Sup35NM
Part name: BBa_K1739000

(Figure 1. Illustrates the construction of our plasmid containing pSBIC3 and Sup35NM. The image was created using SnapGene Viewer.)

We have improved a previously designed BioBrick (Part:BBa_K401001) from the 2010 Valencia iGEM team that encoded the Sup35 protein from Saccharomyces cerevisiae. The previously designed BioBrick contained two illegal cut sites for Pstl and one for Bsal within the coding region that reduce compatibility for digestion and modification of the part. Our improved BioBrick has used genome optimisation in order to remove these cut sites, producing a part compatible with the iGEM part submission standards.

Validation

Cytochrome b₅₆₂
Part name: BBa_K1739001

(Figure 2. Illustrates the construction of our plasmid, containing pSBIC3 and Cytochrome b₅₆₂. The image was created using SnapGene Viewer.)

This part uses the BBa_J23104 and encodes Cytochrome b₅₆₂ in a pSB1C3 backbone. This part has been validated by digestion and quantification of the presence of the cytochrome gene on a diagnostic gel. Cytochrome b₅₆₂ is a single subunit, four-helix bundle protein containing a non-covalently bound b-type haem group with a molecular weight of 25kDa (Fujiwara, Fnkumori, and Yamanaka, 1993; Robinson et al., 1997).

Validation

Sup35NM with N-terminal CsgAss signal sequence
Part name: BBa_K1739002

(Figure 3. Illustrates the construction of our plasmid containing pSBIC3 and Sup35 with an N-terminal CsgAss signal sequence. The image was created using SnapGene Viewer.)

This part uses the BBa_J23104 and has been inserted into the pSB1C3 backbone. The design includes the bipartite csgA signal sequence that targets the protein to the Sec-export pathway and subsequently to the curli export pathway via interaction with csgG (Sivanathan and Hochschild, 2012; Sivanathan and Hochschild, 2013). Sup35-NM is derived from the yeast prion protein Sup35p and excludes the C-terminal domain with the N-terminal domain allowing self-assembly of functional amyloid (Frederick et al., 2014; Glover et al. 1997). This has previously been discussed by Tessier and Lindquist (2009) who show that two beta-sheets bond together in a self-complimenting ‘steric zipper’ that excludes water, leaving a highly stable parallel beta-sheet with one molecule every 4.7 Angstroms. The particular advantage of using Sup35-NM is that in its native state Sup35p has two functional domains, the N and C terminal, separated by the highly charged M domain (Frederick et al., 2014; Glover et al. 1997; Wickner et al., 2007) allowing the fusion of a new functional domain.

Validation

This BioBrick has been validated using a diagnostic Congo Red agar plate. Our plasmid was transformed into the E.coli strain VS45 and compared to a negative control strain, VS45 with PVS105. The results (shown in Fig.4) confirmed that our plasmid induced amyloid formation due to the growth of red colonies of VS45 with PVS72 in comparison to the white VS45 with PVS105, demonstrating no presence of amyloid fibers.

(Figure 4. Shows our Congo red plates. The top plate depicts both our negative control plasmid, PVS105 and our BioBrick containing CsgAss and Sup35NM. Whereas the bottom plate shows only VS45 with our BioBrick. The presence of red colonies illustrates the successful formation of amyloid.)

Further validation was achieved by observing the topography of our cell suspension using Atomic Force Microscopy (AFM). As shown in in Fig.X there is clear amyloid formation in the induced VS45 sample. In contrast, there was no amyloid formation in the sample containing the negative control strain VS105.

The combination of these results demonstrates that our BioBrick facilitates the export of Sup35 and the subsequent formation of amyloid.

Envirowire: Sup35NM with an N-terminal CsgAss signal sequence and a C-terminal Cytochrome b₅₆₂
Part name: BBa_K1739003

(Figure 5. Illustrates the construction of our Envirowire plasmid containing pSBIC3 and Sup35 with an N-terminal CsgAss signal sequence and a C-terminal Cytochrome b₅₆₂. This image was created using SnapGene Viewer.)

This BioBrick contains the constitutive promoter BBa_J23104 and uses the pSB1C3 backbone. It consists of three genes, a csgA signal sequence, Sup35-NM, and Cytochrome b₅₆₂. The bipartite csgA signal sequence targets the Sec protein export pathway followed by the endogenous curli export system of E.coli allowing our protein to be easily exported into an external medium (Sivanathan and Hochschild, 2012; Sivanathan and Hochschild, 2013). Sup35-NM is derived from the yeast prion protein Sup35p and excludes the C-terminal domain. The N-terminal domain allows self-assembly of functional amyloid (Frederick et al., 2014; Glover et al. 1997). This has previously been discussed by Tessier and Lindquist (2009) who show that two beta-sheets bond together in a self-complimenting ‘steric zipper’ that excludes water, leaving a highly stable parallel beta-sheet with one molecule every 4.7Å. The particular advantage of using Sup35-NM is that in its native state Sup35p has two functional domains, the N and C terminal, separated by the highly charged M domain (Frederick et al., 2014; Glover et al. 1997; Wickner et al., 2007). Thus facilitating the removal of one functional domain in order to add our own functional protein, in this case Cytochrome b₅₆₂ to form a fusion protein.

