Sup35NM
Part name: BBa_K1739000

(Figure 1. Illustrates the construction of BBa_K1739000. 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 BBa_K1739001. 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 BBa_K1739002. The image was created using SnapGene Viewer.)

This part 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]. Sup35NM is derived from the yeast prion protein Sup35p from S. cerevisiae (bakers yeast) 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 Sup35NM 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. This part has been inserted into the pSB1C3 backbone and uses the promoter BBa_J23104.

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 BBa_K1739003. This image was created using SnapGene Viewer.)

This BioBrick encodes for the Envirowire fusion protein. It contains three segments, CsgA signal sequence, Sup35NM ( BBa_K1739000 and BBa_K1739002) and Cytochrome b₅₆₂ (BBa_K1739001) 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]. Sup35NM 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]. Here, with the removal of the functional C domain allows us 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 heme binds in a non-ionic fashion to the two ligands Methionine-7 and Histidine-106 ^[10]. Heme 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 heme to be added exogenously to initiate correct folding of the cytochrome by burying hydrophobic side chains ^[2]. Furthermore, heme binding to cytochrome b₅₆₂ has a high affinity interaction with a dissociation constant (Kd) of 9nM at 25°C ^[2]. This BioBrick part has been inserted into the pSB1C3 backbone and uses the promoter BBa_J23104. This part can be used in the VS45 strain of E.colicontaining 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.