sbi.2017.03.010</a></p>
 +
  <p>Orth, P., Schnappinger, D., Hillen, W., Saenger, W., &amp; Hinrichs, W. (2000). Structural basis of gene regulation
 +
    by the tetracycline inducible Tet repressor-operator system. <em>Nature Structural Biology, 7</em>(3), 215–219. <a
 +
      href="https://doi.org/10.1038/73324" target="_blank">https://doi.org/10.1038/73324</a></p>
 +
  <p>Shaner, N. C., Lambert, G. G., Chammas, A., Ni, Y., Cranfill, P. J., Baird, M. A., Sell, B. R., Allen, J. R., Day,
 +
    R. N., Israelsson, M., Davidson, M. W., &amp; Wang, J. (2013). A bright monomeric green fluorescent protein derived
 +
    from <em>Branchiostoma lanceolatum</em>. <em>Nature Methods, 10</em>(5), 407–409. <a
 +
      href="https://doi.org/10.1038/nmeth.2413" target="_blank">https://doi.org/10.1038/nmeth.2413</a></p>
 +
  <p>Wu, P. G., &amp; Brand, L. (1994). Resonance Energy Transfer: Methods and Applications. <em>Analytical
 +
      Biochemistry, 218</em>(1), 1-13. <a href="https://doi.org/10.1006/abio.1994.1134"
 +
      target="_blank">https://doi.org/10.1006/abio.1994.1134</a></p>
 +
</section>
  
  <p>Bindels, D. S., Haarbosch, L., van Weeren, L., Postma, M., Wiese, K. E., Mastop, M., Aumonier, S., Gotthard, G., Royant, A., Hink, M. A., & Gadella, T. W. J. (2017). mScarlet: A bright monomeric red fluorescent protein for cellular imaging. <em>Nature Methods, 14</em>(1), 53–56. <a href="https://doi.org/10.1038/nmeth.4074" target="_blank">https://doi.org/10.1038/nmeth.4074</a></p>
 
 
 
  <p>Hochreiter, B., Kunze, M., Moser, B., & Schmid, J. A. (2019). Advanced FRET normalization allows quantitative analysis of protein interactions including stoichiometries and relative affinities in living cells. <em>Scientific Reports, 9</em>(1), 8233. <a href="https://doi.org/10.1038/s41598-019-44650-0" target="_blank">https://doi.org/10.1038/s41598-019-44650-0</a></p>
 
 
 
  <p>Kisker, C., Hinrichs, W., Tovar, K., Hillen, W., & Saenger, W. (1995). The Complex Formed Between Tet Repressor and Tetracycline-Mg<sup>2+</sup> Reveals Mechanism of Antibiotic Resistance. <em>Journal of Molecular Biology, 247</em>(2), 260–280. <a href="https://doi.org/10.1006/jmbi.1994.0138" target="_blank">https://doi.org/10.1006/jmbi.1994.0138</a></p>
 
 
 
  <p>McCullock, T. W., MacLean, D. M., & Kammermeier, P. J. (2020). Comparing the performance of mScarlet-I, mRuby3, and mCherry as FRET acceptors for mNeonGreen. <em>PLOS ONE, 15</em>(2), e0219886. <a href="https://doi.org/10.1371/journal.pone.0219886" target="_blank">https://doi.org/10.1371/journal.pone.0219886</a></p>
 
 
 
  <p>Okamoto, K., & Sako, Y. (2017). Recent advances in FRET for the study of protein interactions and dynamics. <em>Current Opinion in Structural Biology, 46</em>, 16–23. <a href="https://doi.org/10.1016/j.sbi.2017.03.010" target="_blank">https://doi.org/10.1016/j.sbi.2017.03.010</a></p>
 
 
 
  <p>Orth, P., Schnappinger, D., Hillen, W., Saenger, W., & Hinrichs, W. (2000). Structural basis of gene regulation by the tetracycline inducible Tet repressor-operator system. <em>Nature Structural Biology, 7</em>(3), 215–219. <a href="https://doi.org/10.1038/73324" target="_blank">https://doi.org/10.1038/73324</a></p>
 
 
 
  <p>Shaner, N. C., Lambert, G. G., Chammas, A., Ni, Y., Cranfill, P. J., Baird, M. A., Sell, B. R., Allen, J. R., Day, R. N., Israelsson, M., Davidson, M. W., & Wang, J. (2013). A bright monomeric green fluorescent protein derived from <em>Branchiostoma lanceolatum</em>. <em>Nature Methods, 10</em>(5), 407–409. <a href="https://doi.org/10.1038/nmeth.2413" target="_blank">https://doi.org/10.1038/nmeth.2413</a></p>
 
 
 
