1. Thaw competent cells on wet ice. Place the required number of 1.5 mL eppendorfs on wet ice, then make 100 μL aliquots of competent cells in the chilled 1.5 mL microcentrifuge tubes.

2. Add 10 μL of sample DNA directly into a tube of competent cells. Mix well by gently flicking tube several times.

3. Incubate the cells on ice for 30 min.

4. Heat-shock the cells for exactly 30 s in a 42 ℃ water bath.

5. Incubate the cells on ice for 2 min.

6. Add 900 μL of LB Liquid Medium.

7. Shake the tube at 200 rpm for 1 hour at 37 ℃.

8. Centrifugate eppendorfs at 12000rpm for 3 min and discard 900 μL supernatant. Resuspend cells with the remaining liquid.

9. Spread plates with bacterial suspension and incubate for the night.

(According to FastPure^® Plasmid Mini Kit)

1. Take 1-5 mL bacterial solution into a centrifuge tube, centrifuge at 12,000 rpm for 1 min and remove supernatant.

2. Add 250 μL Solution Ⅰ in centrifuge tube, using the pipet or vortex oscillator to suspend the cells.

3. Add 250 μL Solution II in centrifuge tube and gently flip upside down for 6-8 times to make sure the germ is full cracked.

4. Add 350 μL Solution III, gently flip upside down for 6-8 times to mix until white, flocculent precipitate appears and centrifuge at 12,000 rpm for 10 min.

5. Add the supernatant to the adsorption column in step 5, centrifuge at 12,000 rpm for 1 min, discard the filtrate and reuse collection tube.

6. Add 700 μL Wash solution to the adsorption column, centrifuge at 12,000 rpm for 1 min, discard the filtrate and reuse collection tube.

7. Add 500 μL Wash solution to the adsorption column, centrifuge at 12,000 rpm for 1 min, discard the filtrate and reuse collection tube.

8. Centrifuge the empty adsorption column at 12,000 rpm for 2 min to dry the column matrix. (Residual ethanol may impact downstream application)

9. Transfer the adsorption column into a clean 1.5 mL centrifuge tube, add 50-100 μL Elution buffer to the center of the column membrane, let sit at room temperature for 2 min and centrifuge at 12,000 rpm for 1 min, collect the plasmid solution in the centrifuge tube.

10. Store the plasmid at -20 ℃.

1. Place the gel tray in the appropriate position in the gel cartridge and place the comb in the correct position.

2. Measure 0.5 g agarose, put it in a 250 mL Erlenmeyer flask, add 50 mL 1 × TAE buffer and mix, then put the Erlenmeyer flask in the oven and heat to boil until the agarose is completely dissolved.

3. Add 5 μL GelRed to the solution.

4. Pour the solution into the gel casting tray.

5. After the gel cools to solid, pull out the comb.

6. Place the gel in the electrophoresis chamber with enough TAE buffer.

7. Add 10 × loading buffer to the sample and mix, then transfer the mixture to the well on the gel with a pipette.

8. Power on, run at 120 V for half an hour.

(According to FastPure^® Gel DNA Extraction Mini Kit)

1. Perform agarose gel/ethidium bromide electrophoresis to fractionate DNA fragments. Any type or grade of agarose may be used. However, it is strongly recommended that fresh TAE buffer or TBE buffer be used as running buffer. Do not reuse running buffer as its pH will increase and reduce yields.

2. When adequate separation of bands has occurred, carefully excise the DNA fragment of interest using a wide, clean, sharp scalpel. Minimize the size of the gel slice by removing extra agarose.

3. Determine the appropriate volume of the gel slice by weighing it in a clean 1.5 mL microcentrifuge tube. Assuming a density of 1 g/mL, the volume of gel is derived as follows: a gel slice of mass 0.3 g will have a volume of 0.3 mL.

4. Add 1 volume Binding Buffer (XP2).

5. Incubate at 50-60℃ for 7 min or until the gel has completely melted. Vortex or shake the tube every 2-3 min.

6. Insert a HiBind^® DNA Mini Column in a 2 mL Collection Tube.

7. Add no more than 700 μL DNA/agarose solution from Step 5 to the HiBind^® DNA Mini Column. Centrifuge at 10,000 x g for 1 min at room temperature. Discard the ltrate and reuse collection tube.

8. Repeat Steps 7 until all of the sample has been transferred to the column.

9. Add 300 μL Binding Buffer (XP2). Centrifuge at maximum speed (≥13,000 x g) for 1 min at room temperature. Discard the ltrate and reuse collection tube.

