Construction of expression vector
1. Culture medium
LB (Luria-Bertani)
| Component | Amount |
|---|---|
| NaCl | 10g/L |
| Tryptone | 10g/L |
| Yeast Extract | 5g/L |
| Agar(solid) | 20g/L |
2. E.coli competence preparation
1 Select the single colonies of Escherichia coli and inoculate in 20mL LB medium cultured overnight at 37 ℃.
2 1% inoculum inoculated in 20mL LB medium, and shake the culture for 3 h at 37 ℃ for 230rpm / min.
3 Take 1mlculture, ice30min, then contrifuge at 4℃ 4,000 r/min for 3 min to remove the supernatant
4 Add 500ul cold Cacl2 solution, re-suspension bacteria precipitation at 4 ℃,4000 r/min centrifugation for 3 min, to remove the supernatant.
5 Add 50ul cold 0. 1 mol/L CaCl2 solution, re-suspension bacteria precipitation, ice 3 h-24 h , That is, the competence of Escherichia coli.
3. E.coli heat shock transformation
1 Take the competent Escherichia melted on 20ul ice to the precooled 1.5 mlEP tube.
2 Add 2 ul plasmid, pay attention!Hands do not touch the bottom of the EP tube!Then slowly blow and stir (gently! The competence is fragile) mix well. Rest on the ice for 30 min.
3 Melt the LB solid medium,cool it slowly in an oven at 60℃ for about 1h.
4 Heat shock at 42 ℃ for 90 s, immediately ice bath 2-3min.
5 Add 500 ul non-resistant liquid LB and incubate in shaker at 37 ℃ for 1 h.
6 Take solid LB , add antibiotics and pour the plate.
7 After 5000 r/min centrifugating for 2 min,pour out part of the supernatant, re-suspend it and coated with plate, and cultured at 37 ℃.
Note: this protocol needs to be modified according to the actual situation. The plasmid used in the first round of experiment is the plasmid synthesized by the company, and it is enough to add 2 ul. In the follow-up experiment, if the concentration of recombinant plasmid is low, it needs to be changed to 10 ul. At the same time, 50 ul receptor state is used to improve the success rate of screening. The quality of the competence prepared by ourselves during the heat shock time is poor, the heat shock time is 90 s, and the competence purchased by the company is in accordance with the instructions of the company.
4. Escherichia coli conservation
Add 800 ul 50% glycerol aqueous solution to the seed preservation tube for high-pressure steam sterilization, mixed with 800ul bacteria solution, and stored at-80 ℃.
5. Extract plasmid (use OMEGA plasmid extraction kit)
1 Take 2 ml bacteria centrifuge at 12000 rpm for 1 min, pour out the supernatant, if the plaque is small, then add 2 ml bacteria solution to repeat the operation.
2 Add 250ul solution I (ingredient: EDTA, glucose, EDTA is the chelating agent of divalent metal, chelate divalent metal ion to inhibit the activity of DNase, protect the extracted DNA from degradation, some manufacturers' kit has glucose, which is in order to make the suspended Escherichia coli not to settle quickly), blow and mix repeatedly to ensure that there is no bacterial mass, otherwise the inside of the bacterial mass can not be cleaved.
3 Add 250 ul solution II (ingredients: NaOH and SDS;NaOH dissolve cells, NaOH will react with carbon dioxide in the air, so close the lid in time after using solution II ;SDS enhances the alkalinity of sodium hydroxide, and combines with protein to precipitate), turn the test tube upside down slowly, turn the test tube several times to gently mix, and rest for two minutes to get transparent pyrolysis products. At this step, the DNA is already in our solution, and the operation must be very gentle, otherwise the chain will break, and the genomic DNA will also break under alkaline conditions, so the rest time can not be too long, otherwise the genomic DNA will be broken, and it will not be eazy to separate from the plasmid DNA after the break.
