Notes

7/8

1. Prepare 100ml liquid LB medium

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

(b) Autoclave 121℃ for 20min

2. Formulation of antibiotics

(a) Ampicillin sodium (Amp) 100mg/ ml

(b) Dilute at 1:1000 when using

3. Passage culture of $E. coli$ (pET-21a and GT6CGT)

(1) Liquid LB 15mL plus 15μL ampicillin sodium (Amp, 100mg/mL)

(2) Add 150μL $E. coli$ strain (1:100 dose)

(3) Overnight culture at 220rpm at 37℃

7/9

1. Plasmid extraction

(a) Take 15ml 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

(c) Blow the precipitation repeatedly with the pipette gun until the bacteria are completely suspended

(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)

2. PCR

(1) The PCR system was formulated according to the table below

(2) Put in PCR instrument, PCR program

3. 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

(c) Microwave until completely melted

(d) Cool to 50 ° C to 60 ° C and add 3μL DNA stain

(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

7/10

1. PCR amplification of target gene Gt6CGT

(1) After preparing the following systems, centrifuge and mix them with a palm centrifuge

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

37℃ 30min

2. Agarose gel electrophoresis

(a) Weigh 0.5g agarose and add it to 50ml TAE

(b) Microwave until completely melted

(c) Wait until the temperature is 50 to 60 degrees and add DNA stain (nucleic acid dye)

(d) inverted board

(e) After cooling, the PCR products and the products after enzyme digestion were placed on the samples successively

7/11

1. Gel 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)

2. Preparation of solid 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℃

3. Seamless cloning:

4. transformation

(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℃.

7/12

1. Colony PCR

(a) Bacteria selection: Monoclone was selected from solid LB cultured overnight into 20μL fresh liquid LB, which was used as a template for PCR identification

(b) PCR system

(c) PCR procedure Tm: 55℃, extension time: 1min50s

(d) Agarose gel electrophoresis detection: there were bands in the positive control, but no bands in the 8 selected clones, so it was judged that the first vector construction failed

2. Extraction of pET-21a plasmid

(a) Take 15ml 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

(c) Blow the precipitation repeatedly with the pipette gun until the bacteria are completely suspended

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

7/19

1. Liquid medium

(a) Use a weighing balance to weigh 2.5g broth powder and fill it with deionized water to 100ml

(b) Autoclave at 121℃ for 20min, sterilize and close the lid

2. PCR amplification of the target gene (high-fidelity enzyme) and the single enzyme digestion of the carrier

(a) Remove the prepared TOP10 target gene template from the refrigerator

(b) Configure the PCR system and according to the following table: two tubes of 20μL and one tube of 40μL

(c) The tube was amplified by Bio-Rad gradient PCR apparatus Tm: 60℃, cycle: 35

(d) Prepare the enzyme digestion system according to the following table

3. Agarose gel electrophoresis

(a) Gel configuration: Dilute 50xTAE buffer to 1X with deionized water and then add agarose to prepare 25ml 1% solution

(b) Heat in microwave oven, observe boiling, remove, ensure completely dissolved, reheat if necessary

(c) Rinse the outside of the bottle with water and cool to room temperature

(d) Cool to 50 to 60 degrees and add 1.5μL DNA stain (nucleic acid dye)

(e) Insert comb, flip board

(f) Mix the amplified target gene with DNA loading buffer

(g) marker was injected into the first tank, amplified target gene was injected into the second and third tanks, one cell was left empty, and pET-21a vector product after enzyme digestion was injected into the last three cells

(h) After running the glue for 180V 15min, the electrophoretic bands were observed under UV and photographed

4. Cut glue and extract DNA

(a) Click on the software cutting label and take out the gel

(b) Cut off the gel part containing bands to avoid cutting into marker and blank parts

(c) Can be re-illuminated to ensure that there is no strip residue

5. Glue recycling

(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)

6. Seamless cloning: The target DNA fragment and linearized carrier are added to the PCR tube at a certain molar ratio for recombination reaction.

50℃20min

Immediately after the reaction, the centrifuge tube was placed on ice to cool for 2 minutes before conversion.

7. Transformation

(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) Apply 100μL bacterial solution on Amp resistant LB solid medium on a super-clean work table

Overnight culture inverted at 37℃.

