Experiments

1. Preparation of LB (Luria-Bertani) culture medium

2. First round of recombinant plasmid construction

3. Functional testing

3.1 Target protein expression test
3.2 Detection of L-dopa production using an ELISA test kit

4. Second round of recombinant plasmid construction

5. Second round of fermentation testing

5.1 Detection of L-dopa production using an ELISA test kit. (Temperature, time, inducer concentration, glucose concentration）
5.2 HPLC determination of levodopa content in ECN
1. Preparation of LB (Luria-Bertani) culture medium

Goal: Liquid and solid culture media are formulated and optimized to support robust microbial growth and provide essential nutrients. These media are designed to facilitate key processes in controlled bioprocessing applications, including cell activation, proliferation, and fermentation.

Materials:

  1. 1) Conical flask
  2. 2) Autoclave
  3. 3) Tryptone
  4. 4) Yeast Extract
  5. 5) Sodium Chloride
  6. 6) Double distilled H2O
  7. 7) Agar

Procedures:

  1. 1) Add 10 g tryptone, 5 g yeast extract, and 10 g of sodium chloride per liter of the culture medium. (Note: 1.5% agar powder was added to the solid culture medium);
  2. 2) Add into 1L conical flask;
  3. 3) Maintain at 121 ℃ for 20 minutes in an autoclave for sterilization.
2. First round of recombinant plasmid construction

pCDFDuet-tktA-aroF-aroE，pETDuet-TyrA(K12/SO/HI)-hpaB-hpaC

Obtain target genes and Vector fragments by PCR

Goal: To amplify target genes and Vector fragments for Infusion recombination.

Materials:

  1. 1) 2x PrimeStar Mix
  2. 2) Gene template
  3. 3) Double distilled water (ddH2O)
  4. 4) Primer-F
  5. 5) Primer-R
  6. 6) PCR tube
  7. 7) PCR thermal cycler

PrimeStar Mix includes buffer and dNTPs for PCR. The extention rate for PrimeStar Mix (2x) is 1000bp/10 s.

Procedures:

  1. 1) Add 2x PrimerStar Mix 25µl, gene template 1µl, primer-F 1µl, and primer-R 1µl to centrifuge tube. Add ddH2O to the centrifuge tube up to 50µl. If bubbles are observed in the mixture, centrifuge the mixture for a few seconds to remove the bubbles;
  2. 2) Insert PCR tubes into PCR thermal cycler. Pre-denature the fragments at 95˚C for 3 minutes;
  3. 3) Perform 40 cycles of 30 seconds 95˚C denaturing, 30 seconds 55˚C annealing, and 2 minutes extension 72˚C extension, For the two empty vectors pCDFDuet and pETDuet, the extension time was set at 6 minutes due to their longer lengths.
  4. 4) Extend the fragments for 1 minutes at 72˚C;
  5. 5) If not use immediately, preserve the fragments at 4˚C;
  6. 6) Recover PCR fragments by agarose gel electrophoresis.

Agarose gel construction, deployment and recycle

Goal: To prepare an agarose gel with uniform consistency and optimal physical properties suitable for high-resolution electrophoretic separation of nucleic acids or proteins.

Materials:

1) Casting Tray

2) Well comb

3) Microwave

4) 1x TAE buffer

5) Agarose M

6) 10000x Nucleic acid gel stain

7) Conical flask

Procedures:

  1. 1) Dissolve 1.5g Agarose in 200mL Conical flask;
  2. 2) Add 100mL TAE buffer;
  3. 3) Heat the material with microwave;
  4. 4) Melt the mixture at 30 sec interval;
  5. 5) See whether bubbles are forming, if did succesfully mixed;
  6. 6) Add 10 μl nucleic acid gel stain (10000x);
  7. 7) Let it cool for 5~10 minutes;
  8. 8) Pour the agarose mixture into the casting tray;
  9. 9) Place the appropriate well comb to create the wells;
  10. 10) Wait to solidify ~30 minutes;
  11. 11) Remove the comb and place the gel in the gel box.

Agarose gel electrophoresis

Goal: To confirm the sizes of DNA fragments following electrophoresis, enabling precise excision and purification of target bands for subsequent molecular procedures.

