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.

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 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 3.

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

Part:BBa_K5237022