7/20

1. Colony PCR

(a) Bacteria selection: Monoclone was selected from solid LB cultured overnight into 20μL fresh liquid LB, which was used as a template for PCR identification

(b) PCR system

(c) PCR procedure Tm: 55℃, extension time: 1min50s

(d) Agarose gel electrophoresis detection: there were bands in the positive control, and the target bands were found in all the 7 clones selected, but the bands were weak, and it was expected to be followed by bacterial solution PCR

2. Subculture of bacteria

The verified single colony selected above was expanded and added to 1mL fresh LB (including Amp) for overnight culture at 220rpm at 37℃. At the same time,3 single colonies were selected and inoculated into 10ml fresh LB (including Amp) and cultured overnight at 220rpm at 37℃ for subsequent extraction of plasmid bacteria solution

7/21

1. Bacterial solution PCR: 7 monoclonal bacterial solutions cultured overnight were used as PCR templates. The PCR system is shown in the table below

Tm: 55℃, cycle: 40

The PCR products were tested by agarose gel electrophoresis, and all strains were correct

1. pET-21a(+)-Gt6CGT plasmid extraction

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

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

c) Blow the precipitation repeatedly with the pipette gun until the bacteria are completely suspended

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

2. The plasmid is transformed into the Rosetta strain

a) Remove Rosetta cells from the -80℃ refrigerator and place them on ice

b) 1μL of the extracted pET-21a(+)-Gt6CGT plasmid was absorbed into the receptive cells with a pipette gun and placed on ice for 25 minutes

c) Bathe in water at 42 ° C for 1 minute and place on ice for 2 minutes

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

e) Apply 200μL bacterial solution to Amp resistant LB solid medium on a super-clean work surface

f) Overnight inverted culture at 37℃

7/22

1. Colony PCR was used to verify the Rosetta strain

Bacteria selection: Monoclone was selected from solid LB cultured overnight into 20μL fresh liquid LB, which was used as a template for PCR identification. The PCR system is shown in the table below

Tm: 55℃, cycle: 40

Agarose gel electrophoresis showed that there were bands in the positive control, and the target bands were found in all the 7 clones

2. Subculture of bacteria

The verified single colony selected above was expanded and added to 1mL fresh LB (including Amp) for overnight culture at 220rpm at 37℃.

7/23

1. Expanded culture of pET-21a(+)-Gt6CGT Rosetta strain: The Rosetta strain cultured overnight was inoculated into fresh LB medium at the ratio of 2:50, and cultured at 220rpm at 37℃ until OD600 was about 0.6

2. Protein induction: IPTG was added to the above-mentioned bacterial solution with OD600=0.6 so that its final concentration was 0.1mM. The bacterial solution without IPTG was used as the control group, and the bacterial solution was continued to be cultured at 220 RPM at 37℃ overnight

7/24

Experimentor: Jason, Seven, Gabriel

1. 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)

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

2. Collect bacteria and protein samples for preparation: After centrifugation, the bacteria were collected and washed twice with 500μLTris-HCl. Then the bacteria were collected again by centrifugation, and 200μLTris-HCl was added to 1mL of bacteria solution for re-suspension. The bacteria solution after re-suspension was added to 10μL 2XLoading Buffer, and treated at 98℃ for 15min

3. Polyacrylamide gel electrophoresis

(1) Prepare SDS-PAGE electrophoresis buffer: 14.4g glycine, 3gTris, 1 GSDS 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 on the second day, and the results were recorded and analyzed

7/25

1. Add 100ml liquid LB medium

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

(b) Autoclave 121℃ for 20min

2 Transformation

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

(2) Plasmid was added into receptive cells with pipette gun and placed 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) Apply 200μL bacterial solution on Amp resistant LB solid medium on super clean work table

(6) Overnight inverted culture at 37℃.

7/26

1. Colony PCR verification

(a) Bacteria selection: Monoclone was selected from solid LB cultured overnight into 20μL fresh liquid LB, which was used as a template for PCR identification

PCR system

(c) PCR procedure Tm: 55℃, extension time: 1min50s cycle: 40

(d) Agarose gel electrophoresis detection: there were bands in the positive control, and the target bands were found in all the 7 clones selected, and the transformation was successful

7.29

The verified strains of Rosetta and TOP10 strains were inoculated into fresh LB medium, in which 4 tubes of 1mL each were inoculated for Rosetta strain and 2 tubes of 10mL each were inoculated for TOP10 strain and then were cultured overnight under the conditions of 37℃ and 220rpm.

7.30

Due to the wrong temperature setting in the shaker, the bacterial solution wasn’t cultured successfully. Therefore, the Rosetta strain and TOP10 strain of pET-21a(+)-Gt6CGT were inoculated again. Specifically, the Rosetta strain was inoculated with 4 tubes, 1mL each, and the TOP10 strain was inoculated with 2 tubes, 10mL each and were cultured overnight at 37℃ 200rpm.

