Part:BBa_K4247024:Design

orf438-tyrosinase operon

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 37
  Illegal NotI site found at 76
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 896
  Illegal AgeI site found at 798
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 1138
  

Design Notes

The enzyme tyrosinase is an oxidase found across taxa, it contains copper and is well known for its tyrosine modifying step that gives melanin. Our project specifically focused on converting the tyrosines of mfp151 (parts BBa_K4247020, BBa_K4247021) into DOPA in E.coli, a post-translational modification that makes mfp sticky. A way to achieve this dopaquinone conversion is by first producing mfp in vitro and then expose it to tyrosinase, but it has the limitations of having to purify the enzymes and of not allowing the dopa modification to occur in all the tyrosines that are not exposed to the enzyme. For this reason, we focused on a co-expression system where E.coli would have mfp151 on a plasmid and tyrosinase with its copper cofactor (orf438) on another. By inducing with IPTG, the tyrosines incorporated in the mfp151 protein have been hydroxylated to DOPA.

Source

NCBI accession: WP_030787646.1 Tyrosinase NCBI accession: WP_078632176 Orf438 (tyrosinase cofactor)