Difference between revisions of "Part:BBa K5237022"

Revision as of 20:50, 30 September 2024

mCherry Expression Cassette: UAS, Minimal Promoter, mCherry

This composite part features the 5x Gal4 upstream activating sequence (UAS) followed by a minimal promoter (BBa_K3281012) to regulate the expression of mCherry (BBa_J06504). We used this part for a fluorescence readout assay to investigate cathepsin B cleavage of different peptide linkers in vivo: The fusion protein NLS-Gal4-Linker-VP64 (BBa_K5237020) was overexpressed in HEK293T cells. Binding of Gal4 to the 5x Gal4 UAS induces overexpression of mCherry through VP64 trans-activation, resulting in bright red fluorescence, which is useful for visualizing gene expression. Separation of Gal4 and VP64 through cleavage of the linker would consequently reduce mCherry expression.

1 Sequence Overview
2 Usage and Biology
3 Assembly and Part Evolution
4 Results
5 References

The PICasSO Toolbox

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

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

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

At its heart, the PICasSO part collection consists of three categories.
(i) Our DNA-binding proteins include our finalized enhancer hijacking Cas staple as well as half staples that can be used by scientists to compose entirely new Cas staples in the future. We also include our Simple staples that serve as controls for successful stapling and can be further engineered to create alternative, simpler and more compact staples.
(ii) As functional elements, we list additional parts that enhance the functionality of our Cas and Basic staples. These consist of protease-cleavable peptide linkers and inteins that allow condition-specific, dynamic stapling in vivo. Besides staple functionality, we also include the parts to enable the efficient delivery of PICasSO's constructs with our interkingdom conjugation system.
(iii) As the final category of our collection, we provide parts that support the use of our custom readout systems. These include components of our established FRET-based proximity assay system, enabling users to confirm accurate stapling. Additionally, we offer a complementary, application-oriented testing system for functional readouts via a luciferase reporter, which allows for straightforward experimental simulation of enhancer hijacking in mammalian cells.

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

Our part collection includes:

DNA-binding proteins: The building blocks for engineering of custom staples for DNA-DNA interactions with a modular system ensuring easy assembly.
BBa_K5237000	fgRNA Entry vector MbCas12a-SpCas9	Entryvector for simple fgRNA cloning via SapI
BBa_K5237001	Staple subunit: dMbCas12a-Nucleoplasmin NLS	Staple subunit that can be combined with sgRNA or fgRNA and dCas9 to form a functional staple
BBa_K5237002	Staple subunit: SV40 NLS-dSpCas9-SV40 NLS	Staple subunit that can be combined witha sgRNA or fgRNA and dCas12avto form a functional staple
BBa_K5237003	Cas Staple: SV40 NLS-dMbCas12a-dSpCas9-Nucleoplasmin NLS	Functional Cas staple that can be combined with sgRNA or fgRNA to bring two DNA strands into close proximity
BBa_K5237004	Staple subunit: Oct1-DBD	Staple subunit that can be combined to form a functional staple, for example with TetR. Can also be combined with a fluorescent protein as part of the FRET proximity assay
BBa_K5237005	Staple subunit: TetR	Staple subunit that can be combined to form a functional staple, for example with Oct1. Can also be combined with a fluorescent protein as part of the FRET proximity assay
BBa_K5237006	Simple staple: TetR-Oct1	Functional staple that can be used to bring two DNA strands in close proximity
BBa_K5237007	Staple subunit: GCN4	Staple subunit that can be combined to form a functional staple, for example with rGCN4
BBa_K5237008	Staple subunit: rGCN4	Staple subunit that can be combined to form a functional staple, for example with rGCN4
BBa_K5237009	Mini staple: bGCN4	Assembled staple with minimal size that can be further engineered
Functional elements: Protease-cleavable peptide linkers and inteins are used to control and modify staples for further optimization for custom applications
BBa_K5237010	Cathepsin B-cleavable Linker: GFLG	Cathepsin B-cleavable peptide linker that can be used to combine two staple subunits to make responsive staples
BBa_K5237011	Cathepsin B Expression Cassette	Expression Cassette for the overexpression of cathepsin B
BBa_K5237012	Caged NpuN Intein	A caged NpuN split intein fragment that undergoes protein trans-splicing after protease activation. Can be used to create functionalized staples units
BBa_K5237013	Caged NpuC Intein	A caged NpuC split intein fragment that undergoes protein trans-splicing after protease activation. Can be used to create functionalized staples units
BBa_K5237014	fgRNA processing casette	Processing casette to produce multiple fgRNAs from one transcript, that can be used for multiplexed 3D genome reprograming
BBa_K5237015	Intimin anti-EGFR Nanobody	Interkindom conjugation between bacteria and mammalian cells, as alternative delivery tool for large constructs
Readout Systems: FRET and enhancer recruitment to measure proximity of stapled DNA in bacterial and mammalian living cells enabling swift testing and easy development for new systems
BBa_K5237016	FRET-Donor: mNeonGreen-Oct1	FRET Donor-Fluorpohore fused to Oct1-DBD that binds to the Oct1 binding cassette. Can be used to visualize DNA-DNA proximity
BBa_K5237017	FRET-Acceptor: TetR-mScarlet-I	Acceptor part for the FRET assay binding the TetR binding cassette. Can be used to visualize DNA-DNA proximity
BBa_K5237018	Oct1 Binding Casette	DNA sequence containing 12 Oct1 binding motifs, compatible with various assays such as the FRET proximity assay
BBa_K5237019	TetR Binding Cassette	DNA sequence containing 12 Oct1 binding motifs, can be used for different assays such as the FRET proximity assay
BBa_K5237020	Cathepsin B-Cleavable Trans-Activator: NLS-Gal4-GFLG-VP64	Readout system that responds to protease activity. It was used to test cathepsin B-cleavable linker
BBa_K5237021	NLS-Gal4-VP64	Trans-activating enhancer, that can be used to simulate enhancer hijacking
BBa_K5237022	mCherry Expression Cassette: UAS, minimal Promotor, mCherry	Readout system for enhancer binding. It was used to test cathepsin B-cleavable linker
BBa_K5237023	Oct1 - 5x UAS binding casette	Oct1 and UAS binding cassette, that was used for the simulated enhancer hijacking assay
BBa_K5237024	TRE-minimal promoter- firefly luciferase	Contains Firefly luciferase controlled by a minimal promoter. It was used as a luminescence readout for simulated enhancer hijacking