4 Add 350 ul solution III(ingredient:potassium acetate,acetic acid ,SDS encounters otassium ions to produce PDS which is insoluble in water, resulting in a large amount of flocculent precipitation, while the genomic DNA chain is very long, it is easy to be precipitated by these precipitations, but if the genomic DNA breaks into short chains, it is difficult to precipitate.Acetic acid is to neutralize the sodium hydroxide in the previous step), immediately inverted and mixed.
5 centrifugatied at 12000 rpm for 15 min.
6 Take 100 ul 3M NaOH to purification column for column equilibrium, centrifugatied at 12000 rpm for 1 min to discard excess sodium hydroxide. After column balance, the silicon-based plasma membrane can be activated to the maximum and the plasmid yield can be improved.
7 Absorb the supernatant into the purification column carefully , we would rather absorb less supernatant than absorb precipitation. After static placement for 10 minutes, DNA will be fully adsorbed on the membrane, centrifugated at 12000 rpm for 1 min. Repeat, there is no need to place staticly.
8 Add 500 ul HBC washing Buffer,12000 rpm 1min and discard the filtrate.
9 Add 700 ul DNA washing Buffer (80% ethanol) to clean the purification column, 12000 rpm 1 min, discard the filtrate, wash twice. Two cleaning solutions iis to wash away impurities such as small molecular proteins and inorganic ions.
10 Empty column centrifugated at 12000 rpm for 2min
11 Oven drying to no ethanol (15 min), these two steps aim to remove ethanol, otherwise it will have a great impact on the subsequent elution efficiency.
12 Put the purification column into a clean 1.5 ml EP tube and add the elution buffer about 30~100ul preheated at 60 ℃ to the column, place staticly for 2 min. Elution buffer can improve the elution efficiency, and add it to the center of the silicone film to ensure that the eluent will completely cover the surface of the silicone film to achieve the maximum elution efficiency. Centrifuge at 12000 rpm for 1 min.
13 Repeat the elution with the above eluent, but use different placement.The first placement is to let the eluent cover the silicone film and let the DNA off the colu
6. Gel recovery (purification) (using OMEGA gel recovery kit)
1 Cut the gel as thinly as possible and add the same volume of Bind Buffer,58 ℃ water bath sol.
2 Transfer the liquid into the purification column and place for 5 min
3 12000rpm 1 min , back to suction column, 12000rpm 1 min
4 Discard the liquid, add 300 ul Bind Buffer,12000rpm 1 min (purifying:remove this step)
5 Add 700 ul SPW wash Buffer and place it static for 5 min,12000rpm 1 min (purifying:only add 400 ul)
6 Repeat the previous step
7 Abandon the liquid, 12000 rpm idling 2min
8 Discard the collecting tube, open the cover of the column and dry it in the incubator at 37 ℃ for 15 min.
9 Place the purification column on a 1.5 ml EP tube, add ddH2O preheated at 60 ℃ 20ul to the center of the membrane, and place it 5min.12000 rpm 2min
10 12000 rpm 2min
11 return suction column, 12000 rpm 2min
12 Discard the column and measure the concentration and purity
7. Construction of plasmid with Panb1 as promoter, plasmid with Pynr071c as promoter
1 Enzyme digest the vector with BamH I and EcoR I. System:
| --- | --- |
|---|---|
| EcoRI | 2 ul |
| BamHI | 2 ul |
| Buffer | 4 ul |
| Plasmid | 15 ul |
| Dd water | 17 ul |
Amplified fragment
| --- | --- |
|---|---|
| 2 × Phanta Max Master Mix | 10 ul |
| Upstream primer | 1 ul |
| Downstream primer | 1 ul |
| Template DNA | 1 ul |
| Dd water | 7 ul |
1 Digeste the fragment with BamH I and EcoR I for 2 hours.
2 Electrophoresis:purify the correct linear fragment
3 Gel recovery
4 Connect with T4 ligase
5 Transformation of Escherichia coli
6 The concentration of recombinant plasmid is low, so we use 50 ul competence with 10 ul plasmid, the specific steps are shown in 3. Heat shock transformed E.coli.
Pick at least 5 spots per board.
