Difference between revisions of "Part:BBa K5241008"

 
 
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<partinfo>BBa_K5241008 short</partinfo>
 
<partinfo>BBa_K5241008 short</partinfo>
  
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PICZ&#945;" 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). "pPICZ&#945;A-PHDD-6xH" is a vector used for expressing the PHDD protein with a 6xHis tag in Pichia pastoris
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<span style="font-weight: bold;">Short Description:</span>"pPICZ &#945;" 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). "pPICZ&#945;A-PHDD-6xH" is a vector used for expressing the PHDD protein with a 6xHis tag in Pichia pastoris.
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<span style="font-size: 150%; font-weight: bold;">Gene Circuit Diagram:</span>
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<html>
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<figure><img style="width: 65%; padding:28px;"src="https://static.igem.wiki/teams/5241/wiki/parts-static/8/bba-k5241008-img01.png">
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<figcaption>AOX1 promoter α-factor secretion signal pPICZaAPkrhdd-6xH 6xHis AOX1 terminator</figcaption>
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</figure>
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</html>
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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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<html>
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<figure><img style="width: 65%; padding:28px;"src="https://static.igem.wiki/teams/5241/wiki/parts-static/8/bba-k5241008-img02.png"></figure>
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</html>
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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.
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<span style="font-size: 150%; font-weight: bold;">Reference documentation</span>
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[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
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<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 05:25, 30 September 2024


pPICZaAPkrhdd-6xH

Short Description:"pPICZ α" 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:

AOX1 promoter α-factor secretion signal pPICZaAPkrhdd-6xH 6xHis AOX1 terminator

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.

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 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