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.

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
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 493
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

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, resepctively). 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 fluoresence

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 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 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 withŠidák'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 apperent 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

Part:BBa_K5237006