iGEM 2025 — Team sklbe-china

{% extends "layout.html" %}
{% block styles %}{{ super() }}
{% endblock %}
{% block page\_content %}

![]({{ uploads_url }}hero/wl-1.webp)
![]({{ uploads_url }}hero/wl-2.webp)
![]({{ uploads_url }}hero/wl-3.webp)

# notebook

Notebook

* [YES302](#yes302)
  + [Construction of Temperature-Sensitive pKD-Cre
    Knockout Vector](#ts-pkd-cre)
  + [Markerless Construction of asd Auxotrophic
    Strain in E. coli Nissle 1917](#asd-auxotroph)
  + [Construction of YES302](#construct-yes302)
  + [Xanthine Transport Assay of
    YES302](#xanthine-transport-yes302)
  + [Antibiotic
    Susceptibility Test of YES302](#antibiotic-susceptibility-yes302)
  + [Plasmid Stability Test of
    YES302](#plasmid-stability-yes302)
  + [MIC Assay of YES302](#mic-assay-yes302)
  + [Establishment of CFU-OD600 Relationship](#cfu-od600)
  + [Encapsulation of YES302 with
    Eudragit® L100-55 and Simulated Gastrointestinal Fluid Experiment](#encapsulation-yes302)
  + [Peristaltic Pump-Based Xanthine
    Transport Assay](#peristaltic-xanthine)
* [YES303](#yes303)
  + [Uric Acid Transport Module Construction](#ua-module)
  + [YES303 Uric Acid Transport Assay](#ua-transport-assay)
* [YES304](#yes304)
  + [Cloning and Expression of Uricase
    Gene](#uricase-clone-express)
  + [Expression and Activity Assay
    of Structure-Optimized Uricase](#uricase-activity-optimized)
  + [Secretion Assay of UricaseT in M9
    Medium](#uricase-secretion-m9)
  + [Performance Testing of
    Engineered Strain Containing Both Xanthine Transport and Uric Acid Degradation Systems](#dual-system-performance)
* [References](#refer)

### YES302

##### 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

Table 1. Recombination primers

| Primer name | Sequence (5'→3') |
| --- | --- |
| Cre-F | GCTCTAAGGAGGTTATAAAAAATGTCTAACCTGCTGACCGTC |
| Cre-R | GTCATCGCCATTGCTCCCCAACTAGTCACCGTCCTCCAGCAG |
| pKD46-F | TTGGGGAGCAATGGCGATG |
| pKD46-R | TTTTTATAACCTCCTTAGAGCTCGAATTCCC |

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

![]({{ uploads_url }}notebook/notebook-1.webp)

Result of *DH5α*-pKD-Cre colony PCR

![]({{ uploads_url }}notebook/notbook-2.webp)

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

![]({{ uploads_url }}notebook/notebook-3.webp)

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

![]({{ uploads_url }}notebook/notebook-4.webp)

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 Δ*asd*, 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

![]({{ uploads_url }}notebook/notebook-5.webp)

Electrophoresis results of KanaR gene fragment

![]({{ uploads_url }}notebook/notebook-6.webp)

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

![]({{ uploads_url }}notebook/notebook-7.webp)

Sequencing results of *DH5α* and *DH5α*-pKD46 *asd* gene

![]({{ uploads_url }}notebook/notebook-8.webp)

PCR results of *DH5αasd(-) KANR(+)*

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

![]({{ uploads_url }}notebook/notebook-9.webp)

*DH5α*-*asd*(- )KANR(+)

![]({{ uploads_url }}notebook/notebook-10.webp)

1:Marker;2:*asd*;3;pWT021a

Electrophoresis results before homologous recombination of *asd* gene with pWT021a backbone

![]({{ uploads_url }}notebook/notebook-11.webp)

PCR results of *DH5α* *asd*(-)

![]({{ uploads_url }}notebook/notebook-12.webp)

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.

![]({{ uploads_url }}notebook/notebook-13.webp)

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

![]({{ uploads_url }}notebook/notebook-14.webp)

Figure 2: Growth curves in LB with different concentr
![]({{ uploads_url }}notebook/notebook-15.webp)

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

![]({{ uploads_url }}notebook/notebook-16.webp)

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

![]({{ uploads_url }}notebook/notebook-17.webp)

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

![]({{ uploads_url }}notebook/notebook-18.webp)

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

![]({{ uploads_url }}notebook/notebook-19.webp)

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

![]({{ uploads_url }}notebook/notebook-20.webp)

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

![]({{ uploads_url }}notebook/notebook-21.webp)

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

![]({{ uploads_url }}notebook/notebook-22.webp)

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

![]({{ uploads_url }}notebook/notebook-23.webp)

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:

Stability
(
%
)
=

Number of positive clones

Total colonies picked
×
100

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

![]({{ uploads_url }}notebook/notebook-24.webp)

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

![]({{ uploads_url }}notebook/notebook-25.webp)

##### 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:

CFU
/
mL
=

Average colony count

0.1
 mL
×
Total dilution factor

IV. Experimental Results

![]({{ uploads_url }}notebook/notebook-26.webp)

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

![]({{ uploads_url }}notebook/notebook-27.webp)

Figure 14. Survival rate of simulated gastrointestinal fluid experiment

![]({{ uploads_url }}notebook/notebook-28.webp)

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.

