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Revision as of 20:57, 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.

 



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).

Cathepsin B Fluorescence Readout Assay
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.

Micrographs
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.
Fluorescence Readout Assay
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