Difference between revisions of "Part:BBa K5237020"

 
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<partinfo>BBa_K5237020</partinfo>
 
<partinfo>BBa_K5237020</partinfo>
 
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<body>
 
<body>
    <!-- Part summary -->
+
  <!-- Part summary -->
    <section id="1">
+
  <section>
      <h1>Cathepsin B-Cleavable <i>Trans</i>-Activator (NLS-Gal4-GFLG-VP64)</h1>
+
    <h1>Cathepsin B-Cleavable <i>Trans</i>-Activator: NLS-Gal4-GFLG-VP64</h1>
      <p>This composite part encodes a cathepsin B-cleavable <i>trans</i>-activator fusion protein. It is composed of a SV40 nuclear localization sequence (<a href="https://parts.igem.org/Part:BBa_K2549054" target="_blank">BBa_K2549054</a>), the DNA-binding domain of Gal4 (<a href="https://parts.igem.org/Part:BBa_K4585001" target="_blank">BBa_K4585001</a>), a GFLG linker (<a href="https://parts.igem.org/Part:BBa_K5237010" target="_blank">BBa_K5237010</a>), and the VP64 <i>trans</i>-activator domain (<a href="https://parts.igem.org/Part:BBa_K4586021" target="_blank">BBa_K4586021</a>). We used this part to design a fluorescence-based readout assay in HEK293T cells to investigate cathepsin B cleavage of different peptide linkers. Through this assay, we were able to successfully show that the GFLG linker was specifically cleaved in the presence of cathepsin B, while other linkers did not show this effect. Together, these findings enable the functionalization of our PICasSO system for a wide range of therapeutic and synthetic biology applications.</p>
+
    <p>This composite part encodes a cathepsin B-cleavable <i>trans</i>-activator fusion protein. It is composed of an
      <p>&nbsp;</p>
+
      SV40 nuclear localization sequence (<a href="https://parts.igem.org/Part:BBa_K2549054"
    </section>
+
        target="_blank">BBa_K2549054</a>), the DNA-binding domain of Gal4 (<a
   <div id="toc" class="toc">
+
        href="https://parts.igem.org/Part:BBa_K4585001" target="_blank">BBa_K4585001</a>), a GFLG linker (<a
 +
        href="https://parts.igem.org/Part:BBa_K5237010" target="_blank">BBa_K5237010</a>), and the VP64
 +
      <i>trans</i>-activator domain (<a href="https://parts.igem.org/Part:BBa_K4586021"
 +
        target="_blank">BBa_K4586021</a>). We used this part to design a fluorescence-based readout assay in HEK293T
 +
      cells to investigate cathepsin B cleavage of different peptide linkers. Through this assay, we were able to
 +
      successfully show that the GFLG linker was specifically cleaved in the presence of cathepsin B, while other
 +
      linkers did not show this effect. Together, these findings enable the functionalization of our PICasSO system for
 +
      a wide range of therapeutic and synthetic biology applications.
 +
    </p>
 +
  </section>
 +
  <p> </p>
 +
   <div class="toc" id="toc">
 
     <div id="toctitle">
 
     <div id="toctitle">
 
       <h1>Contents</h1>
 
       <h1>Contents</h1>
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     <ul>
 
     <ul>
 
       <li class="toclevel-1 tocsection-1"><a href="#1"><span class="tocnumber">1</span> <span class="toctext">Sequence
 
       <li class="toclevel-1 tocsection-1"><a href="#1"><span class="tocnumber">1</span> <span class="toctext">Sequence
             overview</span></a>
+
             Overview</span></a>
 
       </li>
 
       </li>
       <li class="toclevel-1 tocsection-2"><a href="#2"><span class="tocnumber">2</span> <span class="toctext">Usage and
+
       <li class="toclevel-1 tocsection-2"><a href="#2"><span class="tocnumber">2</span> <span class="toctext">Usage
 +
            and
 
             Biology</span></a>
 
             Biology</span></a>
 
       </li>
 
       </li>
 
       <li class="toclevel-1 tocsetction-3"><a href="#3"><span class="tocnumber">3</span> <span class="toctext">Assembly
 
       <li class="toclevel-1 tocsetction-3"><a href="#3"><span class="tocnumber">3</span> <span class="toctext">Assembly
             and part evolution</span></a>
+
             and Part Evolution</span></a>
 
       </li>
 
       </li>
 
       <li class="toclevel-1 tocsection-5"><a href="#4"><span class="tocnumber">4</span> <span
 
       <li class="toclevel-1 tocsection-5"><a href="#4"><span class="tocnumber">4</span> <span
 
             class="toctext">Results</span></a>
 
             class="toctext">Results</span></a>
 +
        <ul>
 +
          <li class="toclevel-2 tocsection-6"><a href="#4.1"><span class="tocnumber">4.1</span> Mature Cathepsin B Is
 +
              Expressed in HEK293T Cells</a>
 +
          </li>
 +
          <li class="toclevel-2 tocsection-7"><a href="#4.2"><span class="tocnumber">4.2</span> mCherry and eGFP Can
 +
              be Used as a Reporter System to Measure Cleavage Efficiency</a></li>
 +
          <li class="toclevel-2 tocsection-8"><a href="#4.3"><span class="tocnumber">4.3</span> The Peptide Linker GFLG
 +
              Is Cleaved by Cathepsin B <i>in Vivo</i></a>
 +
          </li>
 +
        </ul>
 
       </li>
 
       </li>
       <li class="toclevel-1 tocsection-8"><a href="#5"><span class="tocnumber">5</span> <span
+
       <li class="toclevel-1 tocsection-9"><a href="#5"><span class="tocnumber">5</span> <span
 +
            class="toctext">Conclusion</span></a>
 +
      <li class="toclevel-1 tocsection-10"><a href="#6"><span class="tocnumber">6</span> <span
 
             class="toctext">References</span></a>
 
             class="toctext">References</span></a>
 +
      </li>
 
       </li>
 
       </li>
 
     </ul>
 
     </ul>
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   <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>
    <!-- Picture explaining parts collection -->
+
     <p>At its heart, the PICasSO part collection consists of three categories. <br /><b>(i)</b> Our <b>DNA-binding
    <!-- below text not finished formatting-->
+
        proteins</b>
     <p>At its heart, the PICasSO parts collection consists of three categories. (i) Our <b>DNA-binding 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. (ii) As <b>functional
+
       new Cas staples in the future. We also include our Simple staples comprised of particularly small, simple
        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 with our
+
      <b>(ii)</b> As <b>functional elements</b>, we list additional parts that enhance and expand the
       interkingdom conjugation system.
+
      functionality of our Cas and
    </p>
+
      Basic staples. These
    <p>
+
       consist of staples dependent on
      (iii) As the final component of our collection, we provide parts that support the use of our <b>custom readout
+
       cleavable peptide linkers targeted by cancer-specific proteases or inteins that allow condition-specific,
         systems</b>. These include components of our established FRET-based proximity assay system, enabling users to
+
      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
+
      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>Half-Staple: 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>Half-Staple: 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>Half-Staple: 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>Half-Staple: 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-Staple: 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>Half-Staple: 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>Half-Staple: 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 201: Line 266:
 
           <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 240: Line 320:
 
           <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 246: Line 327:
 
           <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 252: Line 333:
 
           <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 <i>Trans</i>-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><i>Trans</i>-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 - 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>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">
     <h1>1. Sequence overview</h1>
+
     <h1>1. Sequence Overview</h1>
 
   </section>
 
   </section>
 
</body>
 
</body>
  
 
</html>
 
</html>
 
 
<!--################################-->
 
<!--################################-->
 
<!--The followig lines need to be adjusted for each part (exchange hashes for part number)-->
 
<!--The followig lines need to be adjusted for each part (exchange hashes for part number)-->
<span class='h3bb'>Sequence and Features</span>
+
<span class="h3bb">Sequence and Features</span>
 
<partinfo>BBa_K5237020 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K5237020 SequenceAndFeatures</partinfo>
 
<!--################################-->
 
<!--################################-->
 +
<html>
  
<html> 
 
 
 
 
 
<body>
 
<body>
 
   <section id="2">
 
   <section id="2">
 
     <h1>2. Usage and Biology</h1>
 
     <h1>2. Usage and Biology</h1>
     <p>Proteolytic cleavage of pro-biologics allows for the precise temporal and spatial regulation of biopharmaceutical activity in therapeutic applications (Bleuez <i>et al.</i>, 2022). Cathepsin B is a cysteine protease that is significantly overexpressed in different cancer types, including breast and colorectal cancer (Ruan <i>et al.</i>, 2015).<br>
+
     <p>Cathepsin B is a cysteine protease that is significantly overexpressed in various cancer types, including breast
We used a NLS-Gal4-Linker-VP64 fusion protein to investigate cathepsin B cleavage of different peptide linkers. These linkers can be used for the cleavage-induced oligomerization of Cas proteins through protein <i>trans</i>-splicing of caged intein fragments expanding the functionality of our PICasSO toolbox. We identified GFLG out of five peptide linkers previously reported to be cleaved by cathepsin B (GFLG, FFRG, FRRL, VA, FK) (Jin <i>et al.</i>, 2022; Shim <i>et al.</i>, 2022; Wang <i>et al.</i>, 2024) through a fluorescence readout system in HEK293T cells.</p>
+
      and colorectal cancer (Ruan <i>et al.</i>, 2015). Proteolytic cleavage of pro-biologics via cathepsin B activity
 
