Catalogue
1. Plasmid extraction
Using FastPure Plasmid Mini Kit(DC201, Vazyme).
2. Polymerase chain reaction(PCR)
2.1 PCR (P515 system)
(1) To the reaction system, upstream and downstream primers, template DNA, 2xPhenta Max Master Mix(P525-01, Vazyme) and ddH2O are added proportionally;(2) Cycling Conditions: Step 1: 95℃, 10min; Step 2: 95℃, 15s; Step 3: Primer melting point minus 3 degrees, 15s; Step 4: 72℃, 1kb for 30s; Step 5: Go to Step2, repeat for 35 cycles; Step 6: 72℃, 5min; Step 7: 12℃, infinite.
2.2 PCR (KOD system)
(1) To the reaction system, upstream and downstream primers, template DNA, 2xPhenta Max Master Mix(P525-01, Vazyme) and ddH2O are added proportionally;(2) Cycling Conditions: Step 1: 94℃, 10min; Step 2: 98℃, 10s; Step 3: Primer melting point minus 3 degrees, 15s; Step 4: 68℃, 1kb for 30s; Step 5: Go to Step2, repeat for 35 cycles; Step 6: 68℃, 7min; Step 7: 12℃, infinite.
3. Polymerase chain reaction(PCR) purification
Using FastPure Gel DNA Extraction Mini Kit(DC301, Vazyme).
4.Nucleic Acid Electrophoresis
4.1 Preparation
(1)Accurately weigh 0.25 g agarose and then add 25 ml of 1xTAE buffer.
(2)Heat it in a microwave oven to boiling at least 3 times to make sure that the solution is clear and transparent.
(3)Add 2.5μl Nucleic acid dye in the agarose solution when it’s cooled .
(4)After placing the board on the gel slot and inserting the comb, slowly pour the solution into the gel tank until cooling and solidification.
4.2 Load the Samples
(1)Remove the comb, then remove the gel and place it in an electrophoresis apparatus.Add 1xTAE buffer into the electrophoresis apparatus for nucleic acid and make sure it can be submerged by TAE buffer.(2)Add 10 ul DNA marker into the first gel hole.From the second hole ,add the PCR product in the numbered order.
(3)Turn on the instrument,running 30 minutes or appropriately.
4.3 Visualize the DNA
(1)After electrophoresis, turn off the power supply and carefully remove the gel and put it into the side gel imaging system aside.(2)Visualize the DNA bands using UV light.Then take a photograph of the gel to record the results.
5. Construction of Plasmids by Homologous Recombination
(1)To the reaction system, insert fragments, linearized vector, 2x seamless cloning mix(D7010S, Beyotime), ddH2O are added proportionally;
(2) Reaction Condition: 50℃, 15min;
5.1 Nucleic acid dephosphorylation
(This is suitable for removal of 3' and 5'-phosphate groups from DNA and RNA.) (1)Prepare the following reaction mixture:| Components | Volume |
|---|---|
| Linear DNA (~3 kb plasmid) | 1 ug(~1 pmol termini) |
| 10X reaction buffer for AP used in reaction | 2μL |
| FastAPTM Thermosensitive Alkaline Phosphatase | 1μL(1U) |
| Water, nuclease-free | to 20 μL |
| Total volume | 20 μL |
(2) Mix thoroughly, spin briefly and incubate 10 min at 37 ℃C.
(3) Stop reaction by heating for 5 min at 75 ℃C.
5.2 Using T4 DNA Ligase to connect plasmid carriers and exogenous fragments
(1) Reaction system| Components | Volume |
|---|---|
| 10 x ligation buffer*1 | 2 μL |
| Carrier DNA | 50 ng |
| Insert DNA fragments | (The molar ratio with the carrier DNA is about 3)*2 |
| T4 DNA ligase (350 U/ul) | 1μL |
| Sterilized water | |
| Total | 20 μL |
(2) For sticky end connections, 16react for 1-5 hours; for flat non-end connections, 16℃
(3) reacts for 1-24 hours. X3
(4) Take a part of the connecting liquid for conversion*4. The reaction solution can be stored at -20°C.
5.3 Fast Digestion of Different DNA
(1) Combine the following reaction components at room temperature in the order indicated:| Plasmid DNA | PCR product | Genomic DNA | |
|---|---|---|---|
| Water, nuclease-free | 15 μL | 17uL | 30 μL |
| 10X FastDigest or 10X FastDigest Green Buffer | 2μL | 2μL | 5μL |
| DNA | 2uL（up to 1ug） | 10 μL (~0.2 μg) | 10 μL (5 μg) |
| FastDigest enzyme | 1 μL | 1μL | 5μL |
(2) Mix gently and spin down.