Procedures:

  1. 1) Extract 50µl of every sample; add 5µl of loading buffer (10x) to each sample;
  2. 2) Place the gel in the horizontal electrophoresis system;
  3. 3) Extract 50µl of the mixed solution containing the sample, mix them till their colors are almost uniform;
  4. 4) Add the marker to the first well and load 50µl of each sample into the wells;
  5. 5) The procedure runs at 125V for 25min;
  6. 6) Take out the gel from the device and proceed to following steps.

DNA gel extraction

Goal: The target DNA fragments are isolated and purified from the agarose gel matrix by electrophoretic separation.

Materials:

  1. 1) Agarose gel with target genes fragments and linearized pCDFDuet and pETDuet backbone;
  2. 2) 1.5 mL Eppendorf (EP) tube
  3. 3) Agarose Gel Electrophoresis Recovery Kit

Procedures:

  1. 1) Cut the slice of gel containing target genes fragments and linearized pCDFDuet and pETDuet backbone, cutting off as much unneeded gel as possible, and place it in an 1.5 mL EP tube;
  2. 2) Add 500 microliters of buffer B2 and put the tube into a 50°C hot water bath until gel has completely melted;
  3. 3) Transfer the solution containing melted gel into a purification column that's in a collection tube and perform centrifugation at 12000 rpm for 30 seconds;
  4. 4) Empty the collection tube, put the purification column back in, and add 500 microliters of wash solution containing pure ethanol. Perform centrifugation at 12000 rpm for 30 seconds, and empty collection tube again;
  5. 5) Repeat steps 4;
  6. 6) Perform centrifugation one more time at 12000 rpm for 2 minutes, then open the cap of the tube and let it sit for one minute to allow the ethanol to evaporate;
  7. 7) Transfer purification column to a new 1.5 mL EP tube. Add 50 microliters of double distilled water (ddH2O) at the center of the purification column;
  8. 8) Cap the lid and let the tube sit for 1 minute. Perform centrifugation at 12000 rpm for 1 minute;
  9. 9) Discard the purification column. Store DNA fragments in the EP tube at 4°C.

Homologous recombination of linearized pCDFDuet and pETDuet backbone with target gene fragments

Goal: Perform homologous recombination between the linearized pCDFDuet and pETDuet backbone and target gene fragments to generate the complete plasmid.

Materials:

  1. 1) CloneExpress Mix (2x)
  2. 2)PCR products of tktA, aroF, aroE, tyrA，hpaB，hpaC
  3. 3)Linearized pCDFDuet and pETDuet backbone

Procedure:

  1. 1) Measure the concentration of the gel-recovered fragments;
  2. 2) Prepare the system for the construction of pCDFDuet-tktA-aroF-aroE in PCR tubes: 5μl CloneExpress Mix, 2.5μl Linearized pCDFDuet backbone, and 2.5μl tktA or aroF or aroE fragments;
  3. 3) Prepare the system for the construction of pETDuet-TyrA-hpaB-hpaC in PCR tubes: 5μl CloneExpress Mix, 2.5μl Linearized pETDuet backbone, and 2.5μl TyrA or hpaB or hpaC fragments;
  4. 4) Insert PCR tubes into PCR thermal cycler. Ligase the fragments at 50˚C for 30 minutes;

Transformation

Materials:

  1. 1) Ligated recombination plasmid
  2. 2) Liquid LB solution without antibiotics
  3. 3) LB Agar plates with Ampicillin and spectinomycin antibiotics

Procedures:

  1. 1) Add recombination plasmid into E.Coli BL21 strain;
  2. 2) Placed on ice for 20 minutes;
  3. 3) Heat shock at 42˚C for 45 seconds, then immediately put on ice for 2-3 minutes;
  4. 4) Add 400μL liquid LB solution without antibiotics, incubate at 37 ˚C for 30 minutes;
  5. 5) Centrifuge at 5000 rpm for 3 minutes, extract 300μL of supernatant then discard;
  6. 6) Apply bacterial liquid to LB Agar plates with Kanamycin antibiotics, incubate for 12-16 hours at 37˚C.