+
      allows for precise temporal and spatial regulation of biopharmaceutical activity in therapeutic strategies (Bleuez
 +
      <i>et al.</i>, 2022).<br />
 +
      In this context, we introduce the cathepsin B-cleavable peptide linker GFLG as a functional addition to our
 +
      PICasSO toolbox, enabling a wide range of therapeutic and synthetic biology applications. GFLG has been shown to
 +
      be cleaved by cathepsin B (Wang <i>et al.</i>, 2024), with cleavage occurring either between phenylalanine and
 +
      leucine or after the second glycine of the linker (Rejmanová <i>et al.</i>, 1983; <b>Fig. 2</b>).<br />
 +
      The construct also includes Gal4, a transcription factor that binds upstream activation sites (UAS) to drive gene
 +
      expression, and VP64, a strong transcriptional activator that enhances this expression (Muench <i>et al.</i>,
 +
      2023). Additionally, it incorporates the SV40 nuclear localization signal (NLS), a short peptide derived from the
 +
      <i>simian virus 40</i> (SV40) large T-antigen, which facilitates the transport of the fusion protein into the
 +
      nucleus, where it can bind to a UAS (Yoneda, 1997).<br />
 +
      Through a fluorescence readout assay in HEK293T cells, we identified GFLG as the most effective among five peptide
 +
      linkers known to be cleaved by cathepsin B (Jin <i>et al.</i>, 2022; Shim <i>et al.</i>, 2022; Wang <i>et al.</i>,
 +
      2024). This linker facilitates cleavage-induced oligomerization of Cas proteins via protein <i>trans</i>-splicing
 +
      of
 +
      caged intein fragments (<a href="https://parts.igem.org/Part:BBa_K5237012" target="_blank">BBa_K5237012</a>, <a
 +
        href="https://parts.igem.org/Part:BBa_K5237013" target="_blank">BBa_K5237013</a>), further enhancing the
 +
      capabilities of our system.<br />
 +
    </p>
 
   </section>
 
   </section>
 
   <section id="3">
 
   <section id="3">
     <h1>3. Assembly and part evolution</h1>
+
     <h1>3. Assembly and Part Evolution</h1>
     <p>We designed a fluorescence readout assay in HEK293T cells based on expression of mCherry induced by the transactivator VP64. VP64 was conjugated to the DNA-binding domain (DBD) of Gal4 through the GFLG linker. We purchased the nucleotide sequence encoding the GFLG linker as an oligo. After annealing the oligo, we cloned it into a mammalian expression vector between the open reading frames for Gal4-DBD and VP64 via Golden Gate assembly. Binding of Gal4-DBD upstream of a gene encoding the fluorescence protein mCherry induces overexpression of mCherry by VP64. Consequently, separation of Gal4-DBD and VP64 by cathepsin B cleavage of the GFLG linker reduces mCherry expression (see <b>Fig. 2</b>).</p>
+
     <p>We designed a fluorescence readout assay in HEK293T cells based on expression of mCherry induced by the
 
+
      transactivator VP64. VP64 was conjugated to the DNA-binding domain (DBD) of Gal4 through the GFLG linker. We
 +
      purchased the nucleotide sequence encoding the GFLG linker as an oligo. After annealing the oligo, we cloned it
 +
      into a mammalian expression vector between the open reading frames for Gal4-DBD and VP64 via Golden Gate assembly.
 +
      Binding of Gal4-DBD upstream of a gene encoding the fluorescence protein mCherry induces overexpression of mCherry
 +
      by VP64. Consequently, separation of Gal4-DBD and VP64 by cathepsin B cleavage of the GFLG linker reduces mCherry
 +
      expression (Fig. 2).</p>
 
     <div class="thumb">
 
     <div class="thumb">
       <div class="thumbinner" style="width:450px;"><a href="placeholder"
+
       <div class="thumbinner" style="width:450px;"><img alt="Cathepsin B Fluorescence Readout Assay" class="thumbimage"
          class="image"><img alt="Cathepsin B Fluorescence Readout Assay" src="https://static.igem.wiki/teams/5237/wetlab-results/catb-gal4-vp64-mechanism.svg" width="450"
+
          src="https://static.igem.wiki/teams/5237/wetlab-results/catb-gal4-vp64-mechanism.svg" width="450" />
            class="thumbimage"></a>
+
 
         <div class="thumbcaption">
 
         <div class="thumbcaption">
           <i><b>Figure 2: Schematic Illustration of the Cathepsin B Fluorescence Readout Assay.</b></i> The DNA-binding domain (DBD) of Gal4 is conjugated to the transactivator domain VP64 via a cathepsin B-cleavable peptide linker. Binding of the Gal4-DBD to the upstream activating sequence (UAS) in proximity to the mCherry gene induces mCherry overexpression via VP64. Cathepsin B cleavage of the linker separates Gal4-DBD and VP64 and consequently reduces mCherry expression.
+
           <i><b>Figure 2: Schematic Illustration of the Cathepsin B Fluorescence Readout Assay.</b> The DNA-binding
 +
            domain (DBD) of Gal4 is conjugated to the transactivator domain VP64 via a cathepsin B-cleavable peptide
 +
            linker. Binding of the Gal4-DBD to the upstream activating sequence (UAS) in proximity to the mCherry gene
 +
            induces mCherry overexpression via VP64. Cathepsin B cleavage of the linker separates Gal4-DBD and VP64 and
 +
            consequently reduces mCherry expression.</i>
 
         </div>
 
         </div>
 
       </div>
 
       </div>
 
     </div>
 
     </div>
 
+
    <p>We transfected our genetic constructs into HEK293T cells. The negative control was not transfected with the
<p>We transfected our genetic constructs into HEK293T cells. The negative control was not transfected with the plasmid encoding cathepsin B. We investigated two different test conditions, in which we either transfected 30&nbsp;ng or 60&nbsp;ng of the plasmid encoding cathepsin B. The fluorescence intensity of mCherry was measured 48&nbsp;hours after transfection. Our initial tests did not result in the unambiguous identification of a cathepsin B-cleavable peptide linker (see <b>Fig. 3</b>). For all linkers, we did not observe a large decrease in fluorescence intensity between the negative control and test conditions. In some conditions, the fluorescence intensity even increased between the negative control and test conditions.</p>
+
      plasmid encoding cathepsin B. We investigated two different test conditions, in which we either transfected 30 ng
 
+
      or 60 ng of the plasmid encoding cathepsin B. The fluorescence intensity of mCherry was measured 48 hours after
 +
      transfection. Our initial tests did not result in the unambiguous identification of a cathepsin B-cleavable
 +
      peptide linker (Fig. 3). For all linkers, we did not observe a large decrease in fluorescence intensity
 +
      between the negative control and test conditions. In some conditions, the fluorescence intensity even increased
 +
      between the negative control and test conditions.</p>
 
     <div class="thumb">
 
     <div class="thumb">
       <div class="thumbinner" style="width:450px;"><a href="placeholder"
+
       <div class="thumbinner" style="width:450px;"><img alt="Fluorescence Readout" class="thumbimage"
          class="image"><img alt="Fluorescence Readout" src="https://static.igem.wiki/teams/5237/wetlab-results/catb-results/catb-fluorescent-readout-no-dox-w.svg" width="450"
+
          src="https://static.igem.wiki/teams/5237/wetlab-results/catb-results/catb-fluorescent-readout-no-dox-w.svg"
            class="thumbimage"></a>
+
          width="450" />
 
         <div class="thumbcaption">
 
         <div class="thumbcaption">
           <i><b>Figure 3: Fluorescence Readout After 48&nbsp;hours for Five Different Peptide Linkers and Three Different Conditions.</b></i> The fluorescence intensity for mCherry was measured for five different linkers. The negative control was not transfected with the plasmid encoding cathepsin B. The fluorescence intensity of the negative control was set to one. Two different test conditions were investigated, in which either 30&nbsp;ng or 60&nbsp;ng of the plasmid encoding cathepsin B were transfected.
+
           <i><b>Figure 3: Fluorescence Readout After 48 Hours for Five Different Peptide Linkers and Three Different
 +
              Conditions.</b> The fluorescence intensity for mCherry was measured for five different linkers. The
 +
            negative control was not transfected with the plasmid encoding cathepsin B. The fluorescence intensity of
 +
            the
 +
            negative control was set to one. Two different test conditions were investigated, in which either 30 ng or
 +
            60 ng of the plasmid encoding cathepsin B were transfected.</i>
 
         </div>
 
         </div>
 
       </div>
 
       </div>
 
     </div>
 
     </div>
 
+
    <p>Since cathepsin B is a lysosomal protease that is normally only active in the lysosome and the extracellular
<p>Since cathepsin B is a lysosomal protease that is normally only active in the lysosome and the extracellular environment but not in the cytosol, we decided to change the native amino acid sequence of cathepsin B. Upon further investigation of the lysosomal maturation process of cathepsin B, we chose to express a truncated and mutated version of cathepsin B. We truncated the native cathepsin B amino acid sequence N-terminally by the first twenty amino acids. This N-terminally truncated version of cathepsin B lacks a signal peptide that normally facilitates translation of cathepsin B in the rough endoplasmic reticulum. Additionally, truncation of the first twenty amino acids of cathepsin B had previously been observed to maintain catalytic activity even in the absence of lysosomal proteases like pepsin (Müntener <i>et al.</i>, 2005). Additionally, we introduced three point mutations (D22A, H110A, R116A) in the amino acid sequence of cathepsin B. This has been shown to increase the activity of cathepsin B at higher pH values by disrupting the interactions of an occluding loop with the substrate binding pocket of cathepsin B (Nägler <i>et al.</i>, 1997).<br>
+
      environment but not in the cytosol, we decided to change the native amino acid sequence of cathepsin B. Upon
We performed the same fluorescence readout assay in HEK293T cells that we also used for wild-type cathepsin B. The fluorescence intensity of mCherry was measured 48&nbsp;hours after transfection. However, we observed no decrease in fluorescence between our negative control and test conditions, indicating that Gal4-DBD-Linker-VP64 was not cleaved (see <b>Fig. 4</b>). Additionally, we performed a western blot, where the bands for the truncated and mutated version of cathepsin B were barely visible or absent altogether, indicating lower protein expression compared to the wild type (see <b>Fig. 8</b>). Another key insight from this experiment was that this version of cathepsin B was not activated by cleavage in the cell, as no additional protein bands were observed.</p>
+
      further investigation of the lysosomal maturation process of cathepsin B, we chose to express a truncated and
 
