Difference between revisions of "Part:BBa K5237004"

Revision as of 21:27, 27 September 2024

Half-Staple: Oct1-DBD

Oct1-DBD (BBa_K5237004) is the DNA-binding domain of the human Oct1 transcription factor (POU2F1), which binds specifically to the octamer motif (5'-ATGCAAAT-3'). This domain is key to regulating gene expression, immune response, and stress adaptation. Oct1-DBD can be readily fused to additional proteins for biotechnological applications.

1 Sequence overview
2 Usage and Biology
3 Assembly and part evolution
4 Results
5 References

The PICasSO Toolbox

Figure 1: Example how the part collection can be used to engineer new staples

The 3D organization of the genome plays a crucial role in regulating gene expression in eukaryotic cells, impacting cellular behavior, evolution, and disease. Beyond the linear DNA sequence, the spatial arrangement of chromatin, influenced by DNA-DNA interactions, shapes pathways of gene regulation. However, the tools to precisely manipulate this genomic architecture remain limited, rendering it challenging to explore the full potential of the 3D genome in synthetic biology. We - iGEM Team Heidelberg 2024 - have developed PICasSO, a powerful molecular toolbox based on various DNA-binding proteins to address this issue.

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

At its heart, the PICasSO parts collection consists of three categories. (i) Our DNA-binding proteins include our finalized enhancer hijacking Cas staple as well as half staples that can be used by scientists to compose entirely new Cas staples in the future. We also include our simple staples that serve as controls for successful stapling and can be further engineered to create alternative, simpler and more compact staples. (ii) As functional elements, we list additional parts that enhance the functionality of our Cas and Basic staples. These consist of protease-cleavable peptide linkers and inteins that allow condition-specific, dynamic stapling in vivo. Besides staple functionality, we also include the parts to enable the efficient delivery of PICasSO's with our interkingdom conjugation system.

(iii) As the final component of our collection, we provide parts that support the use of our custom readout systems. These include components of our established FRET-based proximity assay system, enabling users to confirm accurate stapling. Additionally, we offer a complementary, application-oriented testing system for functional readout via a luciferase reporter, which allows for straightforward experimental simulation of enhancer hijacking.

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

Our parts collection includes:

DNA-binding proteins: The building blocks for engineering of custom staples for DNA-DNA interactions with a modular system ensuring easy assembly.
BBa_K5237000	fgRNA Entryvector MbCas12a-SpCas9	Entryvector for simple fgRNA cloning via SapI
BBa_K5237001	Half-Staple: dMbCas12a-Nucleoplasmin NLS	Staple subunit that can be combined to form a functional staple, for example with fgRNA and dCas9
BBa_K5237002	Half-Staple: SV40 NLS-dSpCas9-SV40 NLS	Staple subunit that can be combined to form a functional staple, for example with our fgRNA or dCas12a
BBa_K5237003	Cas-Staple: SV40 NLS-dMbCas12a-dSpCas9-Nucleoplasmin NLS	Functional Cas staple that can be combined with sgRNA or fgRNA to bring two DNA strands in close proximity
BBa_K5237004	Half-Staple: 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	Half-Staple: 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	Half-Staple: GCN4	Staple subunit that can be combined to form a functional staple, for example with rGCN4
BBa_K5237008	Half-Staple: 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	Cathepsin B which can be selectively express to cut the cleavable linker
BBa_K5237012	Caged NpuN Intein	Undergoes protein transsplicing after protease activation, can be used to create functionalized staple units
BBa_K5237013	Caged NpuC Intein	Undergoes protein transsplicing after protease activation, can be used to create functionalized staple units
BBa_K5237014	fgRNA processing casette	Processing casette to produce multiple fgRNAs from one transcript, can be used for multiplexing
BBa_K5237015	Intimin anti-EGFR Nanobody	Interkindom conjugation between bacteria and mammalian cells, as alternative delivery tool for large constructs
Readout Systems: FRET and enhancer recruitment to measure proximity of stapled DNA in bacterial and mammalian living cells enabling swift testing and easy development for new systems.
BBa_K5237016	FRET-Donor: mNeonGreen-Oct1	Donor part for the FRET assay binding the Oct1 binding cassette. Can be used to visualize DNA-DNA proximity
BBa_K5237017	FRET-Acceptor: TetR-mScarlet-I	Acceptor part for the FRET assay binding the TetR binding cassette. Can be used to visualize DNA-DNA proximity
BBa_K5237018	Oct1 Binding Casette	DNA sequence containing 12 Oct1 binding motifs, can be used for different assays such as the FRET proximity assay
BBa_K5237019	TetR Binding Cassette	DNA sequence containing 12 Oct1 binding motifs, can be used for different assays such as the FRET proximity assay
BBa_K5237020	Cathepsin B-Cleavable Trans-Activator: NLS-Gal4-GFLG-VP64	Readout system that responds to protease activity. It was used to test Cathepsin-B cleavable linker.
BBa_K5237021	NLS-Gal4-VP64	Trans-activating enhancer, that can be used to simulate enhancer hijacking.
BBa_K5237022	mCherry Expression Cassette: UAS, minimal Promotor, mCherry	Readout system for enhancer binding. It was used to test Cathepsin-B cleavable linker.
BBa_K5237023	Oct1 - UAS binding casette	Oct1 and UAS binding cassette, that was used for the simulated enhancer hijacking assay.
BBa_K5237024	Minimal promoter Firefly luciferase	Contains Firefly luciferase controlled by a minimal promoter. It was used as a luminescence readout for simulated enhancer hijacking.

