Half staple: TetR

The Tetracycline Repressor (tetR) is a bacterial transcriptional regulator that binds the tetO operon. TetR can be readily fused with other DNA-binding proteins to form a functional staple for DNA-DNA proximity. We used this part as a component of our simple staple (BBa_K5237006), and also fused it to mNeonGreen as part of a FRET readout system (BBa_K5237007).

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. These1 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:

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
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 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
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE WITH RFC[1000]
Illegal SapI.rc site found at 466

2. Usage and Biology

The tetracycline repressor protein (tetR) is naturally present in gram-negative bacteria and is involved in the resistance mechanism against tetracycline (and derivatives). It tightly controls gene expression of tetA, which encodes an efflux pump responsible for removing tetracycline from the cell. TetR binds selectively to two plaindromic recognition sequences (tetO>1,2) with high affinity. For DNA binding to occur TetR adopts a homodimeric structure and binds with two α-helix-turn-α-helix motifs (HTH) to two tandemly oriented tetO sequences. In the presence of tetracycline, TetR undergoes a conformational change, which prevents it from binding to DNA, therby allowing gene expression (Orth et al. 2000; Kisker et al. 1995).
Due to its robust and highly regulatable DNA-binding properties, TetR has become a widely adopted tool in synthetic biology. Its ease of modification and ability to function in both prokaryotic and eukaryotic systems have made it an essential element in the development of gene regulation systems (Berens & Hillen, 2004).
Because of its well-characterized behavior, TetR was integrated into our design of a modular DNA-stapling system.

3. Assembly and part evolution

TetR was C-terminally fused to create a tetR-mScarlet-I-His₆.

As part of developing a Förster Resonance Energy Transfer (FRET) assay, a modified version of TetR was created. Based on previous studies that succesfully engineered single-chain TetR (scTetR) proteins with unaltered DNA binding, we genetically fused two TetR proteins together with a flexible (G₄S)₆ linker (Krueger et al. 2003; Zhou et al. 2007). Unfortunately, under the T7 promoter system we tested, the expression levels were insufficient for further experimental use. (More information can be found on our Wiki or the tetR-mScarlet-I composite part)

4. Results

4.1 Protein expression and Mobility Shift Assay

The fusion protein was expressed from a T7 based expression plasmid and subsequently purified using metal affinity chromatography with Ni-NTA beads.(Figure 1, left) DNA binding affinity in two different buffer systems was estimated with an electrophoretic mobility shift assay (EMSA) (Binding buffer 1: 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2HPO4, 0.1 % (v/v) IGEPAL® CA-360, 1 mM EDTA; Binding buffer 2: 10 mM Tris, 50 mM KCl).

Figure 2: Expression and DNA binding analysis of tetR-mScarlet-I-His₆ fusion protein.
Left image: SDS-PAGE analysis of protein expression. Lane 1: raw lysate of E. coli expression culture after steril-filtration; Lane 2: Flow through of first wash; Lane 3: Flow through of second wash; Lane 4: Elution of purified protein. The expected band size of the protein is 50 737.60 Da, highlighted with a red box on the gel.
Right image: Qualitative electrophoretic mobility shift assay of TetR in two different buffer systems. 1 µM protein and 0.5 µM DNA containing three TetR binding sites were equilibrated in different buffer sytstems (Binding buffer 1: 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2HPO4, 0.1 % (v/v) IGEPAL® CA-360, 1 mM EDTA; Binding buffer 2: 10 mM Tris, 50 mM KCl). Bands were visualized by SYBR-safe staining after gel electrophoresis

4.2 In Silico Characterization using DaVinci

We developed the in silico model DaVinci for rapid engineering and development of our PiCasSO system. DaVinci acts as a digital twin to PiCasSO, designed to understand the forces acting on our system, refine experimental parameters, and find optimal connections between protein staples and target DNA. We calibrated DaVinci with literature and our own experimental affinity data calculated from EMSA assays with purified proteins DaVinci is divided into three phases: static structure prediction, all-atom dynamics simulation, and long-ranged DNA dynamics simulation. We applied the first two to our parts, characterizing structure and dynamics of the DNA-binding interaction.
The structures shown in Figure 4 were predicted using the AlphaFold server and the protein-DNA interaction further analyzed with all atom dynamics simulations. The depicted structures show favorable DNA binding, and no apperent problems with the fusion protein and DNA binding were detected.

Figure 4: Representations of the Simple Staple constructs Proteins are shown in full color (top row) and by their predicted structural accuracy during DNA interaction. The linkers were selected based on their structural property providing maximal flexibility. All structures were predicted using the AlphaFold server (Google DeepMind, 2024).

5. References

(Kisker et al., 1995; Krueger et al., 2003; Orth et al., 2000; Zhou et al., 2007)

Kisker, C., Hinrichs, W., Tovar, K., Hillen, W., & Saenger, W. (1995). The Complex Formed Between Tet Repressor and Tetracycline-Mg²⁺ Reveals Mechanism of Antibiotic Resistance. Journal of Molecular Biology, 247(2), 260–280. https://doi.org/10.1006/jmbi.1994.0138

Krueger, C., Berens, C., Schmidt, A., Schnappinger, D., & Hillen, W. (2003). Single-chain Tet transregulators. Nucleic Acids Research, 31(12), 3050–3056.

Orth, P., Schnappinger, D., Hillen, W., Saenger, W., & Hinrichs, W. (2000). Structural basis of gene regulation by the tetracycline inducible Tet repressor-operator system. Nature Structural Biology, 7(3), 215–219. https://doi.org/10.1038/73324

Zhou, X., Symons, J., Hoppes, R., Krueger, C., Berens, C., Hillen, W., Berkhout, B., & Das, A. T. (2007). Improved single-chain transactivators of the Tet-On gene expression system. BMC Biotechnology, 7, 6. https://doi.org/10.1186/1472-6750-7-6

Part:BBa_K5237005