+
      mutated version of cathepsin B. We truncated the native cathepsin B amino acid sequence N-terminally by the first
 +
      twenty amino acids. This N-terminally truncated version of cathepsin B lacks a signal peptide that normally
 +
      facilitates translation of cathepsin B in the rough endoplasmic reticulum. Additionally, truncation of the first
 +
      twenty amino acids of cathepsin B had previously been observed to maintain catalytic activity even in the absence
 +
      of lysosomal proteases like pepsin (Müntener <i>et al.</i>, 2005). Additionally, we introduced three point
 +
      mutations (D22A, H110A, R116A) in the amino acid sequence of cathepsin B. This has been shown to increase the
 +
      activity of cathepsin B at higher pH values by disrupting the interactions of an occluding loop with the substrate
 +
      binding pocket of cathepsin B (Nägler <i>et al.</i>, 1997).<br />
 +
      We performed the same fluorescence readout assay in HEK293T cells that we also used for wild-type cathepsin B. The
 +
      fluorescence intensity of mCherry was measured 48 hours after transfection. However, we observed no decrease in
 +
      fluorescence between our negative control and test conditions, indicating that Gal4-DBD-Linker-VP64 was not
 +
      cleaved (Fig. 4>). Additionally, we performed a western blot, where the bands for the truncated and
 +
      mutated version of cathepsin B were barely visible or absent altogether, indicating lower protein expression
 +
      compared to the wild type (Fig. 5). Another key insight from this experiment was that this version of
 +
      cathepsin B was not activated by cleavage in the cell, as no additional protein bands were observed.</p>
 
     <div class="thumb">
 
     <div class="thumb">
       <div class="thumbinner" style="width:450px;"><a href="placeholder"
+
       <div class="thumbinner" style="width:450px;"><img alt="Fluorescence Readout" class="thumbimage"
          class="image"><img alt="Fluorescence Readout" src="https://static.igem.wiki/teams/5237/wetlab-results/catb-results/catb-fluorescent-readout-mutated-and-truncated-w.svg" width="450"
+
          src="https://static.igem.wiki/teams/5237/wetlab-results/catb-results/catb-fluorescent-readout-mutated-and-truncated-w.svg"
            class="thumbimage"></a>
+
          width="450" />
 
         <div class="thumbcaption">
 
         <div class="thumbcaption">
           <i><b>Figure 4: Fluorescence Readout for the Truncated and Mutated Version of Cathepsin B.</b></i>
+
           <i><b>Figure 4: Fluorescence Readout for the Truncated and Mutated Version of Cathepsin B.</b>
 +
            Fluorescence intensity for mCherry was measured across five different linkers. The negative control, which
 +
            was
 +
            not transfected with the plasmid encoding cathepsin B, was assigned a fluorescence intensity value of one.
 +
            Two
 +
            test conditions were explored, where either 30 ng or 60 ng of the plasmid encoding cathepsin B was
 +
            transfected.</i>
 
         </div>
 
         </div>
 
       </div>
 
       </div>
 
     </div>
 
     </div>
 
+
    <p>Since our truncated and mutated version of cathepsin B did not seem to be active in the cytosol, we decided to go
<p>Since our truncated and mutated version of cathepsin B did not seem to be active in the cytosol, we decided to go back to wild-type cathepsin B. Therefore, we focused on improving the activity of wild-type cathepsin B in the cytosol. After consulting the literature, we decided to treat cells with the cytostaticum doxorubicin to induce lysosomal escape of cathepsin B, as had been previously reported (Bien <i>et al.</i>, 2004).</p>
+
      back to wild-type cathepsin B. Therefore, we focused on improving the activity of wild-type cathepsin B in the
 
+
      cytosol. After consulting the literature, we decided to treat cells with the cytostatic agent doxorubicin to
 +
      induce
 +
      lysosomal escape of cathepsin B, as had been previously reported (Bien <i>et al.</i>, 2004).</p>
 
   </section>
 
   </section>
 
   <section id="4">
 
   <section id="4">
 
     <h1>4. Results</h1>
 
     <h1>4. Results</h1>
     <h3>The Peptide Linker GFLG Is Cleaved by Cathepsin B <i>in Vivo</i></h3>
+
     <section id="4.1">
<p>We performed a fluorescence readout assay in HEK293T cells to investigate cathepsin B cleavage of different peptide linkers. 24&nbsp;hours after transfection, we added doxorubicin in a final concentration of 500 nM to the cell supernatant. <b>Figure 5</b> shows the fluorescence intensity of mCherry for five different peptide linkers (GFLG, FFRG, FRRL, VA, FK). The negative control was not transfected with the plasmid encoding cathepsin B. We investigated two different test conditions, in which we either transfected 30&nbsp;ng or 60&nbsp;ng of the plasmid encoding cathepsin B. The fluorescence intensity of mCherry was normalized by the measured fluorescence intensity of eGFP in each condition. Additionally, the values for 30&nbsp;ng and 60&nbsp;ng cathepsin B were normalized against the corresponding negative controls. One data point for the VA linker, transfected with 60&nbsp;ng of the plasmid encoding cathepsin B, was excluded due to severe deviation from the other values. We conducted a two-way analysis of variance (ANOVA) to assess the significance of the observed differences between the negative control and the test conditions for each linker. As the negative control did not contain the plasmid encoding cathepsin B, we expected the measured fluorescence intensity of mCherry to be the highest in these conditions. However, this was only observed for the GFLG and FK linkers. Contrary to our expectations, the fluorescence intensity of the negative control was the lowest out of the three conditions tested for the remaining linkers. It appears that the addition of the plasmid encoding cathepsin B increases mCherry fluorescence intensity when the linker is not cleaved. However, this increase is only significant for the FFRG linker in the 60&nbsp;ng condition. For the GFLG linker, we observed significant decreases in fluorescence intensity between the negative control and both test conditions, with no difference between the 30&nbsp;ng and 60&nbsp;ng conditions. For the FK linker, no significant decreases in fluorescence intensity between the negative control and the test conditions were observed.</p>
+
      <h2>4.1 Mature Cathepsin B Is Expressed in HEK293T Cells</h2>
 
+
      <p>To achieve cathepsin B cleavage-induced Cas stapling, catalytically active cathepsin B needs to be expressed in
    <div class="thumb">
+
        the cytosol. Therefore, we investigated the expression of different cathepsin B constructs under different
      <div class="thumbinner" style="width:450px;"><a href="placeholder"
+
        conditions in HEK293T cells. In addition to wild-type (wt) cathepsin B, we also cloned a truncated and mutated
          class="image"><img alt="Fluorescence Readout" src="https://static.igem.wiki/teams/5237/wetlab-results/catb-results/catb-fluorescent-readout-final-results-w.svg" width="450"
+
        version of cathepsin B (Δ1-20, D22A, H110A, R116A) and compared protein expression of both constructs in
            class="thumbimage"></a>
+
        doxorubicin-treated and untreated conditions.<br />
        <div class="thumbcaption">
+
        <b>Figure 5</b> shows a western blot of the wild-type (wt) version of cathepsin B as well as the truncated and
          <i><b>Figure 5: Fluorescence Readout After 48&nbsp;hours for Five Different Peptide Linkers and Three Different Conditions.</b></i> The fluorescence intensity for mCherry was measured for five different linkers and normalized against a baseline eGFP fluorescence intensity. The negative control was not transfected with the plasmid encoding cathepsin B. The fluorescence intensity of the negative control was set to one. Two different test conditions were investigated, in which either 30&nbsp;ng or 60&nbsp;ng of the plasmid encoding cathepsin B were transfected. The fluorescent readout was analyzed using a two-way ANOVA. Medium: DMEM (10% FCS). P values: ns, P > 0.05; *, P &le; 0.05; **, P &le; 0.01; ***, P &le; 0.001; ****, P &le; 0.0001.
+
        mutated version of cathepsin B (Δ1-20, D22A, H110A, R116A). Cells of both cathepsin B versions were treated with
 +
        500 nM doxorubicin (dox) 24 hours post-transfection and incubated for additional 24 hours. For each condition,
 +
        three replicates were blotted. We observed no differences in protein expression levels between the dox-treated
 +
        and untreated wt versions of cathepsin B. For the truncated and mutated version of cathepsin B, however, only
 +
        the untreated samples showed the corresponding band at approximately 36 kDa expected for this version of
 +
        cathepsin B. Additionally, the bands of the truncated and mutated version appeared much weaker than the ones of
 +
        the wt, indicating poorer protein expression. The household protein β-tubulin is visible in all samples at
 +
        approximately 55 kDa. The wt cathepsin B additionally showed bands for pro-cathepsin B at approximately 42 kDa,
 +
        a mature single-chain version of cathepsin B at approximately 33 kDa and a mature double-chain version at
 +
        approximately 26 kDa.
 +
      </p>
 +
      <div class="thumb">
 +
        <div class="thumbinner" style="width:450px;"><img alt="Fluorescence Readout" class="thumbimage"
 +
            src="https://static.igem.wiki/teams/5237/wetlab-results/catb-wb-w.svg" width="450" />
 +
          <div class="thumbcaption">
 +
            <i><b>Figure 5: Western Blot of Two Versions of Cathepsin B With and Without Doxorubicin.</b> From left
 +
              to right: protein ladder, wild-type (wt) cathepsin B with (+) and without (-) doxorubicin, truncated and
 +
              mutated version of cathepsin B with (+) and without (-) doxorubicin. The household protein β-tubulin is
 +
              visible in all samples at 55 kDa. The wt cathepsin B also shows bands for pro-cathepsin B at 42 kDa,
 +
              mature
 +
              single-chain cathepsin B at 33 kDa and mature double-chain cathepsin B at 26 kDa. The band for the
 +
              truncated
 +
              and mutated version of cathepsin B can be seen in the samples without doxorubicin at 36 kDa.</i>
 +
          </div>
 
