Part:BBa_K1985010

Sequence coding for amyloid Sup35 residues 1-61

This part is an improved version of a previously designed BioBrick (Part:BBa_K1739002) from the Kent 2015 iGEM team. This part contains two segments, the CsgA signal sequence and only the first 61 aminoacids of the prion domain Sup35. 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]. This improved plasmid does not contain the constitutive promoter (Part:BBa_J23104) as was used for (Part:BBa_K1739002), giving the user choice over the promoter that they use.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Validation

The plasmid was analysed through a diagnostic double restriction cut, using the enzymes NdeI and SspI. This was followed by agarose gel electrophoresis. The enzymes cleave the insert and a part of the plasmid at 1618bp, with the remainder plasmid being 790 bp. The sizes of the two fragments were compared with the size of the Invitrogen 1kB DNA marker and it was found that our fragments were the correct sizes.

Figure 1.1% agarose gel of the restriction digest of BBa_K1985010 in pSB1C3 plasmid backbone with NdeI and SspI

Function validation of the Sup35 1-61 sequence was performed on BBa_K1985013 by diagnostic Congo Red plate assay demonstrated the presence of amyloid fibres.

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.