Part:BBa_K5237017
FRET-Donor: mNeonGreen-Oct1
This composite part is a fusion protein of Oct1-DBD and mNeonGreen. It was used as a FRET donor in combination with tetR-mScarlet-I as an acceptor. This part was used to measure the proximity of two DNA strands by FRET fluoresence measurements.
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, 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 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 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
(Parker 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.
2. 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.
Fluorescence intensity, normalized to cell count, of mNeonGreen and mScarlet-I was measured 18 h
after induction with varying IPTG concentration (Figure 3). 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. (Figure 3)
5. References
None |