Team:NWU-CHINA-B/Engineering

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  • ⅠConstruction of Pichia pastoris recombinant strain expressing β-glycosidase



    1. Design

      2. 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.



      ② The signal peptide


      We chose 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 motor instruments





    2. Build


      3. Construction of a recombinant strain expressing β-glycosidase in Pichia pastoris.




      Delete the bases


      Insert target gene





    3. Test


      4. ① Extraction of recombinant plasmid pPIC9K-SS-bgly (BBa_K3779009)


      ② Linearization of recombinant plasmids


      ③ Linearized recovery


      ④ Preparation of Pichia pastoris competent cells


      ⑤ Transformation of Pichia pastoris with linearized plasmid


      ⑥ Screening of positive transformants


      ⑦Shaking flask fermentation of recombinant strains


      ⑧The expression products were detected by SDS polyacrylamide gel electrophoresis


      ⑨ The growth curve, enzyme activity and protein content of recombinant strain were determined





    4. Learn


      5. ① Construction of recombinant strain GS115-pPIC9K-SS-bgly


      After code optimization, GC content of SS-bgly gene sequence decreased from 39.7% to 37.3 %, and codon adaptation index (CAI) increased from 0.74 to 0.93. We extracted pPIC9K-SS-bgly plasmid after extracted after activating the strains which had been successfully constructed by the company. After the linearization of Sal I enzyme, the plasmid electricity transformed into a receptive cell and we built MD plate by dilution plating procedure. Repeated freeze-thaw method could be used to release the genome genes of the single colony on the plate, and the supergene was taken for PCR verification. 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 500 bp band. It proved that the recombinant strain was successfully constructed.





      MD plate screening converters

      PCR validation of partial converters




      ② Screening of high enzyme activity inverters





      Standard curve of pNP concentration and absorbance value

      Enzyme activity of partial converters


      In order to obtain inverters with high enzyme activity, we selected 6 transformants successful verified by PCR for shaker fermentation screening. The enzyme activity ranged from 32-70 U/mL and the enzyme activity of 6# inverters was the highest (70 U/mL).




      ③ Determination of growth curve, enzyme activity and protein content


      GS115-pPIC9K-SS-bgly 6# inverter was activated on YPD plate. After the single colony was grown, fermentation was carried out and samples were taken every 24 h. The samples were diluted with ultra-pure water and OD600 was determined. Results As shown in the figure below, the recombinant strain grew rapidly in the first 72 h, then the growth rate slowed down and reached a stable level at 120 h. The activity of iβ-glycosidase kept increasing from 0 to 120 h, and reached the highest value of 70 U/mL at 120 h, then decreased gradually. The protein content increased steadily and reached 1.5 mg/mL after 120 h induction. The protein content continued to increase but the enzyme activity decreased after methanol induction, which was speculated to be caused by the accumulation of protease.





      Fermentation process curve


      Standard curve between standard protein concentration and absorbance value




      ④ SDS-PAGE analysis




      After methanol induction for 120 h, 10μL 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.







    5. Conclusion


      6. In this part, the recombinant Pichia pastoris strain GS115-pPIC9K-SS-bgly was constructed. First,the target gene SS-bgly optimized by codon was submitted to the company for synthesis, and then we constructed the target gene SS-bgly and E.coli TOP10 into a plasmid containing pPIC9K-SS-bgly. After the strain was activated, the plasmid was extracted, linearized by Sal I enzyme, and transferred to Pichia pastoris GS115 by electricity. MD plate and colony PCR were used to preliminarily screen the positive transformants. The successful transformants were screened by shaking flask fermentation to obtain a strain of 6# transformants with high enzyme activity. After methanol induction for 120 h, the enzyme activity and protein content reached 70 U/mL and 1.5 mg/mL, respectively. SDS-PAGE analysis of 10μL supernatant showed that the target band was found at 57 kDa, which proved that SS-bgly gene was successfully expressed in Pichia pastoris.







  • Ⅱ  Construction of recombinant strain with high expression of β-glycosidase



    1. Design


      2. Gene copy number
          Within a certain range, the expression level of target protein increased with the increase of copy number, but when it reached a certain value, the expression level of target protein decreased with the increase of copy number. We screened for high copies by increasing the resistance concentration, and the copy number was determined by fluorescence quantification.




    2. Build


      3. Construction of recombinant strain with high expression of β-glycosidase.




      Delete the bases


      Insert target gene





    3. Test


      4. ① Preparation of medium and solution


      ② SS-bgly gene was amplified


      ③ Construction of recombinant expression vector


      ④ The recombinant plasmid was electrically transferred to Pichia pastoris


      ⑤ Screening of positive transformants


      ⑥ Screening of high-activity strains


      ⑦ The copy number was determined by fluorescence quantitative PCR


      ⑧β-glycosidase activity and protein content were determined






    4. Learn


      5. ① Gene amplification


      Using the plasmid pPIC9K-SS-bgly extracted and preserved in the first part as a template, the SS-bgly gene fragment containing Kpn Ⅰ and Not Ⅰ was amplified by primers with a size of about 1500 bp. Electrophoresis results showed that the amplification was successful.