(3) Incubate at 37°C in a heat block or water thermostat for 5 min. Optional: Inactivate the enzyme by heating for 5 min at 80℃.
5.4 DPnI Digestion
(1) Added system| Components | Volume |
|---|---|
| nuclease-free water | 16 μL |
| 10X Buffer Tango | 2μL |
| DNA (0.5-1 μg/μL) | 1 μL |
| Dpnl | 0.5-2 μL |
(4) Mix gently and spin down for a few seconds.
(5) Incubate at 37°C for 1-16 hours.
6. One Step Cloning and transformation
6.1 One Step Cloning
Using ClonExpress UItra One Step Cloning Kit(C115, Vazyme)6.2 Plasmid Transformation (usingTOP10/BL21 competent cell)
(1) Place the bacterial liquid on ice for 8min to melt;(2) Mix 50μL bacterial liquid with around 100ng plasmid and place on ice for 25min;
(3) 42℃ metal bath heat hit 45s;
(4) Place the mixture on the ice again for 2-3min;
(5)Add 500-700uL Autoclaved bacterial culture medium, shake on the shaker for 1h (37℃, 230rpm).
6.3 Plasmid Transformation(using GS115 competent cell)
(1) Place the bacterial liquid on ice for 8min to melt;(2) Mix 50μL bacterial liquid with around 100ng plasmid and place on ice for 25min;
(3) 42℃ metal bath heat hit 45s;
(4) Place the mixture on the ice again for 2-3min;
(5)Add 500-700uL Autoclaved bacterial culture medium, shake on the shaker for 1h (37℃, 230rpm).
7. Colony PCR
(1) Pick the monoclonal with the pipet tip;
(2) Streak on a blank bacterial culture plate using the pipet tip with colony on;
(3) Add the remaining bacteria on the pipet tip to the pre-prepared PCR system as the template;
(4) Cycling Conditions: Step 1: 95℃, 5min; Step 2: 95℃, 15s; Step 3: Primer melting point minus 3 degrees, 15s; Step 4: 72℃, 1kb for 30s; Step 5: Go to Step2, repeat for 35 cycles; Step 6: 72℃, 5min; Step 7: 4℃, infinite.
8. DNA Gel Extraction and DNA product purification
Using FastPure Gel DNA Extraction Mini Kit(DC301-01, Vazyme)
9. Competent cells preparation (TOP10/BL21/GS115 competent cell)
(1) Spread competent cells onto the bacterial culture plate;
(2) Take single clone in 3mL liquid medium for small amount amplification;
(3) 1:100 inoculation of bacterial solution from shaking tube into 1000ml shaking bottle with 200mL liquid medium for bulk amplification;
(4) Cultivate the bacteria on the shaker until the OD600 of the bacterial solution is about 1.0-1.2 and to 0.5-0.7;
(5) Centrifuge the bacterial solution at 4℃, 5000xg for 5min, and collect all the bacteria into a 50mL centrifuge tube in several times;
(6) Gently resuspend the bacteria with 40 ml of pre-colded 10% glycerol and centrifuge again(4℃, 5000xg for 5min);
(7) Repeat the operation(6) again;
(8) Resuspend the bacteria with 16mL of 10% glycerol and dispense 50ul per tube into 1.5mL centrifuge tubes, freeze in liquid nitrogen and transfer to -80℃ refrigerator.
10. Removing the N-glycosylations by Endo H treatment
Using Endo H Kit(20414ES92 YEASEN)
11. Measurement of protein concentration（BCA method）
Using BCA Protein Assay Kit(BL521S biosharp)
12. SDS-PAGE
12.1 Gel Preparation
Using 12% PAGE Color (Red) Gel Ultra-Rapid Preparation Kit(G2400 Servicebio)12.2 Load the Samples
(1)Mix protein samples with SDS-PAGE loading buffer.(2)Heat the samples at 95-100°C for 5 minutes to denature the proteins.
(3)Add 10 μL protein marker into the first gel hole.From the second hole ,add the protein samples in the numbered order.
(4)Turn on the instrument,running until the samples reaches the bottom of the gel.
12.3 Coomassie Brilliant Blue Staining
(1)Rinse the gel 3 times for 5 minutes with 100 ml deionized water to remove SDS(2)Stain the gel with a solution of Coomassie Brilliant Blue to cover the gel.
