Part:BBa_K3598011

AOX1 Promoter_fp1_mfp5_fp1_AOX1 Terminator

The circuit we transformed into Pichia Pastoris to produce fp151 protein.

Demonstration

Figure 1. Part demonstration

This part is a composite part consisting of an AOX1 Promoter, an fp1-mfp5-fp1 recombinant protein sequence, and an AOX1 Terminator. The cooding sequence of fp1-mfp5-fp1 recombinant protein were code optimised 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 fp1-mfp5-fp1 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 any bands in gel results, which means no protein is synthesized. That may due the formation of inclusion bodies with fp1-mfp5-fp1 recombinant protein.

Figure 2. SDS-PAGE gel analysis of supernatant samples during fp1-mfp5-fp1 fermentation

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NotI site found at 1896
  
• 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 1962
  
• 1000
  COMPATIBLE WITH RFC[1000]