Difference between revisions of "Part:BBa K3779009:Experience"

(→‎Applications of BBa_K3779009)
(→‎Applications of BBa_K3779009)
 
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===Applications of BBa_K3779009===
 
===Applications of BBa_K3779009===
 
① Codon optimization
 
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.
 
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
 
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.
 
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
 
③ AOX1 promoter
 +
 
We used methanol-induced promoters and designed 5 'and 3' primers BBa_K3779000 and BBa_K3779003.
 
We used methanol-induced promoters and designed 5 'and 3' primers BBa_K3779000 and BBa_K3779003.
−
Integration of foreign genes
+
 
 
④ Integration of foreign genes
 
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.
 
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.
 +
 +
https://2021.igem.org/wiki/images/2/29/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.
 +
 +
https://2021.igem.org/wiki/images/2/2a/T--NWU-CHINA-B--GS115-pPIC9K-SS-bgly_strains.png
 +
https://2021.igem.org/wiki/images/7/75/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.
 +
 +
https://2021.igem.org/wiki/images/d/db/T--NWU-CHINA-B--_t9K_12_.png
 +
https://2021.igem.org/wiki/images/6/66/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.
 +
 +
https://2021.igem.org/wiki/images/7/70/T--NWU-CHINA-B--SDS-PAGE_electrophoresis_figure%284%29.png
  
 
===User Reviews===
 
===User Reviews===

Latest revision as of 06:23, 13 October 2021


This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_K3779009

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

User Reviews

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