notebook

Construction of Temperature-Sensitive pKD-Cre Knockout Vector

I. Objective

To construct a temperature-sensitive vector (designated pKD-Cre) on the backbone of pKD46 that expresses Cre recombinase, for subsequent Cre-lox markerless knockout of resistance genes.

II. Design Strategy

·Amplify the cre ORF by PCR and add appropriate Gibson assembly overlap sequences to both ends.

·Perform homologous recombination between the pKD46 backbone and the cre gene fragment.

·Propagate, screen, and verify correct constructs in E. coli DH5α by sequencing.

·Use pKD-Cre in subsequent λ-Red operations and Cre-lox markerless knockout workflow.

III. Specific Procedures

From UniProt data, the cre protein gene from Escherichia phage P1 (accession P06956) was selected. Recombination primers (see Table 1) were designed to replace the araBAD promoter sequence of pKD46.

Cre-F and Cre-R were used to amplify the Cre protein gene; pKD46-F and pKD46-R were used to amplify the pKD46 backbone.

PCR system: 50 μL reaction mixture: 25 μL buffer, 19 μL ddH₂O, 1 μL forward primer, 1 μL reverse primer, 2 μL DNA template, 1 μL high-fidelity polymerase, and 1 μL dNTP.

PCR conditions: initial denaturation at 95 °C for 5 min; 34 cycles of 95 °C for 20 s, 56 °C for 20 s, and 72 °C (30 s per 1 kb extension); final extension at 72 °C for 10 min; hold at 12 °C.

PCR products were analyzed by 1.5% agarose gel electrophoresis, and fragment sizes were confirmed under UV. Target bands were excised and recovered. DNA was quantified using a microplate reader.

Recombination system: 12 μL total volume: 6 μL recombinase, 3 μL target gene, 3 μL plasmid backbone. Reaction at 50 °C for 30 min, then placed on ice.

Transformation: 10 μL of recombination product was added to DH5α competent cells, incubated on ice for 30 min, heat-shocked at 42 °C for 90 s, and placed on ice for 2-3 min.

In a biosafety cabinet, 600 μL sterile LB medium was added to the EP tube, followed by recovery at 30 °C, 220 rpm for 60 min.

The culture was spread evenly on LB agar plates containing 100 μg/mL ampicillin and incubated overnight at 30 °C. Positive colonies were picked and verified by sequencing.

Correct clones were inoculated into 5 mL LB liquid medium containing 100 μg/mL ampicillin and cultured overnight at 30 °C. The extracted plasmid was identified as the temperature-sensitive pKD-Cre.

IV. Experimental Results

Result of DH5α-pKD-Cre colony PCR

Sequencing result of DH5α-pKD-Cre

Note: sequencing confirmed correctness, indicating successful construction.

Markerless Construction of asd Auxotrophic Strain in E. coli Nissle 1917

I. Objective

To construct an asd gene-deficient strain in Escherichia coli Nissle 1917(EcN) using λ-Red homologous recombination combined with the Cre-lox system, thereby generating an exogenous nutrient (DAP)-dependent chassis strain to enhance biosafety.

II. Design Strategy

·Replace the asd gene of E. coli Nissle 1917 with a kanamycin resistance gene (Kana) to improve the screening efficiency of asd knockout strains.

·Remove the resistance cassette via Cre-lox system to achieve a markerless knockout.

III. Specific Procedures

Table 2. Primers used for λ-Red-mediated knockout of the asd gene in EcN

Table 3. Primers for constructing EcN strains carrying lox71 recognition sites

(1) Replacement of asd with Kana resistance gene

From NCBI database, the asd gene of E. coli Nissle 1917, which encodes a key enzyme in the diaminopimelate(DAP) synthesis pathway, was selected.

The asd locus was replaced by a kanamycin resistance gene (with promoter and RBS) using λ-Red homologous recombination.

Primers (Table 2) were designed to amplify the Kana resistance gene. PCR-amplified fragments were obtained from laboratory strain 1917-pET28a. A 10 μL DNA fragment was electroporated into EcN competent cells harboring plasmid pKD46.

Cells were incubated on ice for 10 min, then electroporated at 3 kV, 4 ms. Immediately after, 800 μL LB containing 100 μg/mL DAP was added, and cells were recovered at 37 °C for 2 h.

The culture was plated on LB agar containing 50 μg/mL kanamycin and 100 μg/mL DAP, and incubated overnight at 30 °C. Positive colonies were picked and verified by sequencing, yielding resistant, auxotrophic EcN strains. Glycerol stocks (final 25%) were prepared and stored at -80 °C.

(2) Markerless deletion of resistance gene via Cre-lox system

Primers (Table 3) were designed to insert the Cre recombinase recognition site lox71 flanking the resistance cassette by λ-Red recombination. Positive colonies were verified by sequencing, obtaining EcN auxotrophic strains carrying Kana resistance and lox71 sites.

The previously constructed temperature-sensitive plasmid pKD-Cre was introduced into verified strains. Following overnight incubation at 30 °C, positive transformants were selected and confirmed by sequencing.

Induction of Cre recombinase: strains were cultured in 24-well plates with 800 μL LB containing 100 μg/mL DAP and 1 mM arabinose, at 30 °C, 1000 rpm, for 12 h. Cultures were plated on LB agar containing 100 μg/mL DAP. Colonies growing on DAP-supplemented plates but not on DAP-free plates were screened by colony PCR and sequencing to confirm successful markerless knockout. PCR mix: 1 μL forward primer, 1 μL reverse primer, 2 μL template, 15 μL 2x EasyTaq PCR SuperMix, 11 μL ddH₂O.

