Difference between revisions of "Part:BBa K3779009"

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.

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