Part:BBa_K5237006
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.
Contents
The PICasSO Toolbox
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 constructs 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 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 Entryvector MbCas12a-SpCas9 | Entryvector for simple fgRNA cloning via SapI |
| BBa_K5237001 | Staple subunit: dMbCas12a-Nucleoplasmin NLS | Staple subunit that can be combined to form a functional staple, for example with fgRNA and dCas9 |
| BBa_K5237002 | Staple subunit: SV40 NLS-dSpCas9-SV40 NLS | Staple subunit that can be combined to form a functional staple, for example with our fgRNA or dCas12a |
| 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 in 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 taple: 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 | Cathepsin B which can be selectively express to cut the cleavable linker |
| BBa_K5237012 | Caged NpuN Intein | Undergoes protein transsplicing after protease activation, can be used to create functionalized staple units |
| BBa_K5237013 | Caged NpuC Intein | Undergoes protein transsplicing after protease activation, can be used to create functionalized staple units |
| BBa_K5237014 | fgRNA processing casette | Processing casette to produce multiple fgRNAs from one transcript, can be used for multiplexing |
| BBa_K5237015 | Intimin anti-EGFR Nanobody | Interkindom conjugation between bacteria and mammalian cells, as alternative delivery tool for large constructs | 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 | Donor part for the FRET assay binding 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, can be used for different 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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 493
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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. 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.
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.
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.
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. 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/10.1186/1472-6750-7-62. Usage and Biology
3. Assembly and part evolution
4. Results
In vitro DNA binding
In vivo DNA binding
5. Conclusion
6. References