![]({{ uploads_url }}notebook/notebook-29.webp)

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

![]({{ uploads_url }}notebook/notebook-30.webp)

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.

### YES303

##### 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

![]({{ uploads_url }}notebook/notebook-31.webp)

Colony PCR of *DH5α*-UacT

![]({{ uploads_url }}notebook/notebook-32new.webp)

Colony PCR of *DH5α*-PucJ and *DH5α*-PucK

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

![]({{ uploads_url }}notebook/notebook-33.webp)

Sequencing Results of *DH5α*-UacT

![]({{ uploads_url }}notebook/notebook-34.webp)

Sequencing Results of *DH5α*-PucJ

![]({{ uploads_url }}notebook/notebook-35.webp)

Sequencing Results of *DH5α*-PucK

![]({{ uploads_url }}notebook/notebook-36.webp)

Colony PCR Identification of Nissle 1917-UacT

![]({{ uploads_url }}notebook/notebook-37.webp)

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)

![]({{ uploads_url }}notebook/notebook-38.webp)

Sequencing Results of Nissle 1917-UacT

![]({{ uploads_url }}notebook/notebook-39.webp)

Sequencing Results of *E. coli* 1917-PucJ

![]({{ uploads_url }}notebook/notebook-40.webp)

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.

![]({{ uploads_url }}notebook/notebook-41.webp)

### YES304

##### 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

![]({{ uploads_url }}notebook/notebook-42.webp)

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.

![]({{ uploads_url }}notebook/notebook-43.webp)

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.

![]({{ uploads_url }}notebook/notebook-44.webp)

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.

![]({{ uploads_url }}notebook/notebook-45.webp)

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

![]({{ uploads_url }}notebook/notebook-46.webp)

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.

![]({{ uploads_url }}notebook/notebook-47.webp)

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

![]({{ uploads_url }}notebook/notebook-48.webp)

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

![]({{ uploads_url }}notebook/notebook-49.webp)

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*.

![]({{ uploads_url }}notebook/notebook-50.webp)

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.

##### References

[1]Terkeltaub R, Dodd D. The Gut Microbiome in Hyperuricemia and Gout. Arthritis Rheumatol. 2025 Aug;77(8):955-965. doi: 10.1002/art.43118. Epub 2025 Feb 13. PMID: 39829115; PMCID: PMC12276925.

[2]Gulig P, Swindle S, Fields M, Eisenman D. A Review of Clinical Trials Involving Genetically Modified Bacteria, Bacteriophages and Their Associated Risk Assessments. Appl Biosaf. 2024 Dec 16;29(4):186-206. doi: 10.1089/apb.2024.0002. PMID: 39735407; PMCID: PMC11669762.

[3]Palmada, N.; Hosseini, S.; Avci, R.; Cater, J.E.; Suresh, V.; Cheng, L.K. A Systematic Review of Computational Fluid Dynamics Models in the Stomach and Small Intestine. Appl. Sci. 2023, 13, 6092. https://doi.org/10.3390/app13106092

[4]Chen Y, Ji P, Ma G, Song Z, Tang BQ, Li T. Simultaneous determination of cellular adenosine nucleotides, malondialdehyde, and uric acid using HPLC. Biomed Chromatogr. 2021 Oct;35(10):e5156. doi: 10.1002/bmc.5156. Epub 2021 May 19. PMID: 33955024.

[5]He L, Tang W, Huang L, et al. Insulated expression of periplasmic uricase in*E. coli*Nissle 1917 for the treatment of hyperuricemia. bioRxiv; 2022. DOI: 10.1101/2022.04.17.488604.

[6]Barbe V, Cruveiller S, Kunst F, Lenoble P, Meurice G, Sekowska A, Vallenet D, Wang T, Moszer I, Médigue C, Danchin A. From a consortium sequence to a unified sequence: the Bacillus subtilis 168 reference genome a decade later. Microbiology (Reading). 2009 Jun;155(Pt 6):1758-1775. doi: 10.1099/mic.0.027839-0. Epub 2009 Apr 21. PMID: 19383706; PMCID: PMC2885750.

