Coding
R body

Part:BBa_K5121023:Design

Designed by: Carlo Famularo   Group: iGEM24_Sydney-Australia   (2024-09-28)


Reb1: RebB N-terminal Cys


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 806
    Illegal NheI site found at 868
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 748
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

Although in vivo, the reb genes are transcriptionally independent, here they are encoded in polycistronic fashion. Since wild type rebB contains no cysteines (and neither does rebA), this composite part contains a modified version of rebB with an added N-terminal cysteine (BBa_K5121015). This modification was made using an extension overlap PCR of the original reb1 plasmid (BBa_K5121011) with overlapping primers as described in the design notes for the basic part (BBa_K5121015). Since the R-body is a large protein polymer, we sought to enhance the accessibility of our introduced cysteine to reacting maleimides. We achieved this with a flexible poly-glycine linker placed in between the introduced N-terminal cysteine and the remainder of the rebB coding region (N-CGGGGS-C).

Source

E. coli