Part:BBa_K5237009
Mini staple:
The Mini staple is a fusion of GCN4 and rGCN4, engineered to bind two DNA strands simultaneously and induce proximity. With the Mini staples, we aimed to engineer a smaller alternative to the simple staples.
Contents
Next to the well-studied linear DNA sequence, the 3D spatial organization of DNA plays a crucial role in gene
regulation,
cell fate, disease development and more. 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 constructs
with our
interkingdom conjugation system.
(iii) As the final category 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
readouts via a luciferase reporter, which allows for straightforward experimental simulation of enhancer hijacking
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.
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 Entry vector MbCas12a-SpCas9 | Entryvector for simple fgRNA cloning via SapI |
| BBa_K5237001 | Staple subunit: dMbCas12a-Nucleoplasmin NLS | Staple subunit that can be combined with sgRNA or fgRNA and dCas9 to form a functional staple |
| BBa_K5237002 | Staple subunit: SV40 NLS-dSpCas9-SV40 NLS | Staple subunit that can be combined witha sgRNA or fgRNA and dCas12avto form a functional staple |
| 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 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. Can be used to create functionalized staples units |
| BBa_K5237013 | Caged NpuC Intein | A caged NpuC split intein fragment that undergoes protein trans-splicing after protease activation. Can be used to create functionalized staples units |
| BBa_K5237014 | fgRNA processing casette | Processing casette to produce multiple fgRNAs from one transcript, that can be used for multiplexed 3D genome reprograming |
| BBa_K5237015 | Intimin anti-EGFR Nanobody | Interkindom conjugation between bacteria and mammalian cells, as 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 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 | FRET Donor-Fluorpohore fused to Oct1-DBD that binds to 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, 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. 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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 175
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Small basic-region leucine zipper (bZip) proteins are characterized by their DNA-binding. The bZip motif
consists of a coiled-coil leucine zipper dimerization domain, and a highly charged basic region that directly
contacts and binds to DNA (Hollenbeck & Oakley, 2000). One well characterized example is the General Control
Protein
4 (GCN4), a well-characterized transcriptional activator from yeast (Arndt & Fink, 1986). At its N-terminus,
GCN4
contains basic residues, the so-called bZip domain, through which it binds specifically to the CRE (cyclic AMP
response element) DNA sequence (5' ATGACGTCAT 3') (Hollenbeck et al., 2002). A variant of GCN4 with the DNA
binding bZip-domain
at the C-terminus (rGCN4) has been engineered to bind to the inverted CRE sequence, INV2 (5' GTCAtaTGAC 3', upper
case letters indicate direct interaction between protein and DNA) with similar affinity
(Hollenbeck et al., 2001). By genetically fusing GCN4 to rGCN4, we created a small bivalent DNA binding
staple with less than 150 amino acids, which was for its DNA binding and stapling capabilities.
The amino acid sequence for GCN4 and rGCN4 was obtained from literature (Hollenbeck et al. 2001), and
codon-optimized for Escherichia coli.
The two leucine zipper were combined with a GSG linker harbouring a BamHI site to adapt the construct with different
linker designs.
A FLAG-tag (DYKDDDDK) was added to the N-terminus for protein purification. The FLAG-tag can be cleaved off using an
Enterokinase, if necessary.
The FLAG-GCN4 sequence was cloned into a T7 expression vector and expressed using E. coli BL21 (DE3) cells.
The current version of the part with GCN4-rGCN4 fusion, linked by a GSG peptide linker has not demonstrated any DNA
binding in the tests conducted thus far.
Nevertheless, we belive the part to still be a valuable addition, as it can be further engineered with different
linker types to
create a functioning staple. Through dry lab modelling, various linker designs have already been simulated to
predict
improved dimerization and DNA binding.
The bZip proteins GCN4, rGCN4 and their fusion bGCN4 were fused N-terminally to a FLAG-tag (DYKDDDDK). All
proteins could be readily
expressed under the T7 promoter in E. coli BL21 DE3 and purified with Anti-FLAG affinity
columns. The purity of the proteins was confirmed by SDS-PAGE (Figure 2).
