Part:BBa_K5241006

pBAD-6xH-pelBPkrhdd-6xH

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).

Source:Pseudomonas koreensis,

Description：

Using the designed primers, perform PCR reactions with templates containing the target genes (pelB, Pkhdd) from Pseudomonas koreensis to amplify the required gene fragments. Select appropriate restriction enzymes to digest the pBAD plasmid, creating sticky ends that match the target gene fragments. Purify the target gene fragments and ligate them with the digested pBAD plasmid using DNA ligase. The target gene fragments combine with the plasmid backbone through complementary sticky ends to form the complete plasmid pBAD-pelBPkhdd. Transform the ligation product into the suitable host cells - Escherichia coli TOP10.

Result:

The PBAD vector contains the arabinose operon, and the absence of melanin production after induction with L-arabinose indicates that the construction of the PBAD-6XH-PelBPkrhdd plasmid has failed.

Reference documentation

[1] Clark, M. A., Hammond, F. R., Papaioannou, A., Hawkins, N. J., & Ward, R. L. (1997). Design of a novel switchable antibody display system in Pichia pastoris. Immunotechnology, 3(4), 215-223. https://doi.org/10.1016/s1380-2933(97)00016-x

[2] Schuller, Artur, et al. "Escherichia coli σ70 promoters allow expression rate control at the cellular level in genome-integrated expression systems." Microb. Cell Fact., vol. 19, no. 1, 5 Mar. 2020, p. 6209-6224, https://doi.org/10.1186/s12934-020-01311-6

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 NgoMIV site found at 1140
  Illegal AgeI site found at 319
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 316