iGEM 2021 — Team NWU-CHINA-B

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# Team:NWU-CHINA-B/Notebook

NWU-CHINA-B

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* **TEAM**

  [Team members](https://2021.igem.org/Team%3ANWU-CHINA-B/Team)
  [Attributions](https://2021.igem.org/Team%3ANWU-CHINA-B/Attributions)
* **PROJECT**

  [Contribution](https://2021.igem.org/Team%3ANWU-CHINA-B/Contribution)
  [Project Description](https://2021.igem.org/Team%3ANWU-CHINA-B/Description)
  [Engineering Success](https://2021.igem.org/Team%3ANWU-CHINA-B/Engineering)
  [Experiments](https://2021.igem.org/Team%3ANWU-CHINA-B/Experiments)
  [Notebook](https://2021.igem.org/Team%3ANWU-CHINA-B/Notebook)
  [Proposed Implementation](https://2021.igem.org/Team%3ANWU-CHINA-B/Implementation)
  [Proof Of Concept](https://2021.igem.org/Team%3ANWU-CHINA-B/Proof_Of_Concept)
  [Project Modeling](https://2021.igem.org/Team%3ANWU-CHINA-B/Model)
  [Results](https://2021.igem.org/Team%3ANWU-CHINA-B/Results)
* **PARTS**

  [Parts](https://2021.igem.org/Team%3ANWU-CHINA-B/Parts)
* **SAFETY**

  [Safety](https://2021.igem.org/Team%3ANWU-CHINA-B/Safety)
* **HUMAN PRACTICES**

  [Collaboration](https://2021.igem.org/Team%3ANWU-CHINA-B/Collaborations)
  [Partnership](https://2021.igem.org/Team%3ANWU-CHINA-B/Partnership)
  [Communication](https://2021.igem.org/Team%3ANWU-CHINA-B/Communication)
  [Human Practice(Integrated)](https://2021.igem.org/Team%3ANWU-CHINA-B/Human_Practices)
* **AWARDS**

  [Education](https://2021.igem.org/Team%3ANWU-CHINA-B/Education)
* **JUDGING FORM**

  [Judging Form](https://igem.org/2021_Judging_Form?id=3779)

>

![](https://static.igem.org/mediawiki/2021/e/e2/T--NWU-CHINA-B--notebook1.png)

[![](https://static.igem.org/mediawiki/2021/4/49/T--NWU-CHINA-B--team%E5%9B%BE%E7%89%872.png)](#yemianbeijing)

![](https://static.igem.org/mediawiki/2021/2/21/T--NWU-CHINA-B--contribution.png)

7 July ,2021

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.

![](https://static.igem.org/mediawiki/2021/b/b8/T--NWU-CHINA-B--notebook2.png)

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℃.

![](https://static.igem.org/mediawiki/2021/2/20/T--NWU-CHINA-B--notebook3.png)

13 July ,2021

14 July ,2021

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.

![](https://static.igem.org/mediawiki/2021/5/52/T--NWU-CHINA-B--notebook4.png)

15 July ,2021

16 July ,2021

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.

![](https://static.igem.org/mediawiki/2021/b/b7/T--NWU-CHINA-B--notebook5.png)

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.

![](https://static.igem.org/mediawiki/2021/2/24/T--NWU-CHINA-B--notebook6.png)

17 July ,2021

18 July ,2021

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.

![](https://static.igem.org/mediawiki/2021/0/0a/T--NWU-CHINA-B--notebook7.png)

19 July ,2021

28 July ,2021

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.

30 July ,2021

31 July ,2021

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.

5 August ,2021

6 August ,2021

After the completion of culture, we select the colonies, put them into the prepared LB medium and put them into the shaker culture.

![](https://static.igem.org/mediawiki/2021/9/92/T--NWU-CHINA-B--notebook9.png)
![](https://static.igem.org/mediawiki/2021/f/f7/T--NWU-CHINA-B--notebook10.png)

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.

![](https://static.igem.org/mediawiki/2021/c/c8/T--NWU-CHINA-B--notebook11.png)

7 August ,2021

8 August ,2021

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.

![](https://static.igem.org/mediawiki/2021/5/53/T--NWU-CHINA-B--notebook12.png)

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.

![](https://static.igem.org/mediawiki/2021/f/fd/T--NWU-CHINA-B--notebook13.png)

11 August ,2021

12 August ,2021

We measure the OD of the recombinant strain after shaking flask fermentation and extract the plasmid GS115-9K-ZαA.

![](https://static.igem.org/mediawiki/2021/9/96/T--NWU-CHINA-B--notebook14.png)

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.

13 August ,2021

14 August ,2021

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.

15 August ,2021

16 August ,2021

Plasmid is amplified by PCR. Plasmid digestion was performed at 9 PM and methanol induction is performed at 11 PM.

Methanol induction ended.

18 August ,2021

19 August ,2021

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.

20 August ,2021

21 August ,2021

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.

![](https://static.igem.org/mediawiki/2021/a/a9/T--NWU-CHINA-B--notebook16.png)
![](https://static.igem.org/mediawiki/2021/d/d1/T--NWU-CHINA-B--notebook17.png)

Electrophoresis is performed after the recombinant plasmid was digested, and methanol induction was performed at night to measure the OD of 9K and ZαA.

![](https://static.igem.org/mediawiki/2021/5/56/T--NWU-CHINA-B--notebook18.png)

23 August,2021

24 August,2021

Lyophilized crude enzymes.

![](https://static.igem.org/mediawiki/2021/2/23/T--NWU-CHINA-B--notebook19.png)

MD plate culture is completed.

25 August,2021

26&27 August,2021

MD plate culture is completed.

Begin linearization of plasmid into Pichia Pastoris, prepare plate, inoculate.

1 September,2021

2 September,2021

Pick Colony, shake flask fermentation, plasmid extraction.

![](https://static.igem.org/mediawiki/2021/c/cb/T--NWU-CHINA-B--notebook20.png)
![](https://static.igem.org/mediawiki/2021/8/85/T--NWU-CHINA-B--notebook21.png)

ELECTROPHORESIS, recycling.

![](https://static.igem.org/mediawiki/2021/9/9f/T--NWU-CHINA-B--notebook22.png)

3 September,2021

4 September,2021

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.

7 September,2021

8 September,2021

SS-BGLY-F, SS-bgly-r and Gap-f, gap-r primers were amplified by RT-PCR

![](https://static.igem.org/mediawiki/2021/0/06/T--NWU-CHINA-B--notebook23.png)
![](https://static.igem.org/mediawiki/2021/2/2c/T--NWU-CHINA-B--notebook24.png)

Positive transformants are screened and amplified by PCR.

10 September,2021

11 September,2021

Shake Flask fermentation of recombinant strains.

SDS-PAGE electrophoresis was used to detect the expressed product.

12 September,2021

13 September,2021

Od, methanol induced Pichia pastoris.

Od, methanol induced Pichia pastoris.

14 September,2021

15 September,2021

Od, methanol induced Pichia pastoris.

Od, methanol induced Pichia pastoris.

16 September,2021

17 September,2021

Od, methanol induced Pichia pastoris.

Od, methanol induced Pichia pastoris.

18 September,2021

19 September,2021

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.

20 September ,2021

21 September ,2021

The supernatant was obtained by centrifugation and concentrated by ultrafiltration in a 10kda ultrafiltration centrifuge tube.

![](https://static.igem.org/mediawiki/2021/9/90/T--NWU-CHINA-B--notebook25.png)

Freeze-dry with a Lyophilizer to obtain crude enzymes and refrigerate at 4 °C.

22 September,2021

23 September,2021

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.

24 September,2021

25 September,2021

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.

26 September,2021

27 September,2021

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.

![](https://static.igem.org/mediawiki/2021/1/1d/T--NWU-CHINA-B--T--NWU-CHINA-B--notebook27.jpg)

29 September,2021

1 October,2021

View results.

To improve accuracy, repeat 9.23-9.29.