1. Sequence overview

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

2. Usage and Biology

Oct1-DBD is the DNA-binding domain of the human transcription factor Oct1 (POU2F1), which plays a key role in gene regulation, immune response, and stress adaptation in eukaryotic cells. This domain specifically binds to the octamer motif (5’-ATGCAAAT-3’) within promoter and enhancer regions, influencing transcriptional activity (Lundbäck et al., 2000). The Oct1-DBD consists of both a POU-specific domain and a POU homeodomain, which work together to form a stable complex with DNA (Park et al., 2013, Stepchenko et al. 2021).

In synthetic biology, Oct1-DBD was previously used for plasmid display technology due to its strong binding affinity (K_D = 9 × 10^-11 M). Proteins fused with Oct1-DBD showed increased expression and protein solubility (Parker et al. 2020).

This part was further used in BBa_K5237002 as a fusion with tetR, resulting in a bivalent DNA binding staple, and also fused to mNeonGree, as part of a FRET readout system (BBa_K5237016).

3. Assembly and part evolution

The Oct1-DBD amino acid sequence was obtained from UniProt (P14859, POU domain, class 2, transcription factor 1) and DNA binding domain extracted based on information given from Park et al. 2013 & 2020. An E. coli codon optimized DNA sequence was obtained through gene synthesis and used to clone further constructs

4. Results

Oct1 was N-terminally fused to the His6-mNeonGreen, and expressed under a T7 expression protein and subsequently purified using metal affinity chromatography with Ni-NTA beads.(Figure 1, left) DNA binding affinity was estimated with an electrophoretic mobility shift assay (EMSA). For this, three different buffer conditions were tested (Binding buffer 1: 137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂HPO₄, 0.1 % (v/v) IGEPAL® CA-360, 1 mM EDTA; Binding buffer 2: 10 mM Tris, 50 mM KCl; NaP250: Na₂HPO₄, 150 mM NaCl, 250 mM Imidazol). DNA binding could only be detected for Binding buffer 1. (Figure 1, right)

Figures 1: Figure 1: SDS-PAGE analysis of His₆-mNeonGreen-Oct1-DBD.
Lane 1: raw lysate of E. coli expression culture after steril-filtration; Lane 2: Flow through of first wash (10 bed volumes of NaP10 (Na₂HPO₄, 150 mM NaCl, 10 mM Imidazol)); Lane 3: Flow through of second wash (10 bed volumes of NaP20 (Na₂HPO₄, 150 mM NaCl, 20 mM Imidazol)); Lane 4: Elution of purified protein.
1 µL of each fraction was loaded after mixing and heating with 4x Laeemli buffer, on a 4-15% TGX-Gel. The expected band size of the protein is 56 840.23 Da, highlighted in red on the gel.

