Part:BBa_K1985014

Sequence coding for Sup35(residues 1-61) and Cytochrome b562 with an arabinose inducible promoter

This part is an improved version of the fusion protein (Part:BBa_K1739003) designed by the Kent 2015 iGEM team. It contains four segments, an arabinose inducible promoter designed by the Imperial 2014 iGEM team (Part:BBa_K1321333), the CsgA signal sequence, the first 61 aminoacids of the prion domain Sup35 and the electron transfer protein cytochrome 562. Our improved BioBrick aims to optimize the self-assembly process of amyloid fibrils with the addition of these residues as they have been considered to be a suitable building block for the assembly of functional nanostructures[1]. The addition of the arabinose inducible promoter allows for tighter control for the expression of amyloid fibres rather than a constitutive promoter as was previously used. This part was inserted into part (Part:BBa_K1985016), which is a pSB1A3 backbone with (Part:BBa_K1321333).

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 1205
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1144
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 979
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 961
  

Validation

The improved plasmid was validated in three ways:

Restriction Digest

The plasmid was analysed through a diagnostic double restriction cut, using the enzymes EcoRI and BamHI. This was followed by agarose gel electrophoresis. The enzymes cleave the pBAD Ara-C promoter at 1165 bp, with the remainder plasmid and the insert being 2876 bp. The size of the two fragments were compared with size of the Invitrogen 1kB DNA marker and was found that our fragments were the correct.

Figure 1.1% agarose gel of the restriction digest of BBa_K1985014 in pSB1A3 plasmid backbone with EcoRI and BamHI. Single digest with EcoRI shows undigested plasmid, as two bands can be seen

Congo Red Assay

This assay was used for validation as it confirms the presence of self assembled amyloid that our BioBrick encodes for. The Congo red plates were compared to a negative control strain, VS45 with pVS105. pVS105 contains CsgAss with Sup35M, that does not produce a protein product that self assembles into amyloid. The results (shown in Fig.2) confirmed that our protein self-assembled into amyloid nanostructures due to the red color of the colonies, in comparison to the white negative control colonies, which demonstrates no presence of amyloid. The Sup35-1-61_Cytb cells are a similar colour to the positive control PVS72. These results confirmed that our protein was being produced and targeted to the curli export pathway by CsgAss, as well as self-assembling into amyloid fibres.

Figure 2 shows our Congo Red plate. PVS105 indicates the negative control and PVS72 indicates the positive control. The section labelled cytb shows this BioBrick, indicating that amyloid is being formed as red colonies can be seen

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 the Sup35-1-61_Cytb prion forming domain. The image was compared with samples without amyloid fibers being produced by PVS105 VS45 cells (figure 4) and PSV72 VS45 cells (figure 3) which produced amyloid fibers. From figure 5 it can be seen that there is a similar structure beside the cells to the fiber found on the PSV72 image. This strongly indicates that the sup35-1-61_cytb protein does in fact produce amyloid fibers.

Figure 3.Shows PSV72 which is the positive control. The image shown above shows that there is a presence of amyloid fibers

Figure 4.Shows the negative control PSV105. It is evident that are no fibers present, which was expected

Figure 5.Shows the VS45 cells with the Sup35-1-61_cytb prion forming domain. From the image there are structures beside the cells which resemble the fibers found in the positive control PSV72.

References

[1]Men, D., Zhou, J., Li, W., Leng, Y., Chen, X., Tao, S., and Zhang, X. "Fluorescent protein nanowire-mediated protein microarrays for multiplexed and highly sensitive pathogen detection". ACS Appl. Mater. Interfaces (June 2016) DOI: 10.102.