Team:NWU-CHINA-B/Notebook
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We prepare some medium and put them into sterilizing pot for sterilizing. After that, we put E. coli TOP10-pPIC9K-SS-bgly strain on LB plate and then put them in a 37℃ incubator.
We remove the colonies already grown in the incubator，and pick single colonies with sterile gun heads under the super clean workbench.Then we feed them into a LB liquid containing ,and Develop colonies 16 hours under 25ug/mL ana resistance at 37℃.
We take out the grown plasmids in the shake,and then perform the plasmid for plasmid extraction.We put the extracted plasmids in the-20℃ refrigerator.
We perform the recombinant plasmid step and heat the experimental product in a 37℃ water bath pan for 3 hours which is the monomeric digestion step.Then, we confirm the linearized results by agarose gel electrophoresis，and obtaine our experimental product.Finally we perform linearized recycling using a kit to purify recycling linearized pPIC9K-SS-bgly recombinant plasmids.
We configure with fresh YPD plates and remove the laboratory-originally preserved P. from the liquid nitrogen tankpastoris GS115 species,which are growen at 30℃ on a freshly prepared YPD plate.
We pick single colonies on YPD plates appearing at the underlined position and being in the intermediate position with sterile gun heads.We put them into 5 mL of fresh YPD liquid，and perform them at a temperature of 30℃ and at a rotational speed of 250 rpm for 24 h.
We pick out the colonies on the YPD plates and place them in a fresh-configured YPD liquid for 24 h at 30℃, 250rpm。
We measure the OD values of the YPD liquid and then we repare the LDST solution.
We re-formulate LB medium and medium and then sterilize the culture medium.We then underline the LB plate,and incubate the LB plates in a 37℃ incubator.
We prepare the LB liquid and pick out the colonies.Then we place the strains into the culture medium and incubate them in a shaker for 12h.
We remove the grown plasmids and performe plasmid extraction.Exextracted plasmids are kept in the-20℃ refrigerator.
LB medium is reformulated and colony culture is conducted.
After the completion of culture, we select the colonies, put them into the prepared LB medium and put them into the shaker culture.
We take out the plasmids that have been grown, extract the plasmids and measure the plasmids concentration. After that, we find that the plasmid concentration increased.
After the digestion in the water bath is completed, we perform electrophoresis and gel cut recovery operations. But we find that the recovery concentration is still low.
We reformulated YPD plate and after thawing P. Pastoris GS115 strains at room temperature, inoculated the strains on YPD plate and cultured them in a 30℃ incubator.
We measure the OD of the recombinant strain after shaking flask fermentation and extract the plasmid GS115-9K-ZαA.
The long colony of P. pastoris GS115 is selected and directly shot into 5mL fresh YPD liquid. The culture condition is 30 ℃, 250rpm, for 24h.
Absorb a small amount of bacterial liquid and dilute it with ultra-pure water to measure the OD, prepare LDST solution, and then measure the OD value of BMGY.
BMGY medium is prepared, BMGY OD is measured at 9:00 PM, and methanol induction is started at 11:00 PM.
Plasmid is amplified by PCR. Plasmid digestion was performed at 9 PM and methanol induction is performed at 11 PM.
Methanol induction ended.
We prepare 0.5M PH6.0 sodium acetate buffer
Buffer solution was prepared at 10 am, crude enzymes were dialysis, YPD liquid was prepared, sterilized for 2 hours, bacterial colonies (GS-9K-bgly and GS/9K-ZaK-bgly) were picked out at 1 noon.We put into liquid YPD at 30℃, 220R shaker for 24h.
Dialysis, at 1:30 pm. culture bacterial solution on BMGY medium; OD of 9K and ZαA are measured at 5 PM and 9 PM and methanol induction is performed at 10 PM.
Electrophoresis is performed after the recombinant plasmid was digested, and methanol induction was performed at night to measure the OD of 9K and ZαA.
Lyophilized crude enzymes.
MD plate culture is completed.
MD plate culture is completed.
Begin linearization of plasmid into Pichia Pastoris, prepare plate, inoculate.
Pick Colony, shake flask fermentation, plasmid extraction.
ELECTROPHORESIS, recycling.
The plasmid (linearized) is transferred into Pichia pastoris and coated on the MD plate. During which the experiment made a critical error and was restarted.
The plasmids (linearized) were transferred into Pichia pastoris and coated on MD plate.
SS-BGLY-F, SS-bgly-r and Gap-f, gap-r primers were amplified by RT-PCR
Positive transformants are screened and amplified by PCR.
Shake Flask fermentation of recombinant strains.
SDS-PAGE electrophoresis was used to detect the expressed product.
Od, methanol induced Pichia pastoris.
Od, methanol induced Pichia pastoris.
Od, methanol induced Pichia pastoris.
Od, methanol induced Pichia pastoris.
Od, methanol induced Pichia pastoris.
Od, methanol induced Pichia pastoris.
The supernatant is obtained by centrifugation and then ammonium sulfate is added to the supernatant until the saturation is 60% and the temperature is 4 °C overnight.
Centrifugally collected precipitates were diluted with 0.05 m sodium acetate buffer at Ph 6.0 and precipitated to 1/10 of the original volume. The precipitates were put into dialysis bags. Dialysis was performed with sodium acetate buffer of the same concentration and Ph.
The supernatant was obtained by centrifugation and concentrated by ultrafiltration in a 10kda ultrafiltration centrifuge tube.
Freeze-dry with a Lyophilizer to obtain crude enzymes and refrigerate at 4 °C.
5 MG/ML SS-bgly and 5 MG/ML ginsenoside substrate were used for enzymatic reaction in sodium acetate buffer solution. The reaction was stopped by adding 1.5 times methanol after 4 hours at a Ph value of 6 and a temperature of 50-90 °C. The content of Ginsenoside enzymatic reaction product (CK) was determined by HPLC to determine the optimal temperature of enzyme reaction
5 MG/ML SS-bgly and 5 MG/ML ginsenoside substrates were used for enzymatic reaction in Sodium Acetate buffer solution. The temperature of the reaction system was controlled at 4.0-8.0, respectively, after 4 hours of reaction, adding 1.5 times methanol to stop the reaction, the yield of Ginsenoside enzymatic reaction product CK was determined by HPLC to determine the optimal Ph of enzyme reaction.
Different Metal Ions were added to the above optimized enzyme reaction system. The final concentration was 5 mM. After 4 hours of reaction, 1.5 times of methanol was added to stop the reaction. The yield of ginsenoside enzyme reaction product CK was determined by HPLC, to determine the effects of metal ions on the transformation of ginsenoside substrate to CK.
After selecting the metal ions that will promote the most production of CK, a series of concentration gradients are set up to determine the optimum ion concentration.
9 MG/ML SS-bgly and different concentration of ginsenoside substrate were used for enzymatic reaction in sodium acetate buffer solution. Under the optimum conditions, the concentration of substrate was controlled to 10 MG/ML-50 MG/ML respectively, adding 1.5 times methanol to the reaction, the yield of enzymatic reaction product CK of Ginsenoside was determined by HPLC, and the optimum substrate concentration was determined.
The SS-bgly with different enzyme content and the optimum ginsenoside substrate concentration were used for enzymatic reaction in Sodium Acetate buffer solution. Under the above optimum conditions, the enzyme content was controlled to 0-12 MG/ML, respectively, the yield of Ginsenoside enzymatic reaction product CK was determined by HPLC with addition of 1.5 times methanol to determine the optimal enzyme quantity.
View results.
To improve accuracy, repeat 9.23-9.29.