Part:BBa_K1739000

Sequence coding for amyloid Sup35NM

Sup35 is derived from the yeast prion protein Sup35p and excludes the C-terminal domain. The N-terminal domain allows self-assembly of functional amyloid [1][2]. This BioBrick is an improved version of a previously designed BioBrick (Part:BBa_K401001) from the [http://2010.igem.org/Team:Valencia Valencia 2010 iGEM team] that encoded the Sup35 protein from Saccharomyces cerevisiae. The previously designed BioBrick contained illegal BioBrick restriction sites. Our improved BioBrick has optimized in order to remove these cut sites and we have produced a part compatible with the iGEM part submission standards.

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
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 522
  

Validation

To validate our plasmid, we analyzed it through a diagnostic double restriction cut, followed by agarose gel electrophoresis. A restriction digest of our plasmid was carried out using EcoRI and PstI. These enzymes cleave pSBIC3 at 2029bp, with our insert being 780bp. By comparing the sizes of our inserts to the Invitrogen 1kB DNA Marker, we found that our fragments were the correct sizes.

Figure 1. Agarose gel of the restriction digest of BBa_K1739000 in pSCB13 plasmid backbone with EcoRI and PstI.

Function Validation of the Sup35NM sequence was performed on (BBa_K1739002) by diagnostic Congo Red plate assay and AFM imaging that demonstrated the presence of amyloid by formation of red colonies.

[1] 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.

[2] 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.