8. Construction of plasmid for pigment synthesis
1 Digest the vector with EcoR I and Avr II for two hours.
2 Electrophoretic gel recovery to obtain linearized vector.
3 PCR amplification of two fragments to be inserted.
4 Purify the fragment
5 Configuration of recombination reaction system:The optimum dosage of 5 × CE MultiS Buffer 4ul, Exnase MultiS 2 ul, carrier and fragment is 0.03pmol, adding dd water to 20ul system.
6 React at 37 ℃ for 30 min; And then reduce to 4 ℃ or immediately cool it on ice.
7 Transforme the recombinant product into E.coli TOP10
Heterologous expression of Pichia pastoris GS115
1. Culture medium
YNB
| Component | Amount |
|---|---|
| 10×YNB | 134g/L |
MD medium
| Component | Amount |
|---|---|
| Agar | 15g/L |
| After sterilization, add untill the temperature drop below 60 ℃ | |
| 10×YNB | 100mL/L |
| 500×B(biotin) | 2mL/L |
| 50% glucose | 40mL/L |
YPD medium
| Component | Amount |
|---|---|
| yeast extract | 10g/L |
| peptone | 20g/L |
| Agar(solid) | 20g/L |
| After sterilization, add untill the temperature drop below 60 ℃ | |
| 50%glucose | 20ml/L |
BMGY medium
| Component | Amount |
|---|---|
| glycerol | 10g/L |
| yeast extract | 10g/L |
| peptone | 20g/L |
| 1mol/L phosphate buffer (pH 6.0) | 100mL/L |
| After sterilization, add untill the temperature drop below 60 ℃ | |
| 10×YNB | 100mL/L |
| 500×B | 2mL/L |
BMMY medium
| Component | Amount |
|---|---|
| yeast extract | 10g/L |
| peptone | 20g/L |
| 1mol/L phosphate buffer (pH 6.0) | 100mL/L |
| After sterilization, add untill the temperature drop below 60 ℃ | |
| 10×YNB | 100mL/L |
| 500×B | 2mL/L |
| 10×M(methanol) | 50mL/L |
BMM medium
| Component | Amount |
|---|---|
| 1mol/L phosphate buffer (pH 6.0) | 100mL/L |
| After sterilization, add untill the temperature drop below 60 ℃ | |
| 10×YNB | 100mL/L |
| 500×B | 2mL/L |
| 10×M | 50mL/L |
Among them, YNB and B are living active substances, which need to be filtered and sterilized rather than sterilized at high temperature.B should be preserved away from light. Glucose can not be mixed with YE, peptone and so on to sterilize, need to be sterilized as 50% glucose separately, and add when we need. Because MD board contains B, it needs to be preserved and cultured from light.
Configuration of 0.1M sodium phosphate Buffer (pH=7.2): 77.4 ml 0.1M Na2HPO4 + 22.6ml 0.1M NaH2PO4.
Configuration of 1M sodium phosphate Buffer (pH=6.0): 12.3 ml 1M Na2HPO4 + 87.7 ml 1M NaH2PO4.
The two reagents in the laboratory may have crystal water, and the specific mass is calculated according to the molecular weight on the reagent bottle.
Sorbitol Buffer configuration: configure 1.2M sorbitol with 0.1M sodium phosphate buffer. High pressure steam sterilization.
2.Preparation of competence of Pichia pastoris
(must be used on the same day after production)
a) Two days in advance: pick out the single yeast colony / conservation tube for 10 ul, inoculate it to the 50 mL conical bottle of 5mLYPD, and culture overnight at 28 ℃. (strain activation).
b) One day in advance: take 200 ul (if grow too slowly, take 100 ul more) to 500 ml conical bottl of100 ml YPD and culture for about 20 h. (OD needs to be measured in the first culture, which needs to be between 1.3 and 1.5. The culture time is determined for the first time,which will be followed )
c) Use the new electric rotary cup(if the electric rotary cup can not be washed clean and there will be DNA residue. )
d) Two ice boxes: one ice bath placed in the super clean bench and one carried the competence to operate out the super clean table. Water, 1M sorbitol, electric cup ice bath; add 40 ml bacterial liquid to 50 mlEP tube, install two tubes for convenient balancing, put it in another ice box ice bath.