2. Usage and Biology
3. Assembly and part evolution
4. Results
4.1 Protein Expression and Purification
4.2 Electrophoretic Mobility Shift Assay
4.2.1 Qualitative DNA binding analysis
To asses possible DNA binding, a qualitative EMSA was performed with the purified proteins. The same binding buffer conditions were used, as previously desribed for GCN4 and rGCN4 (Hollenbeck et al. 2001). DNA binding could only be shown for GCN4 and rGCN4, no visible band was observed for the bGCN4 fusion protein (Figure 4).
4.2.2 Quantitative DNA binding analysis
To further analyze DNA binding, quantitative shift assays were performed for GCN4 and rGCN4. Here
0.5 µM DNA were incubated with varying concentrations of protein until equilibration. After
electrophoresis, bands were stained with SYBR-Safe and quantified based on pixel intensity. The
obtained values were fitted to equation 1, describing formation of a 2:1 protein-DNA complex:
Θapp = Θmin + (Θmax - Θmin) ×
(Ka2 [L]tot2) / (1 + Ka2
[L]tot2)
Equation 1
Here [L]tot describes the total protein monomer concentration, Ka
corresponds
to the apparent monomeric equilibration constant. The Θmin/max values are the
experimentally
determined site saturation values (For this experiment 0 and 1 were chosen for min and max
respectively). GCN4 binds to its optimal DNA binding motif with an apparent dissociation
constant Kk of (0.2930.033)×10-6 M, which is almost identical to the
rGCN4 binding
affinity to INVii a d of (0.2980.030)×10-6 M.
To better understand the proteins, quantitative analysis was done to determine the apparent dissociation
constant
for GCN4 and rGCN4.
For the purified proteins GCN4, rGCN4 and the fusion bGCN4 a qualitative gel was run with high protein
concentration (Figure 3).
Bands for GCN4 and rGCN4 were visible but no band for bGCN4 could be detected, indicating a
lack of DNA binding. This suggests that the dimerization, necessary for DNA binding, is disrupted by the
GSG-linker (Ellenberger et al., 1992; Liu et al., 2006; Lupas
et al., 2017; Woolfson, 2023). To better understand possible problems in dimerization
circular dichroism can be used to analyze secondary structure
and proper coiled coil formation (Greenfield, 2006). Further engineering can be done by testing out
various linkers with specific properties to ensure correct folding and dimerization (Chen et
al., 2013). The apparent binding kinetics calculated for GCN4 ((0.2930.033) × 10-6 M) and
rGCN4
((0.2980.030) × 10-6 M) are
approximately a factor 10 higher then those described in literature ((96) × 10-8 M for
GCN4 and (2.90.8) × 10-8 M for rGCN4) (Hollenbeck et al., 2001). The
differences could be due to the lower sensitivity of SYBR-Safe
staining compared to radio-labeled oligos.
The FLAG-tag fusion to the N-terminus of proteins could potentially decrease binding affinity, likely due
to steric hindrance affecting the interaction with DNA. Interestingly, the differences in binding affinity
between GCN4 and rGCN4 appear negligible. Since GCN4 binds to DNA via its N-terminus and rGCN4 binds
C-terminally, the FLAG-tag likely does not directly influence DNA binding. However, it may influence the
dimerization of the proteins, which is necessary for DNA binding. To further investigate this, the
FLAG-tag can be cleaved using an enterokinase and potential changes in binding affinity analyzed
4.3 In Silico Characterization using DaVinci
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 obtained via EMSA assays and purified proteins. This enabled us to simulate enhancer hijacking in silico, providing valuable input for the design of further experiments. Additionally, we apply the same approach to our part collection. 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.
When selecting a peptide linker, both length and rigidity are important considerations. These linkers can determine function and dynamics of the whole construct. We selected seven peptide linkers from the literature to cover a broad range of physical properties (Chen et al., 2013). We tested these linkers first on our mini staples, which allowed for quicker and easier implementation before moving to the more complex Cas staples.
We assessed the flexibility and rigidity of our constructs through the pLDDT values assigned during the predictions. Figure 6 illustrates the variation in linkers using the ('GGGGS')n sequence for flexible linkers and the ('EAAAK')n sequence for rigid linkers (Arai et al., 2001). The predictions are colored by their pLDDT scores, which act as a surrogate measure of chain rigidity (Akdel et al., 2022; Guo et al., 2022). The construct C was tested as part BBa_K5237009 in the Wetlab - it did not bind DNA on any side as the structure is to rigid which hinders dimerisation of the two subunits.