1. Sequence Overview

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 120
Illegal XhoI site found at 138
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

2. Usage and Biology

This composite part utilizes the 5x Gal4 Upstream Activating Sequence (UAS) to regulate mCherry expression. When Gal4 is present in the cell, its DNA-binding domain (DBD) binds to the UAS, promoting overexpression of mCherry via VP64 (Muench et al., 2023). This results in enhanced production of the mCherry protein, which emits bright red fluorescence, making it an effective reporter for gene expression. This construct enriches our part collection, as it can be used in fluorescence readout assays, such as the one depicted in Figure 2, where it reports the activity of our cathepsin B-cleavable linker (BBa_K5237020).

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.

3. Assembly and Part Evolution

The plasmid was sourced from a plasmid bank, with the mCherry coding sequence located downstream of the 5x Gal4 UAS promoter. No additional modifications were made to the construct, ensuring standard functionality for use in synthetic biology applications.

4. Results

Fluorescence readout assays were performed in HEK293T cells transfected with plasmids encoding eGFP, this composite part, and a Gal4-VP64 construct (BBa_K5237020). The null control was not transfected with a plasmid encoding the Gal4-VP64 construct. As can be seen in Figures 3 and 4, there was a large increase in red fluorescence intensity compared to the null control, confirming successful expression of mCherry under the control of the UAS promoter. This demonstrates the part’s effectiveness as a tool for monitoring Gal4-mediated gene expression in mammalian cells.

Figure 3: Micrographs of HEK293T Cells in Two Conditions. Pictures were taken with a fluorescence microscope 48 hours after transfection. An overlay of brightfield, eGFP and mCherry is shown. Both samples were transfected with plasmids encoding eGFP and the composite part. In the null control (left), no plasmid encoding the Gal4-VP64 construct was transfected, while in the negative control (right), the Gal4-VP64 construct was included. The test sample is not displayed here, as it is irrelevant to this specific comparison.

Figure 4: Fluorescence Readout After 48 Hours for Two Conditions of GFLG Linker. The fluorescence intensity for mCherry was measured for the GFLG linker and normalized against a baseline eGFP fluorescence intensity. Both samples were transfected with plasmids encoding eGFP and the composite part. In the null control (left), no plasmid encoding the Gal4-VP64 construct was transfected, while in the negative control (right), the Gal4-VP64 construct was included. The bars correspond to the micrographs in figure 2.