We chose Cytochrome b₅₆₂ as the electron carrier to make our amyloid conductive. The structure of Cytochrome b₅₆₂ consists of a single 24kDa subunit containing four nearly parallel alpha helices (Fujiwara, Fnkumori, and Yamanaka, 1993; Mathews et al., 1979). B-type cytochromes are a favourable choice because haem binds in a non-ionic fashion to the two ligands Methionine-7 and Histidine-106 (Xavier et al., 1978). Haem binding has been shown to occur in both the native protein and the denatured protein, although the latter exhibits a modest affinity with a dissociation constant (Kd) of 3μM. This allows the cytochrome to be exported in an unfolded state and haem to be added exogenously to initiate correct folding of the cytochrome by burying hydrophobic side chains (Robinson et al., 1997). Furthermore, haem binding to Cytochrome b₅₆₂ has a high affinity interaction with a dissociation constant (Kd) of 9nM at 25°C (Robinson et al., 1997). This BioBrick has been optimised for use in the VS45 strain of E.coli containing deletions that prevent amyloid from binding to the outside of the cell and increase the rate of protein exiting the cell via the curli export system.

Validation

Validation of our fusion protein’s export was achieved using two techniques. Firstly, using a segmented diagnostic Congo Red agar plate with the antibiotics chloramphenicol and ampicillin present in order to select the VS45 strains transformed with our plasmid. As a negative control, we used VS45 with PVS105, as it does not have the ability to export amyloid-forming proteins. These strains were plated in 2 quarter segments of the plate and left to incubate for 24 hours at 37°C. This resulted red VS45 with PVS72 colonies due to binding of Congo Red to the amyloid fibres produced by the colonies, and white colonies of VS45 with PVS105 showing no export of amyloid-forming protein (shown in fig.4). These results confirmed that our protein was being produced and targeted to the curli export pathway by csgA, as well as self-assembling into amyloid fibres.

Further validation was required to ensure that our cytochrome was attached to the Sup35-NM monomers and had bound to the haem. This was achieved by carrying out a conductivity test. Firstly a biofilm formation was induced on a piece of acrylic before the conductance was measured over the length of the biofilm [insert length. The test demonstrated a conductance of [insert number and discussion of resistance]. Future testing could be done to assess the affect of biofilm length and diameter on conductance and resistance to optimise electron transmission efficiency.

(Figure 6. Shows the conductivity testing of the biofilm on the haem plates.)

The third method of validation for protein export and amyloid formation was achieved by atomic force microscopy (AFM) imaging to provide a topography of our samples. Using the aforementioned E.coli strains a, 5 day incubation at 25°C was carried out to make sure that the amyloid fibres were stable for the AFM protocol, as suggested by Sivanathan and Hochschild (2012). The resulting images clearly showed the presence of amyloid fibres in the VS45 sample with PVS72 and no amyloid fibres in the VS45 with PVS105 sample.

References

Fujiwara, T., Fnkumori,, Y. and Yamanaka, T. (1993). Halobacterium halobium Cytochrome b-558 and Cytochrome b-562: Purification and Some Properties. J. Biochem., 113, pp.48-54.

Robinson, C., Liu, Y., Thomson, J., Sturtevant, J. and Sligar, S. (1997). Energetics of Heme Binding to Native and Denatured States of Cytochrome b 562 †. Biochemistry, 36(51), pp.16141-16146.

Frederick, K., Debelouchina, G., Kayatekin, C., Dorminy, T., Jacavone, A., Griffin, R. and Lindquist, S. (2014). Distinct Prion Strains Are Defined by Amyloid Core Structure and Chaperone Binding Site Dynamics. Chemistry & Biology, 21(2), pp.295-305.

Glover, J., Kowal, A., Schirmer, E., Patino, M., Liu, J. and Lindquist, S. (1997). Self-Seeded Fibers Formed by Sup35, the Protein Determinant of [PSI+], a Heritable Prion-like Factor of S. cerevisiae. Cell, 89(5), pp.811-819.

Mathews, F. S., Bethge, P. H., & Czerwinski, E. W. (1979). The structure of cytochrome b562 from Escherichia coli at 2.5 A resolution. Journal of Biological Chemistry, 254(5), 1699-1706.

Sivanathan, V. and Hochschild, A. (2012). Generating extracellular amyloid aggregates using E. coli cells. Genes & Development, 26(23), pp.2659-2667

Sivanathan, V. and Hochschild, A. (2013). A bacterial export system for generating extracellular amyloid aggregates. Nat Protoc, 8(7), pp.1381-1390.

Tessier, P. and Lindquist, S. (2009). Unraveling infectious structures, strain variants and species barriers for the yeast prion [PSI+]. Nat Struct Mol Biol, 16(6), pp.598-605.

Wickner, R., Edskes, H., Shewmaker, F. and Nakayashiki, T. (2007). Prions of fungi: inherited structures and biological roles. Nature Reviews Microbiology, 5(8), pp.611-618.

Xavier, A., Czerwinski, E., Bethge, P. and Mathews, F. (1978). Identification of the haem ligands of cytochrome b562 by X-ray and NMR methods. Nature, 275(5677), pp.245-247.