Generator

Part:BBa_K638201:Design

Designed by: Matthew Jones   Group: iGEM11_Cambridge   (2011-09-19)


Arabinose inducible Poly-His Reflectin A1 generator


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
    Illegal BamHI site found at 1260
  • 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


Design Notes

The aim of this design was to produce a his-tagged variant of reflectin A1 so that the protein could be purified by his-affinity column purification. In addition, pBAD was used to allow us to induce expression of the protein with arabinose. The promoter and his-tag sequences were taken from the registry (see subpart list) as were the RBS and the terminator. We used Gibson Assembly to insert the reflectin gene, after we had checked for illeagal restriction sites. We found none, and thanks to the sequence being codon optimised for expression in E.coli had little trouble expressing and purifying the protein.


Source

This part was built from a codon optimised variant of the Reflectin A1 gene provided by Wendy Crookes. We added a his-tag by incorporating the part Part:BBa_K128005 into Gibson Assembly primers.

References