         </div>
 
         </div>
 
       </div>
 
       </div>
     </div>
+
     </section>
 
+
    <section id="4.2">
<h3>mCherry and eGFP are Both Expressed in HEK293T Cells</h3>
+
      <h2>4.2 mCherry and eGFP Can be Used as a Reporter System to Measure Cleavage Efficiency</h2>
<p><b>Figure 6</b> shows micrographs taken with a fluorescence microscope of three different conditions: the null control, the negative control and the test sample. <b>Figure 7</b> shows the corresponding graphs quantifying the fluorescence intensity in the different conditions. All samples were transfected with plasmids encoding eGFP and mCherry. The null control and the negative control were not transfected with the plasmid encoding cathepsin B. The null control was also not transfected with any of the plasmids encoding Gal4-Linker-VP64 constructs. The test sample was transfected with 30&nbsp;ng of the plasmid encoding cathepsin B and with the plasmid encoding Gal4-GFLG-VP64. As expected, the null control showed no detectable mCherry signal, since no plasmid encoding a Gal4-V64 construct was transfected. Consequently, mCherry overexpression via VP64 could not be induced. However, we observed a high fluorescence intensity for eGFP, indicating that the transfection was successful. The negative control showed strong signals of both mCherry and eGFP. Therefore, it can be assumed that the transfection was successful and that our mCherry readout system is functional. Interestingly, there are some cells which either seem to only express mCherry or eGFP and some cells that show no fluorescence signal. The test sample showed less eGFP and mCherry fluorescence compared to the negative control. We expected to observe reduced fluorescence intensity of mCherry, as the transfected cells would express cathepsin B, which cleaves the linker, thereby decreasing mCherry expression.</p>
+
      <p>In this experiment, mCherry and eGFP were evaluated as reporters to quantify the efficiency of cathepsin
 
+
        B-mediated cleavage of Gal4-Linker-VP64 constructs in HEK293T cells.<br />
    <div class="thumb">
+
        <b>Figure 6</b> shows micrographs taken with a fluorescence microscope of three different conditions: the null
      <div class="thumbinner" style="width:700px;"><a href="placeholder"
+
        control, the negative control and the test sample. <b>Figure 7</b> shows the corresponding graphs quantifying
          class="image"><img alt="Fluorescence Readout" src="https://static.igem.wiki/teams/5237/wetlab-results/catb-fluorescence-microscope-w.png" width="700"
+
        the fluorescence intensity in the different conditions. All samples were transfected with plasmids encoding eGFP
            class="thumbimage"></a>
+
        and mCherry. The null control and the negative control were not transfected with the plasmid encoding cathepsin
        <div class="thumbcaption">
+
        B. The null control was also not transfected with any of the plasmids encoding Gal4-Linker-VP64 constructs. The
          <i><b>Figure 6: Micrographs of HEK293T Cells in Two Control Conditions and One Test Condition.</b></i> Micrographs were taken with a fluorescence microscope 48&nbsp;hours after transfection. An overlay of brightfield, eGFP and mCherry is shown. The null control and the negative control were not transfected with the plasmid encoding cathepsin B. The Null Control was also not transfected with any of the plasmids encoding Gal4-Linker-VP64 constructs. The test sample was transfected with 30&nbsp;ng of the plasmid encoding cathepsin B and with the plasmid encoding Gal4-GFLG-VP64. The micrograph of the test sample is not from the same biological replicate as the micrographs of the two controls.
+
        test sample was transfected with 30 ng of the plasmid encoding cathepsin B and with the plasmid encoding
 +
        Gal4-GFLG-VP64. As expected, the null control showed no detectable mCherry signal, since no plasmid encoding a
 +
        Gal4-V64 construct was transfected. Consequently, mCherry overexpression via VP64 could not be induced. However,
 +
        we observed a high fluorescence intensity for eGFP, indicating that the transfection was successful. The
 +
        negative control showed strong signals of both mCherry and eGFP. Therefore, it can be assumed that the
 +
        transfection was successful and that our mCherry readout system is functional. Interestingly, there are some
 +
        cells which either seem to only express mCherry or eGFP and some cells that show no fluorescence signal. The
 +
        test sample showed less eGFP and mCherry fluorescence compared to the negative control. We expected to observe
 +
        reduced fluorescence intensity of mCherry, as the transfected cells would express cathepsin B, which cleaves the
 +
        linker, thereby decreasing mCherry expression.
 +
      </p>
 +
      <div class="thumb">
 +
        <div class="thumbinner" style="width:700px;"><img alt="Fluorescence Readout" class="thumbimage"
 +
            src="https://static.igem.wiki/teams/5237/wetlab-results/catb-fluorescence-microscope-w1.png" width="700" />
 +
          <div class="thumbcaption">
 +
            <i><b>Figure 6: Micrographs of HEK293T Cells in Two Control Conditions and One Test Condition.</b>
 +
              Micrographs were taken with a fluorescence microscope 48 hours after transfection. An overlay of
 +
              brightfield, eGFP and mCherry is shown. The null control and the negative control were not transfected
 +
              with
 +
              the plasmid encoding cathepsin B. The Null Control was also not transfected with any of the plasmids
 +
              encoding Gal4-Linker-VP64 constructs. The test sample was transfected with 30 ng of the plasmid encoding
 +
              cathepsin B and with the plasmid encoding Gal4-GFLG-VP64. The micrograph of the test sample is not from
 +
              the
 +
              same biological replicate as the micrographs of the two controls.</i>
 +
          </div>
 
         </div>
 
         </div>
 
       </div>
 
       </div>
    </div>
+
      <div class="thumb">
 
+
        <div class="thumbinner" style="width:450px;"><img alt="Fluorescence Readout" class="thumbimage"
    <div class="thumb">
+
            src="https://static.igem.wiki/teams/5237/wetlab-results/catb-results/catb-fluorescence-readout-null-negative-test-w.svg"
      <div class="thumbinner" style="width:450px;"><a href="placeholder"
+
            width="450" />
          class="image"><img alt="Fluorescence Readout" src="https://static.igem.wiki/teams/5237/wetlab-results/catb-results/catb-fluorescence-readout-null-negative-test-w.svg" width="450"
+
          <div class="thumbcaption">
            class="thumbimage"></a>
+
            <i><b>Figure 7: Fluorescence Readout After 48 Hours for Two Control Conditions and One Test
        <div class="thumbcaption">
+
                Condition.</b> The fluorescence intensity for mCherry was measured for the GFLG linker and
          <i><b>Figure 7: Fluorescence Readout After 48&nbsp;Hours for Two Control Conditions and One Test Condition.</b></i> The fluorescence intensity for mCherry was measured for the GFLG linker and normalized against a baseline eGFP fluorescence intensity. The null control and the negative control were not transfected with the plasmid encoding cathepsin B. The null control was also not transfected with any of the plasmids encoding Gal4-Linker-VP64 constructs. The test sample was transfected with 30&nbsp;ng of the plasmid encoding cathepsin B and with the plasmid encoding Gal4-GFLG-VP64.  
+
              normalized against a baseline eGFP fluorescence intensity. The null control and the negative control were
 +
              not transfected with the plasmid encoding cathepsin B. The null control was also not transfected with any
 +
              of
 +
              the plasmids encoding Gal4-Linker-VP64 constructs. The test sample was transfected with 30 ng of the
 +
              plasmid
 +
              encoding cathepsin B and with the plasmid encoding Gal4-GFLG-VP64.</i>
 +
          </div>
 