10. Add 700 μL SPW Wash Buffer. Centrifuge at maximum speed for 1 min at room temperature. Discard the ltrate and reuse collection tube.

11. Centrifuge the empty HiBind® DNA Mini Column for 2 min at maximum speed to dry the column matrix. Transfer the HiBind^® DNA Mini Column to a clean 1.5 mL microcentrifuge tube.

12. Add 50 μL deionized water directly to the center of the column membrane. Centrifuge at maximum speed for 1 min.

13. Store DNA at -20 ℃.

1. Prepare the reaction system in a PCR tube on ice, thaw the components and mix well. After use, put them back in -20 ℃.

2. Gently centrifuge to collect the liquid at the bottom of the tube.

3. Transfer the PCR tube to the PCR machine, set the parameters and start the thermal cycle.

(According to ClonExpress® Ultra One Step Cloning Kit)

1. Obtain linear vectors and inserts by PCR.

2. Purify PCR products, and then detect concentration.

3. The optimum cloning vector usage and insert fragment usage for ClonExpress® II recombination reaction system is 0.03 pmol and 0.06 pmol, respectively. According to this introduction, the corresponding DNA quality can be roughly calculated by following formulas.

① Optimal cloning vectors usage= [0.02* Base pairs number of cloning vectors] ng;

② Optimal inserts usage = [0.04 * Base pairs number of inserts] ng.

③ Note: Linear cloning vectors should be used 50-200 ng; insert amplification products should be used between 10 and 200 ng. Select the lowest/highest amount directly when the optimal amount of DNA used exceeds this range by using the above formula.

4. Arrange the following reaction systems on ice.

1. Wash glass plates and spacers, place rubber seal in bottom of gel kit and put the spacers between the two pieces of glass, making sure they are level.

2. Add the clamps and plugs, dispense 12.5% separation gel. Shake well immediately after adding TEMED to fill the gel. When filling the gel, 5 mL of gel can be sucked out along the glass by the gun, and the gel surface can be raised to a height of 1 cm under the rubber comb. Then add a layer of ethanol to the gel, and the gelation after liquid sealing is faster.

3. Placed at room temperature for 30 min to be solidified, when there is a line of refraction between ethanol and gel, the gel has been condensed. When the gel is fully solidified, the upper layer of ethanol can be poured off and the ethanol is blotted dry with absorbent paper.

4. Dispense 5% concentrated gel. Immediately after adding TEMED, the mixture can be filled. Fill the remaining space with the concentrated gel and insert the comb into the concentrate. When filling the gel, the gel should also flow down along the glass plate to avoid bubbles in the gel. Keep the comb level when inserting the comb. Since the volume shrinks and shrinks when the gel solidifies, the loading volume of the sample hole is reduced, so the gel is often applied on both sides during the solidification process of the concentrated gel. After the gel has solidified, pinch the sides of the comb and pull them straight up.

5. Make up running buffer (1 in 10 dilution of stock). Need around a liter.

6. Remove comb and place gel in opposite side of apparatus.

7. Load samples and molecular weight markers. 15 μL of protein from supernatant and 5 μL marker.

8. Put on grey plastic top (make sure rubber seal is in place).

9. Add the plugs and slowly add running buffer.

10. Put on front plastic cover and run at 120 V for 1 h.

(According to High Affinity Ni-NTA Resin)

1.Inoculate recombinant E. coli BL21 (DE3) in 20 mL of LB liquid medium containing 50 μg/mL kanamycin, and cultivate them overnight at 37℃ with shaking at 180 rpm. Inoculate 2% of the overnight cultured bacteria in 100 mL of LB liquid medium containing 50 μg/mL kanamycin, and culture them with shaking at 25℃ for 24 h.

2.The bacterial cultures were harvested and centrifuged at 12000 rpm for 10 min at 4℃.

3.For each culture supernatant, 300 mL of them was concentrated using MilliporeSigmaTM AmiconTM Ultra Centrifugal Filter Units (3 kDa) to about 10 mL in total.

4.Wash the Ni-NTA column with 50 mL PBS.

5.Load the concentrated sample in the Ni-NTA column for several times.

6.Wash the Ni-NTA column with 50 mL PBS.

7.Elute the Ni-NTA column with 200 mM 1.5 mL 250 mM imidazole.

8.Wash the Ni-NTA column with 50 mL PBS and store it at 4℃ by adding 10 mL ethanol.

9.Store the purified protein at -20℃.

1.Wash glass plates and spacers, place rubber seal in bottom of gel kit and put the spacers in between the two pieces of glass, making sure they are level.