4--11 October,2021

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# Team:NWU-CHINA-B/Engineering

NWU-CHINA-B

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* **TEAM**

  [Team members](https://2021.igem.org/Team%3ANWU-CHINA-B/Team)
  [Attributions](https://2021.igem.org/Team%3ANWU-CHINA-B/Attributions)
* **PROJECT**

  [Contribution](https://2021.igem.org/Team%3ANWU-CHINA-B/Contribution)
  [Project Description](https://2021.igem.org/Team%3ANWU-CHINA-B/Description)
  [Engineering Success](https://2021.igem.org/Team%3ANWU-CHINA-B/Engineering)
  [Experiments](https://2021.igem.org/Team%3ANWU-CHINA-B/Experiments)
  [Notebook](https://2021.igem.org/Team%3ANWU-CHINA-B/Notebook)
  [Proposed Implementation](https://2021.igem.org/Team%3ANWU-CHINA-B/Implementation)
  [Proof Of Concept](https://2021.igem.org/Team%3ANWU-CHINA-B/Proof_Of_Concept)
  [Project Modeling](https://2021.igem.org/Team%3ANWU-CHINA-B/Model)
  [Results](https://2021.igem.org/Team%3ANWU-CHINA-B/Results)
* **PARTS**

  [Parts](https://2021.igem.org/Team%3ANWU-CHINA-B/Parts)
* **SAFETY**

  [Safety](https://2021.igem.org/Team%3ANWU-CHINA-B/Safety)
* **HUMAN PRACTICES**

  [Collaboration](https://2021.igem.org/Team%3ANWU-CHINA-B/Collaborations)
  [Partnership](https://2021.igem.org/Team%3ANWU-CHINA-B/Partnership)
  [Communication](https://2021.igem.org/Team%3ANWU-CHINA-B/Communication)
  [Human Practice(Integrated)](https://2021.igem.org/Team%3ANWU-CHINA-B/Human_Practices)
* **AWARDS**

  [Education](https://2021.igem.org/Team%3ANWU-CHINA-B/Education)
* **JUDGING FORM**

  [Judging Form](https://igem.org/2021_Judging_Form?id=3779)

>

![](https://static.igem.org/mediawiki/2021/e/eb/T--NWU-CHINA-B--engineering1.png)

[![](https://static.igem.org/mediawiki/2021/4/49/T--NWU-CHINA-B--team%E5%9B%BE%E7%89%872.png)](#yemianbeijing)

![](https://static.igem.org/mediawiki/2021/2/21/T--NWU-CHINA-B--contribution.png)

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

1. Design
![](https://static.igem.org/mediawiki/2021/7/79/T--NWU-CHINA-B--engeering1.png)

***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.

![](https://static.igem.org/mediawiki/2021/e/e0/T--NWU-CHINA-B--teamengerring1.png)

**The motor instruments**

2. Build

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

![](https://static.igem.org/mediawiki/2021/c/cc/T--NWU-CHINA-B--engeering3.png)

**Delete the bases**

**Insert target gene**

3. Test

**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

***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.

![](https://static.igem.org/mediawiki/2021/9/9b/T--NWU-CHINA-B--engeering4.png)

***MD* plate screening converters**

![](https://static.igem.org/mediawiki/2021/8/82/T--NWU-CHINA-B--teamengerring2.png)

***PCR* validation of partial converters**

***② Screening of high enzyme activity inverters***

![](https://static.igem.org/mediawiki/2021/f/fe/T--NWU-CHINA-B--engeering6.png)

**Standard curve of pNP concentration and absorbance value**

![](https://static.igem.org/mediawiki/2021/0/0c/T--NWU-CHINA-B--engeering7.png)

**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.

![](https://static.igem.org/mediawiki/2021/a/aa/T--NWU-CHINA-B--engeering8.png)

**Fermentation process curve**

![](https://static.igem.org/mediawiki/2021/0/0c/T--NWU-CHINA-B--engeering9.png)

**Standard curve between standard protein concentration and absorbance value**

***④ SDS-PAGE analysis***

![](https://static.igem.org/mediawiki/2021/e/e3/T--NWU-CHINA-B--teamengerring3.png)

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

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

**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

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

![](https://static.igem.org/mediawiki/2021/9/95/T--NWU-CHINA-B--engeering11.png)

**Delete the bases**

**Insert target gene**

3. Test

**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**

**⑧*β-glycosidase* activity and protein content were determined**

4. Learn

***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.

![](https://static.igem.org/mediawiki/2021/1/11/T--NWU-CHINA-B--engeering12.png)

**MD plate screening converters**

![](https://static.igem.org/mediawiki/2021/e/eb/T--NWU-CHINA-B--engeering5.png)

***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.

![](https://static.igem.org/mediawiki/2021/2/28/T--NWU-CHINA-B--engeering13.png)

**Colony *PCR* was used to verify positive inverters**

![](https://static.igem.org/mediawiki/2021/b/b7/T--NWU-CHINA-B--teamengerring6.png)

**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.

![](https://static.igem.org/mediawiki/2021/0/09/T--NWU-CHINA-B--teamengerring7.png)

***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.

![](https://static.igem.org/mediawiki/2021/2/24/T--NWU-CHINA-B--engeering16.png)

**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.

![](https://static.igem.org/mediawiki/2021/5/5c/T--NWU-CHINA-B--engeering18.png)
![](https://static.igem.org/mediawiki/2021/c/cf/T--NWU-CHINA-B--engeering19.png)
![](https://static.igem.org/mediawiki/2021/9/91/T--NWU-CHINA-B--engeering20.png)

**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**

![](https://static.igem.org/mediawiki/2021/4/4f/T--NWU-CHINA-B--teamengerring8.png)

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

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*.

**Reference**

[1] Yadav S, Pandey A K, Dubey S K. Molecular modeling, docking and simulation dynamics of beta-glucosidase reveals high-efficiency, thermo-stable, glucose tolerant enzyme in Paenibacillus lautus BHU3 strain[J]. International Journal of Biological Macromolecules, 2021, 168: 371-382.
[2] Akcapinar G B, Gul O, Sezerman U. Effect of codon optimization on the expression of Trichoderma reesei endoglucanase 1 in Pichia pastoris[J]. Biotechnology Progress, 2011, 27(5): 1257-1263.
[3] Yang J K, Liu L Y. Codon optimization through a two-step gene synthesis leads to a highlevel expression of Aspergillus niger lip2 gene in Pichia pastoris[J]. Journal of Molecular Catalysis B: Enzymatic, 2010, 63(3-4): 164-169.
[4] Kim S A, Jeong E B, Oh D K. Complete Bioconversion of Protopanaxadiol-Type Ginsenosides to Compound K by Extracellular Enzymes from the Isolated Strain Aspergillus tubingensis[J]. Journal of Agricultural and Food Chemistry, 2021, 69(1): 315-324.
[5] Ma Z, Mi Y, Han X, et al. Transformation of ginsenoside via deep eutectic solvents based on choline chloride as an enzymatic reaction medium[J]. Bioprocess and Biosystems Engineering, 2020, 43(7): 1195-1208.
[6] Jeong E B, Kim S A, Shin K C, et al. Biotransformation of Protopanaxadiol-Type Ginsenosides in Korean Ginseng Extract into Food-Available Compound K by an Extracellular Enzyme from Aspergillus niger[J]. Journal of Microbiology and Biotechnology, 2020, 30(10): 1560-1567.
[7] Yao L, Wang J, He J P, et al. Endophytes, biotransforming microorganisms, andengineering microbial factories for triterpenoid saponins production[J]. Critical Reviews in Biotechnology, 2021, 41(2): 249-272.
[8] Yang H Y, Jin H D, Fu Y, et al. Production of Rare Ginsenosides Rg3 and Rh2 by Endophytic Bacteria from Panax ginseng[J]. Journal of Agricultural and Food Chemistry, 2019, 67(31): 8493-8499.
[9] Fan W X, Huang Y L, Zheng H, et al. Ginsenosides for the treatment of metabolic syndrome and cardiovascular diseases: Pharmacology and mechanisms[J]. Biomedicine & Pharmacotherapy, 2020, 132: 110915.

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# Team:NWU-CHINA-B/Experiments

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![](https://static.igem.org/mediawiki/2021/b/bb/T--NWU-CHINA-B--experiments1.png)

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![](https://static.igem.org/mediawiki/2021/2/21/T--NWU-CHINA-B--contribution.png)

* Chapter one:The experiment design

1.1 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.

1.2 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.