e) Take the ice box with EP tube out of the super clean bench, level the EP tube centrifuge, centrifuged at 4 ℃ and 5000 rpm for 3min with a high-speed freezing centrifuge , and put it into the ice box immediately after it is taken out.
f) Enter the super clean bench , pour out the supernatant and add 40 ml water to resuscitate.
g) Pour out the supernatant and add 20 ml of water to re-suspension.
h) Pour out the supernatant and add 10 ml sorbitol to resuscitate.
i) Leveling centrifuge.
j) Pour out the supernatant and add 5 ml sorbitol resuspension.
k) Balance centrifugate, suck out the supernatant, ensure that the supernatant is completely sucked out, and thecompetence is pure, so suct out.
l) Add 200 ul sorbitol, pack every 80 ul to 2 mlEP tube, EP tube in ice bath.
3.Preparation of high concentration linearized plasmid.
a) plasmid linearization: select the appropriate enzyme digestion to linearize the plasmids by single enzyme digestion.
b) plasmid concentration (ethanol precipitation).
i. In the enzyme digestion system, add 2 times volume of anhydrous ethanol and 10 volume of NaAc (pH=5.2) and mix well.
ii. Precipitate at-20 ℃ for 20 min and precool centrifuge at 4 ℃.
iii. 13200 rpm, 20 min, 4℃ centrifuge and abandon supernatant
iv. 75% ethanol (not for disinfection, mixed by anhydrous ethanol and up water), 300 ul gently purge(can not be blown away),ditto centrifuge for 10 min, discard the supernatant.Wash it twice.
v. Dry in a 37-degree incubator until there is no smell of ethanol.
vi. 20 ul up water re-dissolving
4.Yeast electroconversion.
a) Add 20ug linear particles to the sub-packaged EP tube and control the volume at 5~10 ul (that is, the concentration should reach 2000 ng/ul). Suck them all out and add them to the electric rotary cup and place on the ice for 5 min.
b) Electric shock: 1500 V, 5 ms,200Ω, 25 uF.
c) Add 1 ml sorbitol immediately after electric shock,and left in ice for 5 min.
d) Sucked all out and add to 2 ml EP tube, incubated in 30 ℃ incubator for 1 h, do not shake.
e) Take 1 mlYPD from the super clean bench and put it into the above 2 mlEP tube and culture it in a shaker at 30 ℃ for 2 h.
f) Centrifugating 5000 rpm for 1 min (add time if the plaque is not firm), pour out the supernatant, mix the rest, coat with MD plate, and culture without light (with biotin) for about two days.
5.Identification of yeast recombinant by Colony PCR.
a) Repackage 1 ml YPD.
b) Add 8 ul sterile dd water to the sterilized pcr tube.
c) Pick a single colony with the end of a gun and dip it in 8 ul water (do not add too many yeast cells as templates, some components of its cell wall will hinder pcr reaction). Pay attention that a group of unloaded yeast is needed as a negative control.
d) Then insert the gun tip into the YPD, and save the clone, and make sure that the pcr tube corresponds to the 2 mlEP tube.
e) Bacterial liquid PCR.
f) The stripes of each lane are compared with the predicted length one by one, and the consistency indicates a successful reorganization.
6.Yeast conservation.
0.3 ml of 50% glycerol aqueous solution + 0.7 ml of bacterial solution, vortex mix well and then storage at-80 ℃.
7.Yeast expression.
a) Pick a single colony into 5 ml YPD and culture for 24 h.
b) Take 5ml,inoculate into BMGY liquid medium for 16-18 hours. OD:4~6
c) BMGY be induced 2 hours in advance with 0.2% methanol before the transfer of BMMY/BMM.
d) Centrifugate for at 5000rpm for 3 min,and collect all the bacteria,re-suspended with BMMY/BMM liquid culture medium, and add the fermentation volume 1% of methanol every 24 hours
8.Protein purification.
Sample preparation
a) 6500rpm 5min centrifuged at 4 ℃.
b) Take the supernatant and add to the ultrafilter tube. Centrifuge at 4000rpm at 4 ℃ for 15min, and obtain the supernatant.
