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) resulting in a bivalent DNA binding staple, and also fused to mNeonGreen, as part of a FRET readout system (BBa_K5237007).

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:

1. Sequence overview

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 does so by tightly controlling the 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 or its analogs, 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).
In our project, tetR was integrated into the design of a modular DNA-stapling system because of its well-characterized behavior, ensuring reliable DNA interactions.

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. This was achieved by fusing two tetR proteins using a flexible (G₄S)₆ linker. Previous reports in literature engineered single chain (scTetR) with unaltred DNA binding effiency by fusing to tetR proteins with a (G₄S)₆ linker, also reported in literature (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 EMSA

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 (10 bed volumes of NaP10 (Na₂HPO₄, 150 mM NaCl, 10 mM Imidazol)); Lane 3: Flow through of second wash (10 bed volumes of NaP20 (Na₂HPO₄, 150 mM NaCl, 20 mM Imidazol)); 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

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