Revision as of 13:39, 18 September 2015

Sequence coding for Envirowire: Sup35NM with N-terminal CsgAss and C-terminal cytb562

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.

Sequence and Features

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.1) 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.1"".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.

References

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.

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.

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.

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.

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.