2.Add the clamps and plugs, dispense 12.5% separation gel. Shake well immediately after adding TEMED to fill the gel. When filling the gel, 5 mL of gel can be sucked out along the glass by the gun, and the gel surface can be raised to a height of 1 cm under the rubber comb. Then add a layer of ethanol to the gel, and the gelation after liquid sealing is faster.

3.Placed at room temperature for 30 min to be solidified, when there is a line of refraction between ethanol and gel, the gel has been condensed. When the gel is fully solidified, the upper layer of ethanol can be poured off and the ethanol is blotted dry with absorbent paper.

4.Dispense 5% concentrated gel. Immediately after adding TEMED, the mixture can be filled. Fill the remaining space with the concentrated gel and insert the comb into the concentrate. When filling the gel, the gel should also flow down along the glass plate to avoid bubbles in the gel. Keep the comb level when inserting the comb. Since the volume shrinks and shrinks when the gel solidifies, the loading volume of the sample hole is reduced, so the gel is often applied on both sides during the solidification process of the concentrated gel. After the gel has solidified, pinch the sides of the comb and pull them straight up.

5.Make up running buffer (1 in 10 dilution of stock). Need around a liter.

6.Remove comb and place gel in opposite side of apparatus.

7.Load samples and molecular weight markers. 15 μL of protein with SDS-PAGE sample loading buffer(4X) from supernatant and 5 μL marker.

8.Put on grey plastic top (make sure rubber seal is in place).

9.Add the plugs and slowly add running buffer.

10.Put on front plastic cover and run at 120 V for 1 h.

1.We activated BBa_J04450 obtained from the kit and transformed it into E. coli DH5α, followed by overnight incubation at 37°C. The subsequent day, we chose 3 colonies and cultured them in 5 mL LB medium for approximately 12 h. This genetic segment was expressed within the plasmid pSB1C3; consequently, both our LB agar plates and culture media were supplemented with chloramphenicol (50 μg/mL).

2.Plasmid extraction was performed using XbaI and SpeI enzymes to obtain the empty vector pSB1C3(-).

3.The pSB1C3(-) plasmid was transformed into Escherichia coli DH5α, followed by overnight incubation at 37°C. The next day, three colonies were selected and cultured in 5 mL LB medium for approximately 12 h.

4.The plasmids pSB1C3(+) containing BBa_J04450 and pSB1C3(-) were separately transformed into E.coli DH5α, followed by overnight cultivation in 5 mL LB medium.

5.The overnight cultures of E. coli DH5α containing pSB1C3(+) and pSB1C3(-) were individually diluted into three fresh 50 mL LB media, and co-cultivated for sixteen hours.

6.Over this sixteen-hour period, we systematically harvested 200 μL samples of the cultures at half-hour intervals during the initial eight hours and at one-hour intervals throughout the subsequent eight hours. Throughout the collection process, we transferred the cultured samples into Costar 96 Flat Bottom Polystyrene Cat with black and transparent bottoms. Notably, each uniquely labeled culture was subjected to three replicates within both the black and transparent groups.

7.After the sampling was completed, we promptly measured the data using an microplate reader(Tecan Spark)and conducted subsequent analysis.

1. Induced by IPTG---- The bacteria solution need to be cultured for 12 h, while its OD600 should no less than 0.8 Add 1 M IPTG, makeits volume 1/2000 of the liquid volume of bacteria solution After inducer added, cultured it at lower temperature (usually 26°C) Sometimes, the not-induced experiment group is used as control.

2. Regulated by light/dark-- After introduced by IPTG, the experiment groups are wrapped by tinfoil before put into a black nonwovens bag. Cultured at 28°C for 12 h.

1. Firstly, 25 μL of the linear, phosphorylated padlock DNA (100 μM) and 20 μL ligation template (200 μM) were hybridized in the ligation solution (10 mM Tris-HCl, pH = 7.4, 200 mM NaCl,10 mM MgCl₂) at 70°C for 10 min, then cooling down to room temperature to make them hybridize with each other.

2. Second, 12 μL T4 DNA ligase (350 U/μL), 16 μL 0.05 % BSA, and 12 μL of 10 × ligase buffer was added respectively, and the mixture was incubated overnight at 16°C.

3. Next, 8 μL Exo I (20 U/μL) and 16 μL Exo III (100 U/μL) were added, and the mixture was incubated at 37°C for 2 h to degrade the excess ssDNA together with the nicked dsDNA to obtain the circular template.

