Intimin anti-EGFR nanobody

This part contains the N-terminus of intimin harbouring the anti-EGFR nanobody (wild-type 7D12) and can be used for surface display of anti-EGFR nanobodies on E.coli. With this part, we sought to enhance bacterial binding to mammalian cells and potentially improve the chances of DNA delivery via conjugation by the bacterial Type IV secretion system (T4SS). As part of the PICasSO toolbox, we seek to use anti-EGFR adhesins to promote delivery of large DNA constructs encoding the various protein staples (presented in our parts collection) from bacteria to mammalian cells via conjugation to enable controlled modulation of chromatin organization by the delivered staples.

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
INCOMPATIBLE WITH RFC[10]
Illegal EcoRI site found at 105
Illegal EcoRI site found at 2259
Illegal PstI site found at 401
Illegal PstI site found at 649
Illegal PstI site found at 1096
12
INCOMPATIBLE WITH RFC[12]
Illegal EcoRI site found at 105
Illegal EcoRI site found at 2259
Illegal PstI site found at 401
Illegal PstI site found at 649
Illegal PstI site found at 1096
Illegal NotI site found at 2410
21
INCOMPATIBLE WITH RFC[21]
Illegal EcoRI site found at 105
Illegal EcoRI site found at 2259
Illegal BamHI site found at 1983
23
INCOMPATIBLE WITH RFC[23]
Illegal EcoRI site found at 105
Illegal EcoRI site found at 2259
Illegal PstI site found at 401
Illegal PstI site found at 649
Illegal PstI site found at 1096
25
INCOMPATIBLE WITH RFC[25]
Illegal EcoRI site found at 105
Illegal EcoRI site found at 2259
Illegal PstI site found at 401
Illegal PstI site found at 649
Illegal PstI site found at 1096
Illegal NgoMIV site found at 2004
1000
COMPATIBLE WITH RFC[1000]

2. Usage and Biology

This part contains the N-terminus of intimin harbouring the anti-EGFR (wild-type 7D12) adhesin and can be used for surface display of anti-EGFR nanobodies on E.coli. Salema et al., (2013) showed efficient presentation of nanobodies on the surface of E.coli K-12 cells by fusing them to the β domain of intimin. The β domain of intimin comes from the pNeae2 plasmid (addgene #168300) and encodes an N-terminal signal peptide for Sec-dependent translocation into the periplasm, a LysM domain that interacts with the peptidoglycan and provides anchoring, and a β-barrel that inserts into the outer membrane. The coding sequence of the anti-EGFR nanobody is located in the C-terminus (that is exposed to the extracellular milieu) between the E tag (GAPVPYPDPLEP) and the myc tag (EQKLISEED). These tags can be utilized for purification or detection of the protein.

The idea behind engineering this part was to use it for increasing cell-cell contact between bacteria and mammalian cells and thereby potentially enhancing the chances of inter-kingdom conjugational DNA transfer. The inspiration to test inter-kingdom conjugation came from the fact that all the events leading to DNA transfer by conjugation are driven by the donor bacterium and the protein components are typically plasmid encoded, making it possible for any type of cell to serve as the recipient (Waters, 2001). Furthermore, as the primary trigger for conjugation remains obscure, we hypothesized that cell-cell contact might be one of the main determinants for conjugation to occur efficiently. (Robledo et al., 2022) showed that enhanced cell-cell contact mediated by synthetic adhesins led to a 100-fold increase in conjugation efficiency between bacteria in liquid media, where the RP4 conjugative system is particularly ineffective. This combination of knowledge motivated us to engineer conjugation as a generalized DNA delivery tool for large plasmid constructs (around ~100 kb). Figure 2 below presents an illustration of our idea.

Figure 2. Illustration of DNA Delivery from Bacteria to Mammalian Cells via Conjugation.A conjugative helper plasmid catalyzes transfer of an oriT carrying mobilizable plasmid from bacteria to mammalian cells. Anti-EGFR adhesins are shown to stabilize the mating pair.

This part is a member of the PICasSO toolbox and can be used to promote adhesion to mammalian cell surfaces as EGFR is a common mammalian surface receptor. This part finds its application as an alternative DNA delivery tool to mammalian cells for parts in the PICasSO Toolbox such as the dCas-based staples ( BBa_K5237001, BBa_K5237002, BBa_K5237003 ) along with fgRNAs, which are encoded by rather large plasmids. It is known that lipofection efficiency decreases with increasing plasmid size (Kreiss et al., 1999), so conjugation may be employed as an alternative tool to deliver such large plasmids to mammalian cells in vitro, to not only enable engineering of chromatin conformations, but also to allow for its controlled modulation in a stimulus-responsive manner. Moreover, the anti-EGFR nanobody can easily be swapped with other nanobodies to alter the target specificity or binding affinities.