7.31

1. Plasmid extraction

(a) Take 10ml overnight culture bacterial solution, centrifuge at 9000 rpm for 2 minutes, and collect all thallus by multiple centrifuges.

(b) Remove the supernatant and add 250μL buffer P1 to the precipitation.

(c) Resuspend the precipitation repeatedly with the pipette until the pellet disappear.

(d) Add 250μL Buffer P2, and immediately gently reverse the centrifugal tube for 5-10 times to mix well. Let stand at room temperature for 2-4 minutes until the liquid becomes 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 separately.

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

(h) Place the empty adsorption column 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 centrifuge tube.

2. Agarose gel electrophoresis

(a) Gel configuration: Dilute 50xTAE buffer to 1X with deionized water，then add agarose to prepare 25ml 1% solution.

(b) Heat the solution in microwave oven, remove when observing boiling to ensure the completely the solute dissolved, reheat if necessary.

(c) Rinse the outside of the bottle with water and cool it to room temperature.

(d) Cool to 50 ° C to 60 ° C and add 1.5 µl DNA stain.

(e) Insert the comb and cast the gel.

(f) Mix the plasmid with the DNA loading buffer.

(g) Inject the marker into the first tank, and inject the extracted plasmid into the second, third and fourth tanks.

(h) After running the glue for 180V 15min, observe the electrophoretic bands under ultraviolet light and take photos for record.

3. Protein induction

(a) Inoculation at 4% dose.

(b) Expand the culture at 37 degrees Celsius and at 100 RPM for 3h, so that OD600=0.6.

(c) Add the inducer IPTG, construct the concentration gradients, which were 0.1mM, 0.2mM, 0.3mM, 0.4mM,0.5mM, 1mM and 2mM, under fixed temperature. One with no inducer was added as control. Continue the induction at 37℃ and 220rpm for 15h

4. Inoculation of pET-21a(+)-Gt6CGT Rosetta strain: The previously stored pET-21a(+)-Gt6CGT Rosetta was inoculated into 1mL fresh LB medium (Amp 100mg/L) at a ratio of 1% and cultured overnight at 37℃ and 200rpm

8.1

1. Collect bacteria and protein samples for preparation: After centrifugation, the bacteria were collected and washed twice with 500μLTris-HCl. Then the bacteria were collected again by centrifugation, and 200μLTris-HCl was added to 1mL of bacteria solution for re-suspension. The bacteria solution after re-suspension was added to 10μL 2X Loading Buffer, and treated at 98℃ for 15min

2. Polyacrylamide gel electrophoresis

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

(2) Dyeing: After the SDS-PAGE glue running, take out the protein glue and cut off the concentrated glue, 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 clear water, and then add 100mL decoloring solution to decolor on room temperature horizontal shaker overnight.

(3) The protein tape was observed on the second day, photographed and recorded, and the results were analyzed

3. Protein induction

(d) Inoculation at 4% dose.

(e) Expand the culture at 37 degrees Celsius and 100 RPM for 3h, so that OD600=0.6.

(f) The inducer IPTG was added, the temperature was fixed, and the concentration gradients were constructed, which were 0.1mM, 0.2mM, 0.3mM, 0.4mM,0.5mM, 1mM and 2mM, respectively. The one without inducer was added as control. Continue induction at 37℃ and 220rpm for 15h

8.2

Protein glue production

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

Ratio:

Distilled water: 3.4mL

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

Gel buffer A: 2.5mL

10% APS: 0.1 mL

TEMED: 0.006 mL

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

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 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.

2. Collect bacteria and protein samples for preparation: After centrifugation, the bacteria were collected and washed twice with 500μLTris-HCl. Then the bacteria were collected again by centrifugation, and 200μLTris-HCl was added to 1mL of bacteria solution for re-suspension. The bacteria solution after re-suspension was added to 10μL 2XLoading Buffer, and treated at 98℃ for 15min

3. Polyacrylamide gel electrophoresis

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

(2) Dyeing: After the SDS-PAGE glue running, take out the protein glue and cut off the concentrated glue, 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 clear water, and then add 100mL decoloring solution to decolor on room temperature horizontal shaker overnight.

(3) The protein tape was observed on the second day, photographed and recorded, and the results were analyzed

8.5

Polyacrylamide gel electrophoresis

(a) Take out the bacteria samples frozen at -30℃ after induction of 7.31 and 8.1, absorb 20 μL and add 20μL 2xLoading Buffer to mix, and treat at 98℃ for 15min to prepare the protein sample.

(b) Assemble the instrument for SDS-PAGE and prepare the protein glue two days in advance.