  <p>Wu, P. G., & Brand, L. (1994). Resonance Energy Transfer: Methods and Applications. <em>Analytical Biochemistry, 218</em>(1), 1-13. <a href="https://doi.org/10.1006/abio.1994.1134" target="_blank">https://doi.org/10.1006/abio.1994.1134</a></p>
 
 
 
</section>
 
</body>
 
 
</html>
 
</html>

Latest revision as of 13:19, 2 October 2024

BBa_K5237017

FRET-Acceptor: TetR-mScarlet-I

This composite part is a fusion protein of TetR and the red fluorescent protein mScarlet-I. It was used as a FRET acceptor in combination with mNeonGreen-Oct1 as the donor. This part was used to measure the proximity of two DNA strands by FRET fluorescence measurements.

 



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
    INCOMPATIBLE WITH RFC[12]
    Illegal NotI site found at 1149
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 623
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 466

2. Usage and Biology

2.1 TetR DNA Binding Protein

The tetracycline repressor protein (tetR) is naturally present in gram-negative bacteria and is involved in the resistance mechanism against tetracycline (and derivatives). It does so by tightly controlling the gene expression of tetA, which encodes an efflux pump responsible for removing tetracycline from the cell. TetR binds selectively to two plaindromic recognition sequences (tetO>1,2) with high affinity. For DNA binding to occur tetR adopts a homodimeric structure and binds with two α-helix-turn- α-helix motifs (HTH) to two tandemly oriented tetO sequences. In the presence of tetracycline or its analogs, tetR undergoes a conformational change, which prevents it from binding to DNA, therby allowing gene expression(Orth et al. 2000; Kisker et al. 1995).
Due to its robust and highly regulatable DNA-binding properties, tetR has become a widely adopted tool in synthetic biology. Its ease of modification and ability to function in both prokaryotic and eukaryotic systems have made it an essential element in the development of gene regulation systems (Berens & Hillen, 2004).
In our project, tetR was integrated into the design of a modular DNA-stapling system because of its well-characterized behavior, ensuring reliable DNA interactions.

2.2 mScarlet-I

mScarlet-I is a rapidly-maturing monomeric red fluorescent protein designed by Bindels et al. in 2017. It is a derivative of the original mScarlet, with a single amino acid substitution (T74I) that enhances its maturation speed and fluorescence properties (Bindels et al., 201). mScarlet-I absorbs at 569 nm and emits at 594 nm.

mScarlet-I stands out among red fluorescent proteins for its high quantum yield (0.54), long fluorescence lifetime (3.1 ns), and rapid maturation time of approximately 36 minutes. These features ensure strong fluorescence intensity and make it an ideal candidate for Förster Resonance Energy Transfer assays (McCullock et al. 2020). When paired with mNeonGreen, mScarlet-I

2.3 Förster Resonance Energy Transfer (FRET)

Förster Resonance Energy Transfer (FRET) is a distance-dependent physical process where energy is transferred non-radiatively from an excited donor fluorophore to an acceptor fluorophore via dipole-dipole coupling. The efficiency of energy transfer is highly sensitive to the distance between the donor and acceptor, typically in the range of 1-10 nm, making FRET ideal for studying molecular proximity (Hochreiter et al., 2019). This proximity sensitivity is governed by the Förster radius (R₀), which is the distance at which 50% energy transfer occurs. Factors affecting FRET efficiency include the overlap of the donor's emission spectrum with the acceptor's absorption spectrum, the quantum yield of the donor, and the relative orientation of the fluorophores (Wu & Brand, 1994). These characteristics allow FRET to detect interactions such as protein-DNA binding or DNA proximity in real time. For our assay, we selected mNeonGreen and mScarlet-I as donor and acceptor, respectively, due to their strong fluorescence, spectral overlap, and minimal photobleaching, ensuring high FRET efficiency in our system (Bindels et al., 2017; Shaner et al., 2013). FRET's sensitivity to small changes in distance makes it especially powerful for analyzing molecular interactions in living cells (Okamoto & Sako, 2017).

Figure 2: Overview of Excitation and Emission Spectrum of mNeonGreen and m-Scarlet and it's Properties as a FRET Pair

3. Assembly and Part Evolution

As part of our engineering efforts an engineered tetR-mScarlet was tested, that binds DNA as a monomer. This scTetR is a fusion of two tetR proteins with a flexible linker which was described to maintain the same DNA-binding affinity and specificity as wild-type tetR (Kaminoka et al. 2006; Krueger et al.2003)

Our experiments with the scTetR-mScarlet-I fusion protein showed no significant expression. Since the scTetR-mScarlet-I fusion protein was expressed as part of a polycistronic transcript, the results should be taken with caution. Nonetheless we belive that qualitative assesements can still be drawn, because the same construct with tetR-mScarlet-I showed a good expression (Figure 2, 3).

pic
Figure 3: Fluoresence Measurement of scTetR with Mutant T7 Polymerase. Fluorescence intensity of mScarlet-I (ex. 560 nm, em. 600 nm) was measured 18 h after induction with IPTG and normalized to cell count (OD600). Positive control consists of a tetR-mNeonGreen fusion protein.