5. References

Muench, P., Fiumara, M., Southern, N., Coda, D., Aschenbrenner, S., Correia, B., Gräff, J., Niopek, D., & Mathony, J. (2023). A modular toolbox for the optogenetic deactivation of transcription. bioRxiv, 2023.2011.2006.565805. https://doi.org/10.1101/2023.11.06.565805

@@ Line 69: / Line 69: @@
    <section>
+<p><br><br></p>
      <font size="5"><b>The PICasSO Toolbox </b> </font>
-    <p><br></p>
-     <div class="thumb"></div>
+     <div class="thumb" style="margin-top:10px;"></div>
        <div class="thumbinner" style="width:550px"><img alt="" src="https://static.igem.wiki/teams/5237/wetlab-results/registry-part-collection-engineering-cycle-example-overview.svg" style="width:99%;" class="thumbimage">
          <div class="thumbcaption">
-           <i><b>Figure 1: Example how the part collection can be used to engineer new staples</b></i>
+           <i><b>Figure 1: How our part collection can be used to engineer new staples</b></i>
          </div>
        </div>
@@ Line 82: / Line 83: @@
      <p>
        <br>
-       The 3D organization of the genome plays a crucial role in regulating gene expression in eukaryotic cells,
+       Next to the well-studied linear DNA sequence, the 3D spatial organization of DNA plays a crucial role in gene regulation,
-       impacting cellular behavior, evolution, and disease. Beyond the linear DNA sequence, the spatial arrangement of
+       cell fate, disease development and more. However, the tools to precisely manipulate this genomic architecture remain limited, rendering it challenging to explore the full potential of the
-      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
 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>
@@ Line 97: / Line 95: @@
        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
+       parts crucial for testing every step of the cycle (design, build, test, learn) when engineering new parts.
      </p>
@@ Line 103: / Line 101: @@
        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
+       new Cas staples in the future. We also include our Simple staples that serve as controls for successful stapling
        and can be further engineered to create alternative, simpler and more compact staples. <br>
        <b>(ii)</b> As <b>functional elements</b>, we list additional parts that enhance the functionality of our Cas and Basic staples. These
@@ Line 110: / Line 108: @@
        Besides staple functionality, we also include the parts to enable the efficient delivery of PICasSO's constructs with our
        interkingdom conjugation system. <br>
-       <b>(iii)</b> As the final component of our collection, we provide parts that support the use of our <b>custom readout
+       <b>(iii)</b> As the final category of our collection, we provide parts that support the use of our <b>custom readout
          systems</b>. These include components of our established FRET-based proximity assay system, enabling users to
        confirm
        accurate stapling. Additionally, we offer a complementary, application-oriented testing system for functional
-       readout via a luciferase reporter, which allows for straightforward experimental simulation of enhancer hijacking.
+       readouts via a luciferase reporter, which allows for straightforward experimental simulation of enhancer hijacking in mammalian cells.
      </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 style="background-color: #FFD700; color: black;">The highlighted parts showed
-       exceptional performance as described on our iGEM wiki and can serve as a reference. The other parts in the
+       exceptional performance as described on our iGEM wiki and can serve as a reference.</mark> The other parts in the
        collection are versatile building blocks designed to provide future iGEMers with the flexibility to engineer their
        own custom Cas staples, enabling further optimization and innovation.<br>
@@ Line 126: / Line 124: @@
      </p>
-     <table style="width: 90%;">
+     <table style="width: 90%; padding-right:10px;">
        <td colspan="3" align="left"><b>DNA-binding proteins: </b>
          The building blocks for engineering of custom staples for DNA-DNA interactions with a modular system ensuring
@@ Line 133: / Line 131: @@
          <tr bgcolor="#FFD700">
            <td><a href="https://parts.igem.org/Part:BBa_K5237000" target="_blank">BBa_K5237000</a></td>
-           <td>fgRNA Entryvector MbCas12a-SpCas9</td>
+           <td>fgRNA Entry vector MbCas12a-SpCas9</td>
            <td>Entryvector for simple fgRNA cloning via SapI</td>
          </tr>
-         <tr>
+         <tr bgcolor="#FFD700">
            <td><a href="https://parts.igem.org/Part:BBa_K5237001" target="_blank">BBa_K5237001</a></td>
            <td>Staple subunit: dMbCas12a-Nucleoplasmin NLS</td>
-           <td>Staple subunit that can be combined to form a functional staple, for example with fgRNA and dCas9 </td>
+           <td>Staple subunit that can be combined with sgRNA or fgRNA and dCas9 to form a functional staple</td>
          </tr>
-         <tr>
+         <tr bgcolor="#FFD700">
            <td><a href="https://parts.igem.org/Part:BBa_K5237002" target="_blank">BBa_K5237002</a></td>