4. The enzymes of Exo I and Exo III were denatured by heating at 80°C for 20 min.

5. Finally, the mixture was cooled down to room temperature and stored at 4°C fridge until use.

1. Add 20 μL dumbbell template (200 μM) into 25 μL buffer solution (10 mM Tris-HCl, pH = 7.4, 200 mM NaCl, 10 mM MgCl₂)

2. Annealing it at 70°C for 10 min, then cooling down to room temperature.

3、Afterward, 12 μL T4 DNA ligase (350 U/μL), 16 μL 0.05% BSA, and 12 μL of 10 × ligase buffer was added respectively, and the mixture was incubated overnight at 16°C.

4、Next, 8 μL Exo I (20 U/μL) and 16 μL Exo III (100 U/μL) were added, and the mixture was further incubated at 37°C for 2 h to degrade the excess ssDNA.

5、The above mixture was then cooled down to room temperature and stored at 4°C fridge until use.

1. 45 μL of BB buffer (10 mM Tris-HCl, 120 mM NaCl, 20 mM CaCl₂, 5 mM KCl, 0.05% tween-20, pH = 8.5), 2 μL of streptavidin-coated magnetic bead suspension, 5 μL of OTA and 1 μL of circular template were added to a tube and incubated at 37°C for 60 min.

2. Then, 5 μL of 10 mM dNTPs, 1 μL of 1% BSA, 5 μL of 10 × phi29 DNA polymerase buffer and 0.5 μL of phi29 DNA polymerase (10 U/μL) were added and mixed, and incubated at 30°C for 60 min.

3. The mixture was then heated at 65°C for 10 min, and the beads were washed 3 times with 300 μL of hybridization buffer (10 mM Tris-HCl, 300 mM NaCl, 1 mM EDTA, 0.05% tween-20, pH = 7.4) and resuspended in hybridization buffer.

4. 5 μL of molecular beacon MB (10 μM) was added to the amplification products, incubated at 90°C for 5 min and then slowly cooled to 25°C (room temperature), and the fluorescent signal was quantified using a quantitative PCR fluorescence instrument.

5. Fluorescence quantitative PCR instrument detection method: The reaction was carried out in 8 tubes, the 8 tubes containing the molecular beacon (MB) were placed in the instrument, and the reporter was changed to FAM on the Plate Setup page. The program was Holding Stage: 30°C 10 s; Cycling Stage: 30°C 2 min, 60 cycles. The fluorescence plateau value at the end of the run was taken.

Principle: ADH3 or M-CPA hydrolyzes N-methyl-L-phenylalanine to produce methyl hippuric acid, causing a change in absorbance at 254 nm in the reaction system. This alteration in 254 nm absorbance can be used to assess the enzymatic activity of ADH3 or M-CPA.

To a 96-well plate, 10 μL of the previously prepared protein (ADH3 or M-CPA) was added, followed by the addition of 290 μL of HLP substrate. The reaction was conducted at 25°C, with a pH of 7.5. The absorbance at 254 nm was measured at 30-second intervals over a 5-minute period.

1. Prepare the buffer solution: 25 mM Tris-HCl buffer containing 500 mM sodium chloride, pH7.5, 25°C. Use pure water to prepare a solution containing 3.03 mg/mL Trizma base and 29.2 mg/mL sodium chloride. Adjust the pH to 7.5 with 1 N hydrochloric acid at 25°C. Store the solution at room temperature.

2. Prepare a 1.0 mM hippuryl-L-phenylalanine solution (Hippuryl-L-Phe). Use 200-proof ethanol to prepare a solution containing 32.6 mg/mL hippuryl-L-phenylalanine. Once the sample is completely dissolved in ethanol, further dilute it to 0.326 mg/mL with the buffer solution prepared in step 1. Do not adjust the pH of this solution. Store the solution at room temperature and use it within three hours after preparation.

3. Prepare a 1.0 M sodium chloride solution (enzyme dilution solution). Use pure water to prepare a solution containing 58.4 mg/mL sodium chloride. Store the solution at room temperature.

4. Prepare the ADH3 or M-CPA solution (enzyme). Before use, prepare a dilution at room temperature containing 4-8 units/mL of the enzyme. For each sample, create a fresh dilution using the enzyme dilution solution prepared in step 3. Note: Do not dilute the enzyme with a cold dilution solution. Dilute each sample separately and verify once more that aliquots from the same container or batch are consistent. Before pipetting the sample, ensure thorough mixing to confirm that all materials settled at the bottom of the tube are in a homogeneous solution.

