In our project, The purpose of this study is to improve the biosynthesis yield of L-DOPA, mainly through the following methods : Three exogenous tyrA genes were screened for L-DOPA synthesis, and three RBS sequences in the selected tyrA gene were tested to identify key regulatory elements that can increase yield. The optimized L-DOPA production pathway was transferred to probiotics for overexpression. Combined with its probiotic characteristics, new therapies and other health interventions for host-intestinal microbial drug metabolism were developed. Throughout out engineering cycle, we designed two major quantitative experiments requiring categorical measurements:
HPLC assay was critical to the ‘Build’ phase of our Engineering Cycle, whereas sandwich ELISA assay was crucial to the ‘Test’ phase. First, The HPLC experiment is to analyze the fermentation samples by high performance liquid chromatography to determine the yield and purity of L-DOPA, so as to evaluate the efficiency and optimization results of the fermentation process. Second, the sandwich ELISA assay requires a standard curve to quantify the concentrations of target proteins. Accordingly, our measurement protocols were the following:
- HPLC assay: Obtaining the content of each component in the sample, separation and purification effect, retention time and other information.
- Sandwich ELISA assay: The production of L-DOPA was detected by ELISA kit.
HPLC assay[1], which is affected by factors such as the complexity of the sample, the performance of the chromatographic column, the composition of the mobile phase, the type of the detector, and the contamination during the sample treatment process. These factors interact with each other, affecting the separation effect, detection sensitivity and accuracy, and then determine the reliability and effectiveness of HPLC experiments. Through this experiment, we can obtain the purity and concentration of the product, so as to determine the fermentation effect.
In HPLC assay, the procedures are briefly described as following. After centrifugation, the supernatant ( 8000 r / min, 5 min ) was collected and filtered through a 0.22 μm membrane. Chromatographic column C18 ; column temperature 30 °C ; the wavelength was 280 nm. The mobile phase was 0.08 % formic acid / acetonitrile. The flow rate was 1 mL / min. A series of L-DOPA standard samples with different concentrations were prepared and detected under the same conditions to determine the peak time and make the standard curve of peak area-concentration.
- HPLC system (with autosampler, UV detector; optional: electrochemical detector (ECD))
- Reversed-phase C18 column (4.6 × 150 mm, 5 µm)
- Mobile phase A: 50 mM phosphate buffer ( NaH2PO4/Na2HPO4 ), pH 2.5 (adjusted with phosphoric acid), with 0.1% antioxidant (optional)
- Mobile phase B: Acetonitrile (HPLC grade) or methanol (HPLC grade)
- L-DOPA standard (accurately weighed)
- Internal standard (optional, a compound with known concentration that does not overlap with the sample)
- Antioxidant: Ascorbic acid (analytical grade) to prevent oxidation of catechols
- Acidifier: 1.0 M HCl or 0.1 M HCl (for sample stabilization)
- Organic solvent: Methanol (HPLC grade) for protein precipitation; acetonitrile for mobile phase and rinsing
- 0.22 µm syringe filter (polytetrafluoroethylene/polyether or PTFE/nylon) and syringe
- Autosampler vials (amber, light-proof) and silicone caps
- Micropipette and pipette tips, sterile centrifuge tubes (1.5 mL/2 mL), centrifuge, vortex mixer, ultrasonic cleaner (optional)
- Spectrometer (for preparing standards), ice bath and cold chain equipment (for keeping samples cold)
- Waste container and chemical waste labeling
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 (Figure 1)
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).
Figure 1 HPLC standard curve.
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.
e. After IPTG induction for 48 h, the product concentration ( g / L ) was measured at different concentration gradients of IPTG and glucose (Figure 2).
Figure 2 The concentration of L-DOPA under different concentrations of IPTG and glucose.
f. According to Figure 2, the corresponding curve is made. (Figure 3).
Figure 3 The production of L-DOPA under different concentrations of IPTG and glucose
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.