Colony PCR verification

Goal: Following successful transformation and growth of the recombinant E. coli BL21 strain, colony PCR was employed to screen for individual colonies containing the correctly constructed plasmid.

Materials:

  1. 1) Taq Master Mix (2x)
  2. 2) Double distilled water (ddH2O)
  3. 3) Culture plates post-transformation

Procedures:

  1. 1) Add 10μl Taq Master Mix, 1μl primer-F, 1μl primer-R, and 8μl ddH2O into PCR tubes;
  2. 2) Pick a single colony with a 10μl sterile pipette tip and perform aspiration and dispensing in PCR tubes;
  3. 3) Insert mixture into a PCR thermal cycler, design the PCR program according to the manufacturer's instructions.
  4. 4) Perform agarose gel electrophoresis, and the presence of the correct bands indicates the successful construction of recombination plasmids.
3. Functional testing
3.1 Target protein expression test

Ⅰ. Amplification culture for correct colony

Goal: To acquire large amounts of target proteins for SDS-PAGE

Materials:

  1. 1) Liquid LB solution
  2. 2) pCDFDuet-tktA-aroF-aroE，pETDuet-TyrA(K12/SO/HI)-hpaB-hpaC bacterial fluid
  3. 3) Ampicillin and spectinomycin antibiotics
  4. 4) Inducer: IPTG
  5. 5) Iso-thermic shaker

Procedures:

  1. 1) Add 1000μl of pCDFDuet-tktA-aroF-aroE，pETDuet-TyrA(K12/SO/HI)-hpaB-hpaC bacterial fluid and 100μl of Ampicillin and spectinomycin antibiotics into 100mL of liquid LB solutions, respectively;
  2. 2) Culture bacterial liquid in iso-thermic shaker for 3 hours until absorbance OD600nm to 0.4-0.6;
  3. 3) Add IPTG into bacterial liquid with 1mM final concentration;
  4. 4) Sample for each bacterial liquid cultured at 37°C for 6h, respectively.

Ⅱ: Protein crude extraction and SDS-PAGE

Goal: To verify the expression of desired proteins.

Materials for protein crude extraction:

  1. 1) 500μl bacterial liquid for different induced conditions
  2. 2) Centrifuge
  3. 3) Double distilled water (ddH2O)
  4. 4) 6x Protein loading buffer

Procedures for protein crude extraction:

  1. 1) Centrifuge samples at 12000 rpm for 1 minute;
  2. 2) Discard supernatant and resuspend precipitation with 50μl 1x Protein loading buffer diluted by ddH2O;
  3. 3) Boiled samples at 95°C for 15 minutes in PCR thermal cycler.

Materials for SDS-PAGE:

  1. 1) Sangon 12.5% SDS-PAGE Color Preparation kit
  2. 2) Electrophoresis buffer (Tris-Glycine)
  3. 3) Protein ladder
  4. 4) 6x Protein loading buffer
  5. 5) Vertical electrophoresis system
  6. 6) Coomassie blue staining solution

Procedures for SDS-PAGE：

  1. 1) Add and mix 2.2mL 2x separating gel solution, 2.2mL 2X separating gel buffer, and 44μL catalyst in centrifugation tubes for separating gel;
  2. 2) Slowly pipetting mixture into casting stand and frame to avoid bubbles;
  3. 3) Add 1 mL ddH2O at the top of separating gel;
  4. 4) Wait the separating gel to solidify for 8 minutes;
  5. 5) Discard the water that was added previously;
  6. 6) Add and mix 825μL 2x stacking gel solution, 825μL 2X stacking gel buffer, and 11μL catalyst in centrifugation tubes for stacking gel;
  7. 7) Add stacking gel mixture until the cast is completely filled, then slowly insert comb without producing any air bubbles;
  8. 8) Wait the stacking gel to solidify for 12 minutes, then carefully remove the comb, and wash the wells with electrophoresis buffer;
  9. 9) Add 10μL protein ladder into the first well, then load samples in each successive well;
  10. 10) Transfer gel into vertical electrophoresis system;
  11. 11) Run at 120V for 90 minutes;
  12. 12) Stain the gel with Coomassie blue staining solution for 10 minutes, then destain the gel using destaining solution repeatedly until gel background become transparent.
3.2 Detection of L-dopa production using an ELISA test kit

