Difference between revisions of "Part:BBa K3598012"
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The circuit we transformed into Pichia Pastoris to produce AMP CEN1HC-Br.
 
The circuit we transformed into Pichia Pastoris to produce AMP CEN1HC-Br.
  
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===Usage and Biology===
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===Demonstration===
 
[[File:T--BEIJING 4ELEVEN--Acf151 1.png|600px|thumb|center|Figure 1. Part demonstration]]
 
[[File:T--BEIJING 4ELEVEN--Acf151 1.png|600px|thumb|center|Figure 1. Part demonstration]]
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===Usage and Biology===
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Our AOX1-csgA-mfp5-mfp5 is a composite part consisting of an AOX1 Promoter, an csgA-mfp5-mfp5 recombinant protein sequence, and an AOX1 Terminator. It regulates the expression of our recombinant cohesive protein. We inserted the sequence of the system onto vector pPIC9K and transferred the resulting plasmid into Pichia pastoris inoculated on BMMY culture, then added 5% methanol every day to induce its expression. Then we verified the production of adhesive proteins by SDS-PAGE electrophoresis. However, the bands in the results are larger than they should be. This might be because that plasmids are integrated into the yeast genome with multiple copies, producing proteins that fold together.
 
Our AOX1-csgA-mfp5-mfp5 is a composite part consisting of an AOX1 Promoter, an csgA-mfp5-mfp5 recombinant protein sequence, and an AOX1 Terminator. It regulates the expression of our recombinant cohesive protein. We inserted the sequence of the system onto vector pPIC9K and transferred the resulting plasmid into Pichia pastoris inoculated on BMMY culture, then added 5% methanol every day to induce its expression. Then we verified the production of adhesive proteins by SDS-PAGE electrophoresis. However, the bands in the results are larger than they should be. This might be because that plasmids are integrated into the yeast genome with multiple copies, producing proteins that fold together.
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[[File:T--BEIJING 4ELEVEN--Acf 2.png|600px|thumb|center|Figure 2. SDS-PAGE gel analysis of supernatant samples during csgA-mfp5-mfp5 fermentation]]
 
[[File:T--BEIJING 4ELEVEN--Acf 2.png|600px|thumb|center|Figure 2. SDS-PAGE gel analysis of supernatant samples during csgA-mfp5-mfp5 fermentation]]
 
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Revision as of 15:24, 27 October 2020


AOX1 Promoter_CsgA_mfp5_mfp5_AOX1 Terminator

The circuit we transformed into Pichia Pastoris to produce AMP CEN1HC-Br.

Demonstration

Figure 1. Part demonstration

Usage and Biology

Our AOX1-csgA-mfp5-mfp5 is a composite part consisting of an AOX1 Promoter, an csgA-mfp5-mfp5 recombinant protein sequence, and an AOX1 Terminator. It regulates the expression of our recombinant cohesive protein. We inserted the sequence of the system onto vector pPIC9K and transferred the resulting plasmid into Pichia pastoris inoculated on BMMY culture, then added 5% methanol every day to induce its expression. Then we verified the production of adhesive proteins by SDS-PAGE electrophoresis. However, the bands in the results are larger than they should be. This might be because that plasmids are integrated into the yeast genome with multiple copies, producing proteins that fold together.

Figure 2. SDS-PAGE gel analysis of supernatant samples during csgA-mfp5-mfp5 fermentation

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NotI site found at 2127
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 937
    Illegal XhoI site found at 1191
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 2193
  • 1000
    COMPATIBLE WITH RFC[1000]