5. References

Lundbäck, T., Chang, J.-F., Phillips, K., Luisi, B., & Ladbury, J. E. (2000). Characterization of Sequence-Specific DNA Binding by the Transcription Factor Oct-1. Biochemistry, 39(25), 7570–7579. https://doi.org/10.1021/bi000377h

Park, J. H., Kwon, H. W., & Jeong, K. J. (2013). Development of a plasmid display system with an Oct-1 DNA-binding domain suitable for in vitro screening of engineered proteins. Journal of Bioscience and Bioengineering, 116(2), 246-252. https://doi.org/10.1016/j.jbiosc.2013.02.005.

Park, Y., Shin, J., Yang, J., Kim, H., Jung, Y., Oh, H., Kim, Y., Hwang, J., Park, M., Ban, C., Jeong, K. J., Kim, S.-K., & Kweon, D.-H. (2020). Plasmid Display for Stabilization of Enzymes Inside the Cell to Improve Whole-Cell Biotransformation Efficiency. Frontiers in Bioengineering and Biotechnology, 7. https://doi.org/10.3389/fbioe.2019.00444

Stepchenko, A. G., Portseva, T. N., Glukhov, I. A., Kotnova, A. P., Lyanova, B. M., Georgieva, S. G., & Pankratova, E. V. (2021). Primate-specific stress-induced transcription factor POU2F1Z protects human neuronal cells from stress. Scientific Reports, 11(1), 18808. https://doi.org/10.1038/s41598-021-98323-y

