Experiments

Plasmid Preparation

1. Harvest Cells: Transfer the overnight cultured medium to a 1.5 mL EP tube using a pipette. Centrifuge at 10,000×g for 1 min to collect the cell pellet. Discard the supernatant. Repeat this step to enrich the cell pellet.

2. Resuspend Cells: Add 250 μL of Solution I (containing RNase A and stored at 4°C) to the cell pellet. Use a pipette to resuspend the cells until no clumps are visible.

3. Lysis: Add 250 μL of Solution II and gently invert the tube 4-7 times. Incubate for 2-3 min to lyse the cells.

4. Neutralization: Add 350 μL of Solution III and gently invert the tube 4-7 times. Centrifuge at maximum speed (15,000×g) for 10-15 min.

5. Bind DNA: Carefully transfer the supernatant to a blue adsorption column and centrifuge at maximum speed for 1 min. Discard the waste liquid and return the column to an empty collection tube.

6. Wash: Add 500 μL of HBC Buffer (containing isopropanol) to the adsorption column. Centrifuge at maximum speed for 1 min, discard the waste liquid, and return the column to an empty collection tube.

7. Wash Again: Add 700 μL of DNA Wash Buffer(add absolute ethanol) to the adsorption column. Centrifuge at maximum speed for 1 min, discard the waste liquid, and repeat this step twice.

8. No-Load Operation: Place the adsorption column in an empty collection tube and centrifuge at maximum speed for 2 min to remove any residual wash buffer.

9. Ethanol removal: Transfer the adsorption column to a clean 1.5 mL EP tube. Place the tube in a metal bath at approximately 55°C for 3-5 min to dry.

10. Elution: Add 30-50 μL of distilled water to the center of the adsorption membrane. Let it stand at room temperature for 1 min, then centrifuge at maximum speed for 1 min to elute the DNA.

11. Storage: Store the extracted DNA at -20°C.

Transformation

Electroporation Procedure

1. Add the target fragment (The target practice fragment should not exceed 500 ng, the plasmid should not exceed 300 ng, and the total volume added should not exceed 10 μL) to 100 μL of competent cells and place on ice for 10-30 min.

2. Sterilize and dry 2 mm electroporation cuvettes under UV in a laminar flow hood for 20 min, pre-chill on ice, and quickly transfer the competent cells to the cuvette, ensuring cells are at the bottom.

3. Perform the following steps quickly: Wipe the outside of the cuvettes dry. For 2 mm cuvettes, use electroporation program Ec2; for 1 mm cuvettes, use program Ec1. After electroporation, immediately add 900-1000 µL of pre-warmed LB at 37°C, gently pipette, and transfer to a 1.5 mL centrifuge tube. Incubate at 37°C on a shaking incubator at 200 rpm for 45-60 min.

4. Plate an appropriate volume onto Kana+Str double antibiotic plates and incubate at 37°C.

Chemical Transformation

1. Thaw competent cells (stored in a -80°C freezer) on ice, add the ligation product/plasmid, and incubate on ice for 20 min.

2. Heat shock at 42°C for 45 sec, then let the cells rest on ice for 2 min.

3. Add 600 µL of LB medium without antibiotics and incubate at 37°C with shaking for 45 min.

4. Centrifuge at 4200 rpm for 3 min, discard an appropriate amount of supernatant, and plate the cells (using both diluted and concentrated samples if needed). Label the plates accordingly.

5. Incubate the plates upside down at 37°C for 12 h.

PCR

1. Prepare the reaction mixture in a PCR tube on ice and mix thoroughly using a vortex mixer.

2. Collect any residual liquid on the tube walls using a mini centrifuge.

3. Transfer the PCR tube to the thermal cycler and set the program to initiate the PCR thermal cycling process.

x=10~50ng (the addition volume depends on the concentration)

SDS-PAGE

1. Gel preparation:

Assemble the module and secure the gel mold. Prepare the lower gel according to the following recipe, pour it into the mold, and add water to level the surface. Let it sit for 40-60 min until the gel solidifies. Once solidified, discard the water on the surface. Prepare the upper gel according to the recipe, pour it into the mold, insert the comb, and allow it to solidify for approximately 1 h. After solidification, dismantle the mold to complete the gel preparation.

