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

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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