Goal: The kit uses a double-antibody one-step sandwich enzyme-linked immunosorbent assay (ELSA). To a coating medium pre-coated with a levodopa antibody, the standard, then the HBP-labeled detection antibody are added sequentially. After incubation and washing, the sample is assayed using the substrate TMB. TMB is converted to blue under the catalysis of a peroxidase and to yellow under the action of acid. The intensity of the color is positively correlated with the levodopa content of the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the levodopa concentration in the sample.

Materials：

  1. 1) Microwell ELISA plate
  2. 2) Standards
  3. 3) Sample diluent
  4. 4) Detection antibody-HRP
  5. 5) 20x wash buffer
  6. 6) Substrate A
  7. 7) Substrate B
  8. 8) Stop solution
  9. 9) Seal film
  10. 10) Instructions
  11. 11) Ziplock bag

Procedures:

Note: The concentrations of the standards (S0-S5) are: 0, 10, 20, 40, 80, and 160 pg/mL. Reagent Preparation: 20x Wash Buffer Dilution: Dilute distilled water 1:20, i.e., 1 part 20x Wash Buffer with 19 parts distilled water.

Plate Washing Method：

1. Manual Plate Washing: Shake off all liquid from the wells, fill each well with wash buffer, let stand for 1 minute, shake off all liquid from the wells, and tap onto absorbent paper. Wash the plate five times.

2. Automatic Plate Washer: Add 350μL of wash buffer to each well, soak for 1 minute, and wash the plate five times.

Specific Steps：

  1. Remove the desired plate strips from the aluminum foil bag after equilibration at room temperature for 20 minutes. Seal the remaining strips in a ziplock bag and return them to 4°C.
  2. Set up standard and sample wells. Add 50 μL of the standard of different concentrations to each standard well.
  3. Add 10 μL of the sample to be tested to the sample well, followed by 40 μL of the sample diluent. Leave blank wells empty.

4. Add 100 μL of horseradish peroxidase (HRP)-labeled detection antibody to each well, including the standard and sample wells, except for the blank well. Seal the wells with sealing film and incubate at 37°C in a waterbath or incubator for 60 minutes.

5. Discard the liquid, pat dry on absorbent paper, fill each well with wash solution, let stand for 1 minute, discard the wash solution, pat dry on absorbent paper, and repeat this process five times (a plate washer can also be used).

6. Add 50μL each of substrates A and B to each well and incubate at 37°C in the dark for 15 minutes.

7. Add 50μL of stop solution to each well. Within 15 minutes, measure the OD value of each well at a wavelength of 450 nm.

8. Draw a standard curve: In Excel spreadsheet, plot the standard concentration on the horizontal axis and the corresponding OD value on the vertical axis. Calculate the concentration of each sample using the curve equation.

4. Second round of recombinant plasmid construction

Based on the first round of plasmid construction, two new recombinant plasmids were constructed, namely pETDuet-TyrA(K12)-RRS2-hpaB-hpaC and pETDuet-TyrA(K12)-RRS3-hpaB-hpaC，The only difference between these two plasmids and the first round of construction is the sequence of RBS, So the materials and steps are the same as step 2.

5. Second round of fermentation testing
5.1 Detection of L-dopa production using an ELISA test kit. (Temperature, time, inducer concentration, glucose concentration）

This step is consistent with the content of Section 3.2

5.2 HPLC determination of levodopa content in ECN

Goal: Quantitatively detect levodopa (L-DOPA) in samples, generate a linear calibration curve, and determine the concentration of L-DOPA in the sample. This method utilizes a reversed-phase C18 column using HPLC-UV (or optional electrochemical detection (ECD)). The steps include mobile phase preparation, standard preparation, sample pretreatment, sample loading and testing, data processing, and post-column maintenance.

