Simple staple: TetR-Oct1

The Simple staple (Oct1-DBD-TetR fusion) is a bivalent DNA-binding protein designed to bring two DNA sequences into close proximity. The Oct1 DNA-binding domain (Oct1-DBD) recognizes the octamer motif, while the tetracycline repressor protein (TetR) binds specifically to the tetO operator sequences. This Simple Staple was applied to establish a Förster Resonance Energy Transfer (FRET)-based assay, which was used to monitor DNA-DNA proximity in bacterial systems.

Figure 1: How our part collection can be used to engineer new staples

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:

1. Sequence overview

2. Usage and Biology

The Simple Staple (TetR-Oct1-DBD fusion) combines the well-characterized bacterial transcriptional repressor TetR with the human transcription factor Oct1-DBD, creating a versatile DNA-binding protein capable of bringing two DNA sequences into proximity. TetR naturally functions in gram-negative bacteria by regulating the expression of the tetA gene in response to tetracycline (and derivatives). It binds selectively to the palindromic tetO sequences with high affinity, forming a homodimer that dissociates upon exposure to tetracycline, allowing gene expression (Berens & Hillen, 2004). Its well-understood DNA-binding properties make it a reliable component in synthetic biology, particularly in systems where controllable DNA interactions are crucial.

Oct1-DBD is a component of the human transcription factor Oct1, involved in immune regulation and stress responses. It binds specifically to the octamer motif (5'-ATGCAAAT-3') in promoter and enhancer regions, stabilizing DNA binding through its POU-specific and POU homeodomains (Lundbäck et al., 2000). Previous studies have demonstrated that Oct1-DBD can be readily fused to other proteins, increasing solubility whilst preserving DNA-binding capabilities (Park et al., 2013; Stepchenko et al., 2021).

The Simple staple was developed by fusing TetR to Oct1-DBD, is capable of bridging two DNA sequences carrying their specific binding sequences, and thus bringing them into close proximity. This bivalent DNA-binding system was successfully applied in our project to establish a FRET-based proximity assay, enabling real-time monitoring of DNA interactions in bacterial systems. This versatile and modular approach opens up new possibilities for synthetic gene regulation and spatial genome organization.

3. Assembly and Part Evolution

The amino acid sequence of TetR and Oct1 were obtained from the UniProt database (P04483 and P14859, respectively). The DNA binding domain for Oct1-DBD was extracted based on information given from Park et al. 2013 & 2020. Coding sequences were codon optimized for E. coli and obtained through gene synthesis. The proteins were genetically linked with a short GSGGS linker.

4. Results

4.1 In Vitro DNA Binding

The Simple staple construct was modified with a C-terminal His₆-tag and expressed under the T7 promoter. The protein was purified with a Ni-NTA affinity column and fractions were analysed on a 4-15 % SDS-Page (Fig. 2, left). Strong bands for the protein of interest were visible in the raw lysate indicating strong expression. Even though a strong band was seen in the flow-through, indicating unbound protein of interest, the purified fraction had a strong band with almost no unspecific proteins co-purified. The eluate contained 1.5 mg/mL protein, resulting in a total of ~ 3.34 mg purified protein.

For the Electrophoretic Mobility Shift Assay (EMSA), varying concentration of the purified protein (15 µM, 7.5 µM, 3.75 µM, 1.875 µM, 0.9375 µM) were incubated with 0.5 µM of annealed oligos containing either the Oct1 (5'-ATGCAAAT-3') or tetO (5'TCCCTATCAGTGATAGAGA3') binding site. A clear, concentration dependant, shift could be detected for both target sites. Indicating that the Simple staple is able to bind both DNA sequences in vitro. Incubation of the protein with both DNA sequences did not result in slower migration speed compared to the single binding sites (data not shown).

Figure 2: SDS-PAGE and EMSA Analysis of the TetR-Oct1 Fusion Protein. Left: Fractions were loaded on a 4-15 % SDS-PAGE gel and stained with coomassie blue. Lane 1: raw lysate, Lane 2: flow through, Lane 3: purified protein. Right: Electrophoretic Mobility Shift Assay of TetR-Oct1 in PBS (15 µM, 7.5 µM, 3.75 µM, 1.875 µM, 0.9375 µM protein with 0.5 µM DNA), post stained with SYBR-safe.

