Coding
PafA

Part:BBa_K5172000:Design

Designed by: Katie Oxley   Group: iGEM24_Sheffield   (2024-09-29)


PafA alkaline phosphatase from Flavobacterium johnsoniae


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 529
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 529
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 529
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 529
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 529
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

The WT PafA had its signalling peptides removed, and 5x His tags were added (confirmed via sequencing). It lost a His tag due to mutation, however this did not impact purification.

For the 6 redesigned PafA variants, a percentage of the residues were fixed during redesign (30, 50 or 70%) using ConSurf, with two variants ordered for each percentage conserved. In all redesigns, residues identified on ChimeraX as within 5Å from the ligand or the two Zn2+ ions were fixed as well.

The signal peptides of the PafA were removed to prevent it from being expressed into the periplasmic membrane for easier protein purification.



Source

This sequence comes from Flavobacterium johnsoniae genomic DNA. It was provided to us in a Pet21a(+) backbone. From sequencing, it was identified that there are only 5x histidine residues in the tag due to mutation, however, there were no issues regarding protein purification.


References