Difference between revisions of "Part:BBa K5241008"
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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). "pPICZαA-PHDD-6xH" is a vector used for expressing the PHDD protein with a 6xHis tag in Pichia pastoris
 
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). "pPICZαA-PHDD-6xH" is a vector used for expressing the PHDD protein with a 6xHis tag in Pichia pastoris
  
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Gene Circuit Diagram:
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Result:
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<span style="font-size: 150%; font-weight: bold;">Description:</span>
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Choose the appropriate restriction enzymes to perform double digestion on the pPICZaA plasmid, generating sticky ends that match the target gene fragment (Pkrhdd) and the 6xHis tag sequence. Separate and purify the digested plasmid backbone. Perform a ligation reaction with the purified target gene fragment (Pkrhdd), the 6xHis tag sequence, and the digested pPICZaA plasmid backbone.
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<span style="font-size: 150%; font-weight: bold;">Result:</span>
  
 
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Revision as of 09:03, 27 September 2024


pPICZaAPkrhdd-6xH

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). "pPICZαA-PHDD-6xH" is a vector used for expressing the PHDD protein with a 6xHis tag in Pichia pastoris

Gene Circuit Diagram:

Description:

Choose the appropriate restriction enzymes to perform double digestion on the pPICZaA plasmid, generating sticky ends that match the target gene fragment (Pkrhdd) and the 6xHis tag sequence. Separate and purify the digested plasmid backbone. Perform a ligation reaction with the purified target gene fragment (Pkrhdd), the 6xHis tag sequence, and the digested pPICZaA plasmid backbone.

Result:

We are using chemical and electroporation methods to transform the pPICZaAPkrhdd-6xH into Pichia pastoris, and colonies have formed after cultivation, but there is no melanin expression after induction with methanol. This could be due to a failure in plasmid construction, or it could be due to other reasons.

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 XbaI site found at 5174
    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 NheI site found at 4469
    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 BglII site found at 1089
    Illegal BglII site found at 2620
    Illegal BamHI site found at 2977
    Illegal XhoI site found at 1075
    Illegal XhoI site found at 2284
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 43
    Illegal EcoRI site found at 52
    Illegal EcoRI site found at 967
    Illegal XbaI site found at 5174
    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 XbaI site found at 5174
    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 site found at 5330
    Illegal BsaI.rc site found at 316
    Illegal BsaI.rc site found at 3129