2. Sample preparation:/h3>

Collect the induced and uninduced bacterial cultures, measure and obtain 6 OD units of bacterial suspension. Centrifuge at 10,000 g for 10 min, discard the supernatant, and resuspend the pellet in 600 µL of sterile water. Use an ultrasonic cell disruptor to lyse the cells. Centrifuge the lysate at 10,000 g for 10 min to separate the supernatant and pellet. Resuspend the pellet with 600 μL sterile water. Completing the sample preparation.

3. Sample loading:

Prepare the protein buffer solution according to the table below. Place the gel into the electrophoresis apparatus. After checking for leaks with the buffer solution, mix 16 µL of the prepared sample with 4 µL of loading buffer. The mixed sample was put into the PCR instrument and heated at 100 ℃ for 5min to denature it. After cooling, load 16 µL of the mixture into the wells of the gel, followed by 4 µL of the protein marker. Then, sample loading is complete.

4. Electrophoresis:

Connect the power supply and run the gel at 80 V for 30 min, followed by 120 V for about 1 h.

5. Staining:

Carefully peel the gel off in water, then place the gel into the staining solution. Boil the solution and then use the heated staining solution to stain the gel on a small shaker at room temperature for about 45min.

6. Destaining:

Rinse the gel with clean water, wash the staining solution on the surface, add protein decolorizing solution, boil the solution, and decolorize the heated staining solution on a small shaker at room temperature for 30 minutes.

7. Composition:

Upper gel:

Lower Gel:

Protein Destaining Solution (500 mL system):

Anhydrous ethanol (75 mL), acetic acid (50 mL), distilled water (375 mL).

5× Protein Buffer Solution (5× Tris-Glycine Buffer; 1 L system):

Tris base (15.1 g), glycine (94 g), SDS (5 g); adjust the pH to 8.3 with precise measurements.

SDS-PAGE Staining Solution (400 mL system):

Coomassie Brilliant Blue R-250 (0.4 g), isopropanol (100 mL), glacial acetic acid (40 mL), distilled water (260 mL); stir thoroughly and filter using filter paper, frequently replacing the paper to remove particulate matter. Prepare two bottles at once, which can be reused.

Preparation of Electroporation Competent Cells

1. Transfer 200 μL of the glycerol stock into 5 mL of LB liquid medium and culture at 30°C, 200 rpm for approximately 12 h. Subsequently, inoculate into LB medium at a 2% concentration and incubate for 0.5 h before adding arabinose to a final concentration of 0.2%.

2. After culturing at 30°C for approximately 2 to 2.5 h, achieving an OD₆₀₀ of about 0.55-0.6 (values significantly higher or lower can severely affect electroporation efficiency), remove the culture and incubate on ice for 30 min.

3. Pre-cool the centrifuge rotor in a freezer for 10 min. Collect the cells by centrifugation using pre-cooled centrifuge tubes and keep on ice. Centrifuge at 4000 rpm for 10 min, aliquoting 50 mL per tube.

4. Using 20 mL of pre-cooled, sterile 10% glycerol per tube, gently pipette while keeping the tube on ice. Aliquot into two 10 mL centrifuge tubes and centrifuge at 4200 rpm for 10 min in a low-temperature centrifuge.

5. Immediately after centrifugation, discard the supernatant. Add pre-cooled, sterile 10% glycerol and then pipette gently while keeping the tube on ice.

6. Repeat steps 4 and 5 three times. For the first two washes, use 10 mL of pre-cooled, sterile 10% glycerol. For the final wash, resuspend in 0.5 mL of 10% glycerol and combine the two tubes into one.