@@ Line 43: / Line 43: @@
      <p>Oct1-DBD (BBa_K5237004) is the DNA-binding domain of the human Oct1 transcription factor (POU2F1), which binds
        specifically to the octamer motif (5'-ATGCAAAT-3'). This domain is key to regulating gene expression, immune
-       response, and stress adaptation. Oct1-DBD can be readily fused to additional proteins for biotechnological applications.</p>
+       response, and stress adaptation. Oct1-DBD can be readily fused to additional proteins for biotechnological
+      applications.</p>
      <p>&nbsp;</p>
    </section>
@@ Line 70: / Line 71: @@
    <section>
      <font size="5"><b>The PICasSO Toolbox </b> </font>
+    <p><br></p>
+    <div class="thumb"></div>
+      <div class="thumbinner" style="width:550px"><img alt="" src="https://static.igem.wiki/teams/5237/wetlab-results/registry-part-collection-engineering-cycle-example-overview.svg" style="width:99%;" class="thumbimage">
+        <div class="thumbcaption">
+          <i><b>Figure 1: Example how the part collection can be used to engineer new staples</b></i>
+        </div>
+      </div>
+    </div>
-     <p>The 3D organization of the genome plays a crucial role in regulating gene expression in eukaryotic cells,
+     <p>
+      <br>
+      The 3D organization of the genome plays a crucial role in regulating gene expression in eukaryotic cells,
        impacting cellular behavior, evolution, and disease. Beyond the linear DNA sequence, the spatial arrangement of
-       chromatin, influenced by DNA-DNA interactions, shapes pathways of gene regulation. However, tools to precisely
+       chromatin, influenced by DNA-DNA interactions, shapes pathways of gene regulation. However, the tools to precisely
-       manipulate this genomic architecture remain limited, making it challenging to explore the full potential of the 3D
+       manipulate this genomic architecture remain limited, rendering it challenging to explore the full potential of the
-       genome in synthetic biology. To address this issue, team Heidelberg developed PICasSO.
+D genome in synthetic biology. We - iGEM Team Heidelberg 2024 - have developed PICasSO, a powerful molecular
+      toolbox based on various DNA-binding proteins to address this issue.
      </p>
-     <p>The <b>PICasSO part collection</b> offers a comprehensive, modular platform for precise manipulation of DNA-DNA
+     <p>
-      proximity in living cells, enabling researchers to recreate natural 3D genomic interactions, such as enhancer
+      The <b>PICasSO</b> part collection offers a comprehensive, modular platform for precise manipulation and
-      hijacking, or design entirely new spatial architectures for gene regulation. Beyond its versatility, PICasSO
+      re-programming
-      includes robust measurement systems to support the engineering, optimization, and testing of new staples, ensuring
+      of DNA-DNA interactions using protein staples in living cells, enabling researchers to recreate natural 3D genomic
-       functionality both in vivo and in vitro.
+      interactions, such as enhancer hijacking, or to design entirely new spatial architectures for gene regulation.
+      Beyond its versatility, PICasSO includes robust assay systems to support the engineering, optimization, and
+      testing of new staples, ensuring functionality <i>in vitro</i> and <i>in vivo</i>. We took special care to include
+       parts crucial for testing every step of the cycle (design, build, test, learn) when engineering new parts
+    </p>
+    <!-- Picture explaining parts collection -->
+    <!-- below text not finished formatting-->
+    <p>At its heart, the PICasSO parts collection consists of three categories. (i) Our <b>DNA-binding proteins</b>
+      include our
+      finalized enhancer hijacking Cas staple as well as half staples that can be used by scientists to compose entirely
+      new Cas staples in the future. We also include our simple staples that serve as controls for successful stapling
+      and can be further engineered to create alternative, simpler and more compact staples. (ii) As <b>functional
+        elements</b>, we list additional parts that enhance the functionality of our Cas and Basic staples. These
+      consist of
+      protease-cleavable peptide linkers and inteins that allow condition-specific, dynamic stapling <i>in vivo</i>.
+      Besides staple functionality, we also include the parts to enable the efficient delivery of PICasSO's with our
+      interkingdom conjugation system.
+    </p>
+    <p>
+      (iii) As the final component of our collection, we provide parts that support the use of our <b>custom readout
+        systems</b>. These include components of our established FRET-based proximity assay system, enabling users to
+      confirm
+      accurate stapling. Additionally, we offer a complementary, application-oriented testing system for functional
+      readout via a luciferase reporter, which allows for straightforward experimental simulation of enhancer hijacking.
