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, combining the human Oct1 DNA-binding domain (Oct1-DBD) and the bacterial tetracycline repressor protein (TetR). Oct1-DBD recognizes the octamer motif, while 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: Example how the part collection can be used to engineer new staples

The 3D organization of the genome plays a crucial role in regulating gene expression in eukaryotic cells, impacting cellular behavior, evolution, and disease. Beyond the linear DNA sequence, the spatial arrangement of chromatin, influenced by DNA-DNA interactions, shapes pathways of gene regulation. 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 with our interkingdom conjugation system.

(iii) As the final component 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 readout via a luciferase reporter, which allows for straightforward experimental simulation of enhancer hijacking.

The following table gives a complete 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 parts collection includes:

1. Sequence overview

2. Usage and Biology

The Simple Staple (Oct1-DBD-TetR 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. It binds selectively to 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 controlled 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 and preserving DNA-binding capabilities (Park et al., 2013; Stepchenko et al., 2021).

By fusing these two proteins, the Simple staple was developed to bridge DNA sequences carrying their respective binding motifs. 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 Oct1-DBD amino acid sequence was obtained from UniProt (P14859, POU domain, class 2, transcription factor 1) and DNA binding domain extracted based on information given from Park et al. 2013 & 2020. TetR amino acid sequence was obtaine from UniProt(P04483). 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

In vitro DNA binding

The Simple staple construct was modified with a C-terminal His₆-tag and expressed under T7 promoter. Protein was purified with a Ni-NTA affinity column. Fractions were analysed on a 4-15 % SDS-Page (Fig. 2, left). Strong bands of the protein of interest are 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 showed 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.

Electrophoretic Mobility Shift Assay (EMSA) was performedn. Varying concentration of the purified Simple staple (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. This shows that the Simple staple is able to bind both DNA sequences in vitro.

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 fraction. 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)

In vivo DNA binding

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 fusion protein that acts as a bivalent DNA-binding protein, tethering two plasmids via 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 an Oct1 binding site for the staple and FRET donor binding.

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 fluoresence 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 3: 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 withŠidák's multiple comparison test, with a single pooled variance. *p < 0.05, ****p < 0.001. Data is depicted as mean (n=3) ± SD

5. References

Berens, C., & Hillen, W. (2004). Gene regulation by tetracyclines. In J. K. Setlow (Ed.), Genetic engineering: Principles and methods (pp. 255–277). Springer US. https://doi.org/10.1007/978-0-306-48573-2_13

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/10.1038/73318

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

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

Wu, W., Zhang, L., Yao, L., Tan, X., Liu, X., & Lu, X. (2015). Genetically assembled fluorescent biosensor for in situ detection of bio-synthesized alkanes. Scientific Reports, 5, 10907. https://doi.org/10.1038/srep10907

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.1995.0130