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.

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

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NotI site found at 1149
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 623
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE WITH RFC[1000]
Illegal SapI.rc site found at 466

2. Usage and Biology

2.1 TetR DNA binding protein

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).
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.

2.2 mScarlet-I

mScarlet-I is a rapidly-maturing monomeric red fluorescent protein designed by Bindels et al. in 2017. 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., 201). 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

2.3 Förster Resonance Energy Transfer (FRET)

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).

Figure 2: Overview of excitation and emission spectrum of mNeonGreen and m-Scarlet and it's properties as a FRET pair

3. Assembly and part evolution

As part of our engineering efforts an engineered tetR-mScarlet was tested, that binds DNA as a monomer. This scTetR is a fusion of two tetR proteins with a flexible linker which was described to maintain the same DNA-binding affinity and specificity as wild-type tetR (Kaminoka et al. 2006; Krueger et al.2003)

Our experiments with the scTetR-mScarlet-I fusion protein showed no significant expression. Since the scTetR-mScarlet-I fusion protein was expressed as part of a polycistronic transcript, the results should be taken with caution. Nonetheless we belive that qualitative assesements can still be drawn, because the same construct with tetR-mScarlet-I showed a good expression (Figure 2, 3).

Figure 3: Fluoresence measurement of scTetR with mutant T7 polymerase. Fluorescence intensity of mScarlet-I (ex. 560 nm, em. 600 nm) was measured 18 h after induction with IPTG and normalized to cell count (OD₆₀₀). Positive control consists of a tetR-mNeonGreen fusion protein.

4. Results

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.

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.

Figure 4: Overview FRET assay

Fluorescence intensity, normalized to cell count, of mNeonGreen and mScarlet-I was measured 18 h after induction with varying IPTG concentration (Figure 5). 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 5)

Figure 5: Fluorescence of mNeonGreen, mScarlet-I and FRET. Fluorescence intensity of mNeonGreen (ex. 490 nm, em. 530 nm), mScarlet-I (ex. 560 nm, em. 600 nm), and FRET (ex. 490 nm, em. 600 nm) was measured 18 h after induction with IPTG and normalized to cell count (OD₆₀₀). A), B) Fluorescence intensity of mNeonGreen and mScarlet-I after induction with different IPTG concentrations. C) Fluorescence intensity of FRET pair induced with 0.025 mM IPTG. Statistical significance was tested for with Ordinary two-way ANOVA with Šidák's multiple comparison test, with a single pooled variance. *p < 0.05, ****p < 0.001. Only significant results, within groups are shown. Data is depcited as mean ± SD.

5. Conclusion

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.

6. References

Berens, C., & Hillen, W. (2004). Gene Regulation By Tetracyclines. In J. K. Setlow (Ed.), Genetic Engineering: Principles and Methods (pp. 255–277). Springer US. https://doi.org/10.1007/978-0-306-48573-2_13

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

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

McCullock, T. W., MacLean, D. M., & Kammermeier, P. J. (2020). Comparing the performance of mScarlet-I, mRuby3, and mCherry as FRET acceptors for mNeonGreen. PLOS ONE, 15(2), e0219886. https://doi.org/10.1371/journal.pone.0219886

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

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

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

Wu, P. G., & Brand, L. (1994). Resonance Energy Transfer: Methods and Applications. Analytical Biochemistry, 218(1), 1-13. https://doi.org/10.1006/abio.1994.1134

Part:BBa_K5237017