Loss of pKD-Cre plasmid: verified knockout strains were serially passaged under non-selective conditions (LB + 100 μg/mL DAP, 42 °C, 1000 rpm) for six generations. Colonies were then plated on LB agar with/without ampicillin. Colonies growing in DAP-supplemented plates but not in ampicillin-containing plates were confirmed by colony PCR and sequencing.

Finally, positive clones of EcN asd-deficient auxotrophic strain were inoculated into LB liquid medium containing 100 μg/mL DAP, cultured overnight at 37 °C, and stored at -80 °C in 25% glycerol.

IV. Experimental Results

Colony PCR results of EcN-LoxKana(+)-asd(-)

Sequencing results of EcN-LoxKana(+)-asd(-)

Note: sequencing confirmed correctness, indicating successful construction.

Construction of YES302

I. Objective

On the basis of the previously obtained auxotrophic strain E. coli Nissle 1917 Δasd, introduce the optimized xanthine transporter protein XanQ to obtain a safe engineered probiotic strain capable of efficiently transporting xanthine without carrying any antibiotic resistance genes.

II. Design Strategy

·Construct a DH5α asd auxotrophic strain to serve as host for transformation and screening of plasmids carrying the asd gene and XanQ protein.

·Integrate the asd gene into the pWT021a expression vector carrying the optimized XanQ protein gene, then transform into DH5α asd auxotrophic strain. After screening and verification, electroporate into EcN Δasd.

III. Specific Procedures

Table 4. Primers for λ-Red homologous recombination to knock out asd> in DH5α

Table 5. Primers for construction of recombinant strains

(1) Construction of DH5α asd auxotrophic strain

From NCBI database, the asd gene of E.coli K12 DH5α (ATCC 53868), encoding a key enzyme in the diaminopimelate (DAP) biosynthesis pathway, was selected. The asd locus was replaced with a kanamycin resistance (Kana) gene using λ-Red homologous recombination.

Primers (Table 4) were designed for amplification of the Kana resistance gene. The PCR fragment was obtained from laboratory strain 1917-pET28a. A 10 μL fragment was introduced into DH5α competent cells harboring plasmid pKD46. After overnight incubation at 30 °C, colonies were selected on LB plates containing 100 μg/mL DAP. Positive clones were confirmed by sequencing.

Selected clones were cultured at 42 °C to eliminate the temperature-sensitive pKD46 plasmid. Colonies were picked from LB plates supplemented with 100 μg/mL DAP and verified by sequencing. The confirmed strain was designated DH5α asd auxotrophic strain.

(2) Construction of XanQ mutant recombinant strain

The asd gene from EcN, encoding a key enzyme in the DAP pathway, was amplified with primers (Table 5) and integrated into the pWT021a expression vector carrying the XanQ F94Y88 mutant.

The expression plasmid was transformed into competent cells of DH5α asd auxotrophic strain. Colonies were plated on antibiotic-free LB agar and incubated overnight at 37 °C. Positive clones were picked and confirmed by sequencing. Plasmids were extracted from positive clones.

The extracted plasmids were electroporated into the constructed EcN asd auxotrophic strain. Colonies were plated on antibiotic-free LB agar and incubated overnight at 37 °C. Positive clones were picked and confirmed by sequencing.

The resulting positive clone was designated as recombinant strain EcN asd(-)/pWT021a-XanQF94Y88-asd(+), named antibiotic-sensitive engineered probiotic YES302.

IV. Experimental Results

Electrophoresis results of KanaR gene fragment

1-4: DH5αasd(-) KANR(+);5: DH5α;6: DH5α + pKD46

Sequencing results of DH5α and DH5α-pKD46 asd gene

PCR results of DH5αasd(-) KANR(+)

Note: Cannot grow in medium without DAP; sequencing confirmed correctness; knockout was successful.

DH5α-asd(- )KANR(+)

1:Marker;2:asd;3;pWT021a

Electrophoresis results before homologous recombination of asd gene with pWT021a backbone

PCR results of DH5α asd(-)

KANR(+)-pWT021a-asd(+) SMR(-)

Sequencing results of DH5α asd(-)

KANR(+)-pWT021a-asd(+) SMR(-)

Note: Sequencing confirmed correctness; can grow in medium without DAP; construction successful.

Xanthine Transport Assay of YES302

I. Objective

To test the xanthine transport capacity of the antibiotic-sensitive engineered probiotic YES302.

II. Design Strategy

·In this experiment, recombinant E. coli Nissle (YES302), carrying the XanQ mutant and free of antibiotic resistance markers, was used as the transporter strain. With xanthine as substrate, the assay aimed to optimize the conditions in M9 medium to reduce extracellular xanthine levels.

III. Specific Procedures

Plate cultivation: Inoculate E. coli stored at -80 °C onto LB agar plates and incubate at 37 °C for 12 h for plate activation.

Liquid activation: Pick colonies and inoculate into 5 mL LB broth, incubate at 37 °C for 10 h.

Growth culture: Transfer activated culture into M9 medium at 1% (v/v) inoculum, incubate at 37 °C for 12-14 h until OD600 reaches 0.4-0.6. Harvest cells by centrifugation and resuspend in M9 medium, adjusting OD600 to 1.0.

Xanthine transport efficiency assay of YES302:Reaction system: In 1 mL total volume, add xanthine as substrate at a final concentration of 100 μg/mL and YES302 cells at OD₆₀₀ = 1.

Incubation: Shake at 37 °C, 220 rpm, and sample at 5, 10, 15, 20, 30, 45, and 60 min.

Sample processing: Centrifuge at 8000 rpm for 1 min 30 s, collect the supernatant, filter through a 0.22 μm aqueous membrane, and analyze filtrates by HPLC to determine residual xanthine content.

