Sup35NM
Part name: BBa_K1739000

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

Sup35 is derived from the yeast prion protein Sup35p and excludes the C-terminal domain. The N-terminal domain allows self-assembly of functional amyloid [3][4]. This BioBrick is an improved version of a previously designed BioBrick (Part:BBa_K401001) from the Valencia 2010 iGEM team that encoded the Sup35 protein from Saccharomyces cerevisiae. The previously designed BioBrick contained illegal BioBrick restriction sites. Our improved BioBrick has optimized in order to remove these cut sites and we have produced a part compatible with the iGEM part submission standards.

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 encodes Cytochrome b562 in a pSB1C3 backbone. 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 ^[1][2].

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 ^[6][7]. 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 ^[3][4]. This has previously been discussed by Tessier and Lindquist (2009)^[8] 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 ^[3][4][9] allowing the fusion of a new functional domain.

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 ^[6][7]. 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 ^[3][4]. This has previously been discussed by Tessier and Lindquist (2009)^[8] 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 ^[3][4][9]. 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 ^[1][5]. 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 ^[2]. Furthermore, haem binding to Cytochrome b₅₆₂ has a high affinity interaction with a dissociation constant (Kd) of 9nM at 25°C ^[2]). 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.

References

[1] 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.

[2]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.

[3] 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.

[4] 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.

[5] 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.

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

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

[8]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.

[9] 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.

[10]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.