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	<section>		<section>
−	<p><br><br></p>	+	<p><br><br></p>
	<font size="5"><b>The PICasSO Toolbox </b> </font>		<font size="5"><b>The PICasSO Toolbox </b> </font>
	<div class="thumb" style="margin-top:10px;"></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="thumbinner" style="width:550px"><img alt=""
−	<div class="thumbcaption">	+	src="https://static.igem.wiki/teams/5237/wetlab-results/registry-part-collection-engineering-cycle-example-overview.svg"
−	<i><b>Figure 1: How our part collection can be used to engineer new staples</b></i>	+	style="width:99%;" class="thumbimage">
−	</div>	+	<div class="thumbcaption">
		+	<i><b>Figure 1: How our part collection can be used to engineer new staples</b></i>
	</div>		</div>
	</div>		</div>
−		+	</div>
		+
	<p>		<p>
	<br>		<br>
−	Next to the well-studied linear DNA sequence, the 3D spatial organization of DNA plays a crucial role in gene regulation,	+	Next to the well-studied linear DNA sequence, the <b>3D spatial organization</b> of DNA plays a crucial role in
−	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	+	gene regulation,
−	3D genome in synthetic biology. We - iGEM Team Heidelberg 2024 - have developed PICasSO, a powerful molecular	+	cell fate, disease development and more. However, the <b>tools</b> to precisely manipulate this genomic
−	toolbox based on various DNA-binding proteins to address this issue.	+	architecture <b>remain limited</b>, rendering it challenging to explore the full potential of the
		+	3D genome in synthetic biology. We - iGEM Team Heidelberg 2024 - have developed PICasSO, a <b>powerful
		+	molecular toolbox</b> based on various DNA-binding proteins to address this issue.
	</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 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.		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	+	Specifically, the fusion of two DNA binding proteins enables to artifically bring distant genomic loci into
		+	proximty.
		+	To unlock the system's full potential, we introduce versatile chimeric CRISPR/Cas complexes, connected either on
		+	the protein or the guide RNA level. These complexes are reffered to as protein- or Cas staples. 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		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>		</p>
−	<p>At its heart, the PICasSO part collection consists of three categories. <br><b>(i)</b> Our <b>DNA-binding proteins</b>	+	<p>At its heart, the PICasSO part collection consists of three categories. <br><b>(i)</b> 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 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>		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	+	<b>(ii)</b> As <b>functional elements</b>, we list additional parts that enhance the functionality of our Cas and
		+	Basic staples. These
	consist of		consist of
	protease-cleavable peptide linkers and inteins that allow condition-specific, dynamic stapling <i>in vivo</i>.		protease-cleavable peptide linkers and inteins that allow condition-specific, dynamic stapling <i>in vivo</i>.
−	Besides staple functionality, we also include the parts to enable the efficient delivery of PICasSO's constructs with our	+	Besides staple functionality, we also include the parts to enable the efficient delivery of PICasSO's constructs
		+	with our
	interkingdom conjugation system. <br>		interkingdom conjugation system. <br>
−	<b>(iii)</b> As the final category of our collection, we provide parts that support the use of our <b>custom readout	+	<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		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 for functional
−	readouts via a luciferase reporter, which allows for straightforward experimental simulation of enhancer hijacking in mammalian cells.	+	readouts via a luciferase reporter, which allows for straightforward experimental simulation of enhancer hijacking
		+	in mammalian cells.
	</p>		</p>
	<p>		<p>
−	The following table gives a comprehensive overview of all parts in our PICasSO toolbox. <mark style="background-color: #FFD700; color: black;">The highlighted parts showed	+	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.</mark> The other parts in the	+	style="background-color: #FFD700; color: black;">The highlighted parts showed
		+	exceptional performance as described on our iGEM wiki and can serve as a reference.</mark> The other parts in
		+	the
	collection are versatile building blocks designed to provide future iGEMers with the flexibility to engineer their		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>		own custom Cas staples, enabling further optimization and innovation.<br>
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	<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 into close proximity	+	<td>Functional Cas staple that can be combined with sgRNA or fgRNA to bring two DNA strands into close
		+	proximity
	</td>		</td>
	</tr>		</tr>
Line 209:		Line 225:
	<td><a href="https://parts.igem.org/Part:BBa_K5237012" 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>A caged NpuN split intein fragment that undergoes protein <i>trans</i>-splicing after protease activation. Can be used to create functionalized staples	+	<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>		units</td>
	</tr>		</tr>
Line 215:		Line 232:
	<td><a href="https://parts.igem.org/Part:BBa_K5237013" 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>A caged NpuC split intein fragment that undergoes protein <i>trans</i>-splicing after protease activation. Can be used to create functionalized staples	+	<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>		units</td>
	</tr>		</tr>
Line 221:		Line 239:
	<td><a href="https://parts.igem.org/Part:BBa_K5237014" 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>fgRNA processing casette</td>
−	<td>Processing casette to produce multiple fgRNAs from one transcript, that can be used for multiplexed 3D genome reprograming</td>	+	<td>Processing casette to produce multiple fgRNAs from one transcript, that can be used for multiplexed 3D
		+	genome reprograming</td>
	</tr>		</tr>
	<tr>		<tr>
Line 232:		Line 251:
	<td><a href="https://parts.igem.org/Part:BBa_K4643003" target="_blank">BBa_K4643003</a></td>		<td><a href="https://parts.igem.org/Part:BBa_K4643003" target="_blank">BBa_K4643003</a></td>
	<td>incP origin of transfer</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>	+	<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>
Line 242:		Line 262:
	<td><a href="https://parts.igem.org/Part:BBa_K5237016" 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>FRET Donor-Fluorpohore fused to Oct1-DBD that binds to 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>		proximity</td>
	</tr>		</tr>

---

Revision as of 17:35, 1 October 2024

BBa_K5237022

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.

 

Contents

• 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. Specifically, the fusion of two DNA binding proteins enables to artifically bring distant genomic loci into proximty. To unlock the system's full potential, we introduce versatile chimeric CRISPR/Cas complexes, connected either on the protein or the guide RNA level. These complexes are reffered to as protein- or Cas staples. 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:

Sequence and Features

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.

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.