HPLC conditions:

Instrument: Shimadzu LC-2030

Column: Diamonsil Plus 5 μm C18-A, 250x4.6 mm

Mobile phase: 50 mmol/L ammonium acetate buffer (pH 4.60): acetonitrile = 95:5 (v/v)

Flow rate: 0.7 mL/min

Detection wavelength: 254 nm

Column temperature: 30 °C

Injection volume: 10 μL

Retention time: 10 min

Standard curve preparation: Xanthine standards of 6.25, 12.5, 25, 50, 100, and 120 μg/mL were analyzed by HPLC to generate a calibration curve of peak area versus concentration.

Calculation formula:X=(X1-X2)/X2x100%

where X1X_1 is the initial xanthine concentration, and X2X_2 is the residual xanthine concentration in the supernatant.

IV. Experimental Results

As shown in Figure 1, YES302 exhibited a significantly enhanced xanthine transport capacity compared to wild-type EcN 1917.. YES302 was able to effectively transport 90% of extracellular xanthine within 60 min.

Figure 1. Xanthine transport efficiency of YES302.

Antibiotic Susceptibility Test of YES302

I. Objective

To evaluate the susceptibility of the engineered probiotic YES302 to common antibiotics, and to verify that it does not carry any unintended antibiotic resistance, thereby ensuring biosafety.

II. Design Strategy

·Multiple antibiotic classes covered

Five commonly used laboratory/clinical antibiotics were selected:

Ampicillin (β-lactam class)

Streptomycin (aminoglycoside class)

Kanamycin (aminoglycoside class)

Chloramphenicol (protein synthesis inhibitor)

Apramycin (commonly used laboratory screening antibiotic)

These drugs have distinct mechanisms of action, allowing a comprehensive assessment of YES302 susceptibility.

·Different culture media

The assay was conducted in both rich medium (LB) and minimal medium (M9). This was designed to confirm whether the strain's susceptibility to antibiotics is consistent under different nutrient conditions, ruling out medium-related interference.

·Gradient concentration design

For each antibiotic, low, medium, and high concentrations were tested in both media.Growth curves were recorded, enabling evaluation of inhibition profiles and estimation of the minimum inhibitory concentration (MIC) trend.

III. Specific Procedures

Activated YES302 cultures were inoculated at 1% (v/v) into media containing different concentrations of each antibiotic. A volume of 200 μL per condition was added into clear flat-bottom 96-well plates. The plates were incubated at 37 °C, 1000 rpm on a plate shaker, and OD600 was measured every 1 h using a microplate reader.

IV. Experimental Results

Figure 2: Growth curves in LB with different concentr

Figure 3: Growth curves in LB with different concentrations of streptomycin

igure 4: Growth curves in LB with different concentrations of kanamycin

Figure 5: Growth curves in LB with different concentrations of chloramphenicol

Figure 6: Growth curves in LB with different concentrations of apramycin

Figure 7: Growth curves in M9 with different concentrations of ampicillin

Figure 8: Growth curves in M9 with different concentrations of streptomycin

Figure 9: Growth curves in M9 with different concentrations of kanamycin

Figure 10: Growth curves in M9 with different concentrations of chloramphenicol

Figure 11: Growth curves in M9 with different concentrations of apramycin

As shown in Figures 2-11, YES302 exhibited no detectable growth in LB or M9 media supplemented with any of the tested antibiotics, across all concentration gradients.

Conclusion: These results demonstrate that YES302 is susceptible to all the commonly used antibiotics tested, confirming its high biosafety profile and eliminating potential risks of inducing antibiotic-resistant strains.

Plasmid Stability Test of YES302

I. Objective

To evaluate the plasmid retention rate and functional stability of the engineered strain YES302 after continuous passages under non-antibiotic conditions, and to verify the feasibility and safety of maintaining the functional plasmid (containing the XanQ mutant and complementary asd) without antibiotic selection.

II. Design Strategy

·Environmental setting: Continuous passages in liquid LB medium without antibiotics, simulating clinical/food application scenarios; subcultured every 12 h.

·Two levels of detection:

Genetic stability: At each passage, samples were plated on non-antibiotic LB plates. Colony PCR was performed to verify the presence of the plasmid. Stability was calculated as (number of positive colonies / total colonies picked) x 100%. At the 20th generation, sequencing validation was performed.

Functional stability: Using HPLC to measure the residual xanthine concentration, the transport efficiency of the 1st generation (initial) and the 20th generation were compared under identical conditions to check whether the functional phenotype was maintained.

III. Specific Procedures

YES302 was cultured in liquid LB medium without antibiotics. Every 12 h, fresh non-antibiotic LB was used for subculture. Samples were taken, serially diluted, and plated on LB agar plates without antibiotics. Colony PCR was performed, and plasmid stability was calculated as:

At the 20th generation, single colonies were sequenced for verification. For functional analysis, extracellular xanthine transport efficiency was measured: in a 1 mL reaction system, 100 μg/mL xanthine (substrate) was added along with cells adjusted to OD₆₀₀ = 1.0. The reaction was carried out at 37 °C for 5, 10, 20, 30, and 60 min. After centrifugation, the supernatant was analyzed by HPLC to quantify the remaining xanthine.

IV. Experimental Results

Figure 12

As shown in Figure 12, after 20 passages, YES302 retained 95% of its xanthine transport activity. These results indicate that YES302 exhibits strong plasmid stability, maintaining efficient xanthine transport after multiple passages. Furthermore, even after co-incubation in simulated gastrointestinal fluid, its survival rate was about half of the control group, yet it still maintained a high xanthine transport capacity.