1.3 AOX1 promoter

We used methanol-induced promoters and designed 5 'and 3' primers BBa\_K3779000 and BBa\_K3779003.

1.4 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.

* Chapter two: Construction of Pichia pastoris recombinant  *β -glycosidase*  strain

2.1 Experimental reagents and instruments

2.1.1 Strain, plasmid and primer

(1) E.coli TOP 10 strain, plasmid pPIC9K purchased from Invitrogen Company;
(2) P. pastoris GS115 strain was preserved in our laboratory;
(3) Primer sequences used in this chapter are shown in the table below, synthesized by Shanghai Shenggong Bioengineering Technology Service Co.,LTD.

![](https://static.igem.org/mediawiki/2021/0/0e/T--NWU-CHINA-B--experiment9.png)

2.1.2 Main reagents

![](https://static.igem.org/mediawiki/2021/d/d7/T--NWU-CHINA-B--experiment10.png)
![](https://static.igem.org/mediawiki/2021/4/42/T--NWU-CHINA-B--experiment11.png)

2.1.3 Main instruments and equipment

![](https://static.igem.org/mediawiki/2021/f/f6/T--NWU-CHINA-B--experiment12.png)

2.1.4 Preparation of medium and solution

LB medium: 1 % yeast extract, 2 % tryptone, 2 % sodium chloride, and 1.5 %-2 % AGAR can be prepared LB plate was prepared and sterilized at 121 ℃ for 20 min. The plate could be labeled and stored at 4 ℃.

1M phosphate buffer (PPB, pH 6.0) : Dipotassium hydrogen phosphate (174 g/mol) 11.484 g, trihydrate Potassium dihydrogen phosphate (228.22g /mol) 15.06g, add 400 mL ultra-pure water, ultrasonic stirring to dissolve, add hydrogen oxidation Potassium was adjusted to pH 6.0 and kept at room temperature at a constant volume of 500 mL.

BMGY medium: 5 g yeast extract, 10 g tryptone, 1 M phosphate buffer (pH 6.0) 50 mL, 6.7 g yeast without amino nitrogen source, 5 mL glycerin, supplemented to 500 mL.

BMMY medium: Methanol was used to replace glycerin in BMGY medium

*4-nitrophenyl -β-d-glucopyranoside* (pNPG) solution: Weigh 15.05 mg pNPG in 6 mL ultra pure water was dissolved in a 10 mL clean centrifuge tube by ultrasound, then the volume was fixed to 10 mL, and divided into 200 μL, using tin foil paper.Store in refrigerator at 4 ℃ under shading for later use.

2.2 Experimental methods

2.2.1 Gene acquisition

The target gene *SS-bgly*  sequence was found in NCBI database (GenBank Login Number, WP 009992676).
After code optimization, *pPIC9K-SS-bgly*  plasmid was synthesized by Shanghai Engineer (as shown in Figure 2.1) and stored in E.coli TOP 10 cells. The optimized gene sequences are shown in appendix.

[![](https://static.igem.org/mediawiki/2021/0/05/T--NWU-CHINA-B--experiment13.png)](https://static.igem.org/mediawiki/2021/0/05/T--NWU-CHINA-B--experiment13.png)

2.2.2 Construction of recombinant strain *GS115-pPIC9K-SS-bgly*

2.2.2.1 Extraction of recombinant plasmid

*E.coli* TOP *10-pPIC9K-SS-bgly* strain constructed by the company was taken out of the ultra-low temperature refrigerator and solved at room temperature
Frozen. In the ultra-clean table, a small amount of bacterial solution was first sucked out with a sterile spear head on a LB plate containing 25 μg/mL kana-resistant.
Then replace a new spear head to gradually split the bacteria liquid, and culture at 37 ℃ for about 12 h, the colony could be observed.After the single colony growing in the middle position of the lineation position was selected with sterile spear head, the spear head could be directly shot into LB liquid containing 25 μg/mL containing kana-resistance for culture. The culture condition was 37 ℃, 220 rpm, and the culture time was about 16 h. After culture, the plasmid *pPIC9K-SS-bgly*  was extracted, and the extracted plasmid was labeled and stored in the refrigerator at -20 ℃

2.2.2.2 Linearization of recombinant plasmid

Recombinant plasmids can be incorporated into P.pastoris in two different ways: their basic circular structure or linearization. However, studies have shown that the efficiency of circular plasmids is much lower than that of linear plasmids if they are integrated in circular form. Therefore, it is necessary to linearize circular plasmids before homologous integration. According to the restriction site of plasmid pPIC9K and the target gene sequence, the Sal I enzyme was linearized and the integration site was located at His. The single enzyme digestion system of recombinant plasmid can be prepared according to the table below and reacted in 37 ℃ constant temperature water bath for 3-4 h.

![](https://static.igem.org/mediawiki/2021/5/5b/T--NWU-CHINA-B--experiment14.png)

After digestion, the linearization results can be confirmed by agarose gel electrophoresis. The detailed experimental methods are as follows:

(1) Add agarose and 1×TAE buffer in a ratio of 1:1 to a non-polluting triangle bottle, heat it until the powder is completely dissolved and then take it out. Rinse the bottle with running water to make the temperature drop rapidly to the point where the triangle bottle can be held by hand;

(2) Add the dye prepared in advance with a clean spear head, and blow the spear head repeatedly below the liquid level
Beat, until the dyeing agent is evenly mixed, gently pour into the clean rubber making board, insert the comb;

(3) After being placed at room temperature for a period of time and confirming its solidification, carefully remove the comb, put the gel block into the electrophoresis tank, and add 1×TAE buffer until the liquid level does not exceed the gel block;

(4) Take 30 μL samples, add 3 μL 10×loading buffer, mix evenly and add into the sample adding hole of the rubber block;

(5) Turn on the electrophoresis apparatus, set the voltage to 120 V, and start electrophoresis;

(6) After the electrophoresis, close the instrument, remove the glue block, and observe it under the UV lamp.

2.2.2.3 Linearized recovery

After electrophoresis, the linearized *pPIC9K-SS-bgly*  recombinant plasmid was purified and recovered using the kit

2.2.2.4 Preparation of pichia pastoris competent cells

(1) The strain *P.pastoris* GS115 stored in the laboratory was taken out from the liquid nitrogen tank, thawed at room temperature, reversed and mixed repeatedly. A small amount of the strain was first absorbed with a sterile gun in a super-clean table on a freshly prepared YPD plate, and then a new gun was replaced and split in sequence, and cultured at 30 ℃ for about 48 h.

(2) Single colonies appeared in the middle position on the line position of YPD plate were selected with sterile spear head, and the spear head could be directly shot into 5 mL fresh YPD liquid. The culture conditions were 30 ℃ and 250 rpm. After about 24 h of culture, absorb a small amount of bacterial liquid and dilute it with ultrapure water to measure OD600.

(3) According to the measured OD600 value, appropriate amount of bacteria liquid was absorbed, centrifuged, and the bacteria were suspended in 100 mL fresh YPD medium again. The initial OD was 0.5, and the culture conditions were kept unchanged to continue the oscillation culture. During this period, samples were taken until OD600 reached 1.3-1.5, and the culture was finished. It takes about 4-5 hours, during which LDST solution can be prepared;

(4) Collect fresh bacteria liquid into sterile centrifuge tube, centrifuge at 6000 rpm for 5 min to collect bacteria;

(5) 40 mL fresh LDST solution was slowly added to the centrifuge tube with bacterial precipitation, and the sterile gun head was carefully blown several times until the mixture was uniform, and the mixture was incubated at 30℃ for 30 min;

(6) Pour off the supernatant after centrifugation at room temperature, then immediately add 1 mL 1 M sorbitol placed on the ice box with a sterile gun head, carefully and repeatedly blow the thall stuck on the tube wall until homogenized, and then suction out into 1.5 mL sterile centrifuge tube;

(7) Centrifugally collect thallus and repeat washing with sorbitol for 3 times;

(8) Use sterile gun tip to absorb 400 μL 1 M sorbitol placed on the ice box, add to the centrifuge tube carefully and repeatedly until the bacteria completely suspended, then divide according to the amount of 80L per centrifuge tube, take out one tube on ice for reserve, the rest quickly transferred to -80 ℃ refrigerator, can be stored for half a year.