Measurement Discussions
We used the optimal conditions obtained in BL21 ( DE3 ) host strain to ferment the successfully transformed ECN. After the fermentation was completed, we used high performance liquid chromatography to perform three parallel analyses on the fermented samples. Through these three independent analyses, we obtained consistent results, indicating that our fermentation process was stable and repeatable. The results of HPLC analysis showed that the expression and purity of the recombinant protein ECN in the fermentation samples reached the expected goal, which provided a solid foundation for further research and application. As shown in Figures 2 and 3, when the glucose concentration was 40 g / L and the IPTG concentration was 0.6 or 0.8 mM, the yield of L-DOPA was the highest.
Sandwich ELISA assay[2], a highly versatile and widely adopted technique in antibody-antigen detection, is a crucial tool for measuring target protein concentration. This assay relies on building a standard curve for quantifying the expression level of target protein in unknown samples.
Measurement Principles
ELISA (Enzyme-Linked Immunosorbent Assay) is a widely used method for detecting and quantifying proteins. The mechanism involves the binding of specific antibody to its target protein, followed by a secondary enzyme-linked antibody that catalyzes a colorimetric reaction. In order to detect the concentrations of target protein in samples, we need firstly to construct a standard curve. Standard known concentrations of substance are prepared and assayed in parallel with the samples. Then, the biotin-labeled antibody is incubated with samples simultaneously. After washing, add avidin-labeled HRP. After incubation and washing, the unbound enzyme binding is removed, and then substrates are added to act with the enzyme binding at the same time. The optical density (OD) is measured at a specific wavelength (i.e., 450 nm) after adding the substrate for the enzyme. A plot of OD values against the corresponding concentrations generates the standard curve. Fitting this curve, typically using linear regression methods, allows for the determination of unknown sample concentrations based on their OD values.
Measurement Protocols
Materials:
- Microwell ELISA plate
- Standards
- Sample diluent
- Detection antibody-HRP
- 20x wash buffer
- Substrate A
- Substrate B
- Stop solution
- Seal film
- Instructions
- 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：
- 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℃.
- Set up standard and sample wells. Add 50μL of the standard of different concentrations to each standard well.
- Add 10μL of the sample to be tested to the sample well, followed by 40μL of the sample diluent. Leave blank wells empty.
- Add 100μL 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℃ in a waterbath or incubator for 60 minutes.
- 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).
- Add 50μL each of substrates A and B to each well and incubate at 37℃ in the dark for 15 minutes.
- Add 50μL of stop solution to each well. Within 15 minutes, measure the OD value of each well at a wavelength of 450 nm.
- 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 (Figure 4-7).
Figure 4. A. Fermentation test of strain 123 (three replicates), B. Sample preparation, C. Reaction, D. End of reaction, E. Absorbance test
Figure 5. A. Standard curve of the ELISA test kit; B. L-DOPA production by three strains, where Strain 1 contains TyrA from K12, Strain 2 contains TyrA from SO, and Strain 3 contains TyrA from HI.
Figure 6. A Fermentation test of different RBS strains (three replicates), BC is divided into reaction progress and reaction completion
Figure 7. The yield of levodopa corresponding to different RBS sequences
In this study, we conducted fermentation tests on recombinant strains carrying three different sources of TyrA genes to screen out the optimal TyrA. The fermentation conditions were consistent with the protein expression conditions. The fermentation time was 48 hours, and three repeated fermentation experiments were set up for each strain. After fermentation, L-DOPA ELISA kit was used to measure the samples. We first established a standard curve and calculated the L-DOPA content in the sample according to the standard curve and absorbance value. The results showed that strain 1 had the highest L-DOPA production, indicating that TyrA from K12 showed better enzyme activity in the improved strain. For this improved strain, genes from similar species have better adaptability, so we selected this gene for subsequent RBS screening.
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- Tabatabaei MS, Ahmed M. Enzyme-linked immunosorbent assay (ELISA). Methods Mol Biol. 2022;2508:115-134. doi: 10.1007/978-1-0716-2376-3_10. PMID: 35737237.