+    </p>
+    <p>
+      The following table gives a complete 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
      </p>
-    <p>With its combination of staple systems, functionalization options, and measurement tools, PICasSO provides a
-      complete solution for designing, testing, and refining new systems for spatial DNA organization. This toolbox
-      unlocks new possibilities in synthetic biology, from studying fundamental genomic interactions to creating
-      innovative gene therapies.</p>
      <p>
        <font size="4"><b>Our parts collection includes:</b></font><br>
      </p>
-     <table style="width: 65%;">
-       <td colspan="2" align="left"><b>DNA-binding proteins: </b>
+     <table style="width: 90%;">
+       <td colspan="3" align="left"><b>DNA-binding proteins: </b>
          The building blocks for engineering of custom staples for DNA-DNA interactions with a modular system ensuring
          easy assembly.</td>
@@ Line 98: / Line 138: @@
            <td><a href="https://parts.igem.org/Part:BBa_K5237000" target="_blank">BBa_K5237000</a></td>
            <td>fgRNA Entryvector MbCas12a-SpCas9</td>
+          <td>Entryvector for simple fgRNA cloning via SapI</td>
          </tr>
          <tr>
            <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 that can be combined to form a functional staple, for example with fgRNA and dCas9 </td>
          </tr>
          <tr>
            <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 that can be combined to form a functional staple, for example with our fgRNA or dCas12a
+          </td>
          </tr>
          <tr>
            <td><a href="https://parts.igem.org/Part:BBa_K5237003" target="_blank">BBa_K5237003</a></td>
            <td>Cas-Staple: SV40 NLS-dMbCas12a-dSpCas9-Nucleoplasmin NLS</td>
+          <td>Functional Cas staple that can be combined with sgRNA or fgRNA to bring two DNA strands in close proximity
+          </td>
          </tr>
          <tr>
            <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 that can be combined to form a functional staple, for example with TetR.<br>
+            Can also be combined with a fluorescent protein as part of the FRET proximity assay</td>
          </tr>
          <tr>
            <td><a href="https://parts.igem.org/Part:BBa_K5237005" target="_blank">BBa_K5237005</a></td>
            <td>Half-Staple: TetR</td>
+          <td>Staple subunit that can be combined to form a functional staple, for example with Oct1.<br>
+            Can also be combined with a fluorescent protein as part of the FRET proximity assay</td>
          </tr>
          <tr>
            <td><a href="https://parts.igem.org/Part:BBa_K5237006" target="_blank">BBa_K5237006</a></td>
            <td>Simple-Staple: TetR-Oct1</td>
+          <td>Functional staple that can be used to bring two DNA strands in close proximity</td>
          </tr>
          <tr>
            <td><a href="https://parts.igem.org/Part:BBa_K5237007" target="_blank">BBa_K5237007</a></td>
            <td>Half-Staple: GCN4</td>
+          <td>Staple subunit that can be combined to form a functional staple, for example with rGCN4</td>
          </tr>
          <tr>
            <td><a href="https://parts.igem.org/Part:BBa_K5237008" target="_blank">BBa_K5237008</a></td>
            <td>Half-Staple: rGCN4</td>
+          <td>Staple subunit that can be combined to form a functional staple, for example with rGCN4</td>
          </tr>
          <tr>
            <td><a href="https://parts.igem.org/Part:BBa_K5237009" target="_blank">BBa_K5237009</a></td>
            <td>Mini-Staple: bGCN4</td>
+          <td>
+            Assembled staple with minimal size that can be further engineered</td>
          </tr>
        </tbody>
-       <td colspan="2" align="left"><b>Functional elements: </b>
+       <td colspan="3" align="left"><b>Functional elements: </b>
          Protease cleavable peptide linkers and inteins are used to control and modify staples for further optimization
          for custom applications.</td>
@@ Line 143: / Line 198: @@
            <td><a href="https://parts.igem.org/Part:BBa_K5237010" target="_blank">BBa_K5237010</a></td>
            <td>Cathepsin B-Cleavable Linker (GFLG)</td>
+          <td>Cathepsin B cleavable peptide linker, that can be used to combine two staple subunits ,to make responsive
+            staples</td>
          </tr>
          <tr>
            <td><a href="https://parts.igem.org/Part:BBa_K5237011" target="_blank">BBa_K5237011</a></td>
            <td>Cathepsin B Expression Cassette</td>
+          <td>Cathepsin B which can be selectively express to cut the cleavable linker</td>
          </tr>
          <tr>
            <td><a href="https://parts.igem.org/Part:BBa_K52370012" target="_blank">BBa_K5237012</a></td>
            <td>Caged NpuN Intein</td>
+          <td>Undergoes protein transsplicing after protease activation, can be used to create functionalized staple