5. Spectral analysis. Run one sample at a time because the maximum rate usually occurs in the first minute. Provide a spectral analysis blank sample for the hippuryl-L-phenylalanine solution. Since the hippuryl-L-phenylalanine solution has high absorbance, detection needs to be performed with an accuracy greater than 2.0 absorbance units. Add the following components to an appropriate quartz culture dish:

Adjust the suitable constant temperature spectrometer to 25°C, and then add the following components (in mL):

Invert and mix thoroughly, then record the absorbance at 254 nm for approximately 5 min. Obtain the fastest linear rate (ΔA(254 nm)/min) of the sample and blank reaction within one minute time intervals. To ensure data validity, the ΔA(254 nm)/min for each reaction must be between 0.05 and 0.1.

1. Dissolve 1 mg of OTA in 10 mL of methanol to create a 100 μg/mL OTA stock solution, storing it at -20°C. Using this stock solution, prepare OTA standard solutions with concentrations of 5, 10, 15, 20, and 25 μg/mL in methanol. Employ established chromatographic conditions to measure the peak areas of these OTA standard solutions, conducting multiple measurements to calculate the average. Construct a standard curve for OTA based on the relationship between chromatographic peak area and OTA mass concentration. Ensure that at least 1 mL of each OTA concentration is prepared.

2. M-CPA Degradation Reaction System: Mix M-CPA, dissolved in 1 M NaCl (pH 7.5), with the OTA stock solution to achieve a final OTA concentration of 2 μg/mL. Incubate this solution at 37°C with agitation at 180 rpm for 24 h. Employ an OTA standard solution of the same concentration as a control. Repeat this procedure three times.

3. ADH3 Degradation Reaction System: Combine ADH3 with the OTA stock solution to reach a final OTA concentration of 50 μg/mL, adjusting the pH to 8.0. Incubate this solution at 40°C with agitation at 180 rpm for 24 h. Employ an OTA standard solution of the same concentration as a control. Repeat this procedure three times.

4. Centrifuge the reaction mixture at 8000 rpm for 10 min, and then sequentially filter the supernatant through a 0.45 μm filter membrane followed by a 0.22 μm filter membrane.

5. Measure the residual amount of OTA and calculate the degradation rate. Liquid chromatography conditions: C18 reverse-phase column, fluorescence detector with excitation wavelength at 333 nm and emission wavelength at 460 nm, injection volume of 20 μL, flow rate of 1.0 mL/min, column temperature at 30°C, mobile phase consisting of water: acetonitrile: methanol = 50:35:15. Degradation rate formula: OTA degradation rate = ((Peak area of OTA in the control group - Peak area of OTA in the sample)/(Peak area of OTA in the control group)) × 100%.

1. Under sterile conditions, inoculate the engineered bacteria onto LB agar plates (approximately 100 μL per plate) and culture at 37°C for 12 h.

2. Pick two individual colonies and shake them in 5 mL of liquid culture medium at 37°C and 200 rpm for 12 h.

3. Centrifuge the cultured bacterial cells at 4°C, 8000 rpm for 10 min in a low-temperature centrifuge (for cell pellet formation). Collect the bacterial cells, wash them three times in PBS (phosphate-buffered saline, pH 7.4, using approximately 10 mL each time), and then centrifuge at the same conditions to obtain bacterial cells for later use.

4. Set the mass concentration (w/v) of sodium alginate to 1.5%.

5. Prepare a 1.5% sodium alginate solution and sterilize it at 121°C for 15 min. After cooling to room temperature, add the bacterial suspension to 20 mL of different concentrations of sodium alginate solution. Mix thoroughly and use a 5 mL syringe to drop into a 2% (w/v) calcium chloride solution for microcapsules formation.

6. After microcapsules formation, continue to incubate the immobilized microcapsules in the above calcium chloride solution at 4°C for 4 h.

7. Collect the immobilized microcapsules and wash them with PBS.

8. Incubate the immobilized microcapsules in a 1.5 mL chitosan solution (0.4% (w/v)) for 15 min with gentle shaking.

9. Collect the immobilized microcapsules obtained and wash them with PBS.

10. After filtering and drying on sterile filter paper (using appropriate-sized sterile filter paper), store them at 4°C for later use.

Our experiment involved three groups: pSB1C3-tra-lysis (with 50μLIPTG), pSB1C3-tra-lysis (without 50μLIPTG), and empty pSB1C3.

1. Add 50μL fresh bacteria solution and 5μL chloramphenicol to 5ml LB medium, culture it at 37℃ with 220 rpm for 2-4 h.

2. Add the bacteria solution made in step 1 and 50μL chloramphenicol to 50ml LB medium, and culture it at 37℃ with 220 rpm.

3. Take samples every 30 minutes and measure its optical density (OD 600).

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