Part:BBa_K5237017
FRET-Acceptor: TetR-mScarlet-I
This composite part is a fusion protein of TetR and the red fluorescent protein mScarlet-I. It was used as a FRET acceptor in combination with mNeonGreen-Oct1 as the donor. This part was used to measure the proximity of two DNA strands by FRET fluorescence 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]
- 12INCOMPATIBLE WITH RFC[12]Illegal NotI site found at 1149
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 623
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 466
The tetracycline repressor protein (tetR) is naturally present in gram-negative bacteria and is involved in the
resistance mechanism against tetracycline (and derivatives). It does so by tightly controlling the gene expression
of tetA, which encodes an efflux pump responsible for removing tetracycline from the cell.
TetR binds selectively to two plaindromic recognition sequences (tetO>1,2) with high affinity. For DNA
binding to occur tetR adopts a homodimeric structure and binds with two α-helix-turn- α-helix motifs
(HTH) to two tandemly oriented tetO sequences. In the presence of tetracycline or its analogs, tetR undergoes a
conformational change, which prevents it from binding to DNA, therby allowing gene expression(Orth et al.
2000; Kisker et al. 1995).
mScarlet-I is a rapidly-maturing monomeric red fluorescent protein designed by Bindels et al. in 2016. It
is a derivative of the original mScarlet, with a single amino acid substitution (T74I) that enhances its
maturation
speed and fluorescence properties (Bindels et al., 2016). mScarlet-I absorbs at 569 nm and emits at 594 nm.
mScarlet-I stands out among red fluorescent proteins for its high quantum yield (0.54), long fluorescence lifetime
(3.1 ns), and rapid maturation time of approximately 36 minutes. These features ensure strong fluorescence
intensity and make it an ideal candidate for Förster Resonance Energy Transfer assays (McCullock et al. 2020). When paired with mNeonGreen, mScarlet-I
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 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.
Wu, W., Zhang, L., Yao, L., Tan, X., Liu, X., & Lu, X. (2015). Genetically assembled fluorescent biosensor
for in situ detection of bio-synthesized alkanes. Scientific reports, 5, 10907. https://doi.org/10.1038/srep109072. Usage and Biology
2.1 TetR DNA binding protein
Due to its robust and highly regulatable DNA-binding properties, tetR has become a widely adopted tool in
synthetic
biology. Its ease of modification and ability to function in both prokaryotic and eukaryotic systems have made it
an essential element in the development of gene regulation systems (Berens & Hillen, 2004).
In our project, tetR was integrated into the design of a modular DNA-stapling system because of its
well-characterized behavior, ensuring reliable DNA interactions.
2.2mScarlet-I
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