Part:BBa_K5237016
FRET-Donor: mNeonGreen-Oct1
This composite part is a fusion protein of our half-staple Oct1-DBD and mNeonGreen. It was used as a FRET donor in combination with tetR-mScarlet-I as the acceptor (BBa_K5237017). Together, they are the foundation of our proximity measurement setup using FRET measurements.
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 710
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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
(Park et al. 2020).
mNeonGreen is a bright, monomeric fluorescent protein from Branchiostoma lanceolatum discovered by Shaner
et al. (2013). It exhibits fast maturation, high photostability, and a high quantum yield. With an
excitation peak
at 506 nm and an emission maximum at 517 nm, mNeonGreen is ideal for bioimaging applications (Shaner et
al.,
2013). Its high quantum yield and stability make it an optimal electron donor for Förster Resonance Energy
Transfer (FRET). When paired with mScarlet-I, it generates three times the intensity compared to mCherry.
Förster Resonance Energy Transfer (FRET) is a distance-dependent physical process where energy is transferred
non-radiatively from an excited donor fluorophore to an acceptor fluorophore via dipole-dipole coupling. The
efficiency of energy transfer is highly sensitive to the distance between the donor and acceptor, typically in the
range of 1-10 nm, making FRET ideal for studying molecular proximity (Hochreiter et al., 2019). This
proximity
sensitivity is governed by the Förster radius (R₀), which is the distance at which 50% energy transfer occurs.
Factors affecting FRET efficiency include the overlap of the donor's emission spectrum with the acceptor's
absorption spectrum, the quantum yield of the donor, and the relative orientation of the fluorophores (Wu &
Brand,
1994). These characteristics allow FRET to detect interactions such as protein-DNA binding or DNA proximity in
real time.
For our assay, we selected mNeonGreen and mScarlet-I as donor and acceptor, respectively, due to their strong
fluorescence, spectral overlap, and minimal photobleaching, ensuring high FRET efficiency in our system (Bindels
et al., 2017; Shaner et al., 2013). FRET's sensitivity to small changes in distance makes it
especially powerful
for analyzing molecular interactions in living cells (Okamoto & Sako, 2017).
The amino acid sequence of mNeonGreen was taken from the fluorescent protein database (FPbase) and codon optimized for use in E. coli.
It was fused to thhe N-terminus of Oct1-DBD (BBa_K52347004)
for protein purification of Oct1-DBD and in vivo FRET measurements.
The FRET assay was developed using a two-plasmid system in bacterial cells. The expression plasmid
contains a tetR binding site (BBa_K5237019)
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; (2)
Oct1-mNeonGreen, serving as the FRET donor; and (3) tetR-mScarlet-I, the FRET acceptor. This
ensures all three proteins are co-expressed in similar stoichiometry. The folding plasmid contains
an Oct1 binding site (BBa_K5237018) for the staple and FRET
donor binding.
Fluorescence intensity, normalized to cell count, of mNeonGreen and mScarlet-I was measured 18 h
after induction with varying IPTG concentration (Fig. 4). An increasing
expression strength
is visible for decreasing IPTG concentrations. Fluorescence intensity of the positive control was
significantly stronger compared to the negative control and staple. The negative control and
staple, which both have the same expression plasmid construct, had similar fluorescence intensity
for mNeonGreen and mScarlet-I down to approximately 0.05 mM. Lower concentrations resulted in
strong discrepancies. To ensure comparability between the negative control and staple, further
fluorescence intensity measurements were conducted after induction with 0.05 mM IPTG. Fluorescence
measurement of the donor and acceptor showed similar intensities, with only a small significant
difference for mNeonGreen. A large difference could be measured between the staple and negative
control, indicating proximity induced FRET. (Fig. 4)
Our FRET assay successfully demonstrated the proximity of two DNA strands in living cells using mNeonGreen and
mScarlet-I as a donor-acceptor pair. The assay was optimized for maximal FRET efficiency and validated with a
positive control. The results showed a significant difference in fluorescence intensity between the staple and
negative control, indicating successful DNA stapling and FRET. This assay provides a powerful tool to engineer and
test out novel Staples.
Future work will focus on further optimizing the assay in mammalian cells and quantifying interactions.
Bindels, D. S., Haarbosch, L., van Weeren, L., Postma, M., Wiese, K. E., Mastop, M., Aumonier, S., Gotthard, G.,
Royant, A., Hink, M. A., & Gadella, T. W. J. (2017). mScarlet: A bright monomeric red fluorescent protein for
cellular imaging. Nature Methods, 14(1), 53–56. https://doi.org/10.1038/nmeth.4074 Hochreiter, B., Kunze, M., Moser, B., & Schmid, J. A. (2019). Advanced FRET normalization allows quantitative
analysis of protein interactions including stoichiometries and relative affinities in living cells. Scientific
Reports, 9(1), 8233. https://doi.org/10.1038/s41598-019-44650-0 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 Okamoto, K., & Sako, Y. (2017). Recent advances in FRET for the study of protein interactions and dynamics.
Current Opinion in Structural Biology, 46, 16–23. https://doi.org/10.1016/j.sbi.2017.03.010 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 Shaner, N. C., Lambert, G. G., Chammas, A., Ni, Y., Cranfill, P. J., Baird, M. A., Sell, B. R., Allen, J. R., Day,
R. N., Israelsson, M., Davidson, M. W., & Wang, J. (2013). A bright monomeric green fluorescent protein derived
from Branchiostoma lanceolatum. Nature Methods, 10(5), 407–409. https://doi.org/10.1038/nmeth.2413 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 Wu, P. G., & Brand, L. (1994). Resonance Energy Transfer: Methods and Applications. Analytical
Biochemistry, 218(1), 1–13. https://doi.org/10.1006/abio.1994.11342. Usage and Biology
2.1 Oct1-DBD
2.2 mNeonGreen
2.3 Förster Resonance Energy Transfer (FRET)
3. Assembly and Part Evolution
4. Results
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.
5. Conclusion
6. References
| None |