Part:BBa_K5237004
Half-Staple: Oct1-DBD
Oct1-DBD (BBa_K5237004) is the DNA-binding domain of the human Oct1 transcription factor (POU2F1), which binds specifically to the octamer motif (5'-ATGCAAAT-3'). This domain is key to regulating gene expression, immune response, and stress adaptation.
Contents
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, tools to precisely manipulate this genomic architecture remain limited, making it challenging to explore the full potential of the 3D genome in synthetic biology. To address this issue, team Heidelberg developed PICasSO.
The PICasSO part collection offers a comprehensive, modular platform for precise manipulation of DNA-DNA proximity in living cells, enabling researchers to recreate natural 3D genomic interactions, such as enhancer hijacking, or design entirely new spatial architectures for gene regulation. Beyond its versatility, PICasSO includes robust measurement systems to support the engineering, optimization, and testing of new staples, ensuring functionality both in vivo and in vitro.
With its combination of staple systems, functionalization options, and measurement tools, PICasSO provides a complete solution for designing, testing, and refining new systems for spatial DNA organization. This toolbox unlocks new possibilities in synthetic biology, from studying fundamental genomic interactions to creating innovative gene therapies.
Our parts 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 Entryvector MbCas12a-SpCas9 |
| BBa_K5237001 | Half-Staple: dMbCas12a-Nucleoplasmin NLS |
| BBa_K5237002 | Half-Staple: SV40 NLS-dSpCas9-SV40 NLS |
| BBa_K5237003 | Cas-Staple: SV40 NLS-dMbCas12a-dSpCas9-Nucleoplasmin NLS |
| BBa_K5237004 | Half-Staple: Oct1-DBD |
| BBa_K5237005 | Half-Staple: TetR |
| BBa_K5237006 | Simple-Staple: TetR-Oct1 |
| BBa_K5237007 | Half-Staple: GCN4 |
| BBa_K5237008 | Half-Staple: rGCN4 |
| BBa_K5237009 | Mini-Staple: bGCN4 | 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) |
| BBa_K5237011 | Cathepsin B Expression Cassette |
| BBa_K5237012 | Caged NpuN Intein |
| BBa_K5237013 | Caged NpuC Intein |
| BBa_K5237014 | fgRNA processing casette |
| BBa_K5237015 | Intimin anti-EGFR Nanobody | 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 |
| BBa_K5237017 | FRET-Acceptor: TetR-mScarlet-I |
| BBa_K5237018 | Oct1 Binding Casette |
| BBa_K5237019 | TetR Binding Cassette | BBa_K5237020 | Cathepsin B-Cleavable Trans-Activator: NLS-Gal4-GFLG-VP64 |
| BBa_K5237021 | NLS-Gal4-VP64 | BBa_K5237022 | mCherry Expression Cassette: UAS, minimal Promotor, mCherry |
| BBa_K5237023 | UAS binding Firefly Luciferase Readout System |
1. Sequence overview
Sequence and Features
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2. Usage and Biology
Oct1-DBD is the DNA-binding domain of the human transcription factor Oct1 (POU2F1), which plays a key role in gene regulation, immune response, and stress adaptation in eukaryotic cells. This domain specifically binds to the octamer motif (5’-ATGCAAAT-3’) within promoter and enhancer regions, influencing transcriptional activity (Lundbäck et al., 2000). The Oct1-DBD consists of both a POU-specific domain and a POU homeodomain, which work together to form a stable complex with DNA (Park et al., 2013, Stepchenko et al. 2021).
In synthetic biology, Oct1-DBD was previously used for plasmid display technology due to its strong binding affinity (KD = 9 × 10-11 M). Proteins fused with Oct1-DBD showed increased expression and protein solubility (Parker et al. 2020).
This part was further used in BBa_K5237002 as a fusion with tetR, resulting in a bivalent DNA binding staple, and also fused to mNeonGree, as part of a FRET readout system (BBa_K5237016).
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. An E. coli codon optimized DNA sequence was obtained through gene synthesis and used to clone further constructs
4. Results
Oct1 was N-terminally fused to the His6-mNeonGreen, and expressed under a T7 expression protein and subsequently purified using metal affinity chromatography with Ni-NTA beads.(Figure 1, left) DNA binding affinity was estimated with an electrophoretic mobility shift assay (EMSA). For this, three different buffer conditions were tested (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; NaP250: Na2HPO4, 150 mM NaCl, 250 mM Imidazol). DNA binding could only be detected for Binding buffer 1. (Figure 1, right)
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.
1 µL of each fraction was loaded after mixing and heating with 4x Laeemli buffer, on a 4-15% TGX-Gel. The expected band size of the protein is 56 840.23 Da, highlighted in red on the gel.
5. References
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
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
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