Difference between revisions of "Part:BBa K3779009"

 
 
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__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K3779009 short</partinfo>
 
<partinfo>BBa_K3779009 short</partinfo>
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① Codon optimization
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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.
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②the signal peptide
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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.
 +
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③ AOX1 promoter
 +
 +
We used methanol-induced promoters and designed 5 'and 3' primers BBa_K3779000 and BBa_K3779003.
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 +
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.
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 +
https://2021.igem.org/wiki/images/2/29/T--NWU-CHINA-B--pPIC9K-SS-bgly.png
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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.
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https://2021.igem.org/wiki/images/2/2a/T--NWU-CHINA-B--GS115-pPIC9K-SS-bgly_strains.png
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https://2021.igem.org/wiki/images/7/75/T--NWU-CHINA-B--PCR_validation_of_inverters.png
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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.
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https://2021.igem.org/wiki/images/d/db/T--NWU-CHINA-B--_t9K_12_.png
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https://2021.igem.org/wiki/images/6/66/T--NWU-CHINA-B--_t9K_13_.png
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sds-page analysis
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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.
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 +
https://2021.igem.org/wiki/images/7/70/T--NWU-CHINA-B--SDS-PAGE_electrophoresis_figure%284%29.png
  
 
By introducing the synthetic gene into the plasmid, we can obtain the desired strain
 
By introducing the synthetic gene into the plasmid, we can obtain the desired strain

Latest revision as of 06:46, 20 October 2021


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.

T--NWU-CHINA-B--pPIC9K-SS-bgly.png

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.

T--NWU-CHINA-B--GS115-pPIC9K-SS-bgly_strains.png T--NWU-CHINA-B--PCR_validation_of_inverters.png

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.

T--NWU-CHINA-B--_t9K_12_.png T--NWU-CHINA-B--_t9K_13_.png 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.

T--NWU-CHINA-B--SDS-PAGE_electrophoresis_figure%284%29.png

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]