MIC Assay of YES302

I. Objective

To determine the minimum inhibitory concentration (MIC) of antibiotics under in vitro conditions (18-24 h) that inhibits visible growth of YES302, in order to evaluate the antibacterial activity of drugs and classify sensitivity (S/I/R).

II. Design Strategy

Broth microdilution (twofold serial dilution) in 96-well plates:

Use CAMHB/MH broth as the medium; prepare a twofold serial dilution of the antibiotic in the wells to generate a concentration gradient.

Prepare bacterial suspension using the 0.5 McFarland standard, then dilute 1:1000 in broth to ensure the inoculum in each well is within the recommended range.

Incubate at 35-37 °C for 16-20 h, record OD600/visible turbidity, and determine the MIC.

III. Specific Procedures

(1) Reagent and plate preparation

Medium: prepare CAMHB/MH broth; alternatively, weigh ingredients according to the formula, dissolve, and sterilize (121 °C, 25 min).

Sterilization: PBS/sterile water, pipette tips, and centrifuge tubes were autoclaved; 96-well plates were UV-sterilized in a biosafety cabinet for 30 min.

(2) Antibiotic concentration gradient (twofold dilution)

Stock solution: generally ≥1000 μg/mL.

On the first plate, perform twofold serial dilution:

Add 190 μL medium to wells A1-H1, and 100 μL medium to the other wells. Add 10 μL antibiotic stock to A1-H1 and mix.

From column 1 to the right, transfer 100 μL from the previous column into 100 μL in the next column, mix, and continue until column 11.

The typical final concentration gradient ranged from 256 → 0.25 μg/mL.

(3) Inoculation

Transfer drug solutions to the second plate: add 20 μL of each working solution to the corresponding wells. Column 12 contained no drug and served as the growth control.

Prepare inoculum: culture bacteria to 0.5 McFarland standard, then dilute 1:1000 with broth.

Add 180 μL diluted bacterial suspension to each well, making a total volume of 200 μL/well.

Controls: negative control for sterility check; positive/growth control to monitor growth.

(4) Incubation and reading

Incubate plates at 35 °C under normal air for 16-20 h; OD600 could also be recorded hourly using a microplate reader to obtain growth curves.

Result interpretation: valid when growth control shows obvious growth and negative control remains clear. MIC was defined as the lowest antibiotic concentration without visible growth.

IV. Experimental Results

Table 1. Antibiotic resistance of YES302 to different antibiotics

Establishment of CFU-OD₆₀₀ Relationship

I. Objective

To establish the correspondence between OD600 and colony-forming units (CFU/mL), providing the basis for subsequent quantitative experiments.

II. Design Strategy

·In viability assays, strict quantification of bacterial cell numbers is required.

·Relying solely on OD₆₀₀ is influenced by culture conditions and cell morphology; therefore, plate counting must be used to establish a mathematical correspondence between OD₆₀₀ and CFU, resulting in a reliable quantitative standard curve.

·This step serves as a baseline for subsequent experiments, ensuring data comparability and reproducibility.

III. Specific Procedures

For the same YES302 bacterial suspension, perform a tenfold serial dilution. Measure OD₆₀₀ at each dilution, and conduct plate counting at selected dilutions. Fit the paired data into a regression relationship of log10(CFU/mL)-OD₆₀₀ or CFU/mL-OD₆₀₀, which can be used to estimate viable cell counts rapidly from OD.

(1) Culture preparation:

Overnight YES302 culture stored in the laboratory was inoculated 1:100 into fresh medium and grown at 37 °C, 220 rpm until OD₆₀₀ ~0.5-1.0.

(2) Tenfold dilution:

Prepare 7-8 sterile tubes with 900 μL sterile diluent each, labeled 10⁻¹ to 10⁻⁷ (as required).

Add 100 μL of original culture to the 10⁻¹ tube, vortex thoroughly; then take 100 μL from the 10⁻¹ tube into the 10⁻² tube, and so on until the desired dilution. Mix thoroughly at each step to avoid wall residue errors.

Measure OD₆₀₀: record OD₆₀₀ for the original suspension and each dilution. Correct OD values were noted as OD₆₀₀_corrected. Each sample was tested with ≥3 technical replicates, and averages were taken.

(3) Plate counting

Select 2-3 dilutions expected to yield 30-300 colonies per plate (empirically 10⁻⁴-10⁻⁶ range). Plate 100 μL of each dilution evenly on agar plates (two groups per plate separated by a midline). Each dilution was plated with ≥3 technical replicates. Incubate at 37 °C, inverted, for 12-18 h.

Select countable plates: prioritize plates/drops with 30-300 colonies, as results outside this range are prone to error. Take the mean across technical replicates for each dilution.

Calculation of CFU/mL:

IV. Experimental Results

Figure 13. CFU-OD relationship curve of YES302

As shown, the CFU-OD curve of YES302 exhibited smoothly distributed data points consistent with the typical exponential growth phase CFU-OD relationship. The curve was nonlinear: at low OD₆₀₀, CFU/mL increased rapidly with OD, while after OD₆₀₀ > 1.5 the rate of increase gradually slowed, and at high OD₆₀₀ (>2.0) the curve tended toward a plateau with weakened linear correlation.

Encapsulation of YES302 with Eudragit® L100-55 and Simulated Gastrointestinal Fluid Experiment

I.Objective

To encapsulate YES302 using Eudragit® L100-55 in order to improve its survival rate under gastric and intestinal fluid conditions.

II. Design Strategy

·Probiotics administered orally must pass through the gastrointestinal tract, where gastric acid and digestive enzymes severely damage the cells, leading to massive bacterial death and reduced therapeutic effect. Therefore, it is necessary to build a delivery system that can protect engineered bacteria under gastric acid conditions and release them under intestinal conditions.

