Revision as of 16:24, 18 September 2015

Sequence coding for Sup35 with a N-terminal CsgA signal sequence for amyloid export 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.

Sequence and Features

Validation

To validate the nature of our part, we first analysed it through gel electrophoresis. We carried out a diagnostic restriction digest using ECORI and PSTI. These enzymes cleave pSBIC3 into a fragment of 2031bp, whereas our insert is 1020bp. We used the gel to check the sizes of our fragments against the Invitrogen 1kB plus DNA marker. We were therefore able to confirm that we had the correct part (see figure 1.)

Figure 1. Shows the agarose gel, following a restrictive digest of our BioBrick containing CsgA_ss and Sup35NM.

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.

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.

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.