4.2 In vivo DNA binding

Figure 3: Schematic of FRET-based Proximity Assay for DNA Stapling. A In the absence of TetR-Oct1 no stapling occurs B Oct1-TetR staples together the plasmids and brings FRET pairs in close proximity, resulting in measurable fluorescence

The Förster Resonance Energy Transfer (FRET) assay was developed using a two-plasmid system in bacterial cells. The expression plasmid contains a TetR binding site and expresses three key proteins under the control of a single T7 promoter in a polycistronic operon: (1) TetR-Oct1, our Simple staple a bivalent DNA-binding fusion protein, tethering together two plasmids by binding the TetR and Oct1 binding sites (BBa_K5237019, BBa_K5237018); (2) Oct1-mNeonGreen (BBa_K2375016), serving as the FRET-donor; and (3) TetR-mScarlet-I (BBa_K2375017), the FRET-acceptor. This ensures all three proteins are co-expressed in similar stoichiometry. The folding plasmid contains 12 repeats of the Oct1 binding site for binding of the staple and FRET-donor.

When TetR-Oct1 binds its respective sites on both plasmids, it brings mNeonGreen and mScarlet-I into close proximity, facilitating FRET between the two fluorophores. Successful stapling of the plasmids results in increased energy transfer from mNeonGreen to mScarlet-I, which can be detected by exciting mNeonGreen and measuring enhanced emission from mScarlet-I. A positive control, consisting of a direct fusion of mNeonGreen and mScarlet-I, ensures maximal FRET efficiency and serves as a benchmark for the assay.

Samples were induced with 0.05 mM IPTG and fluorescence intensity of mNeonGreen, mScarlet-I and FRET was measured after 18 h. he positive control exhibited significantly higher fluorescence intensity for both mNeonGreen and mScarlet-I, indicating stronger expression levels of the FRET pair in this condition. Both the negative control and the staple showed comparable fluorescence for mNeonGreen and mScarlet-I. A small but significant difference was observed for mNeonGreen (p = 0.0416). Importantly the measured FRET signal was significantly higher for the sample compared to the negative control (p < 0.0001). This indicates that the TetR-Oct1 staple successfully brought the two plasmids into close proximity, allowing for FRET to occur.

Figure 4: Fluorescence Measurement of mNeonGreen, mScarlet-I and FRET. Fluorescence intensity of mNeonGreen (ex. 490 nm, em. 530 nm), mScarlet-I (ex. 560 nm, em. 600 nm), and FRET (ex. 490 nm, em. 600 nm) was measured 18 hours after IPTG induction (0.05 mM) and normalized to cell count (OD₆₀₀). Statistical significance was determined with Ordinary two-way ANOVA with Šidák's multiple comparison test, with a single pooled variance. *p < 0.05, ****p < 0.001. Data is depicted as mean (n=3) ± SD

4.3 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 5 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 apparent problems with the fusion protein and DNA binding were detected.

Figure 5: 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. Conclusion

The Simple staple (Oct1-DBD-TetR fusion) is a versatile bivalent DNA-binding protein that brings two DNA sequences into close proximity. It was successfully applied to establish a FRET-based assay to monitor DNA-DNA proximity in bacterial systems. The results demonstrate the Simple Staple's functionality in both in vitro and in vivo settings, highlighting its potential for future applications in gene regulation and spatial genome organization.

6. References

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.

Lundbäck, T., Chang, J.-F., Phillips, K., Luisi, B., & Ladbury, J. E. (2000). Characterization of Sequence-Specific DNA Binding by the Transcription Factor Oct-1. Biochemistry, 39(25), 7570–7579. https://doi.org/10.1021/bi000377h

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

Park, J. H., Kwon, H. W., & Jeong, K. J. (2013). Development of a plasmid display system with an Oct-1 DNA-binding domain suitable for in vitro screening of engineered proteins. Journal of Bioscience and Bioengineering, 116(2), 246–252. https://doi.org/10.1016/j.jbiosc.2013.02.005

Park, Y., Shin, J., Yang, J., Kim, H., Jung, Y., Oh, H., Kim, Y., Hwang, J., Park, M., Ban, C., Jeong, K. J., Kim, S.-K., & Kweon, D.-H. (2020). Plasmid Display for Stabilization of Enzymes Inside the Cell to Improve Whole-Cell Biotransformation Efficiency. Frontiers in Bioengineering and Biotechnology, 7. https://doi.org/10.3389/fbioe.2019.00444

Stepchenko, A. G., Portseva, T. N., Glukhov, I. A., Kotnova, A. P., Lyanova, B. M., Georgieva, S. G., & Pankratova, E. V. (2021). Primate-specific stress-induced transcription factor POU2F1Z protects human neuronal cells from stress. Scientific Reports, 11(1), 18808. https://doi.org/10.1038/s41598-021-98323-y

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