2.2.2.5 Transformation of Pichia pastoris with linearized plasmid

(1) Linearized plasmids of about 0.1-2 μg were added to the competent cells, mixed gently, and then added to the pre-cooled 0.2cm shock cup (the shock cup was soaked in 75% ethanol, taken out with tweezers on the sterile operating table and dried in strong wind under UV), and ice bath for 5 min, with care not to generate bubbles;

(2) Take out the electric shock cup, dry its surface with clean toilet paper, pay attention to no water, and then put it into the electric shock tank of the electric rotary meter, pay attention to clamping, set the parameters of the electric rotary meter as voltage 1.5kV, resistance 200Ω, capacitance 50 μF;

(3) Immediately after the shock, 1 mL of 1 M sorbitol in ice bath was added and gently mixed, then transferred to a sterile 1.5 mL centrifuge tube;

(4) Appropriate amount of bacterial liquid was absorbed and coated on MD plate, and cultured at 30 ℃ until single colony appeared.

2.2.2.6 Screening of positive transformants

(1) Select some inverters on the MD plate, mark them with a marker pen, then select a small amount of the selected inverters, smear them on the bottom of the PCR tube, then add 50 μL of enzyme-free water, blow and mix;

(2) It was heated at 95 ℃ for 10 min in the PCR instrument, and then immediately frozen in the ultra-low temperature refrigerator for 10 min, repeated 4-5 times;

(3) Centrifuged at 12000 rpm for 1 min, 3 μL supernatant was taken for PCR reaction, and the system was prepared as shown in the table below.

![](https://static.igem.org/mediawiki/2021/4/4b/T--NWU-CHINA-B--experiment15.png)

2.2.3 Shake flask fermentation of recombinant strains

According to the results of colony PCR, a small number of bacteria corresponding to the number of inverters on the MD plate were selected with sterile spear head, and the spear head could be directly shot into 20 mL BMGY medium for culture. The culture condition was controlled at 30 ℃ and 220 rpm. During the culture process, samples were continuously sampled until OD600 reached 4-10. The culture was stopped. According to the measured OD600 value, an appropriate amount of bacterial liquid was absorbed and centrifuged to obtain the thunb. Then the thunb was suspended in 20 mL BMMY medium, and its initial OD600 was 1. The activity of OD600 and *β-glycosidase*  was determined by a small amount of bacteria solution every 24 h. Then 200 μL methanol was added to keep the final concentration of methanol in the medium at about 1%. When *β -glycosidase*  activity decreased continuously, the culture could be stopped.

2.2.4 SDS polyacrylamide gel electrophoresis detected the expressed products

(1) Preparation of SDS-PAGE gel.

![](https://static.igem.org/mediawiki/2021/5/52/T--NWU-CHINA-B--experiment16.png)

After mixing the separated glue evenly in a clean small beaker, add it evenly into the glue making tank with 5 mL gun head, then seal it carefully with ultrapure water, and then gently knock out the bubbles. After the bubbles are exhausted, put them into the oven at 37 ℃ for drying. When it is confirmed that it is completely solidified, the water used for liquid sealing can be carefully poured out, and then the concentrated glue can be added slowly and evenly with 5 mL gun head. The comb will be immediately inserted, which has already been prepared. Be careful to insert it vertically, otherwise the sample hole will be tilted, and carefully move it to the oven at 37 ℃ for drying. The finished glue can be carefully removed from the glue making tank and soaked in ultrapure water. If not temporarily, it can be stored in the refrigerator at 4 ℃.

(2) Sample preparation: Mix the fermentation supernatant and 5× protein electrophoresis buffer at 4:1 volume in a 1.5 mL centrifuge tube, boil for 10 min, and cool to room temperature.

(3) Electrophoresis: take 10 μL samples, and use 80 V voltage electrophoresis to the end of the concentrated gel, then use 100 V voltage to separate the protein. The end sign is that a blue indicator can be observed at the bottom of the gel.

(4) Dyeing and decolorization: sdS-PAGE glue is carefully removed and dyed with staining solution, and then decolorized with decolorization solution until the strip shows clearly.

2.2.5 Growth curve, enzyme activity and protein content of recombinant strain were determined

2.2.5.1 Determination of growth curve

When the bacteria were transferred from BMGY medium to BMMY medium, 5 mL blank BMMY medium could be retained and added into the colorimetric dish as a control. For the bacterial solution sampled during fermentation, ultra-pure water could be used to dilute appropriately to ensure that its OD600 value was in the range of 0.2-0.8. Methanol induction time was taken as abscissa and OD600 as ordinate to draw the growth curve

2.2.5.2 Determination of enzyme activity

(1) Determination of pNP standard curve: pNP solutions with final concentrations of 0,0.8, 1.6, 3.2, 4.0, 4.8mM were prepared respectively. Then add 10 μL to each 96-well plate. Add 80 μL sodium acetate buffer (50 mM, pH 6.0) and 10 μL pNPG to each well. Immediately after bathing at 80 ℃ for 30 min, add 100 μL NaOH (0.5 M) solution. The OD value was measured at 400 nm with a microplate reader, and the mean value of the three groups was measured. Taking OD value as ordinate and pNP concentration as abscissa, the standard curves of OD value and pNP concentration were established.

(2) *β-glycosidase*  activity: *β -glycosidase*  hydrolyzed substrate pNPG to produce pNP, and the maximum absorption peak was found at 400 nm. Add 10 μL diluted enzyme solution, 80 μL sodium acetate buffer solution (pH 6.0), 10 μL 5 mM pNPG to the 96-well plate, react in 80 ℃ water bath for 30 min, add 100 μL 0.5 M NaOH solution to stop the reaction. The absorbance was measured at 400 nm with a microplate reader and the hydrolysate pNP was quantified according to the standard curve. An enzyme activity unit was defined as the amount of enzyme required to produce 1 μM pNP in 1 minute under standard conditions. Enzyme activity E (U/ mL) was calculated according to the following formula:

![](https://static.igem.org/mediawiki/2021/5/5d/T--NWU-CHINA-B--experiment17.png)

C: concentration of PNP produced by hydrolysis;
N: dilution ratio of enzyme;
T: reaction time between enzyme and substrate;
V ': volume of reaction system;
V: enzyme volume.

![](https://static.igem.org/mediawiki/2021/f/fe/T--NWU-CHINA-B--experiment18.png)

2.2.5.3 Determination of protein content

Protein content was determined by using BCA kit

- Chapter 3 Construction of recombinant strains with high expression of β -glycoside enzyme

3.1 Experimental Reagents and instruments

3.1.1 Plasmid and Primers

(1) Plasmid *pPICZ A* was purchased from Invitrogen Company, USA;
(2) Host strain *GS115-pPIC9K-SS-bgly* 6# was constructed and preserved in the second chapter
(3) Primers used in this chapter are shown in the table below, synthesized by Optimus

![](https://static.igem.org/mediawiki/2021/2/24/T--NWU-CHINA-B--experment1.png)

3.1.2 Primary Reagent

![](https://static.igem.org/mediawiki/2021/5/5a/T--NWU-CHINA-B--experiment2.png)

3.1.3 Main instruments and equipments

![](https://static.igem.org/mediawiki/2021/b/b1/T--NWU-CHINA-B--experiment3.png)

3.1.4 Medium and solution preparation

Low Salt LB Media: Reduce the quality of sodium chloride in the second chapter in the second chapter of 2.5 g, and the remaining operation methods are the same. To make resistance tablets, it should be added to Zeocin after sterilization, and then shake the slap in the sterile operating table, and then press the slab. There is a 4 ° C refrigerator to prevent light with a sealing membrane.

YPD resistance tablet: Produce it in the production method of YPD tablet in Chapter 2. When the temperature falls to the extent to which you can hold，add to Zeocin in ultra-clean. Then fully shake the slab, and place the front to place the tablet to solidify and then invert. If you don't have a good labeled mark, there is a 4 ℃ refrigerator to prevent light.

