Part:BBa_K5241002

pkrhdd

Description: Encodes 4-hydroxyphenylpyruvate dioxygenase.

Source:Pseudomonas koreensis

Result:

1.Based on Pseudomonas_koreensis_D26 [gene=hppD][protein=4-hydroxyphenylpyruvate dioxygenase], a gene with a PelB signal peptide and 6His tag is synthesized, aiming for the tyrosinase to be localized to the bacterial periplasmic space，After constructing the plasmid and transformation, there was a small amount of melanin expressed.

2.Encodes a fusion protein consisting of the Pkrhdd enzyme from Pseudomonas koreensis, an N-terminal T7 tag for solubility, and a C-terminal 6xHis tag for purification. The fusion protein is under the control of the pBAD promoter, which allows for arabinose-inducible expression, and confers ampicillin resistance (AmpR). We used arabinose induction, and there was no visible melanin, indicating that our plasmid construction was unsuccessful.

3."PICZα" refers to a vector containing the yeast alpha-factor signal peptide sequence, which aids in the proper secretion of proteins. It was developed by Invitrogen (now part of Thermo Fisher Scientific). We used the vector pPICZαA-PHDD-6xH, which expresses the PHDD protein with a 6xHis tag in Pichia pastoris, and although colonies formed, no melanin was produced after induction with methanol, indicating that our plasmid construction failed.

Reference documentation

[1] Gätjen, D., Tomszak, F., Dettmann, J. C., Droste, M., Nölle, V., & Wieczorek, M. (2022). Design of a novel switchable antibody display system in Pichia pastoris. Applied Microbiology and Biotechnology, 106(18), 6209-6224. https://doi.org/10.1007/s00253-022-12108-5

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 43
  Illegal EcoRI site found at 52
  Illegal EcoRI site found at 967
  Illegal PstI site found at 925
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 43
  Illegal EcoRI site found at 52
  Illegal EcoRI site found at 967
  Illegal PstI site found at 925
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 43
  Illegal EcoRI site found at 52
  Illegal EcoRI site found at 967
  Illegal XhoI site found at 1075
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 43
  Illegal EcoRI site found at 52
  Illegal EcoRI site found at 967
  Illegal PstI site found at 925
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 43
  Illegal EcoRI site found at 52
  Illegal EcoRI site found at 967
  Illegal PstI site found at 925
  Illegal NgoMIV site found at 342
  Illegal AgeI site found at 319
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 316