Experiments

1. PCR

(a) Prepare the PCR system according to the following table

(b）Put the prepared PCR system into the account centrifuge and mix

(c）Put it into the PCR instrument. See the following table for PCR procedures，cycle：35

2. Agarose gel electrophoresis

(a) Add 5ml 50X TAE to 245ml deionized water to prepare 250ml 1X TAE

(b) Weigh 0.25g agarose and add to 25ml 1X TAE

(d) Cool to 50 to 60 degrees and add 3μLDNA stain (nucleic acid dye)

(e) Inverted board

(f) After cooling, sample in sequence

(g) 180V 400mA 15min

(h) Observe the strip under the photoscope and take photos for record

3. DNA glue recovery

(1) Observe the strip in the glue illuminator, cut the glue block of the target strip and place it in the new centrifuge tube, try not to cut the excess part

(2) Add Buffer B2 to the centrifuge tube containing glue blocks

(3) Treat at 50-60℃ until the glue is completely dissolved

(4) Take a new adsorption column and place it in the collection tube, add all the sol to the adsorption column, 9000rpm30s

(5) Drain the liquid from the collection pipe, add 600μL Wash Solution, 9000rpm30s, and repeat twice

(6) Drain the liquid in the collection tube and place the adsorption column in the collection tube at 9000rpm for 2min

(7) Place the adsorption column in a new centrifuge tube and place it in the fume hood for 10min to remove residual ethanol

(8) Add 30μL Elution Buffer into the adsorption column, leave at room temperature for 2min, and then centrifuge at 9000rpm for 2min to collect the liquid in the centrifugal tube

(9) To ensure the concentration of glue recovery, repeat step 8)

4. Preparation of LB medium

Prepare 100ml liquid LB medium

(a) Weigh 2.5g LB and fill with deionized water to 100ml

(b) Autoclave 121℃ for 20min

Prepare 200ml solid LB medium

(a) Weigh 5g LB,4g AGAR and fill it with deionized water to 200ml

(b) Autoclave 121℃ for 20min

(c) After taking out the sterilized medium, add 200mL and 100mg/mL of ampicillin when it is cooled to about 60℃ and mix well

d) Evenly pour the culture medium with antibiotics into the petri dishes, each dish about 20mL culture medium, cool at room temperature, seal with a sealing film and store at 4℃.

5. Enzyme Digestion

(1） Put it into the PCR instrument. See the following table for PCR procedures

(2）37℃ 30min

6. Seamless cloning

(1） The target DNA fragment and linearized vector are added to the PCR tube at a certain molar ratio for recombination reaction.

(2）50℃ 20min

(3）Immediately after the reaction, the centrifuge tube was placed on ice to cool for 2 minutes before transfromation.

7. Transfromation

(1) Take the receptive cells (TOP10) from the -80℃ refrigerator and put them on ice;

(2) Add the connected reaction solution into the receptive cells with a pipette gun and place it on ice for 25min;

(3) Water bath at 42 ° C for 1min and ice for 2min

(4) Add 500μL liquid free LB medium and oscillate at 220rpm at 37℃ for 1h

(5) Evenly apply 200μL bacterial solution on Amp resistant LB solid medium on a super-clean work table

(6) Overnight inverted culture at 37℃.

8. Plasmid extraction

(a) Take 10ml overnight culture bacterial solution, centrifuge at 9000 rpm for 2 minutes, and collect all bacterial bodies after multiple centrifugation

(b) The supernatant was removed and 250μL buffer P1 was added to the precipitation

(d) Add 250μL Buffer P2, and immediately gently reverse the centrifugal tube for 5-10 times and mix well. Let stand at room temperature for 2-4 minutes. You'll see the liquid clear and clear

(e) Add 300μL Buffer P3 and immediately gently reverse the centrifuge tube for 5-10 times to mix. At this point you can see the white precipitate forming

(f) Centrifuge at 12000rpm for 5-10 min. Transfer the supernatant into the adsorption column, centrifuge at 12000rpm for 30 seconds, and drain the liquid in the collection tube. If the supernatant liquid is too much, it can be centrifuged

(g) Add 600μL Wash Solution, centrifuge at 9000rpm for 30 seconds, and drain the liquid in the collection tube. Repeat twice

(h) The empty adsorption column is placed in the collection tube at 9000rpm for 2min