3.2 The experimental method

3.2.1 SS- bgly gene was amplified

The plasmid *PPIC9K-SS-bgly* extracted from Chapter 2 was used as a template and amplified by primers on the gradient PCR instrument*SS- bgly* fragment containing *Kpn Ⅰ and Not Ⅰ* restriction sites was obtained. PCR reaction system (100 μL) is as follows:

![](https://static.igem.org/mediawiki/2021/4/4e/T--NWU-CHINA-B--experiment4.png)

Reaction conditions: predenaturation at 95 ℃ for 30 s; 95 ℃ 15 s, 55 ℃ 15 s, 72 ℃ 90 s for 35 cycles extension at 72 ℃ for 5 min; Store at 4 ℃.After completing the designed PCR program, electrophoresis was performed to detect and recycle the gel. For the method, refer to 2.2.2.3.

3.2.2 Construction of recombinant expression vector

3.2.2.1 PCR products were digested and linked to *pPICZαA*

The expression vector *pPICZαA* and the amplified *SS-bgly* sequence were double-amplified with *Kpn I* and *Not I* enzymes, respectively enzyme digestion, enzyme digestion system is as follows:

Enzyme digestion at 37 ℃ for 3-4 h, electrophoresis detection, purification and recovery, the recovered vector and gene fragment were first used micro.The concentration is determined by a nucleic acid quantizer, and then different volumes of gene fragments and vector DNA solutions are drawn to make both.The ratio of the amount of substances between them is limited within the range of 1:10-1:3, and the enzyme-linked system is as follows:

![](https://static.igem.org/mediawiki/2021/f/ff/T--NWU-CHINA-B--experiment5.png)

Connect it at 16 ℃ for 4 h. If it is not needed for the time being, it can be labeled and stored in the refrigerator at -20 ℃.

3.2.2.2 Preparation of TOP 10 competent *E.coli* cells

(1) The purchased *E.coli* TOP 10 strains were taken out from the liquid nitrogen tank, thawed at room temperature, and used in the ultra-clean table.A small amount of bacteria liquid was absorbed by the spear head of bacteria on the low-salt LB plate, and then a new spear head was replaced to split the bacteria liquid successively at 37 ℃ culture for about 12 h. A single colony in the middle of the LB plate at the lineation position was selected and a sterile spear head was used a small amount of thunb can be dipped, and the spear head can be directly shot into 5 mL LB medium for culture. The culture condition is 37 ℃, 220 rpm, and the time is controlled at 16-22 h.

(2)Appropriate amount of fresh bacterial liquid was added to 50 mL LB medium, and the amount of bacterial liquid generally absorbed was controlled at 500 μL, oscillating culture at 37 ℃ and 220 rmp. During the period, it is stopped from being sampling until the OD600 reaches 0.5-0.6.

(3）Collect fresh bacterial liquid into two sterile centrifuge tubes in ice bath for 10 min, then centrifuge
5 min, the bacteria were collected, and the conditions were set as 4 ℃ and 3500 rpm.

(4)Add 15 mL of 0.1 M pre-cooled CaCl2 solution to the centrifuge tube. Be careful with the pipette tip and pipette the bacteria on the tube wall several times until it is completely mixed, then place on ice for 30 min. Then centrifuge for 5 min to collect bacteria, the conditions are set to: 4 ℃, 3500 rpm.

(5)Repeat steps（4）

(6)Slowly add 1 mL of 0.1 M CaCl2 solution in an ice bath to the centrifuge tube. Carefully and repeatedly pipette until the cells are completely suspended, and then aliquot 100 L per tube. If they are not used temporarily, store them in a refrigerator at -80 ℃, which can be stored for half a year, and take one portion for transformation

3.2.2.3 Receptor cell transformation

（1） A certain amount of ligand products were gently added to the newly prepared *E. coli* TOP 10 competent cells *pPICZαA-SS-bgly*, placed in ice for about 30 min;

(2) The operation of hot beating can be completed in a water bath. The condition of hot beating is that the temperature is 42 ℃ and the time is controlled at 45 s.Take it out of the water bath immediately after the hot stroke, and place it on ice for about 2 minutes, pay attention not to shake;

(3)An appropriate amount of low-salt LB liquid medium was absorbed at the head of the gun and added to the centrifuge tube which had just completed the hot shock operation. The mixture was evenly mixed and the volume was generally 700 μL, and then the culture was oscillated at 37 ℃ for 1 hour to restore the normal growth of E. coli.and expressed the Zeocin resistance gene on the plasmid.

(4)Appropriate volume of bacterial liquid can be directly absorbed or part of the supernatant can be discarded after a short centrifugation and then absorbed.It was uniformly coated on low-salt LB resistant plate (containing 25 μg/mL Zeocin) and cultured at 37 ℃ for 12-18 h. Because this antibiotic is easy to decompose when exposed to light, it is necessary to avoid light.

3.2.2.4 Screening of positive clones and plasmid extraction

Transformants spread on the resistant plate after heat shock operation need to do colony PCR to verify whether the target gene is inserted into the vector. On the aseptic operating table, firstly select some transformants, and after marking them, use a sterilized toothpick to gently touch the labeled transformants. Then dip it in a PCR tube containing 50 μL enzyme-free water. Because the wall of E. coli is relatively thin, just boil it in a boiling water bath for 10 minutes to break the wall. Then centrifuge briefly and take the supernatant for colony PCR verification, with 5 'AOX1 and 3' AOX1 as primers, the reaction system is as follows:

![](https://static.igem.org/mediawiki/2021/d/d6/T--NWU-CHINA-B--experiment6.png)

Reaction conditions refer to 2.2.2.6.
The positive transformants were selected and cultured in low-salt LB liquid (containing 25 μg/mL Zeocin).
After the plasmid was extracted, it was verified by double enzyme digestion. After electrophoresis detection, it was sent to Shanghai SENGong for sequencing to ensure that there was no mutation in the gene sequence.

3.2.3 The recombinant plasmid was electrically transferred to Pichia pastoris

3.2.3.1 Linearization of recombinant plasmids

The recombinant strain *E.coli TOP 10-PPiczαA-SS-BGly* was removed from the ultra-low temperature refrigerator.After thawing at room temperature, they were strewed on low-salt LB resistant plate and cultured at 37 ℃ for about 12 h. Sterile toothpicks were selected from a small number of single colonies in the middle position of the lineation position and cultured in low-salt LB liquid containing the corresponding resistance.The conditions were as follows: 37 ℃, 220 rpm, time was controlled at about 16 h, and plasmids were extracted after culture.
According to the restriction site and target gene sequence of recombinant plasmid *pPICZ-A-SS- bgly*, Sac I restriction site was selected for linearization, and the integration site was located at 5 'AOX1. The restriction system was as follows:

![](https://static.igem.org/mediawiki/2021/7/7d/T--NWU-CHINA-B--experiment7.png)

Enzyme digestion at 37 ℃ for 3-4 h, after electrophoresis purification and recovery, and use NanoDrop to determine its concentration, if not used temporarily, it can be labeled and placed in the refrigerator at -20 ℃.

3.2.3.2 Preparation of competent cells

In this study, the transformants *GS115-pPIC9K-SS-bgly* 6# selected in Chapter 2 were used as host bacteria to prepare feelings for the method of state cell, refer to 2.2.2.4.

3.2.3.3 Electrical transformation of receptive cells

2.2.2.5 is the main reference for the method. The difference is that it needs to be placed in a constant temperature incubator at 30 ℃ after the completion of electrical transfer .It was left for about 1 hour, then evenly coated on YPD resistant plate (containing 100 μg/mL Zeocin), and cultured at 30 ℃ under dark conditions until single colonies were observed.

3.2.4 Screening of positive transformants

3.2.4.1 Screening of high copy converters

(1)On a sterile operating table, the strains on YPD resistant plates were carefully rinsed and collected with appropriate amount of enzyme-free water Place in a sterile centrifuge tube for use.

(2)According to the experimental situation, appropriate amount of bacteria can be directly absorbed or diluted and then absorbed, respectively YPD plates containing 200, 300 and 500 μg/mL Zeocin were cultured at 30 ℃ until single colonies appeared.

3.2.4.2 Colony PCR was used to identify the inverters

Methods refer to 2.2.2.6. The difference is that its primers are 5 'AOX1 and F3, in order to identify recombinant plasmids The successful introduction of *pPIC9K-SS-bgly*  was distinguished from the previous introduction of recombinant plasmid *pPIC9K-SS-bgly* . The F3 primers were selected from the PTEF1 sequence on *pPICZαA* vector.

