Half-Staple: Oct1-DBD

Oct1-DBD is the DNA-binding domain of the human Oct1 transcription factor, binding specifically to the octamer motif (5'-ATGCAAAT-3') with high affinity and stability.

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:

1. Sequence overview

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 (K_D = 9 &times 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. [!!Picture of SDS-Page sds-page-mng-oct1-expression.svg] 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 SDS-PAGE analysis of His₆-mNeonGreen-Oct1-DBD
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, which is highlited in red on the gel.