         </div>
 
         </div>
 
       </div>
 
       </div>
     </div>
+
     </section>
 
+
    <section id="4.3">
<h3>Mature Cathepsin B is Expressed in HEK293T Cells</h3>
+
      <h2>4.3 The Peptide Linker GFLG Is Cleaved by Cathepsin B <i>in Vivo</i></h2>
<p><b>Figure 8</b> shows a western blot of the wild-type (wt) version of cathepsin B as well as the truncated and mutated version of cathepsin B (&Delta;1-20, D22A, H110A, R116A). Cells of both cathepsin B versions were treated with 500 nM doxorubicin (dox) 24&nbsp;hours post-transfection and incubated for additional 24&nbsp;hours. For each condition, three replicates were blotted. We observed no differences in protein expression levels between the dox-treated and untreated wt versions of cathepsin B. For the truncated and mutated version of cathepsin B, however, only the untreated samples showed the corresponding band at approximately 36&nbsp;kDa expected for this version of cathepsin B. Additionally, the bands of the truncated and mutated version appeared much weaker than the ones of the wt, indicating poorer protein expression. The household protein &beta;-tubulin is visible in all samples at approximately 55&nbsp;kDa. The wt cathepsin B additionally showed bands for pro-cathepsin B at approximately 42&nbsp;kDa, a mature single-chain version of cathepsin B at approximately 33&nbsp;kDa and a mature double-chain version at approximately 26&nbsp;kDa.</p>
+
      <p>We performed a fluorescence readout assay in HEK293T cells to investigate cathepsin B cleavage of different
 
+
        peptide linkers. 24 hours after transfection, we added doxorubicin in a final concentration of 500 nM to the
    <div class="thumb">
+
        cell supernatant. <b>Figure 8</b> shows the fluorescence intensity of mCherry for five different peptide linkers
      <div class="thumbinner" style="width:450px;"><a href="placeholder"
+
        (GFLG, FFRG, FRRL, VA, FK). The negative control was not transfected with the plasmid encoding cathepsin B. We
          class="image"><img alt="Fluorescence Readout" src="https://static.igem.wiki/teams/5237/wetlab-results/catb-wb-w.svg" width="450"
+
        investigated two different test conditions, in which we either transfected 30 ng or 60 ng of the plasmid
            class="thumbimage"></a>
+
        encoding cathepsin B. The fluorescence intensity of mCherry was normalized by the measured fluorescence
        <div class="thumbcaption">
+
        intensity of eGFP in each condition. Additionally, the values for 30 ng and 60 ng cathepsin B were normalized
          <i><b>Figure 8: Western Blot of Two Versions of Cathepsin B With and Without Doxorubicin.</b></i> From left to right: protein ladder, wild-type (wt) cathepsin B with (+) and without (-) doxorubicin, truncated and mutated version of cathepsin B with (+) and without (-) doxorubicin. The household protein, &beta;-tubulin, is visible in all samples at 55&nbsp;kDa. The wt cathepsin B also shows bands for pro-cathepsin B at 42&nbsp;kDa, mature single-chain cathepsin B at 33&nbsp;kDa and mature double-chain cathepsin B at 26&nbsp;kDa. The band for the truncated and mutated version of cathepsin B can be seen in the samples without doxorubicin at 36&nbsp;kDa.
+
        against the corresponding negative controls. One data point for the VA linker, transfected with 60 ng of the
 +
        plasmid encoding cathepsin B, was excluded due to severe deviation from the other values. We conducted a two-way
 +
        analysis of variance (ANOVA) to assess the significance of the observed differences between the negative control
 +
        and the test conditions for each linker. As the negative control did not contain the plasmid encoding cathepsin
 +
        B, we expected the measured fluorescence intensity of mCherry to be the highest in these conditions. However,
 +
        this was only observed for the GFLG and FK linkers. Contrary to our expectations, the fluorescence intensity of
 +
        the negative control was the lowest out of the three conditions tested for the remaining linkers. It appears
 +
        that the addition of the plasmid encoding cathepsin B increases mCherry fluorescence intensity when the linker
 +
        is not cleaved. However, this increase is only significant for the FFRG linker in the 60 ng condition. For the
 +
        GFLG linker, we observed significant decreases in fluorescence intensity between the negative control and both
 +
        test conditions, with no difference between the 30 ng and 60 ng conditions. For the FK linker, no significant
 +
        decreases in fluorescence intensity between the negative control and the test conditions were observed.</p>
 +
      <div class="thumb">
 +
        <div class="thumbinner" style="width:450px;"><img alt="Fluorescence Readout" class="thumbimage"
 +
            src="https://static.igem.wiki/teams/5237/wetlab-results/catb-results/catb-fluorescent-readout-final-results-w.svg"
 +
            width="450" />
 +
          <div class="thumbcaption">
 +
            <i><b>Figure 8: Fluorescence Readout After 48 Hours for Five Different Peptide Linkers and Three Different
 +
                Conditions.</b> The fluorescence intensity for mCherry was measured for five different linkers and
 +
              normalized against a baseline eGFP fluorescence intensity. The negative control was not transfected with
 +
              the
 +
              plasmid encoding cathepsin B. The fluorescence intensity of the negative control was set to one. Two
 +
              different test conditions were investigated, in which either 30 ng or 60 ng of the plasmid encoding
 +
              cathepsin B were transfected. The fluorescence readout was analyzed using a two-way ANOVA. Medium: DMEM
 +
              (10%
 +
              FCS). P values: ns, P &gt; 0.05; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.</i>
 +
          </div>
 
         </div>
 
         </div>
 
       </div>
 
       </div>
     </div>
+
     </section>
 
+
<h3>Conclusion</h3>
+
<p>All in all, these findings demonstrate that our fluorescence-based readout assay can reliably detect cathepsin B-mediated cleavage of peptide linkers, with the GFLG linker showing particular susceptibility to cleavage. This makes GFLG a promising candidate for targeted applications in environments with upregulated cathepsin B activity, such as in cancerous tissues. Additionally, our cathepsin B-cleavage linker can be combined with caged inteins (Gramespacher <i>et al.</i>, 2017) conjugated to a dead Cas9 to selectively induce Cas-stapling in the presence of cathepsin B.</p>
+
 
   </section>
 
   </section>
 
   <section id="5">
 
   <section id="5">
     <h1>5. References</h1>
+
     <h1>5. Conclusion</h1>
 +
    <p>All in all, these findings demonstrate that our fluorescence-based readout assay can reliably detect cathepsin
 +
      B-mediated cleavage of peptide linkers, with the GFLG linker showing particular susceptibility to cleavage. This
 +
      makes GFLG a promising candidate for targeted applications in environments with upregulated cathepsin B activity,
 +
      such as in cancerous tissues. Additionally, our cathepsin B-cleavable linker can be combined with caged inteins
 +
      (Gramespacher <i>et al.</i>, 2017) conjugated to a dead Cas9 to selectively induce Cas stapling in the presence of
 +
      cathepsin B.</p>
 +
  </section>
 +
  <section id="6">
 +
    <h1>6. References</h1>
 