3.2.5 Screening of high activity strains

The inverters were selected from the resistant plate and verified by colony PCR and then combined with shaker fermentation for high viability .For the screening of potent strains, please refer to 2.2.3.

3.2.6 The copy number was determined by fluorescence quantitative PCR

*GS115/ pPIC9K-SS-bgly*6# constructed in Chapter 2 was extracted using pichia pastoris genomic DNA kit Genomic DNA of strain and high-activity strain constructed and screened in this chapter. *SS- bgly-F* and *SS- bgly-R* were synthesized Primers and gap-f and gap-r primers were used for RT-PCR amplification, where gap was the selected reference gene.The reaction system is as follows:

![](https://static.igem.org/mediawiki/2021/1/19/T--NWU-CHINA-B--experiment8.png)

During this operation, gloves should be worn throughout the whole process, and 3 parallel samples should be made for each sample, which should be avoided as far as possible in the system preparation process .In the presence of bubbles, the liquid left on the tube wall can be collected to the bottom of the tube by centrifugation after the preparation, and the operation process should be completed may be faster. The reaction system was put into fluorescence quantitative pcr instrument, and pcr conditions were set as follows: pre-denaturation at 95 ℃ for 30 s; 95 ℃,30 s, 58 ℃, 30 s, 35 cycles. After the reaction, the data were copied out of the instrument for relative quantitative classical analysis
Method 2-△△CT was used to determine the copy number of target gene.

3.2.7 *β -glycosidase* activity and protein content were determined

Methods refer to 2.2.5.2 and 2.2.5.3.

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# Team:NWU-CHINA-B/Results

NWU-CHINA-B

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* *Ⅰ*

After the *SS-bgly* gene sequence is optimized by codon，GC content decreased from 39.7% to 37.3%，the Codon Adaptation Index number (CAI) increased from 0.74 to 0.93. And then activate the successfully constructed strains and extract *pPIC9K-SS-bgly* plasmid. After Sal Ⅰ enzyme is linearized, it is electroporated into competent cells and spread on the MD plate. For single colonies appearing on the plate, repeated freezing and thawing methods can be used to release genomic genes. Take the supernatant for PCR verification，the result is shown in Figure 2.4. There is a band of interest with the theoretical size at about 2000 bp, while the blank control GS115-pPIC9K only has a band of about 500 bp, which proves that the recombinant strain was successfully constructed.

![](https://static.igem.org/mediawiki/2021/c/cd/T--NWU-CHINA-B--proof0fconcept2.png)

**Figure.Screening transformants by MD plate**

![](https://static.igem.org/mediawiki/2021/b/b2/T--NWU-CHINA-B--teamresults2.png)

**Figure. PCR analysis of partial P. pastoris transformantsLane M: DNA marker; Lanes 1-7: PCR products of transformants; Lane 8: Control**

Different transformants have slightly different enzyme production activities. In order to obtain transformants with high enzyme activity, 6 transformants verified by colony PCR were selected for shake flask fermentation screening, and their enzyme activity was about 32-70 U/mL. Among them, the 6# transformant has the highest enzyme activity:70 U/mL.

![](https://static.igem.org/mediawiki/2021/8/81/T--NWU-CHINA-B--teamresults3.png)

**Figure.The standard curve of pNP concentration and absorbance value**

![](https://static.igem.org/mediawiki/2021/0/0a/T--NWU-CHINA-B--teamresults4.png)

Activate the *GS115-pPIC9K-SS-bgly* 6# transformant on the YPD plate. After the single bacteria grow, proceed to fermentation.Sampling is taken every 24 hours. The sampled bacterial liquid is diluted with ultrapure water and measured for OD600, and then centrifuged to take the supernatant to determine its β-glycosidase activity and protein content. The result is shown in the figure below. In the first 72 hours, the recombinant strain grew rapidly, after which the growth rate slowed down and reached a stable level in 120 hours. The activity of β-glycosidase kept increasing from 0-120 h, reaching the highest value of 70 U/mL at 120 h of fermentation, and then gradually decreasing.The protein content has been increasing steadily, reaching 1.5 mg/mL at 120 h of induction. The methanol continued to induce, and the protein content continued to increase, but the enzyme activity decreased. It is speculated that it may be caused by the accumulation of protease.

![](https://static.igem.org/mediawiki/2021/f/f8/T--NWU-CHINA-B--teamresults5.png)

**Figure. Fermentation process curves**

![](https://static.igem.org/mediawiki/2021/c/ca/T--NWU-CHINA-B--teamresults6.png)

**Figure.Standard curve between standard protein concentration and absorbance value**

After 120 hours of methanol induction, 10 μL supernatant was taken for SDS-PAGE electrophoresis. A clear band was observed near the target molecular weight of 57 kDa (Figure 2.9), indicating that the *SS-bgly* gene was successfully expressed in P. pastoris .

![](https://static.igem.org/mediawiki/2021/1/12/T--NWU-CHINA-B--teamproof_of_concept5.png)

**Figure.SDS-PAGE electrophoresis**

This chapter is mainly to construct *GS115-pPIC9K-SS-bgly* Pichia pastoris recombinant engineering strain. First, after codon optimization of the *SS-bgly* gene, it was sent to Shanghai Shenggong for synthesis, and the E.coli TOP 10 strain containing the *pPIC9K-SS-bgly* plasmid was obtained. After the strain is activated, the plasmid is extracted and linearized by Sal Ⅰ enzyme, and then transferred to Pichia pastoris GS115 by electroporation. Use MD plate and colony PCR to initially screen positive transformants. After the successfully verified transformants are screened by shake flask fermentation, a 6# transformant with high enzyme activity is obtained. During the fermentation process, the recombinant strain grows normally, and the enzyme activity and protein content can reach 70 U/mL and 1.5 mg/mL respectively after 120 h of methanol induction. Take 10 μL supernatant for SDS-PAGE detection, and the band of interest appears at 57 kDa, which proves that the *SS-bgly* gene was successfully expressed in Pichia pastoris.

1. *Ⅱ*

The plasmid pPIC9K-*SS-bgly* was extracted and preserved in the previous step as a template, and the *SS-bgly* gene fragment containing the restriction sites Kpn Ⅰ and Not Ⅰ was amplified by primers. The size is about 1500 bp. And the electrophoresis results showed that it was successfully amplified.

![](https://static.igem.org/mediawiki/2021/4/4c/T--NWU-CHINA-B--teamresults04.png)

**Figure.Agarose gel electropheresis of the *SS-bgly* gene Lane M: DNA marker; Lane 1: *SS-bgly***

The PCR-amplified gene *SS-bgly* introduced restriction sites Kpn Ⅰ and Not Ⅰ at both ends, and double-enzyme digestion with the expression vector pPICZαA. After purification and recovery by electrophoresis, it is connected and transformed into competent large intestine cells.Then evenly spread on the low-salt LB plate with corresponding resistance. The colony PCR result (Figure 3.2) shows that the band size is 2000 bp, which is consistent with theory. The positive clones that have been successfully verified by PCR are selected for further restriction enzyme digestion verification. The results are shown in Figure 3.3. After double digestion of the recombinant vector, pPICZαA and *SS-bgly* bands consistent with the theoretical size appeared. Combined with the sequencing results of Shanghai Shenggong, it was confirmed that the plasmid pPICZαA-*SS-bgly* was successfully constructed.

![](https://static.igem.org/mediawiki/2021/2/2c/T--NWU-CHINA-B--teamresults9.png)

**Figure.Screening transformants by MD plate**

![](https://static.igem.org/mediawiki/2021/1/11/T--NWU-CHINA-B--teamresults10.png)

**Figure. PCR analysis of partial P. pastoris transformantsLane M: DNA marker; Lanes 1-7: PCR products of transformants; Lane 8: Control**

![](https://static.igem.org/mediawiki/2021/6/62/T--NWU-CHINA-B--teamresults28.png)

**Figure .Restriction identification of recombinant plasmid
Lane M: DNA marker; Lane 1: recombinant plasmid; Lane 2: double-digested product of recombinant plasmid**

The strains with successful sequencing verification results were cultured overnight and the plasmid *pPICZA-SS-bgly* was extracted. After linearization with Sac Ⅰ enzyme and purification and recovery, electrotransfer to the *GS115-pPIC9K-SS-bgly* 6# strain selected in the last process. Spread on YPD resistant plate (containing 100 μg/mL Zeocin) and collect the single colonies grown on the plates with sterile water and spread them on YPD plates with different resistances in a gradient. For the finally screened transformants, the genomic genes are released by repeated freezing and thawing operations, and the supernatant is used as a template, and the primers are used for PCR verification. The result is shown in Figure 3.5, there is a clear band around 3000 bp, which is consistent with the theoretical value, indicating that the gene has been successfully inserted into the target strain.