(i) Place the adsorption column in a new centrifuge tube and place it in the fume hood for 10min to remove residual ethanol

(j) Add 30μL Elution Buffer into the adsorption column, leave at room temperature for 2min, and then centrifuge at 9000rpm for 2min to collect the liquid in the centrifugal tube

(k) In order to ensure the concentration of glue recovery, you can repeat the step j)

9. Protein glue production

(1) Make 12% separation glue (8mL)

Ratio:

Distilled water: 3.4 mL

30%Acr-Bis (29:1) : 4.0mL

Gel buffer A: 2.5 mL

10% APS: 0.1 mL

TEMED: 0.006 mL

(2) Preparation of 5% concentrated glue (3mL)

Ratio:

Distilled water: 1 mL

30%Acr-Bis (29:1) : 0.5 mL

Gel buffer A: 1.5mL

10% APS: 0.03mL

TEMED: 0.003 mL

After the mixed solution of 12% separation glue is configured, add it into the glue maker with a pipette gun and add the deionized water seal layer. Wait for about 2 hours after solidification, pour away the upper water, add the prepared 5% concentrated glue, and insert the comb, wait for solidification

10. Polyacrylamide gel electrophoresis

(1) Prepare SDS-PAGE electrophoresis buffer: 14.4g glycine, 3gTris, 1 g SDS with deionized water to 1L

(2) Protein Loading and electrophoresis: First, Protein Marker is applied, and then the prepared protein sample is placed on 10μL successively. Run the glue at 80V voltage for about 30min until the strip enters the separation glue part, and then the voltage is modulated to 120V. Wait for loading to the bottom of the protein glue

(3) Dyeing: After the SDS-PAGE glue running, take out the protein glue and cut the concentrated glue part, put it into the dyeing box and add 100ml dyeing solution. After dyeing at room temperature for 0.5h on the horizontal shaker, pour out the dyeing solution and wash off the excess dyeing solution with clean water, and then add 100mL decoloring solution and decoloring on the normal room temperature horizontal shaker overnight

(4) The protein tape was observed, photographed and recorded and the results were analyzed

11. Prokaryotic expression of Escherichia coli

The correctly sequenced Rosetta strain was cultured at 220rpm at 37℃ overnight.
The strains cultured overnight were inoculated into fresh LB medium at a ratio of 2:50 and cultured at 220 RPM at 37℃ until OD600=0.6;
IPTG was added so that the final concentration was 0.1mM, 0.2mM, 0.3mM, 0.4mM, 0.5mM, 1mM and 2mM, and the culture was continued at 220rpm at 37℃ for 15h；
After centrifugation, the bacteria were collected and washed twice with 500μLTris-HCl. Then the bacteria were collected by centrifugation again, and 200μLTris-HCl was added to 1mL bacterial solution for re-suspension. After re-suspension, the bacteria solution was added to 10μL 2XLoading Buffer and treated at 98℃ for 15min
The protein production was detected by polyacrylamide gel electrophoresis.

12. Enzyme activity detection

(1) Prepare 10mL 0.2M PB buffer with PH=7.6: prepare 0.2M sodium dihydrogen phosphate solution and 10mL disodium hydrogen phosphate solution, respectively, and mix according to 13:87 ratio;

(2) Prepare 1mM of 1-naphthol solution: weigh 15.5mg1-naphthol and dissolve in 1mL anhydrous ethanol, use and mix;

(3) Prepare 20mM UDP-glucose solution: 12.2 mg UDP-glucose is dissolved in 1mL deionized water and stored at -20℃;

(4) Prepare the reaction system according to the following table

Note: The supernatant and precipitation of the protein after ultra-sound crushing were used as the enzyme solution for the reaction. The enzyme solution was replaced by Tris-HCl used in the collection of Escherichia coli in the blank control group.

(5) Finally, 10 μL of 10 μM α-naphthol was added to the reaction mixture and incubated at 37°C for 1 minute. Then, 200 μL of the mixture was transferred to each well of a black microplate, with three replicates for each group.

(6）The black microplate was placed into a microplate reader for detection, with the following settings: temperature at 37°C, excitation wavelength at 287 nm, emission wavelength at 335 nm, and a slit width of 5 nm.

(7）Record data and make graph analysis