·Eudragit® L100-55 was chosen because it is insoluble under pH < 5.5, which effectively resists gastric acid, while it can rapidly dissolve and release bacteria under intestinal conditions (pH> 6).

·Through in vitro simulated gastric fluid/intestinal fluid experiments, compare the survival rates of encapsulated and non-encapsulated groups to evaluate the effect of encapsulation technology on improving delivery efficiency.

III. Specific Procedures

(1) Encapsulation with Eudragit® L100-55

Grow YES302 engineered bacteria in 4 mL LB medium at 37 °C with shaking at 200 rpm for 12 h.

Centrifuge at 4000 rcf for 5 min and wash twice with PBS. Resuspend YES302 in 900 μL ice-cold phosphate-calcium solution containing 12.5 mM CaCl₂ and vortex for 5 min.

Add 500 μL of 1 mg/mL L100-55 solution to the suspension and shake further for 5 min.

Adjust the pH of the 0.1 M HCl suspension to about 5.0 and collect the encapsulated YES302 engineered bacteria by centrifugation.

Wash the obtained YES302 twice and resuspend in 1 mL PBS (pH < 5.0). Store the suspension at -20 °C for later use.

(2) Simulated gastrointestinal fluid experiment

Take engineered bacteria from -80 °C storage, streak 10 μL on LB agar plates for activation, and culture at 37 °C for 10-12 h. Scrape colonies into a 24-well plate containing 700 μL liquid LB and culture at 37 °C, 1000 rpm for 12 h. Transfer 1% (v/v) into 30 mL M9 medium containing 30 μL Smr, and culture at 37 °C, 220 rpm for 12-14 h.

After culture, centrifuge to remove the medium. Add a portion of cells into pre-prepared simulated gastric fluid and incubate at 37 °C for 5 min without shaking.

Centrifuge to collect the bacteria, discard excess simulated gastric fluid, then add simulated intestinal fluid and incubate at 37 °C, 220 rpm for 60 min. After incubation, plate cells to determine colony counts after simulated gastrointestinal fluid treatment.

Resuspend both untreated and simulated gastrointestinal-treated engineered bacteria in M9 medium to unify OD₆₀₀ to 1. Add xanthine to a final concentration of 100 μM. For gastrointestinal-treated bacteria, xanthine was added simultaneously with simulated intestinal fluid to a final concentration of 100 μM. Incubate at 37 °C, 220 rpm for 60 min.

Resuspend gastrointestinal-treated engineered bacteria in M9 medium to OD₆₀₀ = 1, and measure extracellular xanthine transport efficiency using the transport assay method. In a 1 mL reaction system, add 100 μg/mL xanthine as substrate and bacteria with OD₆₀₀ = 1.0. Incubate at 37 °C for 15, 30, 45, and 60 min. Centrifuge and collect the supernatant. Measure remaining xanthine in supernatant using HPLC.

IV. Experimental Results

Figure 14. Survival rate of simulated gastrointestinal fluid experiment

Figure 15. Effect of Eudragit® L100-55 encapsulation: survival rate (A) and xanthine transport efficiency (B)

YES302 did not transport xanthine after encapsulation, possibly because the encapsulation material covered the surface transport proteins.

After gastrointestinal fluid treatment, xanthine levels unexpectedly increased, possibly due to insufficient cell lysis in vitro or excessive bacterial density.

Peristaltic Pump-Based Xanthine Transport Assay

I. Objective

To simulate the peristaltic environment of the small intestine using a peristaltic pump, so that the results of the xanthine transport assay would better reflect in-vivo conditions.

II. Design Strategy

·Encapsulate YES302 cells in hydrogel and fix them inside the tubing.

·Use a peristaltic pump to circulate a mixture of simulated intestinal fluid and xanthine solution through the tubing.

·Monitor changes in xanthine concentration in the circulating medium by HPLC.

III. Detailed Procedures

·Activate the engineered strain YES302 and collect cells; resuspend to OD = 1.3.

·Prepare 0.2% sodium alginate solution, remove air bubbles, mix it 1:1 with the bacterial suspension, introduce the mixture into the tubing, and allow it to gel by contacting it with 0.3% CaCl₂ solution, forming hydrogel beads fixed in place.

·Mix 180 mL simulated intestinal fluid with 20 mL of 1 mM xanthine solution, and circulate the mixture through the tubing using a peristaltic pump for 2 h.

·Collect samples by centrifugation and determine xanthine concentration in the supernatant using HPLC.

IV. Experimental Results

YES302 transported only about 10% of the xanthine in the initial experiment. We hypothesized that the limited transport efficiency was due to the excessive volume of simulated intestinal fluid, insufficient bacterial density, and rapid flow rate. Therefore, we conducted another experiment using smaller vessels and reduced the simulated intestinal fluid volume to 50 mL. However, the results showed no significant improvement compared with the first experiment. We further speculated that this was because the hydrogel beads exposed only their surface to the simulated intestinal fluid, while the encapsulated bacteria inside the hydrogel were unable to contact the substrate and thus failed to transport xanthine efficiently, leading to a substantial performance gap compared with the free-cell transport experiment.

YES302 Bacterial Concentration-Response Curve

I. Objective

Using the established YES302 CFU–OD₆₀₀ standard curve, we prepared bacterial suspensions at various actual cell densities (CFU/mL) and measured the xanthine uptake rates over the same incubation period (1 h). A “bacterial concentration–response curve” was plotted to determine the most economical and effective dosing level as well as the kinetic characteristics

II. Design Strategy

·Based on the CFU–OD₆₀₀ calibration, the target CFU values (e.g., 1×10^9, 8×10^8, 6×10^8, 4×10^8, 3×10^8, 2×10^8, 1×10^8 CFU/mL) were converted into corresponding OD₆₀₀ values.

