Team:NWU-CHINA-B/Experiments

NWU-CHINA-B

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  • 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.

    2.1.2 Main reagents




    2.1.3 Main instruments and equipment


    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.

    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.



    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.


    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.




    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:




    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.





    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





    3.1.2 Primary Reagent




    3.1.3 Main instruments and equipments




    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 instrumentSS- bgly fragment containing Kpn Ⅰ and Not Ⅰ restriction sites was obtained. PCR reaction system (100 μL) is as follows:




    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:




    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:




    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:




    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-bgly6# 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:






    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.