4. Results

The FRET assay was developed using a two-plasmid system in bacterial cells. The expression plasmid contains a tetR binding site (BBa_K5237019) and expresses three key proteins under the control of a single T7 promoter in a polycistronic operon: (1) tetR-Oct1, our simple staple fusion protein that acts as a bivalent DNA-binding protein, tethering two plasmids via tetR and Oct1 binding sites; (2) Oct1-mNeonGreen, serving as the FRET donor; and (3) tetR-mScarlet-I, the FRET acceptor. This ensures all three proteins are co-expressed in similar stoichiometry. The folding plasmid contains an Oct1 binding site (BBa_K5237018) for the staple and FRET donor binding.

When tetR-Oct1 binds its respective sites on both plasmids, it brings mNeonGreen and mScarlet-I into close proximity, facilitating FRET between the two fluorophores. Successful stapling of the plasmids results in increased energy transfer from mNeonGreen to mScarlet-I, which can be detected by exciting mNeonGreen and measuring enhanced emission from mScarlet-I. A positive control, consisting of a direct fusion of mNeonGreen and mScarlet-I, ensures maximal FRET efficiency and serves as a benchmark for the assay.

Figure 4: Overview FRET Assay

Fluorescence intensity, normalized to cell count, of mNeonGreen and mScarlet-I was measured 18 h after induction with varying IPTG concentration (Fig. 5). An increasing expression strength is visible for decreasing IPTG concentrations. Fluorescence intensity of the positive control was significantly stronger compared to the negative control and staple. The negative control and staple, which both have the same expression plasmid construct, had similar fluorescence intensity for mNeonGreen and mScarlet-I down to approximately 0.05 mM. Lower concentrations resulted in strong discrepancies. To ensure comparability between the negative control and staple, further fluorescence intensity measurements were conducted after induction with 0.05 mM IPTG. Fluorescence measurement of the donor and acceptor showed similar intensities, with only a small significant difference for mNeonGreen. A large difference could be measured between the staple and negative control, indicating proximity induced FRET. (Fig. 5)

pic
Figure 5: Fluorescence of mNeonGreen, mScarlet-I and FRET. Fluorescence intensity of mNeonGreen (ex. 490 nm, em. 530 nm), mScarlet-I (ex. 560 nm, em. 600 nm), and FRET (ex. 490 nm, em. 600 nm) was measured 18 h after induction with IPTG and normalized to cell count (OD600). A), B) Fluorescence intensity of mNeonGreen and mScarlet-I after induction with different IPTG concentrations. C) Fluorescence intensity of FRET pair induced with 0.025 mM IPTG. Statistical significance was tested for with Ordinary two-way ANOVA with Šidák's multiple comparison test, with a single pooled variance. *p < 0.05, ****p < 0.001. Only significant results, within groups are shown. Data is depcited as mean ± SD.

5. Conclusion

Our FRET assay successfully demonstrated the proximity of two DNA strands in living cells using mNeonGreen and mScarlet-I as a donor-acceptor pair. The assay was optimized for maximal FRET efficiency and validated with a positive control. The results showed a significant difference in fluorescence intensity between the staple and negative control, indicating successful DNA stapling and FRET. This assay provides a powerful tool to engineer and test out novel Staples. Future work will focus on further optimizing the assay in mammalian cells and quantifying interactions.

6. References

Berens, C., & Hillen, W. (2004). Gene Regulation By Tetracyclines. In J. K. Setlow (Ed.), Genetic Engineering: Principles and Methods (pp. 255–277). Springer US. https://doi.org/10.1007/978-0-306-48573-2_13

Bindels, D. S., Haarbosch, L., van Weeren, L., Postma, M., Wiese, K. E., Mastop, M., Aumonier, S., Gotthard, G., Royant, A., Hink, M. A., & Gadella, T. W. J. (2017). mScarlet: A bright monomeric red fluorescent protein for cellular imaging. Nature Methods, 14(1), 53–56. https://doi.org/10.1038/nmeth.4074

Hochreiter, B., Kunze, M., Moser, B., & Schmid, J. A. (2019). Advanced FRET normalization allows quantitative analysis of protein interactions including stoichiometries and relative affinities in living cells. Scientific Reports, 9(1), 8233. https://doi.org/10.1038/s41598-019-44650-0

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