·Bacterial suspensions were resuspended in M9 medium (or designated medium) to the required OD, then incubated with a fixed xanthine concentration (final 100 μM) at 37 °C, 220 rpm for 60 min

·Samples were collected every 15 min, centrifuged/filtered, and the remaining xanthine in the supernatant was quantified by HPLC.

III. Detailed Procedures

1.Retrieve frozen YES302 cells from –80 °C, activate on LB and further culture in M9 as previously described.

2.Centrifuge at 6000 rpm for 10 min, resuspend cells to the desired OD₆₀₀. Prepare reaction mixtures with final xanthine concentration of 100 μM. Mix quickly and start timing (t = 0) at 37 °C, 220 rpm.

3.Collect samples at 0, 15, 30, 45, and 60 min; immediately centrifuge at 6000 rpm for 3 min to pellet cells. Filter supernatants through a 0.22 μm syringe filter into HPLC vials.

4.Quantify xanthine concentration using HPLC

IV. Results

Xanthine uptake efficiency at different bacterial concentrations

At a concentration of 1×10^9 CFU/mL, YES302 reached near-maximal uptake within 15 min. Strains at 8×10^8 and 6×10^8 CFU/mL showed progressively increasing xanthine uptake over time and were able to reach an uptake level comparable to that of 1×10^9 CFU/mL by 60 min.

Uric Acid Transport Module Construction

I. Objective

To express three potential high-efficiency uric-acid transporters (UacT, PucJ, and PucK) individually in the chassis strain E. coli Nissle 1917 (EcN 1917), in order to screen for the most efficient uric-acid transporter to be incorporated into the engineered probiotic.

II. Design Strategy

·PCR amplification of the coding sequences of the three uric-acid transporter genes and the pWT021a plasmid backbone.

·Homologous recombination of the amplified gene fragments with the plasmid backbone, followed by transformation into DH5α; screening by colony PCR and verification by sequencing.

·After successful verification, plasmids were extracted and electroporated into EcN 1917; recombinant strains were identified by colony PCR and confirmed by sequencing.

III. Detailed Procedures

Primer Information

Procedures:

·The UacT fragment was amplified by PCR from E. coli K-12 MG1655.

·The PucJ and PucK fragments, optimized for E. coli codon usage, were amplified from Bacillus subtilis subsp. subtilis str. 168.

·Using the previously constructed plasmid in YES301 as the template, the plasmid backbone was obtained.

·The amplified UacT / PucJ / PucK genes were assembled with the plasmid backbone by homologous recombination, and the recombined plasmids were transformed into DH5α.

·Recombinant colonies were selected on SMR-containing plates, and validated by colony PCR.

·Successfully validated plasmids were extracted from DH5α.

·The extracted plasmids were then electroporated into competent E. coli Nissle 1917 cells.

·Transformants were selected on SMR-containing plates and verified.

IV. Experimental Results

Colony PCR of DH5α-UacT

Colony PCR of DH5α-PucJ and DH5α-PucK

(1-6: DH5α-pWT-J23100-B0034-PucJ; 7-12:DH5α-pWT-J23100-B0034-PucK)

Sequencing Results of DH5α-UacT

Sequencing Results of DH5α-PucJ

Sequencing Results of DH5α-PucK

Colony PCR Identification of Nissle 1917-UacT

Colony PCR of E. coli 1917-PucJ and E. coli 1917-PucK

(1-6: 1917-pWT-J23100-B0034-PucJ; 7-12: 1917-pWT-J23100-B0034-PucK)

Sequencing Results of Nissle 1917-UacT

Sequencing Results of E. coli 1917-PucJ

Sequencing Results of E. coli 1917-PucK

YES303 Uric Acid Transport Assay

I. Objective

To test the uric-acid transport capacity of the three uric-acid transporters expressed in the chassis strain E. coli Nissle 1917 (EcN 1917).

II. Design Strategy

·Conduct uric-acid transport assays using the three EcN 1917 strains expressing different uric-acid transporters.

·Measure uric-acid concentrations using the Elabscience® Uric Acid (UA) Colorimetric Assay Kit.

III. Detailed Procedures

·Strains were activated in LB medium.

·Cells were harvested at the logarithmic growth phase, and the OD was adjusted to 1.2 using fresh LB medium.

·The bacterial suspension was mixed with uric-acid solution in sterile 15-mL centrifuge tubes to achieve a final uric-acid concentration of 100 μM.

·Each group was set up with three biological replicates.

·Samples were incubated at 37 °C, 220 rpm with shaking for the designated time.

·After incubation, samples were centrifuged at 6000 rpm for 3 min, and the supernatant was collected for further analysis.

·The concentration of uric acid in the supernatant was determined using the Elabscience® Uric Acid (UA) Colorimetric Assay Kit.

IV. Experimental Results

Among the three uric-acid transporters tested, UacT exhibited the highest uric-acid transport efficiency.

Cloning and Expression of Uricase Gene

I. Objective

To verify the uric acid degradation function of the uricase gene expressed by EcN, in order to attempt to endow the engineered probiotic with uric acid degradation capability.

II. Design Strategy

·Codon-optimized Bacillus cereus SKIII uricase gene was recombined into the MCS of pET-28a to be cloned into Escherichia coli BL21 (DE3).

·The protein was purified using the 6xHis tag carried by pET-28a, and the purified uricase was subjected to enzyme activity assay to verify its uric acid degradation activity.

·Western blot (WB) was performed using cell lysates and culture supernatants to determine whether uricase could be expressed in YES301.