12.4 Destaining
For Coomassie staining, destain the gel in a solution of methanol, acetic acid, and water until the background is clear and the protein bands are visible.13. IPTG Induction of Proteins
Using IPTG Induction and Extraction of Proteins (FM-000008 Gold Biotechnology)
IPTG Induction and Extraction of Proteins
14. Extraction of bacterial cellulose
After static culture for 7 days, the cellulose was filtered and collected, and the culture medium was removed by deionizing repeatedly. Soak in 0.2 mol/L Na OH solution and bathe at 70 ~ 80 °C for 2 hours to remove the residue (mainly residual bacteria and culture medium), rinse with deionized water for many times until the pH value is about 7.0 (measured by pH test paper). Finally, the milky white translucent BC gel liquid film can be obtained and then placed in a vacuum oven and dried at 80 °C for 12 hours and cooled to room temperature.
15. High Performance Liquid Chromatography(HPLC)
BUFFER A：KH2PO4（50mM）
BUFFER B：Methanol
I. Sample preparation
We added a plastic sheet to 250μg/mL enzyme solution, and after 24 hours of reaction, took the enzyme solution after reaction through a filter (diameter =0.22μ m), filtered out the solid substances in it, and then injected it into the sample bottle.
II. Program setting
We used the instrument UltiMate 3000 HPLC and C18 column for measurement, using the procedures in the following chart. The temperature of the fixed column temperature chamber was 30℃, and the absorption value at the absorption wavelength of 240nm was detected. According to the corresponding absorption peak area of TPA and MHET, the product concentration was converted to reflect the activity of the enzyme.
| Time | Flow velocity(ml/min) | %A | %B |
|---|---|---|---|
| 0.000 | 0.500 | 80.0 | 20.0 |
| 14.000 | 0.500 | 80.0 | 20.0 |
| 15.000 | 0.500 | 80.0 | 20.0 |
III. Data processing
We prepare standard samples and dilute them each time in order to obtain more accurate fitting curves and accurately calculate the content of each product. Under the current procedure, the peak time of TPA and MHET is 3.5 minutes and 4.7 minutes. We also measured the enzyme solution that did not undergo plastic degradation, calculated the area at the peak time and subtracted it to exclude the influence of other impurities in the enzyme solution, and then added the difference into the fitted equation to determine the degradation rate.
16. Protein purification
-
Design of the enzyme, add an extra 6×his histidine label on the enzyme.
-
Use HyPur T Ni-NTA 6FF (His-Tag) PrePacked Gravity Column, in which Ni ions can adsorb our proteins.
-
Centrifuge the induced expression cells. Then the liquid medium is removed.
-
Add the final concentration of 1 mM PMSF for cleavage, and the cells are mixed at 4℃ for 30 minutes before being ultrasonically broken.
-
Centrifuge the lysate at 12000rpm for 10min, to obtain inclusion bodies, and wash with 1× PBS for 2 to 3 times.
-
Balance the column twice with wash buffer, and the resin is slowly discharged, and then add sample solution to the column to collect the flow solution, and the column is cleaned with twice the column volume of wash buffer to collect the flow solution.
-
Eluted the histidine label protein twice with the column volume of WASH buffer.
-
Balanced the gravity column with 5 times the column volume of wash buffer.
-
Add 20% ethanol protection solution and store in the refrigerator at 4℃.
17. Media component
17.1 Media component
HS medium
| Component | Content |
|---|---|
| Glucose | 20 g/L |
| Peptone | 5 g/L |
| Yeast Extract | 5 g/L |
| Citric acid | 1.15 g/L |
| Na2HPO4 | 2.7 g/L |
BMGY medium
| Component | Content |
|---|---|
| Yeast Extract | 10 g/L |
| Tryptone | 20 g/L |
| Yeast Nitrogen Base(YNB) | 13.4 g/L |
| Glycerol | 10 g/L |
| PBS | 100 mmol/L |
BMMY medium
