Part:BBa_K3598012

AOX1 Promoter_CsgA_mfp5_mfp5_AOX1 Terminator

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

Demonstration

Figure 1. Part demonstration

This part is a composite part consisitiong an AOX1 promter, CsgA-mfp5-mfp5 recombinant protein sequence, and an AOX1 terminator. The coding sequence of CsgA-mfp5-mfp5 has been codon optimized for Pichia.

Experiments and results

This circuit was inserted into the pPIC9K vector through enzyme digestion and ligation, and then transformed to P. Pastoris GS115 through LiCl chemical transformation.

The recombinant strain was is used for the fermentation production of CsgA-mfp5-mfp5 recombinant protein. The fermentation was carried out in BMMY medium and added 5% methanol every day to regulate the inducible system. We took samples every 24 h, the protein exsited in the fermentation supernatant.

We verified the recombinant proteins by SDS-PAGE. However, we did not observe correct bands in the gel results, which means no correct protein was synthesized. In addition, the bands observed in the gel results were much larger than they should be. This might be because that plasmids were 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]