III. Specific Procedures

Table 6. Primers for uricase cloning

(1) Gene cloning

First, the target gene was amplified in vitro from pUC57-uricase. The uricase gene contained 1482 bp and was recombined into the multiple cloning site (MCS) of pET-28a with a 6xHis tag. After colony PCR and sequencing verification, the strain was induced to express protein and purified. Protein samples from each purification stage—including total protein, supernatant, flow-through, and ultrafiltered product—were analyzed by 12.5% SDS-PAGE.

Subsequently, uricase was quantified using a BCA kit. 20 μL of sample was mixed with 200 μL of BCA reagent, incubated for 30 min, and the absorbance value at OD562 was measured against a standard curve to calculate protein concentration.

(2) Uricase activity assay

Uric acid has a maximum absorption peak at 293 nm. Using a UV-transparent flat-bottom 96-well plate, uric acid solutions at 0 μM, 10 μM, 20 μM, 40 μM, 60 μM, 80 μM, 100 μM, 120 μM, 150 μM, and 200 μM were prepared. The absorbance at OD293 was measured using a microplate reader to plot a standard curve. In actual testing, appropriate dilution was applied according to the standard curve range.

Ultrafiltered uricase (200 μL) was mixed with 1800 μL uric acid solution at concentrations of 0 μM, 10 μM, 20 μM, 40 μM, 80 μM, 140 μM, 200 μM, and 500 μM. The mixture was incubated at 37 °C for 10 min, and absorbance was measured. Using the Michaelis-Menten equation for linear fitting, Km and Vmax data were obtained, yielding the kinetic parameters of this uricase.

Subsequently, the uric acid degradation ability of uricase was tested. 200 μL uricase was added to 1800 μL of 500 μM uric acid solution. Uric acid concentration was measured at the initial time point and every 10 min thereafter. A degradation curve of uric acid was plotted.

(3) Uricase secretion assay

To verify whether the uricase expressed in the engineered strain could be secreted extracellularly, the uricase gene was coupled into the pWT plasmid and transformed into EcN 1917. The strain was cultured in LB medium, and both lysed cells and culture supernatant were collected for WB assay to determine whether uricase was secreted outside the cell.

IV. Experimental Results

Figure 16. SDS-PAGE result of uricase(Cloning and Expression of Uricase Gene)

(1) The molecular weight of uricase is 56.3 kDa. Compared with the protein marker, a clear band was observed near 50 kDa with high expression levels. From the supernatant after cell lysis, the protein was mainly expressed intracellularly. After nickel column purification and ultrafiltration, protein purity increased and contaminant bands decreased.

Figure 17. Uric acid quantification standard curve (A) and uricase kinetic curve (B)

(2) Based on absorbance values of uric acid solutions at 293 nm, a linear equation was obtained:

y = 0.009010X + 0.02221, R² = 0.9976.

The regression indicates this method can accurately and effectively measure uric acid concentration in solution. Meanwhile, biochemical parameters of uricase at different uric acid concentrations were obtained:

Km = 163.7 μM, Vmax = 2577 μM·min⁻¹·mg⁻¹, demonstrating strong uric acid degradation activity of the enzyme.

Figure 18. Uric acid degradation curve

(3) According to the uric acid degradation curve, when the uricase concentration was 0.2 mg/mL and the uric acid concentration was 450 μM, the uricase completely degraded uric acid within 80 min. This indicates that uricase shows good activity and stability in vitro. However, due to its intracellular nature, it may not sufficiently contact uric acid in the gut. Therefore, secretion optimization should be considered to increase direct interaction between uricase and uric acid in the intestine.

Figure 19. Uricase secretion analysis (M: protein marker; W: whole protein; L: culture supernatant)

(4) As shown, uricase expressed in YES301 was detected in whole protein (lane 2), but not in supernatant. However, a strong band was observed near 40 kDa. Further analysis revealed the reason. Using Pymol to model the protein structure and performing homology comparison with the PucL protein from Bacillus subtilis (Barbe et al., The Bacillus subtilis 168 Reference Genome a Decade Later. Microbiology-SGM, 2009), high homology was found. Both consist of two structural domains—PucL N-terminal domain (aa 1-169) and the uricase catalytic domain (aa 170 onwards), which oxidizes uric acid to 5-hydroxyisourate. This reaction cannot proceed spontaneously, while subsequent steps converting uric acid to allantoin can proceed slowly and spontaneously.

Therefore, we truncated uricase to retain amino acids 171-494, with a predicted molecular weight of 36.7 kDa, consistent with the observed band. The failure of uricase secretion may be due to its large molecular weight, preventing transport across the cytoplasmic membrane. Thus, we decided to truncate uricase and attempt re-expression and secretion.

Expression and Activity Assay of Structure-Optimized Uricase

I.Objective

To optimize the secretion ability and uric acid degradation capacity of uricase, the uricase gene was further structurally optimized and subjected to site-directed mutagenesis and directed evolution.

II. Design Strategy

·Express and purify the truncated uricase with amino acids 1-169 removed.

·Verify the molecular weight of the three uricases by SDS-PAGE and test their uric acid degradation ability.

III. Specific Procedures

Table 7. Primers for uricase structural optimization

(1) Uricase structural optimization: Using primers uricaseT-R and uricase-F, PCR was performed with pUC57-uricase as a template to obtain the structurally optimized uricase gene. The fragment was cloned into the MCS of pET-28a by homologous recombination and expressed under induction.

(2) Site-directed mutagenesis: Point mutations were introduced into the optimized uricase URIT gene at Q438 and D214, cloned, expressed, and purified using the same method.