            <td>Staple subunit: SV40 NLS-dSpCas9-SV40 NLS</td>
-           <td>Staple subunit that can be combined to form a functional staple, for example with our fgRNA or dCas12a
+           <td>Staple subunit that can be combined witha sgRNA or fgRNA and dCas12avto form a functional staple
            </td>
          </tr>
          <tr>
            <td><a href="https://parts.igem.org/Part:BBa_K5237003" target="_blank">BBa_K5237003</a></td>
-           <td>Cas-Staple: SV40 NLS-dMbCas12a-dSpCas9-Nucleoplasmin NLS</td>
+           <td>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 or fgRNA to bring two DNA strands into  close proximity
            </td>
          </tr>
@@ Line 167: / Line 165: @@
          <tr>
            <td><a href="https://parts.igem.org/Part:BBa_K5237006" target="_blank">BBa_K5237006</a></td>
-           <td>Simple taple: TetR-Oct1</td>
+           <td>Simple staple: TetR-Oct1</td>
            <td>Functional staple that can be used to bring two DNA strands in close proximity</td>
          </tr>
@@ Line 188: / Line 186: @@
        </tbody>
        <td colspan="3" align="left"><b>Functional elements: </b>
-         Protease cleavable peptide linkers and inteins are used to control and modify staples for further optimization
+         Protease-cleavable peptide linkers and inteins are used to control and modify staples for further optimization
-         for custom applications.</td>
+         for custom applications</td>
        <tbody>
          <tr bgcolor="#FFD700">
            <td><a href="https://parts.igem.org/Part:BBa_K5237010" target="_blank">BBa_K5237010</a></td>
-           <td>Cathepsin B-Cleavable Linker (GFLG)</td>
+           <td>Cathepsin B-cleavable 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>
          </tr>
@@ Line 200: / Line 198: @@
            <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>
+           <td>Expression Cassette for the overexpression of cathepsin B</td>
          </tr>
          <tr>
            <td><a href="https://parts.igem.org/Part:BBa_K5237012" target="_blank">BBa_K5237012</a></td>
            <td>Caged NpuN Intein</td>
-           <td>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 activation. Can be used to create functionalized staples
              units</td>
          </tr>
@@ Line 211: / Line 209: @@
            <td><a href="https://parts.igem.org/Part:BBa_K5237013" 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
+           <td>A caged NpuC split intein fragment that undergoes protein <i>trans</i>-splicing after protease activation. Can be used to create functionalized staples
              units</td>
          </tr>
@@ Line 217: / Line 215: @@
            <td><a href="https://parts.igem.org/Part:BBa_K5237014" target="_blank">BBa_K5237014</a></td>
            <td>fgRNA processing casette</td>
-           <td>Processing casette to produce multiple fgRNAs from one transcript, can be used for multiplexing</td>
+           <td>Processing casette to produce multiple fgRNAs from one transcript, that can be used for multiplexed 3D genome reprograming</td>
          </tr>
          <tr>
@@ Line 228: / Line 226: @@
        <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>
+         enabling swift testing and easy development for new systems</td>
        <tbody>
          <tr bgcolor="#FFD700">
            <td><a href="https://parts.igem.org/Part:BBa_K5237016" target="_blank">BBa_K5237016</a></td>
            <td>FRET-Donor: mNeonGreen-Oct1</td>
-           <td>Donor part for the FRET assay binding the Oct1 binding cassette. Can be used to visualize DNA-DNA
+           <td>FRET Donor-Fluorpohore fused to Oct1-DBD that binds to the Oct1 binding cassette. Can be used to visualize DNA-DNA
              proximity</td>
          </tr>
@@ Line 245: / Line 243: @@
            <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
+           <td>DNA sequence containing 12 Oct1 binding motifs, compatible with various assays such as the FRET
              proximity assay</td>
          </tr>
@@ Line 256: / Line 254: @@
          <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>
+         <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>
+           <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>
+         <td>Readout system for enhancer binding. It was used to test cathepsin B-cleavable linker</td>
          </tr>
          <tr>
            <td><a href="https://parts.igem.org/Part:BBa_K5237023" target="_blank">BBa_K5237023</a></td>
            <td>Oct1 - 5x UAS binding casette</td>
-           <td>Oct1 and UAS binding cassette, that was used for the simulated enhancer hijacking assay.</td>
+           <td>Oct1 and UAS binding cassette, that was used for the simulated enhancer hijacking assay</td>
          </tr>
          <tr>
@@ Line 276: / Line 274: @@
            <td>TRE-minimal promoter- firefly luciferase</td>
            <td>Contains Firefly luciferase controlled by a minimal promoter. It was used as a luminescence readout for
-             simulated enhancer hijacking.</td>
+             simulated enhancer hijacking</td>
          </tr>
        </tbody>