     <p>
 
     <p>
Bien, S., Ritter, C. A., Gratz, M., Sperker, B., Sonnemann, J., Beck, J. F., Kroemer, H. K. (2004). Nuclear factor-kappaB mediates up-regulation of cathepsin B by doxorubicin in tumor cells. Molecular Pharmacology 65(5), 1092-102. <a
+
      Bien, S., Ritter, C. A., Gratz, M., Sperker, B., Sonnemann, J., Beck, J. F., Kroemer, H. K. (2004). Nuclear
        href="https://doi.org/10.1124/mol.65.5.1092" target="_blank">https://doi.org/10.1124/mol.65.5.1092</a>  
+
      factor-kappaB mediates up-regulation of cathepsin B by doxorubicin in tumor cells. Molecular Pharmacology 65(5),
</p>
+
      1092-102. <a href="https://doi.org/10.1124/mol.65.5.1092"
<p>
+
        target="_blank">https://doi.org/10.1124/mol.65.5.1092</a>
Bleuez, C., Koch, W. F., Urbach, C., Hollfelder, F., & Jermutus, L. (2022). Exploiting protease activation for therapy. Drug Discov Today, 27(6), 1743-1754. <a
+
    </p>
        href="https://doi.org/10.1016/j.drudis.2022.03.011" target="_blank">https://doi.org/10.1016/j.drudis.2022.03.011</a>  
+
    <p>
</p>
+
      Bleuez, C., Koch, W. F., Urbach, C., Hollfelder, F., &amp; Jermutus, L. (2022). Exploiting protease activation for
<p>
+
      therapy. Drug Discov Today, 27(6), 1743-1754. <a href="https://doi.org/10.1016/j.drudis.2022.03.011"
Gramespacher, J. A., Stevens, A. J.,&nbsp;Nguyen, D. P., Chin, J. W., & Muir, T. W. (2017). Intein Zymogens: Conditional Assembly and Splicing of Split Inteins via Targeted Proteolysis. J Am Chem Soc, 139(24), 8074-8077. <a
+
        target="_blank">https://doi.org/10.1016/j.drudis.2022.03.011</a>
         href="https://doi.org/10.1021/jacs.7b02618" target="_blank">https://doi.org/10.1021/jacs.7b02618</a>  
+
    </p>
</p>  
+
    <p>
<p>
+
      Gramespacher, J. A., Stevens, A. J., Nguyen, D. P., Chin, J. W., &amp; Muir, T. W. (2017). Intein Zymogens:
Jin, C., EI-Sagheer, A. H., Li, S., Vallis, K. A., Tan, W., & Brown, T. (2022). Engineering Enzyme-Cleavable Oligonucleotides by Automated Solid-Phase Incorporation of Cathepsin B Sensitive Dipeptide Linkers. Angewandte Chemie International Edition, 61(13), e202114016. <a
+
      Conditional Assembly and Splicing of Split Inteins via Targeted Proteolysis. J Am Chem Soc, 139(24), 8074-8077. <a
        href="https://doi.org/10.1002/anie.202114016" target="_blank">https://doi.org/10.1002/anie.202114016</a>  
+
         href="https://doi.org/10.1021/jacs.7b02618" target="_blank">https://doi.org/10.1021/jacs.7b02618</a>
</p>  
+
    </p>
<p>
+
    <p>
Müntener, K., Willimann, A., Zwicky, R., Svoboda, B., Mach, L., & Baici, A. (2005). Folding Competence of N-terminally Truncated Forms of Human Procathepsin B*. Journal of Biological Chemistry, 280(12), 11973-11980. <a
+
      Jin, C., EI-Sagheer, A. H., Li, S., Vallis, K. A., Tan, W., &amp; Brown, T. (2022). Engineering Enzyme-Cleavable
         href="https://doi.org/10.1074/jbc.M413052200" target="_blank">https://doi.org/10.1074/jbc.M413052200</a>  
+
      Oligonucleotides by Automated Solid-Phase Incorporation of Cathepsin B Sensitive Dipeptide Linkers. Angewandte
</p>  
+
      Chemie International Edition, 61(13), e202114016. <a href="https://doi.org/10.1002/anie.202114016"
<p>
+
        target="_blank">https://doi.org/10.1002/anie.202114016</a>
Nägler, D. K., Storer, A. C., Portaro, F. C. V., Carmona, E., Juliano, L., & Ménard, R. (1997). Major Increase in Endopeptidase Activity of Human Cathepsin B upon Removal of Occluding Loop Contacts. Biochemistry, 36(41), 12608-12615. <a
+
    </p>
        href="https://doi.org/10.1021/bi971264+" target="_blank">https://doi.org/10.1021/bi971264+</a>
+
    <p>
</p>  
+
      Muench, P., Fiumara, M., Southern, N., Coda, D., Aschenbrenner, S., Correia, B., Gräff, J., Niopek, D., &amp;
<p>
+
      Mathony, J. (2023). A modular toolbox for the optogenetic deactivation of transcription. bioRxiv,
Ruan, H., Hao, S., Young, P., & Zhang, H. (2015). Targeting Cathepsin B for Cancer Therapies. Horiz Cancer Res, 56, 23-40.
+
      2023.2011.2006.565805. <a href="https://doi.org/10.1101/2023.11.06.565805"
</p>
+
        target="_blank">https://doi.org/10.1101/2023.11.06.565805</a>
<p>
+
    </p>
Shim, N., Jeon, S. I., Yang, S., Park, J. Y., Jo, M., Kim, J., Choi, J., Yun, W. S., Kim, J., Lee, Y., Shim, M. K., Kim, Y., & Kim, K. (2022). Comparative study of cathepsin B-cleavable linkers for the optimal design of cathepsin B-specific doxorubicin prodrug nanoparticles for targeted cancer therapy. Biomaterials, 289, 121806. <a
+
    <p>
         href="https://doi.org/10.1016/j.biomaterials.2022.121806" target="_blank">https://doi.org/10.1016/j.biomaterials.2022.121806</a>
+
      Müntener, K., Willimann, A., Zwicky, R., Svoboda, B., Mach, L., &amp; Baici, A. (2005). Folding Competence of
</p>  
+
      N-terminally Truncated Forms of Human Procathepsin B*. Journal of Biological Chemistry, 280(12), 11973-11980. <a
<p>
+
         href="https://doi.org/10.1074/jbc.M413052200" target="_blank">https://doi.org/10.1074/jbc.M413052200</a>
Wang, J., Liu, M., Zhang, X., Wang, X., Xiong, M., & Luo, D. (2024). Stimuli-responsive linkers and their application in molecular imaging. Exploration, 4(4), 20230027. <a
+
    </p>
        href="https://doi.org/10.1002/EXP.20230027" target="_blank">https://doi.org/10.1002/EXP.20230027</a>
+
    <p>
</p>
+
      Nägler, D. K., Storer, A. C., Portaro, F. C. V., Carmona, E., Juliano, L., &amp; Ménard, R. (1997). Major Increase
 +
      in Endopeptidase Activity of Human Cathepsin B upon Removal of Occluding Loop Contacts. Biochemistry, 36(41),
 +
      12608-12615. <a href="https://doi.org/10.1021/bi971264+" target="_blank">https://doi.org/10.1021/bi971264+</a>
 +
    </p>
 +
    <p>
 +
      Ruan, H., Hao, S., Young, P., &amp; Zhang, H. (2015). Targeting Cathepsin B for Cancer Therapies. Horiz Cancer
 +
      Res, 56, 23-40.
 +
    </p>
 +
    <p>
 +
      Shim, N., Jeon, S. I., Yang, S., Park, J. Y., Jo, M., Kim, J., Choi, J., Yun, W. S., Kim, J., Lee, Y., Shim, M.
 +
      K., Kim, Y., &amp; Kim, K. (2022). Comparative study of cathepsin B-cleavable linkers for the optimal design of
 +
      cathepsin B-specific doxorubicin prodrug nanoparticles for targeted cancer therapy. Biomaterials, 289, 121806. <a
 +
         href="https://doi.org/10.1016/j.biomaterials.2022.121806"
 +
        target="_blank">https://doi.org/10.1016/j.biomaterials.2022.121806</a>
 +
    </p>
 +
    <p>
 +
      Wang, J., Liu, M., Zhang, X., Wang, X., Xiong, M., &amp; Luo, D. (2024). Stimuli-responsive linkers and their
 +
      application in molecular imaging. Exploration, 4(4), 20230027. <a href="https://doi.org/10.1002/EXP.20230027"
 +
        target="_blank">https://doi.org/10.1002/EXP.20230027</a>
 +
    </p>
 +
    <p>
 +
      Yoneda,Y. (1997). How Proteins Are Transported from Cytoplasm to the Nucleus. The Journal of Biochemistry, 121(5),
 +
      811 – 817. <a href="https://doi.org/10.1093/oxfordjournals.jbchem.a021657"
 +
        target="_blank">https://doi.org/10.1093/oxfordjournals.jbchem.a021657</a>
 +
    </p>
 
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Latest revision as of 12:28, 2 October 2024


BBa_K5237020

Cathepsin B-Cleavable Trans-Activator: NLS-Gal4-GFLG-VP64

This composite part encodes a cathepsin B-cleavable trans-activator fusion protein. It is composed of an SV40 nuclear localization sequence (BBa_K2549054), the DNA-binding domain of Gal4 (BBa_K4585001), a GFLG linker (BBa_K5237010), and the VP64 trans-activator domain (BBa_K4586021). We used this part to design a fluorescence-based readout assay in HEK293T cells to investigate cathepsin B cleavage of different peptide linkers. Through this assay, we were able to successfully show that the GFLG linker was specifically cleaved in the presence of cathepsin B, while other linkers did not show this effect. Together, these findings enable the functionalization of our PICasSO system for a wide range of therapeutic and synthetic biology applications.



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


While synthetic biology has in the past focused on engineering the genomic sequence of organisms, the 3D spatial organization of DNA is well-known to be an important layer of information encoding in particular in eukaryotes, playing a crucial role in gene regulation and hence cell fate, disease development, evolution, and more. However, tools to precisely manipulate and control the genomic spatial architecture are limited, hampering the exploration of 3D genome engineering in synthetic biology. We - the iGEM Team Heidelberg 2024 - have developed PICasSO, a powerful molecular toolbox for rationally engineering genome 3D architectures in living cells, based on various DNA-binding proteins.

The PICasSO part collection offers a comprehensive, modular platform for precise manipulation and re-programming of DNA-DNA interactions using engineered "protein staples" in living cells. This enables researchers to recreate naturally occurring alterations of 3D genomic interactions, such as enhancer hijacking in cancer, or to design entirely new spatial architectures for artificial gene regulation and cell function control. Specifically, the fusion of two DNA binding proteins enables to artificially bring otherwise distant genomic loci into spatial proximity. To unlock the system's full potential, we introduce versatile chimeric CRISPR/Cas complexes, connected either at the protein or - in the case of CRISPR/Cas-based DNA binding moieties - the guide RNA level. These complexes are referred to as protein- or Cas staples, respectively. Beyond its versatility with regard to the staple constructs themselves, PICasSO includes robust assay systems to support the engineering, optimization, and testing of new staples in vitro and in vivo. Notably, the PICasSO toolbox was developed in a design-build-test-learn engineering cycle closely intertwining wet lab experiments and computational modeling and iterated several times, yielding a collection of well-functioning and -characterized parts.

At its heart, the PICasSO part collection consists of three categories.
(i) Our DNA-binding proteins include our finalized Cas staple experimentally validated using an artificial "enhancer hijacking" system as well as "half staples" that can be combined by scientists to compose entirely new Cas staples in the future. We also include our Simple staples comprised of particularly small, simple and robust DNA binding domains well-known to the synthetic biology community, which serve as controls for successful stapling and can be further engineered to create alternative, simpler, and more compact staples.
(ii) As functional elements, we list additional parts that enhance and expand the functionality of our Cas and Basic staples. These consist of staples dependent on cleavable peptide linkers targeted by cancer-specific proteases or inteins that allow condition-specific, dynamic stapling in vivo. We also include several engineered parts that enable the efficient delivery of PICasSO's constructs into target cells, including mammalian cells, with our new interkingdom conjugation system.
(iii) As the final category of our collection, we provide parts that underlie our custom readout systems. These include components of our established FRET-based proximity assay system, enabling users to confirm accurate stapling. Additionally, we offer a complementary, application-oriented testing system based on a luciferase reporter, which allows for straightforward experimental assessment of functional enhancer hijacking events in mammalian cells.

