Revision as of 20:57, 30 September 2024 (view source) Boninvi (Talk | contribs) ← Older edit		Revision as of 23:25, 30 September 2024 (view source) Boninvi (Talk | contribs) Newer edit →
Line 228:		Line 228:
	<td>Interkindom conjugation between bacteria and mammalian cells, as alternative delivery tool for large		<td>Interkindom conjugation between bacteria and mammalian cells, as 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>

---

Revision as of 23:25, 30 September 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. 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.