7. Aliquot 100 μL per tube into pre-cooled centrifuge tubes. Proceed with electroporation or store immediately at -80°C. The competent cells can be stored at -80°C for up to six months.

Preparation of Competent Cells for Chemical Transformation

1. From -20°C or -80°C glycerol stock, use a sterile loop or white pipette tip to pick up the bacterial culture. Streak it onto an agar plate and incubate for 12 h (to isolate single colonies).

2. Pick single colonies and inoculate into 5 mL of liquid LB medium using a white pipette tip (or by pipetting up and down). Label the tubes and incubate at 37°C with shaking for 12 h.

3. Transfer 1 mL of the LB culture into 100 mL of LB medium and incubate at 37°C with shaking for about 2 h. Measure the OD until it reaches approximately 0.4 (monitor time carefully).

4. Place the culture into a 16°C shaker and incubate for about 1 huntil the OD reaches 0.5.

5. At this stage, do not use the alcohol lamp. Perform all procedures under strict aseptic conditions on ice. Aliquot 100 mL of the culture into four 50 mL centrifuge tubes and let sit on ice for 30 min to stop active growth.

6. Centrifuge at 4°C and 4200 rpm for 10 min. After centrifugation, place the tubes on ice, quickly discard the supernatant, and remove residual liquid using a pre-cooled pipette tip. Add 1 mL of a mixed solution of 80 mM MgCl₂ and 20 mM CaCl₂ to each tube (you may combine centrifuge tubes, ensuring balance).

7. Incubate on ice for 30 min.

8. Centrifuge at 4°C and 4200 rpm for 10 min. After centrifugation, place the tubes on ice, quickly discard the supernatant using aseptic techniques, and remove residual liquid with a pre-cooled pipette tip. Resuspend the pellet in 4 mL of a 15% glycerol and 100 mM CaCl₂ mixture, gently shaking on ice.

9. Aliquot 100 μL into pre-cooled tubes. Record the number of aliquots prepared. Prepare labeling for the storage bags, including preparation date, preparer, competent cell type, and aliquot volume. Place the labeling bags in ice for cooling.

10. Store at -80°C.

Gel Preparation:

1. Prepare agarose gels of appropriate concentration: 1% for detection gels and 2% for recovery gels. Heat in a microwave until the agarose is fully dissolved.

2. Allow the solution to cool to approximately 60°C, then add 1/10,000 Goldview and mix thoroughly.

3. Place an appropriately sized comb into the gel mold. Pour the warm agarose gel into the mold, ensuring the gel thickness is between 3-5 mm. Allow the gel to solidify for approximately 20 min.

4. Once the gel has completely solidified, gently remove the comb and place the gel tray into the electrophoresis chamber. Add 1×TAE buffer so that the buffer level is about 1 mm above the gel surface, ensuring proper conductivity.

Sample Loading:

1. Add the following to the wells: (PCR products + loading buffer) or DNA marker.

Electrophoresis:

1. Cover the electrophoresis chamber and run the gel at 80-120V.

2. When the dye front has migrated an appropriate distance through the gel (approximately 30 min), turn off the power, remove the gel, and observe the DNA bands under a UV lamp.

Gel Imaging:

1. Place the gel onto the gel imaging system and capture an image under ultraviolet light to visualize the gel.

Colony PCR

1. Prepare the colony PCR reaction system. For a 10 µL reaction volume, use: 1 µL of bacterial suspension, 0.4 µL of upstream and downstream primers, 5 µL of PCR enzyme Master Mix, and 3.2 µL of sterile water. If directly picking a colony, add 4.2 µL of sterile water to a final volume of 10 µL. For a 50 µL reaction volume, scale up the components proportionally.

