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

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 (Sivanathan and Hochschild, 2012; Sivanathan and Hochschild, 2013). 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 (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). ). 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 (Fujiwara, Fnkumori, and Yamanaka, 1993; Mathews et al., 1979). B-type cytochromes are a favourable choice because heme binds in a non-ionic fashion to the two ligands Methionine-7 and Histidine-106 (Xavier et al., 1978). 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 (Robinson et al., 1997). Furthermore, heme 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 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.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

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 7
Illegal NheI site found at 30
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE WITH RFC[1000]
Illegal SapI.rc site found at 718

Validation

Validation of our fusion protein’s export was achieved using four techniques.

Plasmid

We first analysed the biobrick by diagnostic restriction digest followed by gel electrophoresis (Fig. 1). By comparing the sizes of the fragments to a marker we validated the size of our fragment (as seen in figure 1) to be consistent with size of 1339 base pairs of the CsgAss, Sup35NM and Cytochrome b₅₆₂ sequences.

Figure 1. Agarose gel of the restriction digest of BBa_K1739003 in pSCB13 plasmid backbone with EcoRI and PstI.

Congo Red plate assay

We then validated our part using a Congo Red agar plate assay with the antibiotics chloramphenicol and ampicillin present in order to select the VS45 strains expression our fusion protein. As a negative control, we used VS45 with pVS105, as this plasmid contains CsgAss and Sup35M, it does not have the ability to self-assemble into amyloid nano-wires. These strains were plated in 2 quarter segments of the plate and left to incubate for 24 hours at 37°C. This resulted red colonies of VS45 with CsgAss-Sup35NM-Cyt b₅₆₂ due to the binding of Congo Red to the amyloid fibres produced by the colonies. White colonies of VS45 with the negative control plasmid, as expected, showed no export of amyloid-forming protein (shown in fig.1). These results confirmed that our fusion protein was being produced and targeted to the curli export pathway by CsgA_ss, as well as self-assembling into amyloid fibres.

Figure 2. 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.

AFM Imaging

Further validation for protein export and amyloid formation by our fusion protein was achieved using atomic force microscopy (AFM) imaging 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 showed the presence of amyloid aggregates in the VS45 sample with our Envirowire construct (figures 3 and 4) compared with samples without amyloid fibres being produced by VS45 (figure 5), although the nano-wire were assembled less efficiently resulting in less straight, shorter and more clumped fibrils than with Sup35NM alone.

Figure 3. Shows AFM imaging of our Envirowire plasmid. The nano-wires are visible although the reduced assembly efficiency of our designed amyloid nano-wire may be due to the incomplete folding of cytochrome b₅₆₂ as the heme needed for complete folding wasn’t present in the growth media.

Figure 4. Illustrates a cluster of amyloid fibers, it is zoomed in from figure 3.

Figure 5. Shows AFM imaging of our negative control. No nano-wires are present.

Heme plate and conductivity assay

Further validation was performed to ensure that our cytochrome attached to the Sup35NM nano-wire had bound to the heme and conferred conductive properties. This was achieved by carrying out a conductivity test. Firstly a biofilm formation was induced on a agar plate before the conductance was measured over the length of the biofilm (1 inch), the results, however, were inconclusive. Future testing on individual nano-wire could be done to assess the affect of biofilm length and diameter on conductance and resistance to optimize assembly and conductivity efficiency.

Part Improvement

Team Kent 2016 improved this part by using Sup-1-61 instead of Sup35NM and using an arabinose inducible promoter to express amyloid fibres. See (Part:BBa_K1985014) for more detail.

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.