(4) Protein Loading and electrophoresis: First, apply the Protein Marker, and place the prepared protein sample on 10μL successively, then apply the 80V voltage to glue for about 30min until the strip enters the separation glue part. Next, modulate the voltage to 120V and wait for loading to the bottom of the protein glue.

(5) 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 to decoloring on room temperature horizontal shaker overnight.

(6) The protein tape was observed on the second day, photographed and recorded and the results were analyzed.

8.6

1. Preparation of medium

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

(b) Autoclave 121℃ for 20min

2. Expanded culture induction of $E. coli$

(a) The pET-21a(+)-Gt6CGT Rosetta strain cultured overnight was inoculated into 90mL fresh LB medium (Amp final concentration 100mg/L) at a ratio of 1:100, and incubated at 37℃ at 200rpm to OD600=0.6

(b) Inducer IPTG was added into the cultured bacterial solution so that its final concentration was 0.1mM, and the culture was continued at 37℃ and 200rpm for 15h

3. Passage culture of $E. coli$: The preserved strain pET-21a(+)-Gt6CGT TOP10 was inoculated into 10mL fresh LB medium (Amp final concentration 100mg/L) and cultured overnight at 37℃ at 200rpm by shock

8/7

1. Ultrasonic breakage of $E. coli$

(1) Centrifuge at 6000rpm at 4℃ for 15min to collect overnight induced bacteria

(2) Discard the supernatant and re-suspend the precipitation with 20 MLTRIS-HCl, centrifuge at 6000rpm for 15min at 4℃, and repeat twice

(3) The precipitation was re-suspended with 5mL Tris-HCl, and then the bacteria were broken at a low temperature with an ultrasonic crusher, the power was 40%, broken for 3s, suspended for 2s, suspended for 2min every 2min, and repeated the step 3 times.

(4) The crushed bacteria were centrifuged at 6000rpm at 4℃ for 15min, the supernatant was placed in a new centrifuge tube, and the precipitation was suspended with 5mL Tris-HCl, all of which were stored at -30℃ for subsequent protein electrophoresis and enzyme activity detection

2. Plasmid extraction

(a) Take 10ml overnight culture bacterial solution, centrifuge at 9000 rpm for 2 minutes, and collect all thallus by multiple centrifuges.

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

(c) Resuspend the precipitation repeatedly with the pipette until the bacteria are completely suspended.

(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 until the liquid becomes 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

(k) 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

At this stage, due to the operation error of the experimenter, the liquid after centrifugation was not stratified, and the experiment failed and waited for subsequent plasmid extraction

3. Production of protein glue

(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)

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.

8/8

1. 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 mgUDp-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) 1-naphthol with a mother liquor concentration of 10μM was added to the prepared system at last, and the amount of 1-naphthol was added at 37℃ for 1min, and then the mixed solution was added to the black enzyme-labeled plate with 200μL per well, and 3 replicates were set in each group.

(6) Put the black label plate into the label meter for detection, and set the conditions as follows: temperature 37℃, excitation wavelength 287nm and emission wavelength 335nm, slit width 5nm

(7) Record data and make graph analysis

2. Polyacrylamide gel electrophoresis

(1) Preparation of protein samples: the supernuant and precipitation after ultrasonic crushing on the previous day were removed from the -30 refrigerator and melted on ice. After all melting, 30μL was absorbed into a new centrifugation tube, and 30μL 2xLoading Buffer was added. Strains without inducer were used as control;

(2) Apply the above samples at 98℃ for 15min

(3) Electrophoresis:

① Glue: Pour an appropriate amount of electrophoresis buffer into the electrophoresis device, check whether there are bubbles at the bottom of the SDS glue, the bubbles will affect the electrophoresis result, after the check, gently pull out the comb on the SDS glue.

② Sampling: Absorb the boiled sample 10µL, align the sample with the glue hole, and be careful not to point the sample outside. Use appropriate molecular weight protein markers.

③ Electrophoresis: the positive and negative electrodes of the electrophoresis device are connected, and the power supply is turned on. Concentrated glue: voltage 100-120V, separation glue: voltage 130-150V.

④ Unglue: the sample in the glue runs to 2-3 mm from the bottom of the rubber plate, and the electrophoresis stops, and the general time is about 1-2 hours. Remove the rubber plate from the electrophoresis device, pry open the rubber plate with a blade, and cut off the concentrated rubber part of the rubber;

⑤ Dyeing: Put into the Coomasi bright blue dyeing solution for dyeing until the protein glue color becomes dark blue;

⑥ decolorization: Put the dyed SDS glue into the decolorization solution until the background is white, and replace the new decolorization solution according to the decolorization situation in the middle.

3. Preparation of medium

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

b) Autoclave 121℃ for 20min