| Component | Content |
|---|---|
| Yeast Extract | 10 g/L |
| Tryptone | 20 g/L |
| Yeast Nitrogen Base(YNB) | 13.4 g/L |
| PBS | 100 mmol/L |
LB liquid medium
| Component | Content |
|---|---|
| Yeast Extract | 5 g/L |
| Peptone | 10 g/L |
| NaCl | 10 g/L |
LB Agar medium
| Component | Content |
|---|---|
| Yeast Extract | 5 g/L |
| Peptone | 10 g/L |
| NaCl | 10 g/L |
| Agar | 20 g/L |
MD Agar medium
| Component | Content |
|---|---|
| Yeast Nitrogen Base(YNB) | 13.4 g/L |
| Glucose | 20 g/L |
| Agar | 20 g/L |
BMMY-glucose medium
| Component | Content |
|---|---|
| Yeast extract | 10 g/L |
| Tryptone | 20 g/L |
| Yeast Nitrogen Base(YNB) | 13.4 g/L |
| Glucose | 20g/L |
BMMY-sucrose medium
| Component | Content |
|---|---|
| Yeast extract | 10 g/L |
| Tryptone | 20 g/L |
| Yeast Nitrogen Base(YNB) | 13.4 g/L |
| Phosphate buffer solution | 100 mmol/L |
| Sucrose | 20 g/L |
BMMY-EG medium
| Component | Content |
|---|---|
| Yeast extract | 10 g/L |
| Tryptone | 20 g/L |
| Yeast Nitrogen Base(YNB) | 13.4 g/L |
| Phosphate buffer solution | 100 mmol/L |
| EG | 20 g/L |
BMMY-TPA medium
| Component | Content |
|---|---|
| Yeast extract | 10 g/L |
| Tryptone | 20 g/L |
| Yeast Nitrogen Base(YNB) | 13.4 g/L |
| Phosphate buffer solution | 100 mmol/L |
| TPA | 20 g/L |
BMMY-EG-TPA medium
| Component | Content |
|---|---|
| Yeast extract | 10 g/L |
| Tryptone | 20 g/L |
| Yeast Nitrogen Base(YNB) | 13.4 g/L |
| Phosphate buffer solution | 100 mmol/L |
| TPA | 20 g/L |
| EG | 20g/L |
BMMY-EG sucrose medium
| Component | Content |
|---|---|
| Yeast extract | 10 g/L |
| Tryptone | 20 g/L |
| Yeast Nitrogen Base(YNB) | 13.4 g/L |
| Phosphate buffer solution | 100 mmol/L |
| Sucrose | 20g/L |
| EG | 20g/L |
BMMY-TPA sucrose medium
| Component | Content |
|---|---|
| Yeast extract | 10 g/L |
| Tryptone | 20 g/L |
| Yeast Nitrogen Base(YNB) | 13.4 g/L |
| Phosphate buffer solution | 100 mmol/L |
| Sucrose | 20g/L |
| TPA | 20g/L |
BMMY-EG-TPA-sucrose medium
| Component | Content |
|---|---|
| Yeast extract | 10 g/L |
| Tryptone | 20 g/L |
| Yeast Nitrogen Base(YNB) | 13.4 g/L |
| Phosphate buffer solution | 100 mmol/L |
| Sucrose | 20g/L |
| EG | 20g/L |
| TPA | 20g/L |
HS liquid medium
| Component | Content |
|---|---|
| Glucose | 20 g/L |
| Protein peptone | 5 g/L |
| Yeast extract | 5g/L |
| Citric acid | 1.15 g/L |
| Disodium hydrogen phosphate | 2.7 g/L |
HS-EG liquid medium
| Component | Content |
|---|---|
| Glucose | 20 g/L |
| Protein peptone | 5 g/L |
| Yeast extract | 5g/L |
| Citric acid | 1.15 g/L |
| Disodium hydrogen phosphate | 2.7 g/L |
| EG | 20 g/L |
HS- sucrose liquid culture medium
| Component | Content |
|---|---|
| Protein peptone | 5 g/L |
| Yeast extract | 5g/L |
| Citric acid | 1.15 g/L |
| Disodium hydrogen phosphate | 2.7 g/L |
| Sucrose | 20 g/L |
HS-EG-TPA liquid culturemedium
| Component | Content |
|---|---|
| Glucose | 20 g/L |
| Protein peptone | 5 g/L |
| Yeast extract | 5g/L |
| Citric acid | 1.15 g/L |
| Disodium hydrogen phosphate | 2.7 g/L |
| EG | 20 g/L |
| TPA | 20 g/L |
Calcium carbonate solid culture medium
| Component | Content |
|---|---|
| Yeast extract | 5 g/L |
| Bacterial peptone | 3 g/L |
| Glucose | 30 g/L |
| Calcium carbonate | 10 g/L |
| Agar | 15 g/L |
YPS medium
| Component | Content |
|---|---|
| Yeast extract | 10 g/L |
| Peptone | 20 g/L |
| Sucrose | 20 g/L |
YPD medium
| Component | Content |
|---|---|
| Yeast extract | 10 g/L |
| Peptone | 20 g/L |
| Glucose | 20 g/L |
（The pH of all medium was adjusted to 6,7; The pH of BMMY medium was adjusted with potassium dihydrogen phosphate and potassium dihydrogen phosphate, and the pH of HS medium was adjusted with phosphate and sodium hydroxide.Sterilize in a high-pressure steam sterilizer at 115°C for 20 minutes. After sterilization, BMMY medium was added with 1% (v/v) methanol.)