2. Set up the PCR program according to the length of the bacterial PCR fragment and run the PCR.

3. Prepare the electrophoresis gel based on the number of bacterial PCR products and the system.

4. Load the bacterial PCR products and an appropriately sized DNA marker into the wells of the gel. Place the gel into the electrophoresis chamber and run the gel at 120-180V.

5. Determine the electrophoresis completion time based on the marker bands. After completion, remove the gel, visualize under UV light, and photograph for analysis.

Restriction Digestion

1. Select appropriate restriction sites based on the plasmid map.

2. Identify the corresponding restriction enzymes and system instructions, and prepare the reaction mixture according to the instructions.

3. Incubate the prepared mixture at the specified temperature in a metal bath or PCR machine for the recommended time.

Microplate Reader Detection

1. Inoculate the bacterial strain into 6 tubes of LB medium. Incubate three tubes in a shaking incubator at 30°C and the other three tubes in a shaker at 37°C, with all tubes cultured for 12 hours.

2. Multi-well plate preparation: Sterilize and label the 96-well plate.

3. Add 200 μL of bacterial solution dissolved in LB to each well.

4. Set the corresponding parameters on the microplate reader to measure the growth and fluorescence intensity of each well strain.

Digestion

1. Add Dpn I enzyme: Add 1 µL of Dpn I enzyme (10 U/µL) to each 50 µL PCR reaction mixture.

2. Gently mix the reaction mixture to avoid vigorous shaking and incubate at 37°C for 1~2 h to ensure complete digestion of the methylated template plasmid by Dpn I.

3. After digestion is complete, proceed with bacterial transformation without the need to deactivate Dpn I.

Induction

1. Turn on the UV light in the laminar flow hood and sterilize for 10-20 min.

2. Turn off the UV light, then turn on the fan and lighting. Sterilize and place the required glycerol stocks, antibiotics, and LB medium into the laminar flow hood.

3. Add 5 µL of the antibiotic corresponding to the bacterial strain (the volume of antibiotic added should be 1/1000 of the medium volume) to 5 mL of LB medium in a test tube.

4. Transfer 50 µL of bacterial culture from the glycerol stock (the volume of glycerol stock added should be 1/100 of the medium volume) into the LB medium with the added antibiotic.

5. Turn off the fan and lighting, remove the test tube, and incubate at 37°C with shaking for 12 h.

6. After incubation, prepare ZY medium (shaking flask), 50×M, 50×5052, trace elements, MgSO₄, and antibiotics, and sterilize them before placing them into the sterilized laminar flow hood.

7. Add the following to the ZY medium (shaking flask) using a pipette: 2 mL of 50×M, 2 mL of 50×5052, 200 µL of trace elements, 200 µL of MgSO₄, 100 µL of the corresponding antibiotic, and 1 mL of the bacterial culture. Seal the flask with parafilm and secure it with a rubber band.

8. Remove the flask and incubate at 37°C with shaking for 1-2 h until the OD600 reaches 0.5.

9. After incubation, prepare 20% arabinose and IPTG as needed, sterilize them, and place them into the sterilized laminar flow hood.

10. Add 1 mL of 20% arabinose (final concentration 0.2%) and 100 µL of IPTG to the ZY medium (shaking flask) using a pipette. Seal the flask with parafilm and secure it with a rubber band.

11. Remove the flask and incubate at 30°C with shaking for 16 h.

Whole-Cell Catalysis

1. Remove the induced shaking flask, take the bacterial culture with the corresponding OD value, and transfer it to a 10 mL centrifuge tube. Centrifuge at 4200 rpm and 4°C for 10 min.

2. Discard the supernatant and add 5 mL of fermentation broth. Mix thoroughly.

3. Transfer the mixed solution to a 250 mL shaking flask.

Fig. 2 Whole-cell catalyzed sample vials made

4. Incubate the shaking flask at 30°C and sample at fixed time intervals. (during the fermentation process, the conical flasks need to be transferred from 30°C to 37°C midway)

Composition of fermentation broth (40 mL system): Na₂HPO₄·12H₂O is 23.88 mM, NaH₂PO₄ is 11.02 mM, NaCl is 4.28 mM, NH₄Cl is 9.35 mM, Tris·HCl (pH = 8.0) is 25 mM, MgCl₂ is 2.5 mM, L-glutamine is 15 mM, and anhydrous glycerol is 2 mL.