The following table gives a comprehensive overview of all parts in our PICasSO toolbox. The highlighted parts showed exceptional performance as described on our iGEM wiki and can serve as a reference. The other parts in the collection are versatile building blocks designed to provide future iGEMers with the flexibility to engineer their own custom Cas staples, enabling further optimization and innovation in the new field of 3D genome engineering.

Our part collection includes:

DNA-Binding Proteins: Modular building blocks for engineering of custom staples to mediate defined DNA-DNA interactions in vivo
BBa_K5237000 Fusion Guide RNA Entry Vector MbCas12a-SpCas9 Entry vector for simple fgRNA cloning via SapI
BBa_K5237001 Staple Subunit: dMbCas12a-Nucleoplasmin NLS Staple subunit that can be combined with crRNA or fgRNA and dSpCas9 to form a functional staple
BBa_K5237002 Staple Subunit: SV40 NLS-dSpCas9-SV40 NLS Staple subunit that can be combined with a sgRNA or fgRNA and dMbCas12a to form a functional staple
BBa_K5237003 Cas Staple: SV40 NLS-dMbCas12a-dSpCas9-Nucleoplasmin NLS Functional Cas staple that can be combined with sgRNA and crRNA or fgRNA to bring two DNA strands into close proximity
BBa_K5237004 Staple Subunit: Oct1-DBD Staple subunit that can be combined to form a functional staple, for example with TetR.
Can also be combined with a fluorescent protein as part of the FRET proximity assay
BBa_K5237005 Staple Subunit: TetR Staple subunit that can be combined to form a functional staple, for example with Oct1.
Can also be combined with a fluorescent protein as part of the FRET proximity assay
BBa_K5237006 Simple Staple: TetR-Oct1 Functional staple that can be used to bring two DNA strands in close proximity
BBa_K5237007 Staple Subunit: GCN4 Staple subunit that can be combined to form a functional staple, for example with rGCN4
BBa_K5237008 Staple Subunit: rGCN4 Staple subunit that can be combined to form a functional staple, for example with rGCN4
BBa_K5237009 Mini Staple: bGCN4 Assembled staple with minimal size that can be further engineered
Functional Elements: Protease-cleavable peptide linkers and inteins are used to control and modify staples for further optimization for custom applications
BBa_K5237010 Cathepsin B-cleavable Linker: GFLG Cathepsin B-cleavable peptide linker that can be used to combine two staple subunits to make responsive staples
BBa_K5237011 Cathepsin B Expression Cassette Expression cassette for the overexpression of cathepsin B
BBa_K5237012 Caged NpuN Intein A caged NpuN split intein fragment that undergoes protein trans-splicing after protease activation, which can be used to create functionalized staple subunits
BBa_K5237013 Caged NpuC Intein A caged NpuC split intein fragment that undergoes protein trans-splicing after protease activation, which can be used to create functionalized staple subunits
BBa_K5237014 Fusion Guide RNA Processing Casette Processing cassette to produce multiple fgRNAs from one transcript, that can be used for multiplexed 3D genome reprogramming
BBa_K5237015 Intimin anti-EGFR Nanobody Interkingdom conjugation between bacteria and mammalian cells, as an alternative delivery tool for large constructs
BBa_K4643003 IncP Origin of Transfer Origin of transfer that can be cloned into the plasmid vector and used for conjugation as a means of delivery
Readout Systems: FRET and enhancer recruitment readout systems to rapidly assess successful DNA stapling in bacterial and mammalian cells
BBa_K5237016 FRET-Donor: mNeonGreen-Oct1 FRET donor-fluorophore fused to Oct1-DBD that binds to the Oct1 binding cassette, which can be used to visualize DNA-DNA proximity
BBa_K5237017 FRET-Acceptor: TetR-mScarlet-I Acceptor part for the FRET assay binding the TetR binding cassette, which can be used to visualize DNA-DNA proximity
BBa_K5237018 Oct1 Binding Casette DNA sequence containing 12 Oct1 binding motifs, compatible with various assays such as the FRET proximity assay
BBa_K5237019 TetR Binding Cassette DNA sequence containing 12 Oct1 binding motifs, can be used for different assays such as the FRET proximity assay
BBa_K5237020 Cathepsin B-Cleavable Trans-Activator: NLS-Gal4-GFLG-VP64 Readout system that responds to protease activity, which was used to test cathepsin B-cleavable linker
BBa_K5237021 NLS-Gal4-VP64 Trans-activating enhancer, that can be used to simulate enhancer hijacking
BBa_K5237022 mCherry Expression Cassette: UAS, minimal Promoter, mCherry Readout system for enhancer binding, which was used to test cathepsin B-cleavable linker
BBa_K5237023 Oct1 - 5x UAS Binding Casette Oct1 and UAS binding cassette, that was used for the simulated enhancer hijacking assay
BBa_K5237024 TRE-minimal Promoter- Firefly Luciferase Contains firefly luciferase controlled by a minimal promoter, which was used as a luminescence readout for simulated enhancer hijacking

1. Sequence Overview

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 254
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 173

2. Usage and Biology

Cathepsin B is a cysteine protease that is significantly overexpressed in various cancer types, including breast and colorectal cancer (Ruan et al., 2015). Proteolytic cleavage of pro-biologics via cathepsin B activity allows for precise temporal and spatial regulation of biopharmaceutical activity in therapeutic strategies (Bleuez et al., 2022).
In this context, we introduce the cathepsin B-cleavable peptide linker GFLG as a functional addition to our PICasSO toolbox, enabling a wide range of therapeutic and synthetic biology applications. GFLG has been shown to be cleaved by cathepsin B (Wang et al., 2024), with cleavage occurring either between phenylalanine and leucine or after the second glycine of the linker (Rejmanová et al., 1983; Fig. 2).
The construct also includes Gal4, a transcription factor that binds upstream activation sites (UAS) to drive gene expression, and VP64, a strong transcriptional activator that enhances this expression (Muench et al., 2023). Additionally, it incorporates the SV40 nuclear localization signal (NLS), a short peptide derived from the simian virus 40 (SV40) large T-antigen, which facilitates the transport of the fusion protein into the nucleus, where it can bind to a UAS (Yoneda, 1997).
Through a fluorescence readout assay in HEK293T cells, we identified GFLG as the most effective among five peptide linkers known to be cleaved by cathepsin B (Jin et al., 2022; Shim et al., 2022; Wang et al., 2024). This linker facilitates cleavage-induced oligomerization of Cas proteins via protein trans-splicing of caged intein fragments (BBa_K5237012, BBa_K5237013), further enhancing the capabilities of our system.

3. Assembly and Part Evolution

We designed a fluorescence readout assay in HEK293T cells based on expression of mCherry induced by the transactivator VP64. VP64 was conjugated to the DNA-binding domain (DBD) of Gal4 through the GFLG linker. We purchased the nucleotide sequence encoding the GFLG linker as an oligo. After annealing the oligo, we cloned it into a mammalian expression vector between the open reading frames for Gal4-DBD and VP64 via Golden Gate assembly. Binding of Gal4-DBD upstream of a gene encoding the fluorescence protein mCherry induces overexpression of mCherry by VP64. Consequently, separation of Gal4-DBD and VP64 by cathepsin B cleavage of the GFLG linker reduces mCherry expression (Fig. 2).

Cathepsin B Fluorescence Readout Assay
Figure 2: Schematic Illustration of the Cathepsin B Fluorescence Readout Assay. The DNA-binding domain (DBD) of Gal4 is conjugated to the transactivator domain VP64 via a cathepsin B-cleavable peptide linker. Binding of the Gal4-DBD to the upstream activating sequence (UAS) in proximity to the mCherry gene induces mCherry overexpression via VP64. Cathepsin B cleavage of the linker separates Gal4-DBD and VP64 and consequently reduces mCherry expression.

We transfected our genetic constructs into HEK293T cells. The negative control was not transfected with the plasmid encoding cathepsin B. We investigated two different test conditions, in which we either transfected 30 ng or 60 ng of the plasmid encoding cathepsin B. The fluorescence intensity of mCherry was measured 48 hours after transfection. Our initial tests did not result in the unambiguous identification of a cathepsin B-cleavable peptide linker (Fig. 3). For all linkers, we did not observe a large decrease in fluorescence intensity between the negative control and test conditions. In some conditions, the fluorescence intensity even increased between the negative control and test conditions.

Fluorescence Readout
Figure 3: Fluorescence Readout After 48 Hours for Five Different Peptide Linkers and Three Different Conditions. The fluorescence intensity for mCherry was measured for five different linkers. The negative control was not transfected with the plasmid encoding cathepsin B. The fluorescence intensity of the negative control was set to one. Two different test conditions were investigated, in which either 30 ng or 60 ng of the plasmid encoding cathepsin B were transfected.

