1. Sequence overview

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 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).

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 (Figure 2). 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 2: 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