Sample Preparation

1. Suck 20 µL of bacterial culture from the shake flask and mix it with 180 µL of water. Transferred to EP tubes and centrifuged at 10000 x g for 10 min.

2. After centrifugation, remove the EP tube and use a 1 mL syringe to aspirate the entire supernatant.

3. Attach the syringe to a 0.22 µm filter membrane, and push the syringe to filter the supernatant into a new EP tube. Repeat the process if necessary to ensure complete filtration.

4. Use a pipette to transfer the filtered supernatant into a brown liquid-phase vial. The sample is now prepared and can be stored at -20°C for short-term use.

Vanillic Acid Detection

HPLC Detection

1.Use a C18 column with a mobile phase of 0.1% phosphoric acid aqueous solution : pure methanol = 70:30.

2.Detect the absorbance peak at a UV wavelength of 254 nm with a column temperature of 30°C.

Attempt to Produce AAP Using Vanillic Acid as Substrate

Preparation of vanillic acid as production matrix

1.Weigh 10mM vanillic acid (standard compound) and put it into a 100ml conical flask.

2.Add 40ml of water to the conical flask and use a magnetic stirrer until the vanillic acid is completely dissolved.

3.Filter with 0.22 micron filter membrane to remove bacteria.

Production of AAP

1.Suck 5 ml of fermentation broth into a 10 ml centrifuge tube containing 125OD volume of engineered E. coli strain (BW25113- ΔnhoA carrying plasmids pYB1a-pabABC-ABH60 and pSB1c-I38-PANAT). Resuspend the cell pellet evenly to initiate biotransformation.

2.pipette 20 μL of resuspended culture and mix it with 180 μL of water and put it into a 1.5 ml EP tube as a 0-hour control sample. The remaining resuspended culture was transferred to a 100ml Erlenmeyer flask containing 5mm l-glutamine and 5mm vanillic acid (final concentration). The flask was sealed with silica gel plugs and shake at 220 rpm for 36 hours at 30 ° C, and then shake at 220 rpm for 60 hours at 37 ° C for fermentation and AAP production. Every 6 h during the fermentation process, 20 μL of sample was taken from the culture solution and transferred to a 1.5 ml EP tube for subsequent HPLC sample preparation.

Sample Preparation for AAP Detection

1.Take samples at 17 fermentation time points in a 100ml Erlenmeyer flask (including the 0-hour control), take 20 μL of each and add 180 μL of water for dilution, mix with a pipette gun.

2.Centrifuge the diluted sample at 10000 g for 10 min to pellet cells and any debris.

3.After centrifugation, carefully aspirate all supernatants using a 1 ml syringe to avoid precipitation.

4.Connect the yellow filter membrane (pore size 0.22 μ m) to a 1 ml syringe, and filter the supernatant into a new clean Brown HPLC vial. If necessary, repeat aspiration and filtration from the same sample to ensure complete transfer and clarification.

5.Seal the vial with a cap. Samples are now prepared for HPLC analysis. (if immediate analysis is not possible, store the sample at -20 ° C to prevent degradation.)

HPLC Detection

1.Use a C18 reverse-phase column for separation. The mobile phase consists of 0.1% phosphoric acid aqueous solution : pure methanol = 70:30(v/v).

2.Detect the absorbance of the sample at a UV wavelength of 254 nm (optimized for AAP detection). Maintain the column temperature at 30°C throughout the analysis.

3.Identify and quantify the AAP peak in the samples by comparing its retention time and peak area to a standard curve generated using pure AAP standard.