      MD plate screening converters

      SS-bgly electrophoresis figure bgly gene amplification




      ② Double digestion validation of recombinant plasmid



      Kpn Ⅰ and Not Ⅰ were introduced into both ends of the PCR amplified gene SS-bgly and the expression vector pPICZαA were double digested, purified and recovered by electrophoresis, then linked and transformed into competent cells, and then evenly coated on the low-salt LB plate with corresponding resistance. The colony PCR results showed that the band size was 2000 bp, which was consistent with the theory. The positive clones successfully verified by PCR were selected for further enzymatic digestion verification. As shown in the figure, pPICZαA and SS-bgly were bands consistent with the theoretical size after the recombinant vector was digested by double enzyme. Combined with the sequencing results of the synthesis company, it was confirmed that the plasmid pPICZαA-SS-bgly was successfully constructed.



      Colony PCR was used to verify positive inverters



      Identification of recombinant plasmid pPICZaA-SS-bgly by enzyme digestion




      ③ Screening of positive transformants


      The plasmid pPICZαA-SS-bgly was extracted from the successful strain after overnight culture. After the Sac ⅰ enzyme was linear and purified, the strain GS115-pPIC9K-SS-bgly 6#, which was optimized in chapter 2, was transferred by power transfer. Coated on YPD resistant plate (containing 100 g/mL Zeocin), sterile water was used to collect single colonies grown on the plate and gradient coated on YPD plates with different resistance. The final screened inverters were released by repeated freeze-thaw and other operations, and the above clear was used as template for PCR verification with primers. As shown in the figure, obvious bands appeared at about 3000 bp, which was consistent with the theoretical value, indicating that the gene had been successfully inserted into the target strain.





      PCR identification of recombinant yeast strains




      ④ Screening of high-activity strains


      Different inverters showed slightly different enzyme activities in shaking flask test. In order to screen for strains with high vitality, the inverters successfully screened by high resistance plate and verified by colony PCR were selected for shaking flask fermentation, and the enzyme activities ranged from 102-152 U/mL, among which the activity of 4# inverters was the highest, 152 U/mL.





      Enzyme activity of inverters




      ⑤ Comparison of fermentation characteristics between GS115/9K-SS-bgly 6# and GS115/9K-ZαA-SS-bgly 4#


      The recombinant strain GS115/9K-ZαA-SS-bgly 4# was fermented and we determined the growth curve, enzyme activity and protein content. As shown in the figure, there was no difference in the growth of recombinant GS115/9K-SS-bgly 6# and GS115/9K-ZαA-SS-bgly 4#. The enzyme activity and protein content of GS115/9K-ZαA-SS-bgly 4# were higher than which of GS115/9K-SS-bgly 6#. The enzyme activity of GS115/9K-SS-bgly 6# kept increasing from 0 to 120 h,and reached the highest value of 152 U/mL at 120 h,and then gradually decreased, which increased by 117 %. The protein content has been increasing steadily, reaching 1.8 mg/mL after methanol induction for 120 h.The protein content is 20% higher than that induced by methanol. The protein content still increased after methanol induction, but the enzyme activity decreased, which may be due to the accumulation of protease, leading to the degradation of the target protein.




      Fermentation process curves (a) Growth curve (b) Enzyme activity (c) Protein content




      ⑥ The copy number of genes was determined by fluorescence quantitative PCR


      △△CT=(CT, BGLY-CT,GAP)Test-(CT, Bgly-ct (GAP)Calibrator calculated the mean CT values of target gene and reference gene and the expression level of target gene SS-bgly in transforant GS115/9K-SS-bgly 6# and transforant GS115/9K-ZαA-SS-bgly 4#. Using plasmid pPIC9KSS-bgly as single copy, the copy numbers of SS-bgly gene in GS115/9K-SS-bgly 6# and GS115/9K-ZαA-SS-bgly 4# were 1.89 and 4.03, respectively, and were round 2 and 4.



      Gene copy number calculation




      Note 2: GAP was the internal reference gene and was represented by Ref; Plasmid pPIC9K-SS-Bgly was used as reference sample and was represented by Cal. SS-bgly stands for Target gene and is represented by Target. Strains GS115/9K-bgly 6# and GS115/9K-ZαA-bgly 4# were used as Test samples.




    5. Conclusion


      6. The SS-bgly gene sequence was amplified from the plasmid pPIC9K-SS-bgly extracted from the first part as a template, and was linked to the vector pPICZαA. E. Coli TOP 10 strain containing pPICZαA-SS-bgly plasmid was successfully constructed. After the strain was activated on the low-salt LB resistance plate, the plasmid was extracted and linearized by restriction endonuclease Sac ⅰ enzyme. Then the plasmid was transferred to GS115-pPIC9K-SS-bgly 6# strain constructed in the first part. The transformants were preliminarily screened by resistance plate and colony PCR with different concentration gradients. The high activity strain GS115/9K-ZαA-SS-bgly 4# was obtained by shaking flask fermentation. After 120 h of methanol induction, its enzyme activity was up to 152 U/mL, which was increased by 117 %, and its protein content was 1.8 mg/mL, which was increased by 20 %. The gene copy number was 4 by fluorescence quantitative PCR.



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