Prepare the Nickel column
c) Make Ni-NTA affinity chromatography column.
d) Use deionized water to elute slowly ,avoid introducing bubbles into the column bed.
e) Pre-equilibrium with a binding buffer of 10 times the volume of the column bed.
Gradient buffer preparation
f) Buffer solution1:50mM pH7.4. PBS buffer
Preparation: 0.5M NaH2PO4 19ml, 0.5M Na2HPO4 81ml ,NaCl 29.3g, add proper amount of water to dissolve and fix the volume to 1000mL.
g) Buffer solution 2:50mM phosphate buffer, pH7.4 PBS solution.
Preparation: 0.5m NaH2PO4 19ml, 0.5m Na2HPO4 81ml, NaCl 29.3g and imidazole 34g, dissolved in proper amount of water, fixed volume to 1000mL.
h) Buffer solution 3:Prepared by buffer B with different imidazole concentration
| Imidazole concentration | Buffer 1volume (ml) | Buffer 2 volume (ml) |
|---|---|---|
| 10 mM | 98 | 2 |
| 20 mM | 96 | 4 |
| 50 mM | 90 | 10 |
| 100 mM | 80 | 20 |
| 200 mM | 60 | 40 |
| 300 mM | 40 | 60 |
| 400 mM | 20 | 80 |
Sample purification
a)Inject the concentrated supernatant into the Ni-NTA affinity chromatography column. Slowly shake the 60min on a side rocker or a horizontal shaker at 4 ℃
b) Rinse with a combined buffer of 10 times the column volume and collect the filtrate.
c) Gradient elution is carried out with buffer 3 containing 10,20,50,100,200,300 and 400mM imidazole respectively, and collect the elution peaks at each stage.
Column recycling
d) Wash 5 column bed volumes with pure water and 3 column bed volumes with 20% ethanol.
9.SDS-PAGE verification analysis.
Make gel:
a) Set up the device, align the lower ends of the two glass plates on the formwork slot, clamp the formwork slot on the in-situ glue maker and clamp it to 1.0 scale.Then,detect leak.
b) Prepare separation gel (in order): 6.9ml resteam water + 4.0ml gel storage solution + 3.8ml separation gel buffer (pH8.8) + 0.15ml10%SDS+0.15ml10%AP+0.009mlTEMED.
c) Perfuse the separation glue, seal it with water, and the gel solidify gradually in 10-30min.
d) Suck out part of the water seal and gently suck out the remaining water with filter paper.
e) Prepare of concentrated gel (in order): 4.1ml resteam water + 1.0ml gel storage solution + 0.75ml separation gel buffer (pH8.8) + 0.06ml10%SDS+0.06ml10%AP+0.006mlTEMED.
f) Pour concentrated gel and insert comb in time. After about 5min, began to solidify, waiting for the gel to polymerize 30min.
If you use prefabricated glue, start directly from here:
g) The protein solution is mixed with Loading buffer at 4:1 proportion, and mixed in the 1.5mlEP tube. Then,seal it and heat in in a metal bath at 95 ℃ for 5min to fully stretch the spatial structure of the protein.
h) Fill the diluted electrophoresis buffer, immerse the glass plate in the groove with a height of about 0.5cm, and take out the comb.
i)Take 30ul the treated sample with a liquid transfer gun and add the sample vertically through the buffer.
j) Add the electrode buffer to the electrophoresis cell to make the liquid level on both sides equal. Connect the positive and negative electrophoretic trough of the electrophoretic instrument , and the voltage is 80V for 30min.
k) Voltage change into 120 V after entering the separation gel, keep constant voltage and continue electrophoresis for 1 h.
l) Stripping out of plate, using 0.1% Coomassie brilliant blue R250 dye solution, cold dye 20min (baking dyeing: microwave boiling 6-7 times).
m) Rinse twice with rinsing solution, each time 10min.Then change into ddH2O and continue to rinse until the strip is clear.
n) Observe the bands and analyze the results.
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