Materials:

1. HPLC system (with autosampler, UV detector; optional: electrochemical detector (ECD))

2. Reversed-phase C18 column (4.6 × 150 mm, 5 µm)

3. Mobile phase A: 50 mM phosphate buffer (NaH2PO4/Na2HPO4), pH 2.5 (adjusted with phosphoric acid), with 0.1% antioxidant (optional)

4. Mobile phase B: Acetonitrile (HPLC grade) or methanol (HPLC grade)

5. L-DOPA standard (accurately weighed)

6. Internal standard (optional, a compound with known concentration that does not overlap with the sample)

7. Antioxidant: Ascorbic acid (analytical grade) to prevent oxidation of catechols

8. Acidifier: 1.0 M HCl or 0.1 M HCl (for sample stabilization)

9. Organic solvent: Methanol (HPLC grade) for protein precipitation; acetonitrile for mobile phase and rinsing

10. 0.22 µm syringe filter (polytetrafluoroethylene/polyether or PTFE/nylon) and syringe

11. Autosampler vials (amber, light-proof) and silicone caps

12. Micropipette and pipette tips, sterile centrifuge tubes (1.5 mL/2 mL), centrifuge, vortex mixer, ultrasonic cleaner (optional)

13. Spectrometer (for preparing standards), ice bath and cold chain equipment (for keeping samples cold)

14. Waste container and chemical waste labeling

Procedures:

1. Mobile Phase Preparation and Handling (Pre-processing)

a. Prepare 50 mM phosphate buffer: Prepare according to the recipe and dissolve in sufficient deionized water. Adjust the pH to 2.5 (adjust slightly with phosphoric acid or dilute HCl).

b. Add 0.1% (w/v) ascorbic acid or 0.1 mM EDTA to inhibit L-DOPA oxidation (recommended for long-term sample storage). c. Mix the buffer and acetonitrile in the desired ratio (recommended starting ratio: A:B = 95:5, isocratic). If using a gradient, design a time-ratio schedule. d. Filter the mobile phase through a 0.22 µm filter and degas by sonication or use an in-line degasser. e. Place the mobile phase in a labeled bottle and place it in the HPLC feed. 2. HPLC Conditions (recommended starting conditions, which can be optimized based on column and detector) a. Column: C18 4.6 × 150 mm, 5 µm; Column temperature: 30°C (can be adjusted to 25–35°C to stabilize retention time). b. Flow rate: 1.0 mL/min (can be adjusted to 0.6–1.2 mL/min for smaller column diameters or detector requirements). c. Injection volume: 10 µL (can be increased to 20 µL for low-concentration samples). d. Detection: UV at 280 nm (L-DOPA absorbs at ~280 nm); if using an ECD, set the operating potential to an appropriate level (typically +0.6–+0.8 V) and use conditions compatible with the deionized mobile phase.

e. Run time: 10–15 min (depending on the retention time setting, to ensure complete peak elution); 10 min is generally sufficient under isocratic conditions; longer may be used if interferences occur.

3. Standard Preparation and Calibration Curve

a. Accurately weigh L-DOPA (e.g., 10.0 mg) and dissolve it in ice-cold 0.1 M HCl + 0.1% ascorbic acid solution to 10.00 mL to prepare a 1000 µg/mL (or 1 mg/mL) stock solution. Protect from light and work on ice.

b. Prepare a series of working standards (e.g., 0.5, 1, 2, 5, 10, 20, 50 µg/mL) from the stock solution using mobile phase or 0.1 M HCl + ascorbic acid. Prepare QC samples (low, medium, and high concentrations, e.g., 1, 10, and 40 µg/mL, respectively). If using an internal standard, add a constant amount of internal standard to all standards.

c. Use the standard solution as soon as possible. For long-term storage, refrigerate and protect from light (recommended for no more than 24–48 hours).

