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. 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.
Contents
While synthetic biology has in the past focused on engineering the genomic sequence of organisms, the 3D
spatial organization of DNA is well-known to be an important layer of information encoding in
particular in eukaryotes, playing a crucial role in
gene regulation and hence
cell fate, disease development, evolution, and more. However, tools to precisely manipulate and control the
genomic spatial
architecture are limited, hampering the exploration of
3D genome engineering in synthetic biology. We - the iGEM Team Heidelberg 2024 - have developed PICasSO, a
powerful
molecular toolbox for rationally engineering genome 3D architectures in living cells, based on
various DNA-binding proteins.
The PICasSO part collection offers a comprehensive, modular platform for precise manipulation and re-programming of DNA-DNA interactions using engineered "protein staples" in living cells. This enables researchers to recreate naturally occurring alterations of 3D genomic interactions, such as enhancer hijacking in cancer, or to design entirely new spatial architectures for artificial gene regulation and cell function control. Specifically, the fusion of two DNA binding proteins enables to artificially bring otherwise distant genomic loci into spatial proximity. To unlock the system's full potential, we introduce versatile chimeric CRISPR/Cas complexes, connected either at the protein or - in the case of CRISPR/Cas-based DNA binding moieties - the guide RNA level. These complexes are referred to as protein- or Cas staples, respectively. Beyond its versatility with regard to the staple constructs themselves, PICasSO includes robust assay systems to support the engineering, optimization, and testing of new staples in vitro and in vivo. Notably, the PICasSO toolbox was developed in a design-build-test-learn engineering cycle closely intertwining wet lab experiments and computational modeling and iterated several times, yielding a collection of well-functioning and -characterized parts.
At its heart, the PICasSO part collection consists of three categories.
(i) Our DNA-binding
proteins
include our
finalized Cas staple experimentally validated using an artificial "enhancer hijacking" system as well as
"half staples" that can be combined by scientists to compose entirely
new Cas staples in the future. We also include our Simple staples comprised of particularly small, simple
and robust DNA binding domains well-known to the synthetic biology community, which 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 and expand the
functionality of our Cas and
Basic staples. These
consist of staples dependent on
cleavable peptide linkers targeted by cancer-specific proteases or inteins that allow condition-specific,
dynamic stapling in vivo.
We also include several engineered parts that enable the efficient delivery of PICasSO's constructs into
target cells, including mammalian cells,
with our new
interkingdom conjugation system.
(iii) As the final category of our collection, we provide parts that underlie 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 based on a
luciferase reporter, which allows for straightforward experimental assessment of functional enhancer
hijacking events
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 in the new field of 3D genome
engineering.
Our part collection includes:
| DNA-Binding Proteins: Modular building blocks for engineering of custom staples to mediate defined DNA-DNA interactions in vivo | ||
| BBa_K5237000 | Fusion Guide RNA Entry Vector MbCas12a-SpCas9 | Entry vector for simple fgRNA cloning via SapI |
| BBa_K5237001 | Staple Subunit: dMbCas12a-Nucleoplasmin NLS | Staple subunit that can be combined with crRNA or fgRNA and dSpCas9 to form a functional staple |
| BBa_K5237002 | Staple Subunit: SV40 NLS-dSpCas9-SV40 NLS | Staple subunit that can be combined with a sgRNA or fgRNA and dMbCas12a to form a functional staple |
| BBa_K5237003 | Cas Staple: SV40 NLS-dMbCas12a-dSpCas9-Nucleoplasmin NLS | Functional Cas staple that can be combined with sgRNA and crRNA 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, which can be used to create functionalized staple subunits |
| BBa_K5237013 | Caged NpuC Intein | A caged NpuC split intein fragment that undergoes protein trans-splicing after protease activation, which can be used to create functionalized staple subunits |
| BBa_K5237014 | Fusion Guide RNA Processing Casette | Processing cassette to produce multiple fgRNAs from one transcript, that can be used for multiplexed 3D genome reprogramming |
| BBa_K5237015 | Intimin anti-EGFR Nanobody | Interkingdom conjugation between bacteria and mammalian cells, as an 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 readout systems to rapidly assess successful DNA stapling in bacterial and mammalian cells |
| BBa_K5237016 | FRET-Donor: mNeonGreen-Oct1 | FRET donor-fluorophore fused to Oct1-DBD that binds to the Oct1 binding cassette, which 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, which 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, which 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 Promoter, mCherry | Readout system for enhancer binding, which 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, which was used as a luminescence readout for simulated enhancer hijacking |
1. Sequence overview
- 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 (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. The amino acid sequence of TetR and Oct1 were obtained from the UniProt database (P04483 and P14859, respectively).
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.
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).
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 fluorescence 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.
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 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
4.1 In Vitro DNA Binding
4.2 In vivo DNA binding
4.3 In Silico Characterization using DaVinci
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 apparent
problems with the fusion protein and DNA binding were detected.
5. Conclusion
6. References
| None |