(3) Activity testing: The three uricase proteins were analyzed by SDS-PAGE, quantified using a BCA assay, and then tested for uric acid degradation ability at the same concentration.

(4) Expression in EcN: The three uricase genes were cloned into the pWT plasmid and expressed under the pj23100 promoter. WB assays were performed using the same method as in the previous experiment.

IV. Experimental Results

Figure 20. SDS-PAGE results of UricaseT, UricaseTD214V, and UricaseTQ438R (L) and their uric acid degradation curves (R)

As shown, the three uricases were successfully expressed and purified, with bands observed around 40 kDa, consistent with their expected molecular weights. At a final uric acid concentration of 450 μM, the truncated uricase and its mutants nearly completely degraded uric acid within 60 min. No significant difference was observed between the mutants and the truncated uricase. However, compared with the initial uricase, the truncated version shortened the reaction time, nearly degrading all uric acid within ~55 min, indicating faster degradation activity. The truncated uricase remained stable and active at 37 °C for prolonged degradation. Therefore, the truncated uricase UricaseT was selected for subsequent experiments.

Figure 21. Secretion of structure-optimized uricase (M: protein marker; W: whole protein; L: culture supernatant)

Compared with the control group, a clear band appeared at ~40 kDa (36.7 kDa) in whole protein, indicating successful expression of truncated uricase in the engineered strain. In the culture supernatant lane, a lighter band was also observed, showing that truncated uricase could be secreted extracellularly. Truncation reduced the molecular weight of the protein, allowing possible secretion into the extracellular environment.

This makes intestinal uric acid degradation by engineered strains feasible. Moreover, the truncation reduced plasmid size, enhancing plasmid stability and decreasing cellular burden and toxicity, which benefits the application of the engineered strain.

Secretion Assay of UricaseT in M9 Medium

I. Objective

To determine whether UricaseT can be effectively secreted in M9 medium.

II. Design Strategy

Repeat the secretion assay of UricaseT in M9 medium when culturing YES301.

III. Specific Procedures

YES301 was cultured in M9 medium, and both lysed cells and culture supernatants were collected for WB assay to determine secretion efficiency.

M9 medium contains only basic inorganic salts and a carbon source. Bacterial cells tend to allocate resources to survival and reproduction, which may reduce expression levels of related enzymes.

IV. Experimental Results

Figure 22. Secretion of structure-optimized uricase in different media (M: protein marker; W: whole protein; L: culture supernatant)

As shown in Figure 21, uricase expressed intracellularly in EcN was nearly unaffected in either LB or M9 media, indicating that truncated uricase was expressed alongside EcN growth and reproduction. However, based on the band intensity of supernatants, extracellular secretion of truncated uricase was influenced by medium composition. In nutrient-rich LB medium, secretion was stronger, while in M9 medium, although secretion was weaker, it was still detectable. This suggests that truncated uricase has the potential to be combined with the xanthine transport system for further study.

Performance Testing of Engineered Strain Containing Both Xanthine Transport and Uric Acid Degradation Systems

I. Objective

To verify the performance of the uric acid degradation system in the presence of the xanthine transport system.

II. Design Strategy

·After ulturing YES301 coupled with the uricase system, collect and lyse the cells.

·Perform both live-cell xanthine transport experiments and uric acid degradation experiments.

III. Specific Procedures

(1) YES301-UricaseT was activated, and 300 μL of culture was inoculated into 30 mL of LB medium containing the corresponding antibiotics, followed by incubation at 37 °C, 220 rpm for 12-14 h.

After culture, cells and supernatant were separated by centrifugation at 4 °C, 3000 rcf for 10 min. Both the cell pellet and the culture supernatant were collected. Cells were resuspended in 30-40 mL PBS and disrupted by ultrasonication with the following parameters: 3 s on, 5 s off, 40% power. Lysis continued until the suspension was clear, not exceeding 40 min. To preserve enzyme activity, sonication was performed at low temperature. After sonication, the lysate was centrifuged at 4 °C, 8000 rpm for 10 min, and the supernatant was collected. The proteins in the culture medium were concentrated using an ultrafiltration tube with a suitable pore size (10 kDa). The crude uricase in the lysate was incubated with uric acid solution at 37 °C, and uric acid concentration was measured at different time intervals.

(2) YES301 and YES301-UricaseT were subjected simultaneously to xanthine transport and uric acid degradation experiments. Samples were taken at 0, 20, 40, and 60 min. Xanthine content was measured by HPLC, and uric acid concentration was determined using a uric acid assay kit.

IV. Experimental Results

Figure 23. Uric acid degradation capacity of whole-cell lysates

(1) Results showed that crude enzyme solution from the strain completely degraded 450 μM uric acid within 70 min, indicating that the uricase expressed in YES301 was active. Together with previous results, this confirmed the successful construction of both a xanthine transport system and a uric acid degradation system in EcN.

Figure 24. Xanthine transport (A) and uric acid degradation (B) of YES301-UricaseT

In the presence of 100 μM xanthine, YES301-UricaseT nearly transported all xanthine within 60 min. Compared with YES301, the transport rate of xanthine decreased at intermediate time points, but there was no significant difference in the total transported amount at 60 min. This suggests that addition of the uricase gene did not significantly affect xanthine transport capability.

At an initial uric acid concentration of 100 μM, YES301-UricaseT degraded nearly 50% of uric acid within 60 min. Although degradation ability was weaker compared with purified or crude enzyme, likely because uricase was still mostly intracellular, the engineered strain still showed uric acid degradation ability compared with YES301. This indicates that after introducing the uric acid degradation system, the strain could potentially not only prevent uric acid accumulation by reducing purine uptake but also degrade uric acid directly, providing a therapeutic function. This lays the foundation for further in vivo experiments.