+            units</td>
          </tr>
          <tr>
            <td><a href="https://parts.igem.org/Part:BBa_K52370013" target="_blank">BBa_K5237013</a></td>
            <td>Caged NpuC Intein</td>
+          <td>Undergoes protein transsplicing after protease activation, can be used to create functionalized staple
+            units</td>
          </tr>
          <tr>
            <td><a href="https://parts.igem.org/Part:BBa_K52370014" target="_blank">BBa_K5237014</a></td>
            <td>fgRNA processing casette</td>
+          <td>Processing casette to produce multiple fgRNAs from one transcript, can be used for multiplexing</td>
          </tr>
          <tr>
            <td><a href="https://parts.igem.org/Part:BBa_K52370015" target="_blank">BBa_K5237015</a></td>
            <td>Intimin anti-EGFR Nanobody</td>
+          <td>Interkindom conjugation between bacteria and mammalian cells, as alternative delivery tool for large
+            constructs</td>
          </tr>
        </tbody>
-       <td colspan="2" align="left"><b>Readout Systems: </b>
+       <td colspan="3" align="left"><b>Readout Systems: </b>
          FRET and enhancer recruitment to measure proximity of stapled DNA in bacterial and mammalian living cells
          enabling swift testing and easy development for new systems.</td>
@@ Line 172: / Line 237: @@
            <td><a href="https://parts.igem.org/Part:BBa_K52370016" target="_blank">BBa_K5237016</a></td>
            <td>FRET-Donor: mNeonGreen-Oct1</td>
+          <td>Donor part for the FRET assay binding the Oct1 binding cassette. Can be used to visualize DNA-DNA
+            proximity</td>
          </tr>
          <tr bgcolor="#FFD700">
            <td><a href="https://parts.igem.org/Part:BBa_K5237017" target="_blank">BBa_K5237017</a></td>
            <td>FRET-Acceptor: TetR-mScarlet-I</td>
+          <td>Acceptor part for the FRET assay binding the TetR binding cassette. Can be used to visualize DNA-DNA
+            proximity</td>
          </tr>
          <tr>
            <td><a href="https://parts.igem.org/Part:BBa_K5237018" target="_blank">BBa_K5237018</a></td>
            <td>Oct1 Binding Casette</td>
+          <td>DNA sequence containing 12 Oct1 binding motifs, can be used for different assays such as the FRET
+            proximity assay</td>
          </tr>
          <tr>
            <td><a href="https://parts.igem.org/Part:BBa_K5237019" target="_blank">BBa_K5237019</a></td>
            <td>TetR Binding Cassette</td>
+          <td>DNA sequence containing 12 Oct1 binding motifs, can be used for different assays such as the FRET
+            proximity assay</td>
          </tr>
          <td><a href="https://parts.igem.org/Part:BBa_K5237020" target="_blank">BBa_K5237020</a></td>
          <td>Cathepsin B-Cleavable Trans-Activator: NLS-Gal4-GFLG-VP64</td>
+        <td>Readout system that responds to protease activity. It was used to test Cathepsin-B cleavable linker.</td>
          </tr>
          <tr>
            <td><a href="https://parts.igem.org/Part:BBa_K5237021" target="_blank">BBa_K5237021</a></td>
            <td>NLS-Gal4-VP64</td>
+          <td>Trans-activating enhancer, that can be used to simulate enhancer hijacking. </td>
          </tr>
          <td><a href="https://parts.igem.org/Part:BBa_K5237022" target="_blank">BBa_K5237022</a></td>
          <td>mCherry Expression Cassette: UAS, minimal Promotor, mCherry</td>
+        <td>Readout system for enhancer binding. It was used to test Cathepsin-B cleavable linker.</td>
          </tr>
          <tr>
            <td><a href="https://parts.igem.org/Part:BBa_K5237023" target="_blank">BBa_K5237023</a></td>
-           <td>UAS binding Firefly Luciferase Readout System</td>
+           <td>Oct1 - UAS binding casette</td>
+          <td>Oct1 and UAS binding cassette, that was used for the simulated enhancer hijacking assay.</td>
+        </tr>
+        <tr>
+          <td><a href="https://parts.igem.org/Part:BBa_K5237024" target="_blank">BBa_K5237024</a></td>
+          <td>Minimal promoter Firefly luciferase</td>
+          <td>Contains Firefly luciferase controlled by a minimal promoter. It was used as a luminescence readout for
+            simulated enhancer hijacking.</td>
          </tr>
        </tbody>
      </table>
@@ Line 266: / Line 348: @@
        <div class="thumbinner" style="display: flex; justify-content: center;">
          <div>
-           <a href="Fig1_left" class="image">
+           <a href="Fig1_left">
              <img alt="" src="https://static.igem.wiki/teams/5237/wetlab-results/sds-page-mng-oct1-expression.svg"
                style="height: 350px; width: auto;" class="thumbimage">
@@ Line 272: / Line 354: @@
          </div>
          <div>
-           <a href="Fig1_right" class="image">
+           <a href="Fig1_right">
              <img alt=""
                src="https://static.igem.wiki/teams/5237/wetlab-results/emsa-oct1-binding-buffer-optimization.svg"