Team:Kent/Basic Part
Basic Parts
Contents
Sup35NMCytochrome b562
Sup35 with N-terminal CsgAss signal sequence
Envirowire
Sup35NM
Part name: BBa_K1739000
We have improved 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 two illegal cut sites for Pstl and one for Bsal.rc within the coding region that reduce compatibility for digestion and modification of the part. Our improved BioBrick has used genome optimisation in order to remove these cut sites, producing a part compatible with the iGEM part submission standards. Validation of this part used a diagnostic Congo Red plate that demonstrated the presence of amyloid by formation of red colonies.
Cytochrome b562
Part name: BBa_K1739001
This part uses the BBa_J23104 and encodes cytochrome b562 in a pSB1C3 backbone. This part has been validated by digestion and quantification of the presence of the cytochrome gene on a diagnostic gel. 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 (Fujiwara, Fnkumori, and Yamanaka, 1993; Robinson et al., 1997).
Sup35 with N-terminal CsgAss signal sequence
Part name: BBa_K1739002
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.
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, VS105. The results (shown in Fig.X) confirmed that our plasmid induced amyloid formation due to the growth of red VS45 colonies in comparison to the white VS105 colonies, demonstrating no presence of amyloid fibers.
Further validation was achieved by observing the topography of our cell suspension using Atomic Force Microscopy (AFM). As shown in in Fig.X there is clear amyloid formation in the induced VS45 sample. In contrast, there was no amyloid formation in the sample containing the negative control strain VS105.
The combination of these results demonstrates that our BioBrick facilitates the export of Sup35 and the subsequent formation of amyloid.
Envirowire
Part name: BBa_K1739003
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.
Validation
Validation of our fusion protein’s export was achieved using two techniques. Firstly, using a segmented diagnostic Congo Red agar plate with the antibiotics chloramphenicol and ampicillin present in order to select the VS45 strains transformed with our plasmid. As a negative control, we used the VS105 strain of E.coli , as it does not have the ability to export amyloid-forming proteins. These strains were plated in 2 quarter segments of the plate and left to incubate for 24 hours at 37°C. This resulted red VS45 colonies due to binding of Congo Red to the amyloid fibres produced by the colonies, and white VS105 colonies showing no export of amyloid-forming protein (shown in fig.X). These results confirmed that our protein was being produced and targeted to the curli export pathway by csgA, as well as self-assembling into amyloid fibres.
The second method of validation for protein export and amyloid formation was achieved by atomic force microscopy (AFM) imaging to provide a topography 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 clearly showed the presence of amyloid fibres in the VS45 sample with PVS72 and no amyloid fibres in the VS105 sample.
Further validation was required to ensure that our cytochrome was attached to the Sup35-NM monomers and had bound to the haem. This was achieved by carrying out a conductivity test. Firstly a biofilm formation was induced on a piece of acrylic before the conductance was measured over the length of the biofilm [insert length. The test demonstrated a conductance of [insert number and discussion of resistance]. Future testing could be done to assess the affect of biofilm length and diameter on conductance and resistance to optimise electron transmission efficiency.