5. References
Akdel, M., Pires, D. E. V., Pardo, E. P., Janes, J., Zalevsky, A. O., Meszaros, B., Bryant, P., Good, L. L., Laskowski, R. A., Pozzati, G., Shenoy, A., Zhu, W., Kundrotas, P., Serra, V. R., Rodrigues, C. H. M., Dunham, A. S., Burke, D., Borkakoti, N., Velankar, S., … Beltrao, P. (2022). A structural biology community assessment of AlphaFold2 applications. Nat Struct Mol Biol, 29(11), 1056–1067. https://doi.org/10.1038/s41594-022-00849-w
Arai, R., Ueda, H., Kitayama, A., Kamiya, N., & Nagamune, T. (2001). Design of the linkers which effectively separate domains of a bifunctional fusion protein. Protein Engineering, Design and Selection, 14(8), 529–532. https://doi.org/10.1093/protein/14.8.529
Arndt, K., & Fink, G. R. (1986). GCN4 protein, a positive transcription factor in yeast, binds general control promoters at all 5’ TGACTC 3’ sequences. Proceedings of the National Academy of Sciences, 83(22), 8516–8520. https://doi.org/10.1073/pnas.83.22.8516
Chen, X., Zaro, J. L., & Shen, W.-C. (2013). Fusion protein linkers: Property, design and functionality. Advanced Drug Delivery Reviews, 65(10), 1357–1369. https://doi.org/10.1016/j.addr.2012.09.039
Google DeepMind. (2024). AlphaFold Server. https://alphafoldserver.com/terms
Guo, H.-B., Perminov, A., Bekele, S., Kedziora, G., Farajollahi, S., Varaljay, V., Hinkle, K., Molinero, V., Meister, K., Hung, C., Dennis, P., Kelley-Loughnane, N., & Berry, R. (2022). AlphaFold2 models indicate that protein sequence determines both structure and dynamics. Scientific Reports, 12(1), 10696. https://doi.org/10.1038/s41598-022-14382-9
Ellenberger, T. E., Brandl, C. J., Struhl, K., & Harrison, S. C. (1992). The GCN4 basic region leucine zipper binds DNA as a dimer of uninterrupted alpha helices: Crystal structure of the protein-DNA complex. Cell, 71(7), 1223–1237. https://doi.org/10.1016/s0092-8674(05)80070-4
Hollenbeck, J. J., Gurnon, D. G., Fazio, G. C., Carlson, J. J., & Oakley, M. G. (2001). A GCN4 Variant with a C-Terminal Basic Region Binds to DNA with Wild-Type Affinity. Biochemistry, 40(46), 13833–13839.
Hollenbeck, J. J., McClain, D. L., & Oakley, M. G. (2002). The role of helix stabilizing residues in GCN4 basic region folding and DNA binding. Protein Science, 11(11), 2740–2747. https://doi.org/10.1110/ps.0211102
Hollenbeck, J. J., & Oakley, M. G. (2000). GCN4 Binds with High Affinity to DNA Sequences Containing a Single Consensus Half-Site. Biochemistry, 39(21), 6380–6389. https://doi.org/10.1021/bi992705n
Liu, J., Zheng, Q., Deng, Y., Cheng, C.-S., Kallenbach, N. R., & Lu, M. (2006). A seven-helix coiled coil. Proceedings of the National Academy of Sciences, 103(42), 15457–15462. https://doi.org/10.1073/pnas.0604871103
Lupas, A. N., Bassler, J., & Dunin-Horkawicz, S. (2017). The Structure and Topology of α-Helical Coiled Coils. Fibrous Proteins: Structures and Mechanisms, 82, 95–129. https://doi.org/10.1007/978-3-319-49674-0_4
Woolfson, D. N. (2023). Understanding a protein fold: The physics, chemistry, and biology of α-helical coiled coils. Journal of Biological Chemistry, 299(4), 104579. https://doi.org/10.1016/j.jbc.2023.104579
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