3. Assembly and Part Evolution

The pNeae2 plasmid was obtained from addgene (#168300). Codon optimized DNA sequence of chain L anti-EGFR (7D12) nanobody (Schmitz et al., 2013) for expression in E.coli was procured as a gBlock containing 5' and 3' restriction sites for SfiI and NotI. Attempts at cloning the anti-EGFR nanobody by restriction ligation (using SfiI and NotI) into the coding sequence of intimin in pNeae2 (suggested cloning strategy by Salema et al., (2013)) were unsuccessful as indicated by test digests and Sanger sequencing. To circumvent vector re-ligation issues that we encountered, Gibson assembly shall be attempted in the near future.

4. Results

**Successful expression of myc-tagged intimin by E.coli 10-beta**

Despite unsuccessful cloning of anti-EGFR adhesin into the coding sequence of intimin, protein expression was tested in E.coli 10-beta since they will be utilized as the donor strain in our upcoming conjugation tests - due to their ability to stably maintain and tolerate large plasmid constructs. Since E.coli 10-beta is not a typical strain used for protein expression, it was important to test their ability to overexpress outer membrane proteins and its potential toxicity to the cells.

E.coli 10-beta were transformed with pNeae2, and protein expression was induced with 50 µM IPTG. A Western Blot against the myc epitope on the C-terminus of intimin revealed expression of myc-tagged intimin of the expected size (~76 kDa) after induction (Fig 3). However, a smear of several bands was noted which could be a consequence of several factors included but not limited to protein degradation, expression of truncated proteins or high SDS-PAGE protein load. Nevertheless, this result still suggests that E.coli 10-beta could be used as the donor strain for our future conjugation assays owing to their ability to express full-length intimin.

Figure 3: Fluorescence Western Blot Scan Showing Expression of Myc-tagged Intimin by E.coli 10-beta after IPTG Induction. E.coli 10-beta were transformed with pNeae2, induced with 50 µM IPTG and lysed. Lane 1 was loaded with 30 µg of total protein from the E.coli lysate after IPTG induction and Lane 2 was loaded with 30 µg of total protein from the uninduced E.coli lysate. The blue arrow indicates the position of the myc-tagged intimin (~76 kDa)

Outlook

Upcoming experiments shall address the interaction between the anti-EGFR adhesin and EGFR in vitro and also examine the increased localization of adhesin-expressing bacteria on the surface of mammalian cells. Ultimately, this part shall be used in conjugation assays between bacteria and mammalian cells to delineate the role of cell-cell contact in promoting inter-kingdom conjugation.

5. References

Kreiss, P., Cameron, B., Rangara, R., Mailhe, P., Aguerre-Charriol, O., Airiau, M., Scherman, D., Crouzet, J., & Pitard, B. (1999). Plasmid DNA size does not affect the physicochemical properties of lipoplexes but modulates gene transfer efficiency. Nucleic Acids Research, 27(19). https://doi.org/10.1093/nar/27.19.3792

Robledo, M., Álvarez, B., Cuevas, A., González, S., Ruano-Gallego, D., Fernández, L. Á., & De La Cruz, F. (2022). Targeted bacterial conjugation mediated by synthetic cell-to-cell adhesions. Nucleic Acids Research, 50(22). https://doi.org/10.1093/nar/gkac1164

Salema, V., Marín, E., Martínez-Arteaga, R., Ruano-Gallego, D., Fraile, S., Margolles, Y., Teira, X., Gutierrez, C., Bodelón, G., & Fernández, L. Á. (2013). Selection of Single Domain Antibodies from Immune Libraries Displayed on the Surface of E. coli Cells with Two β-Domains of Opposite Topologies. PLoS ONE, 8(9). https://doi.org/10.1371/journal.pone.0075126

Schmitz, K. R., Bagchi, A., Roovers, R. C., Van Bergen En Henegouwen, P. M. P., & Ferguson, K. M. (2013). Structural evaluation of EGFR inhibition mechanisms for nanobodies/VHH domains. Structure, 21(7).https://doi.org/10.1016/j.str.2013.05.008

Waters, V. L. (2001). Conjugation between bacterial and mammalian cells. Nature Genetics, 29(4). https://doi.org/10.1038/ng779

Part:BBa_K5237015