Part:BBa_K1739000

Sequence coding for amyloid Sup35NM

We have improved 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 two illegal cut sites for Pstl and one for Bsal 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.

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 the nature of our plasmid, we analysed it through 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, therefore validating our BioBrick.

Figure 1. Shows the agarose gel produced, following the restriction digest of our part.