**Figure.Identification of positive transformants by colony PCR
Lane M: DNA marker; Lane 1-7: PCR products of transformants; Lane 8: Control**

![](https://static.igem.org/mediawiki/2021/4/49/T--NWU-CHINA-B--teamresults12.png)

**Figure 3.5 PCR identification of recombinant P. pastoris strainsLane M: DNA marker; Lanes 1-7: PCR products of transformants**

Different transformants are subjected to shake flask experiments, and their enzyme activities are slightly different. In order to screen high vigorous strains, select transformants that have been successfully screened by high resistance plates and verified by colony PCR for shaking flask fermentation. Its enzyme activity is between 102-152 U/mL, and the 4# transformant has the highest activity, which is 152 U/mL.

![](https://static.igem.org/mediawiki/2021/e/e7/T--NWU-CHINA-B--teamresults13.png)

![](https://static.igem.org/mediawiki/2021/3/31/T--NWU-CHINA-B--teamresults290.png)

**Figure.Enzyme activity of transformants**

Ferment the recombinant strain *GS115/9K-ZαA-SS-bgly* 4# to determine the growth curve, enzyme activity and protein content. As shown in Figure 3.7, there is no difference in the growth of recombinant bacteria *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# are higher than that of *GS115/9K SS-bgly* 6# at all stages. The enzyme activity of *GS115/9K-SS-bgly* 6# kept increasing from 0-120 h, and then gradually decreased. The enzyme activity reached the highest value of 152 U/mL at 120 h, which was an increase of 117%. The protein content has been increasing steadily, reaching 1.8 mg/mL after 120 hours of methanol induction, which is a 20% increase compared to 20%. The methanol continued to induce, but the protein content still increased, but the enzyme activity decreased. It may be due to the accumulation of proteases, which leads to the degradation of the target protein.

![](https://static.igem.org/mediawiki/2021/6/69/T--NWU-CHINA-B--teamresults14.png)

The glycerol phosphate dehydrogenase gene GAP is used as an internal reference gene basis：△CT=(CT, bgly -CT,GAP)Test-(CT, bgly - CT,GAP)Calibrator. Calculate the average CT value of the target gene and the internal reference gene and the expression level of the target gene *SS-bgly* in the transformant *GS115/9K-SS-bgly* 6# and the transformant *GS115/9K-ZαA-SS-bgly* 4#. Using the plasmid *pPIC9K SS-bgly* as a single copy, it was calculated that the chromosome copy numbers of the *SS-bgly* gene in the *GS115/9K-SS-bgly* 6# and *GS115/9K-ZαA SS-bgly* 4# strains were 1.89 and 4.03, rounded to 2 and 4.

**Table. Calculation of gene copy number**

![](https://static.igem.org/mediawiki/2021/3/31/T--NWU-CHINA-B--teamresults290.png)

Use the plasmid *pPIC9K-SS-bgly* extracted in Chapter 2 as a template to amplify the *SS-bgly* gene sequence and connect it to the *pPICZαA vector.The E.coli TOP 10* strain containing *pPICZαA-SS-bgly* plasmid was successfully constructed. After the strain was activated on a low-salt LB resistant plate, the plasmid was extracted, linearized by the restriction enzyme Sac 1, and electrotransferred to the *GS115-pPIC9K-SS-bgly* 6# strain constructed in Chapter 2. After initial screening of the transformants with concentration gradient resistance plates and colony PCR, the high vigor strain *GS115/9K-ZA-SS-bgly* 4# was obtained by shaking flask fermentation. After 120 h of methanol induction, its enzyme activity was as high as 152 U/mL, which is an increase of 117%, and the protein content is 1.8 mg/mL, which is an increase of 20%. The fluorescence quantitative PCR technology determines that its gene copy number is 4.- *Ⅱ*

Temperature and pH are both important parameters that affect enzymatic reactions. Under the condition that other conditions remain the same, we studied the effect of *SS-bgly* on the conversion of ginsenoside substrate to CK activity in the range of 40~90 ℃ . As shown in Figure 4.2, when the temperature is 40~80 ℃, the activity of *SS-bgly* transforming ginsenoside substrate to produce CK increases with the increase of temperature, and when the temperature increases to 90 ℃, the activity is reduced, therefore, 80 ℃ is the best reaction temperature for *SS-bgly* to hydrolyze the ginsenoside substrate. *SS-bgly* transforms ginsenoside substrate to produce CK at a higher temperature than most reported ginsenoside hydrolases, and its high temperature resistance makes it more suitable for industrial conversion of ginsenoside substrate to produce CK.

![](https://static.igem.org/mediawiki/2021/1/12/T--NWU-CHINA-B--teamresults16.png)

**Figure. Effect of temperature on the production of CK**

Similarly, when other conditions were consistent, we studied the effect of *SS-bgly* on the conversion of ginsenoside substrate to CK in the range of pH 4.0~8.0. The results are shown in Figure 4.3. When the pH is 4.0-6.0, the activity of *SS-bgly* transforming ginsenoside substrate to produce CK increases with the increase of pH, and when the pH increases to 7.0, its activity to produce CK decreases. Therefore, the optimal reaction pH for *SS-bgly* hydrolysis of ginsenoside substrate is 6.0. The *SS-bgly* gene is expressed in E. coli, and the optimal pH for its transformation of ginsenoside substrate is also 6.0.

![](https://static.igem.org/mediawiki/2021/e/ed/T--NWU-CHINA-B--teamresults17.png)

**Figure. Effect of pH on the production of CK**

Some metal ions have been found to promote the conversion of ginsenosides. For example, Ca2+ regulates the external calcium signal of microorganisms and participates in the regulation of β-glucosidase activity, thereby promoting the biotransformation of ginsenoside Rd. Mg2+ and Fe3+ also promoted the production of ginsenoside CK, but the mechanism is still unclear. CaCO3 CaCl2 and MnCl2 metal ions have been used to increase the β-glucosidase activity of T. purpureogenus in Microbacterium esteraromaticum， Thermotoga petrophila and T. purpureogenus. This study explored the effect of some metal ions on the activity of *SS-bgly* transformed ginsenoside substrate to produce CK. As shown in Figure 4.4, the addition of ZnCl2, KCl, CaCl2, FeCl3 metal ions in the enzyme reaction system has no effect on the activity of transforming ginsenoside substrates to produce CK, while the addition of CuCl2 metal ions reduces the activity of CK production to 57.2%. The addition of LiCl and MgSO4 metal ions can increase the activity of CK production by 25% and 15%. In order to determine the optimal type and concentration of metal ions, we designed further experiments. The result is shown in Figure 4.5. When LiCl and MgSO4 metal ions are added to the enzyme reaction system at a final concentration of 3 mM, the CK production activity is the highest. Under the optimal metal ion concentration, the activity of LiCl metal ion for CK production is 1.15 times that of MgSO4. Therefore, in the subsequent enzymatic reactions, LiCl metal ions with a final concentration of 3 mM were added to promote the production of CK. In previous reports, LiCl has not been found to promote *SS-bgly* conversion of ginsenoside substrate to produce CK. We speculated that the addition of LiCl metal ions changed the structure of the enzyme and increased the affinity of the enzyme for the ginsenoside substrate, thereby promoting the production of CK.Its specific mechanism needs further analysis.

