Part:BBa_K3779009

pPIC9K-SS-bgly ① Codon optimization

The original target gene BBa_K3779022 was codon optimized according to pichia pastoris preference, and the optimized codon SS-bgly (BBa_K3779001) was used for plasmid construction to improve the activity of our enzyme.

②the signal peptide

We added the signal peptide α-factor (BBa_K3779002) to our plasmid, which contributed to the extracellular expression of our target protein in pichia pastoris.

③ AOX1 promoter

We used methanol-induced promoters and designed 5 'and 3' primers BBa_K3779000 and BBa_K3779003.

④ Integration of foreign genes

Linearized plasmids were introduced into p. Pastoris genome for homologous integration by electrotransformation. This method is simple and efficient in practice. The recombinant expression strain of pichia pastoris β -glycosidase was constructed, as shown in figure.

Results As shown in the figure, there was a target band of about 2000 bp in line with the theoretical size, while the blank control GS115-pPIC9K only had a band of 500 bp, proving that the recombinant strain was successfully constructed.

The GS115-pPIC9K-SS-bgly 6# transformants were activated and fermented on YPD plate.The fermentation process curve and the standard curve between the standard protein concentration and the absorbance value are shown below.

sds-page analysis

After methanol induction for 120 h, 10 L of supernatant was taken for SDS-PAGE electrophoresis. A clear band was observed near the target molecular weight of 57 kDa, indicating that SS-bgly gene was successfully expressed in P. Pastoris.

By introducing the synthetic gene into the plasmid, we can obtain the desired strain

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 7794
  Illegal EcoRI site found at 13316
  Illegal EcoRI site found at 18563
  Illegal EcoRI site found at 27993
  Illegal XbaI site found at 14126
  Illegal XbaI site found at 19373
  Illegal XbaI site found at 28803
  Illegal PstI site found at 1920
  Illegal PstI site found at 3161
  Illegal PstI site found at 5716
  Illegal PstI site found at 7543
  Illegal PstI site found at 8683
  Illegal PstI site found at 11238
  Illegal PstI site found at 13065
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 7794
  Illegal EcoRI site found at 13316
  Illegal EcoRI site found at 18563
  Illegal EcoRI site found at 27993
  Illegal NheI site found at 8365
  Illegal NheI site found at 13887
  Illegal NheI site found at 14834
  Illegal NheI site found at 19134
  Illegal NheI site found at 20081
  Illegal NheI site found at 28564
  Illegal NheI site found at 29511
  Illegal PstI site found at 1920
  Illegal PstI site found at 3161
  Illegal PstI site found at 5716
  Illegal PstI site found at 7543
  Illegal PstI site found at 8683
  Illegal PstI site found at 11238
  Illegal PstI site found at 13065
  Illegal NotI site found at 7806
  Illegal NotI site found at 13328
  Illegal NotI site found at 18575
  Illegal NotI site found at 28005
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 7794
  Illegal EcoRI site found at 13316
  Illegal EcoRI site found at 18563
  Illegal EcoRI site found at 27993
  Illegal BglII site found at 4170
  Illegal BglII site found at 6573
  Illegal BglII site found at 9692
  Illegal BglII site found at 12095
  Illegal BglII site found at 17342
  Illegal BglII site found at 24215
  Illegal BglII site found at 26772
  Illegal BamHI site found at 7510
  Illegal BamHI site found at 13032
  Illegal BamHI site found at 18279
  Illegal BamHI site found at 27709
  Illegal XhoI site found at 1749
  Illegal XhoI site found at 3005
  Illegal XhoI site found at 7764
  Illegal XhoI site found at 13286
  Illegal XhoI site found at 18533
  Illegal XhoI site found at 23050
  Illegal XhoI site found at 27963
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 7794
  Illegal EcoRI site found at 13316
  Illegal EcoRI site found at 18563
  Illegal EcoRI site found at 27993
  Illegal XbaI site found at 14126
  Illegal XbaI site found at 19373
  Illegal XbaI site found at 28803
  Illegal PstI site found at 1920
  Illegal PstI site found at 3161
  Illegal PstI site found at 5716
  Illegal PstI site found at 7543
  Illegal PstI site found at 8683
  Illegal PstI site found at 11238
  Illegal PstI site found at 13065
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 7794
  Illegal EcoRI site found at 13316
  Illegal EcoRI site found at 18563
  Illegal EcoRI site found at 27993
  Illegal XbaI site found at 14126
  Illegal XbaI site found at 19373
  Illegal XbaI site found at 28803
  Illegal PstI site found at 1920
  Illegal PstI site found at 3161
  Illegal PstI site found at 5716
  Illegal PstI site found at 7543
  Illegal PstI site found at 8683
  Illegal PstI site found at 11238
  Illegal PstI site found at 13065
  Illegal AgeI site found at 7872
  Illegal AgeI site found at 13394
  Illegal AgeI site found at 18641
  Illegal AgeI site found at 28071
  
• 1000
  COMPATIBLE WITH RFC[1000]