Since cathepsin B is a lysosomal protease that is normally only active in the lysosome and the extracellular environment but not in the cytosol, we decided to change the native amino acid sequence of cathepsin B. Upon further investigation of the lysosomal maturation process of cathepsin B, we chose to express a truncated and mutated version of cathepsin B. We truncated the native cathepsin B amino acid sequence N-terminally by the first twenty amino acids. This N-terminally truncated version of cathepsin B lacks a signal peptide that normally facilitates translation of cathepsin B in the rough endoplasmic reticulum. Additionally, truncation of the first twenty amino acids of cathepsin B had previously been observed to maintain catalytic activity even in the absence of lysosomal proteases like pepsin (Müntener et al., 2005). Additionally, we introduced three point mutations (D22A, H110A, R116A) in the amino acid sequence of cathepsin B. This has been shown to increase the activity of cathepsin B at higher pH values by disrupting the interactions of an occluding loop with the substrate binding pocket of cathepsin B (Nägler et al., 1997).
We performed the same fluorescence readout assay in HEK293T cells that we also used for wild-type cathepsin B. The fluorescence intensity of mCherry was measured 48 hours after transfection. However, we observed no decrease in fluorescence between our negative control and test conditions, indicating that Gal4-DBD-Linker-VP64 was not cleaved (Fig. 4>). Additionally, we performed a western blot, where the bands for the truncated and mutated version of cathepsin B were barely visible or absent altogether, indicating lower protein expression compared to the wild type (Fig. 5). Another key insight from this experiment was that this version of cathepsin B was not activated by cleavage in the cell, as no additional protein bands were observed.

Fluorescence Readout
Figure 4: Fluorescence Readout for the Truncated and Mutated Version of Cathepsin B. Fluorescence intensity for mCherry was measured across five different linkers. The negative control, which was not transfected with the plasmid encoding cathepsin B, was assigned a fluorescence intensity value of one. Two test conditions were explored, where either 30 ng or 60 ng of the plasmid encoding cathepsin B was transfected.

Since our truncated and mutated version of cathepsin B did not seem to be active in the cytosol, we decided to go back to wild-type cathepsin B. Therefore, we focused on improving the activity of wild-type cathepsin B in the cytosol. After consulting the literature, we decided to treat cells with the cytostatic agent doxorubicin to induce lysosomal escape of cathepsin B, as had been previously reported (Bien et al., 2004).

4. Results

4.1 Mature Cathepsin B Is Expressed in HEK293T Cells

To achieve cathepsin B cleavage-induced Cas stapling, catalytically active cathepsin B needs to be expressed in the cytosol. Therefore, we investigated the expression of different cathepsin B constructs under different conditions in HEK293T cells. In addition to wild-type (wt) cathepsin B, we also cloned a truncated and mutated version of cathepsin B (Δ1-20, D22A, H110A, R116A) and compared protein expression of both constructs in doxorubicin-treated and untreated conditions.
Figure 5 shows a western blot of the wild-type (wt) version of cathepsin B as well as the truncated and mutated version of cathepsin B (Δ1-20, D22A, H110A, R116A). Cells of both cathepsin B versions were treated with 500 nM doxorubicin (dox) 24 hours post-transfection and incubated for additional 24 hours. For each condition, three replicates were blotted. We observed no differences in protein expression levels between the dox-treated and untreated wt versions of cathepsin B. For the truncated and mutated version of cathepsin B, however, only the untreated samples showed the corresponding band at approximately 36 kDa expected for this version of cathepsin B. Additionally, the bands of the truncated and mutated version appeared much weaker than the ones of the wt, indicating poorer protein expression. The household protein β-tubulin is visible in all samples at approximately 55 kDa. The wt cathepsin B additionally showed bands for pro-cathepsin B at approximately 42 kDa, a mature single-chain version of cathepsin B at approximately 33 kDa and a mature double-chain version at approximately 26 kDa.

Fluorescence Readout
Figure 5: Western Blot of Two Versions of Cathepsin B With and Without Doxorubicin. From left to right: protein ladder, wild-type (wt) cathepsin B with (+) and without (-) doxorubicin, truncated and mutated version of cathepsin B with (+) and without (-) doxorubicin. The household protein β-tubulin is visible in all samples at 55 kDa. The wt cathepsin B also shows bands for pro-cathepsin B at 42 kDa, mature single-chain cathepsin B at 33 kDa and mature double-chain cathepsin B at 26 kDa. The band for the truncated and mutated version of cathepsin B can be seen in the samples without doxorubicin at 36 kDa.

4.2 mCherry and eGFP Can be Used as a Reporter System to Measure Cleavage Efficiency

In this experiment, mCherry and eGFP were evaluated as reporters to quantify the efficiency of cathepsin B-mediated cleavage of Gal4-Linker-VP64 constructs in HEK293T cells.
Figure 6 shows micrographs taken with a fluorescence microscope of three different conditions: the null control, the negative control and the test sample. Figure 7 shows the corresponding graphs quantifying the fluorescence intensity in the different conditions. All samples were transfected with plasmids encoding eGFP and mCherry. The null control and the negative control were not transfected with the plasmid encoding cathepsin B. The null control was also not transfected with any of the plasmids encoding Gal4-Linker-VP64 constructs. The test sample was transfected with 30 ng of the plasmid encoding cathepsin B and with the plasmid encoding Gal4-GFLG-VP64. As expected, the null control showed no detectable mCherry signal, since no plasmid encoding a Gal4-V64 construct was transfected. Consequently, mCherry overexpression via VP64 could not be induced. However, we observed a high fluorescence intensity for eGFP, indicating that the transfection was successful. The negative control showed strong signals of both mCherry and eGFP. Therefore, it can be assumed that the transfection was successful and that our mCherry readout system is functional. Interestingly, there are some cells which either seem to only express mCherry or eGFP and some cells that show no fluorescence signal. The test sample showed less eGFP and mCherry fluorescence compared to the negative control. We expected to observe reduced fluorescence intensity of mCherry, as the transfected cells would express cathepsin B, which cleaves the linker, thereby decreasing mCherry expression.

Fluorescence Readout
Figure 6: Micrographs of HEK293T Cells in Two Control Conditions and One Test Condition. Micrographs were taken with a fluorescence microscope 48 hours after transfection. An overlay of brightfield, eGFP and mCherry is shown. The null control and the negative control were not transfected with the plasmid encoding cathepsin B. The Null Control was also not transfected with any of the plasmids encoding Gal4-Linker-VP64 constructs. The test sample was transfected with 30 ng of the plasmid encoding cathepsin B and with the plasmid encoding Gal4-GFLG-VP64. The micrograph of the test sample is not from the same biological replicate as the micrographs of the two controls.
Fluorescence Readout
Figure 7: Fluorescence Readout After 48 Hours for Two Control Conditions and One Test Condition. The fluorescence intensity for mCherry was measured for the GFLG linker and normalized against a baseline eGFP fluorescence intensity. The null control and the negative control were not transfected with the plasmid encoding cathepsin B. The null control was also not transfected with any of the plasmids encoding Gal4-Linker-VP64 constructs. The test sample was transfected with 30 ng of the plasmid encoding cathepsin B and with the plasmid encoding Gal4-GFLG-VP64.

4.3 The Peptide Linker GFLG Is Cleaved by Cathepsin B in Vivo

We performed a fluorescence readout assay in HEK293T cells to investigate cathepsin B cleavage of different peptide linkers. 24 hours after transfection, we added doxorubicin in a final concentration of 500 nM to the cell supernatant. Figure 8 shows the fluorescence intensity of mCherry for five different peptide linkers (GFLG, FFRG, FRRL, VA, FK). The negative control was not transfected with the plasmid encoding cathepsin B. We investigated two different test conditions, in which we either transfected 30 ng or 60 ng of the plasmid encoding cathepsin B. The fluorescence intensity of mCherry was normalized by the measured fluorescence intensity of eGFP in each condition. Additionally, the values for 30 ng and 60 ng cathepsin B were normalized against the corresponding negative controls. One data point for the VA linker, transfected with 60 ng of the plasmid encoding cathepsin B, was excluded due to severe deviation from the other values. We conducted a two-way analysis of variance (ANOVA) to assess the significance of the observed differences between the negative control and the test conditions for each linker. As the negative control did not contain the plasmid encoding cathepsin B, we expected the measured fluorescence intensity of mCherry to be the highest in these conditions. However, this was only observed for the GFLG and FK linkers. Contrary to our expectations, the fluorescence intensity of the negative control was the lowest out of the three conditions tested for the remaining linkers. It appears that the addition of the plasmid encoding cathepsin B increases mCherry fluorescence intensity when the linker is not cleaved. However, this increase is only significant for the FFRG linker in the 60 ng condition. For the GFLG linker, we observed significant decreases in fluorescence intensity between the negative control and both test conditions, with no difference between the 30 ng and 60 ng conditions. For the FK linker, no significant decreases in fluorescence intensity between the negative control and the test conditions were observed.

Fluorescence Readout
Figure 8: Fluorescence Readout After 48 Hours for Five Different Peptide Linkers and Three Different Conditions. The fluorescence intensity for mCherry was measured for five different linkers and normalized against a baseline eGFP fluorescence intensity. The negative control was not transfected with the plasmid encoding cathepsin B. The fluorescence intensity of the negative control was set to one. Two different test conditions were investigated, in which either 30 ng or 60 ng of the plasmid encoding cathepsin B were transfected. The fluorescence readout was analyzed using a two-way ANOVA. Medium: DMEM (10% FCS). P values: ns, P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.

5. Conclusion

All in all, these findings demonstrate that our fluorescence-based readout assay can reliably detect cathepsin B-mediated cleavage of peptide linkers, with the GFLG linker showing particular susceptibility to cleavage. This makes GFLG a promising candidate for targeted applications in environments with upregulated cathepsin B activity, such as in cancerous tissues. Additionally, our cathepsin B-cleavable linker can be combined with caged inteins (Gramespacher et al., 2017) conjugated to a dead Cas9 to selectively induce Cas stapling in the presence of cathepsin B.

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