![](https://static.igem.org/mediawiki/2021/6/64/T--NWU-CHINA-B--teamresults18.png)

**Figure.Effects of metal ions on CK producing activity**

![](https://static.igem.org/mediawiki/2021/e/eb/T--NWU-CHINA-B--teamresults19.png)

**Figure.Effects of LiCl and MgSO4 concentration on CK producing activity**

When the temperature of the enzymatic reaction system is set to 80 ℃ and the pH is 6.0, the final concentration of LiCl metal ions is 3 mM. We have studied the influence of the substrate concentration on the conversion of saponin, and the results are shown in Table 4.4. When the ginsenoside substrate concentration is 10 mg/mL and 20 mg/mL, it can be completely converted into 3.79 mg/mL and 7.58 mg/mL CK within 12 h and 30 h, respectively. When the substrate concentration is 30 mg/mL, At mL, the output of CK reaches the maximum value of 9.39 mg/mL, and the conversion rate is 82.5%. When the substrate concentration continued to increase to 40 mg/mL, the CK yield decreased to 9.08 mg/mL, and the conversion rate decreased to 59.8%. According to the production of CK, it is determined that 30 mg/mL is the optimal substrate concentration. Regarding *SS-bgly*, the highest CK yield previously reported is that when combined with Cs-abf, 4.2 mg/mL CK can be obtained. However, in this study, the yield of CK was as high as 9.39 mg/mL, which is the highest concentration of CK currently reported by *SS-bgly*, which is higher than the yield of CK reported in most of the literature. At present, there are many reports about glycosidase conversion of ginsenoside Rb1 to CK. In this topic, the concentration of substrate Rb1 is 15 mg/mL, which is higher than most of the substrate concentration for glycosidase conversion of ginsenoside Rb1.

![](https://static.igem.org/mediawiki/2021/7/75/T--NWU-CHINA-B--teamresults20.png)

**Figure.Effect of crude enzyme concentration on the production of CK**

![](https://static.igem.org/mediawiki/2021/5/5d/T--NWU-CHINA-B--teamresults21.png)

**Figure.Time course for CK production from ginsenoside substrate**

When the temperature of the enzymatic reaction system was set to 80 ℃, the pH was 6.0, the final concentration of LiCl metal ions was 3 mM, and the substrate concentration was 30 mg/mL, the relationship between CK production and enzyme concentration was studied. The results are shown in the figure. As shown in 4.6, when the enzyme amount is increased from 0 mg/mL to 9 mg/mL, the CK output increases as the enzyme amount increases, and when the enzyme amount increases to 12 mg/mL, the CK output increases. Compared with the previous slowdown, 9 mg/mL was confirmed to be the optimal enzyme concentration.

![](https://static.igem.org/mediawiki/2021/8/86/T--NWU-CHINA-B--teamresults22.png)

Under the above optimized enzymatic reaction conditions, 9 mg/mL *SS-bgly* can convert 30 mg/mL ginsenoside substrate into 9.39 mg/mL CK within 48 hours, with a productivity of 195.6 mg/L/h, molar conversion Rate 82.5%.

![](https://static.igem.org/mediawiki/2021/d/d7/T--NWU-CHINA-B--teamresults23.png)

According to the measured initial reaction velocity of different substrate concentrations, take 1/[S] as the abscissa and 1/V as the ordinate. We can obtain the double reciprocal form of the Michaelis equation, and obtain the maximum reaction rate Vmax and Michaelis constant Km according to the Linweaver-Burk mapping method, and calculate the conversion number kcat. Among them, the Michaelis constant represents the affinity of the enzyme to the substrate. The smaller the Km, the higher the affinity of the enzyme to the substrate. Conversion number kcat is the number of micromoles of substrate converted per micromol of enzyme molecule per second of enzymatic reaction. The larger the kcat, the higher the efficiency of enzyme catalysis.

The kinetic parameters are shown in Table 4.6. It can be seen that the values of Km, kcat and kcat/Km follow the order of Rd> Rb1> F2, F2> Rb1> Rd and F2> Rb1> Rd, respectively. This indicates that the catalytic efficiency of *SS-bgly* on ginsenoside F2 is much higher than that of Rb1 and Rd. F2 is converted to CK as soon as it appears, so the presence of F2 cannot be detected in HPLC. On the contrary, *SS-bgly* has the lowest catalytic efficiency for ginsenoside Rd, which indicates that the conversion of ginsenoside Rd to F2 is a limiting step.

**Table. Kinetic parameters of enzymatic reaction**

![](https://static.igem.org/mediawiki/2021/c/c6/T--NWU-CHINA-B--teamresults30.png)

The intermediate product of *SS-bgly* conversion of ginsenoside substrate to CK was analyzed by HPLC. In the HPLC chromatogram, the ginsenoside Rb1, Rd, F2, and CK standard products showed obvious peaks at about 6 min, 14.5 min, and 17 min. It can be seen from Figure 4.8 that *SS-bgly* converted 5 mg/mL ginsenoside substrate (including 2.5 mg/mL Rb1 and 0.75 mg/mL Rd). After 3 hours, Rb1 disappeared completely and converted to 1.52 mg/mL Rd and 0.87. mg/mL CK, until after 6 h of reaction, Rd disappears completely and all is converted to CK. Therefore, the hydrolysis path of *SS-bgly* to convert ginsenoside substrate to CK is Rb1→ Rd→ F2→ CK. This shows that *SS-bgly* firstly hydrolyzes the 20-O-glycosidic bond to convert Rb1 to Rd, then hydrolyzes the 3-O-glycosidic bond of Rd to become F2, and finally hydrolyzes the 3-O-glycosidic bond of F2 to become CK (Figure 4.9). Ginsenoside F2 cannot be detected in HPLC due to its high substrate specificity. There are many different ways to convert ginsenoside Rb1 into CK using glycosidase. For example, Rb1→Rd→CK,Rb1→XVII→F2→CK, Rb1 → gypenoside XVII → gypenoside LXXV → CK. This is mainly determined by the specificity of the enzyme for the aglycone at the C-3 or C-20 position of ginsenoside Rb1.

**Figure.Transformation pathway of ginsenoside Rb1 to CK**

![](https://static.igem.org/mediawiki/2021/c/c0/T--NWU-CHINA-B--teamresults33.png)
![](https://static.igem.org/mediawiki/2021/9/9b/T--NWU-CHINA-B--teamresults31.png)

In order to improve the catalytic efficiency of *SS-bgly* on ginsenoside Rd, AutoDock version 4.2 was used to dock the structure of ginsenoside Rd on the active center of *SS-bgly* (PDB code 1GOW). The coordination files of protein and ligand required for docking calculation are prepared by AutoDock-Tools, and the docking conformation has been analyzed using Pymol software and LigPlot. The results are shown in Figure 4.9. If you want to improve the catalytic efficiency of *SS-bgly* on ginsenoside Rd, you can choose Tyr322, Ala263, Val209, Leu213, Phe359, Trp361, His342 as possible mutation sites. For example, Kyung-Chul et al. [156] reported that by mutating the Val209 protein, the activity of *SS-bgly* on ginsenoside Rd was increased by 4.2 times, and the catalytic efficiency was increased by 3.7 times.

The constructed strains were fermented, and concentrated crude enzymes were obtained through operations such as salting out, dialysis and ultrafiltration. The enzymatic reaction was carried out in sodium acetate buffer, and we carried out a series of optimizations on the enzymatic reaction conditions. We found that when the temperature of the enzyme reaction system is 80 ℃, the pH is 6.0, and the final concentration of LiCl metal ions is 3 mM, 9 mg/mL *SS-bgly* can completely convert 10 mg/mL and 20 mg /mL of ginsenoside substrate within 12 h and 30 h. Finally, 3.79 mg/mL and 7.58 mg/mL of CK were obtained. When the substrate concentration reached 30 mg/mL, after 48 hours of reaction, the CK yield reached 9.39 mg/mL, the conversion rate reached 82.5%, and when the substrate concentration further increased to 40 mg/mL, the CK yield dropped to 9.08 mg/mL mL, the conversion rate is 59.8%.The conversion pathway of ginsenoside Rb1 analyzed by HPLC is Rb1→ Rd→ F2→ CK. We studied the kinetic parameters of *SS-bgly* catalyzed hydrolysis of ginsenosides and found that the Michaelis constants Km of *SS-bgly* to the substrates Rb1, Rd, F2 are 3.64, 5.08, and 1.86 mM. Through analysis, the biological conversion of ginsenoside Rd to F2 is the rate-limiting step. For this reason, the structure of ginsenoside Rd is docked to the active center of *SS-bgly*, and hydrophobin sites that may improve its catalytic efficiency are given.