4. Sample Pretreatment (for fermentation supernatant/culture medium)

a. Centrifugation: Take a sample (e.g., 1 mL) of fermentation/culture broth, cool it (4°C), and centrifuge it at 10,000 × g for 10 minutes. Collect the supernatant.

b. Acidification Stabilization: Add 1–10 µL of 1.0 M HCl (or an amount to bring the pH to ~2–3) to the supernatant. Add ascorbic acid to a final concentration of 0.1% (w/v) to prevent L-DOPA oxidation. Mix gently.

c. Protein precipitation: Add 3 volumes of cold methanol equal to the sample volume (e.g., 300 µL of cold methanol for 100 µL sample), vortex mix for 30 seconds, place on ice for 10 minutes, and centrifuge at 10,000 × g for 10 minutes.

d. Collect the supernatant. If the concentration is low, perform solid-phase extraction (SPE) or concentrate with nitrogen purge before reconstitution with mobile phase. If using directly on the instrument, filter the supernatant through a 0.22 µm syringe filter into an autosampler vial (amber vials are recommended to prevent photodegradation). If using an internal standard, add a known amount of internal standard before protein precipitation.

e. For tissue or cell samples: Homogenize with ice-cold 0.1 M HCl + 0.1% ascorbic acid, centrifuge to remove cellular debris, and then perform protein precipitation and filtration as above. 5. System Suitability and Sample Loading Order

a. Equilibrate the column in the target mobile phase for 20–30 minutes (or at least 10 column volumes) before loading.

b. Recommended injection sequence: Blank → Solvent Blank → Lowest Standard → Stepping Standard → Intermediate QC → Sample (Batch) → High QC → Duplicate Samples → Final Blank; insert a standard every 10 samples to monitor drift.

c. System Suitability Assessment (Example Criteria): 5 replicate injections of the standard with a relative standard deviation (RSD) of < 2% (or < 5%) area, a retention time RSD of < 0.5%, a number of theoretical plates > 2000, and a peak tailing factor < 2. If these values ​​are not met, adjust the mobile phase or column temperature and re-equilibrate.

6. Data Processing and Quantitation Methods

a. Peak Integration: Manually check the automated integration to ensure a reasonable baseline and no false peaks are misidentified; record peak heights/areas and retention times. b. Calibration curve construction: Plot peak area (or peak area ratio: target/internal standard) against standard concentration. Use linear regression (weighting 1/x or 1/x^2 is recommended to minimize low-concentration bias). Calculate the slope, intercept, and R^2 (target R^2 ≥ 0.995).

c. Sample concentration calculation: Back-calculate the injected sample concentration from the regression equation and multiply by the dilution factor and volume conversion to obtain the actual concentration in the original sample (µg/mL or mg/L).

d. LOD/LOQ assessment (optional): Use the signal-to-noise ratio (S/N = 3 for LOD, S/N = 10 for LOQ) or estimate the standard error of the calibration curve.

7. Quality Control and Reproducibility

a. Each batch of samples should include at least one set of low, mid, and high quality control (QCs). If a QC exceeds the acceptable range (e.g., ±15%), the batch should be retested or reprocessed.

b. It is recommended that samples be run in duplicate and analyzed, with intra- and inter-batch variation documented.

c. Be sure to record sample collection and pretreatment times and temperatures, as catechol compounds are extremely sensitive to the environment (light, oxygen, and metals).

8. Post-column Care and Storage

a. After completing the day's testing, flush the column with a mobile phase containing 50% acetonitrile (or methanol) for 10–15 minutes to remove adhering matter, then flush with the original mobile phase for 5–10 minutes to restore the column to storage conditions.

b. If the column will not be used for an extended period, store it in a buffer containing 20% ​​organic phase as recommended by the column manufacturer (avoid long-term storage in pure water) and store at 4°C in the dark.

c. Regularly check column pressure and background noise (based on frequency of use), record column mileage, and replace as needed.

9. Precautions (Quick Tips)

a. L-DOPA is susceptible to oxidation and polymerization: Maintain the column in the dark, cool, and quickly throughout the entire process, and add ascorbic acid for stabilization.

b. Prepare samples and standards using the same solvent system or acidification conditions to minimize matrix effects. c. Filtration and centrifugation must be thorough; microparticles can clog the injection needle or cause baseline noise.

d. If background interference is severe, consider switching to an electrochemical detector (ECD), adjusting the mobile phase pH, or adding an organic phase to improve separation.

e. All solvents and reagents must be HPLC or analytical grade. Wastewater should be collected separately and disposed of according to laboratory standards.