[7]Li W, Xu S, Zhang B, Zhu Y, Hua Y, Kong X, Sun L, Hong J. Directed evolution to improve the catalytic efficiency of urate oxidase from Bacillus subtilis. PLoS One. 2017 May 22;12(5):e0177877. doi: 10.1371/journal.pone.0177877. PMID: 28531234; PMCID: PMC5439685.

[8]Papakostas K, Botou M, Frillingos S. Functional identification of the hypoxanthine/guanine transporters YjcD and YgfQ and the adenine transporters PurP and YicO of Escherichia coli K-12. J Biol Chem. 2013 Dec 27;288(52):36827-40. doi: 10.1074/jbc.M113.523340. Epub 2013 Nov 8. PMID: 24214977; PMCID: PMC3873542.

[9]Zhao R, Li Z, Sun Y, Ge W, Wang M, Liu H, Xun L, Xia Y. Engineered Escherichia coli Nissle 1917 with urate oxidase and an oxygen-recycling system for hyperuricemia treatment. Gut Microbes. 2022 Jan-Dec;14(1):2070391. doi: 10.1080/19490976.2022.2070391. PMID: 35491895; PMCID: PMC9067508.

[10]Bao R, Chen Q, Li Z, Wang D, Wu Y, Liu M, Zhang Y, Wang T. Eurycomanol alleviates hyperuricemia by promoting uric acid excretion and reducing purine synthesis. Phytomedicine. 2022 Feb;96:153850. doi: 10.1016/j.phymed.2021.153850. Epub 2021 Nov 6. PMID: 34785103.

[11]Cui D, Liu S, Tang M, Lu Y, Zhao M, Mao R, Wang C, Yuan Y, Li L, Chen Y, Cheng J, Lu Y, Liu J. Phloretin ameliorates hyperuricemia-induced chronic renal dysfunction through inhibiting NLRP3 inflammasome and uric acid reabsorption. Phytomedicine. 2020 Jan;66:153111. doi: 10.1016/j.phymed.2019.153111. Epub 2019 Oct 16. PMID: 31790902.

[12]Guo XL, Gao YY, Yang YX, Zhu QF, Guan HY, He X, Zhang CL, Wang Y, Xu GB, Zou SH, Wei MC, Zhang J, Zhang JJ, Liao SG. Amelioration effects of α-viniferin on hyperuricemia and hyperuricemia-induced kidney injury in mice. Phytomedicine. 2023 Jul 25;116:154868. doi: 10.1016/j.phymed.2023.154868. Epub 2023 May 13. PMID: 37209608.

![]({{ uploads_url }}lastlast/gzcgzc.avif)

{% endblock %}

{% extends "layout.html" %}
{% block page\_content %}

![]({{ uploads_url }}hero/p-1.webp)
![]({{ uploads_url }}hero/p-2.webp)
![]({{ uploads_url }}hero/p-3.webp)

# Engineering

Wet Lab

* [Xanthine Transport](#eng-wl-content-pro1)
* [Cycle 1: Knockout of resistance
  genes](#eng-wl-content-pro1-1)
* [Cycle 2: Continuous passage of
  plasmid](#eng-wl-content-pro1-2)
* [Cycle 3: Microcapsule delivery](#eng-wl-content-pro1-3)
* [Cycle 4: Peristaltic pump
  culture](#eng-wl-content-pro1-4)
* [Uric Acid Transport](#eng-wl-content-pro2)
* [Cycle 1: Construction of UacT
  chassis](#eng-wl-content-pro2-1)
* [Cycle 2: Directed evolution by
  point mutations](#eng-wl-content-pro2-2)
* [Uric Acid Metabolism](#eng-wl-content-pro3)
* [Cycle 1: Cloning and expression
  of URI](#eng-wl-content-pro3-1)
* [Cycle 2: URIT after structural
  optimization](#eng-wl-content-pro3-2)
* [Cycle 3: Co-expression of URIT
  and XanQ](#eng-wl-content-pro3-3)

Model

* [Cycle 1: Initial Version](#eng-model-content-1)
* [Cycle 2: Final Version: Dynamic
  model](#eng-model-content-2)
* [Cycle 3: Interactive voice
  assistant](#eng-model-content-3)

IHP

* [Cycle 1: Project Promotion](#eng-ihp-content-pro1)
* [Cycle 2: Expanding the Audience](#eng-ihp-content-pro2)
* [Cycle 3: Inclusivity](#eng-ihp-content-pro3)
* [Cycle 4: Commercial Exploration](#eng-ihp-content-pro4)

Wet Lab
Model
IHP

![]({{ uploads_url }}eng/101.avif)

## Xanthine Transport

### Cycle 1: Knockout of Resistance Genes

#### Design

Core Objective. Address Smr-marker biosafety in YES301 and improve
plasmid stability by deleting chromosomal *asd* (DAP synthesis) in EcN and complementing *asd* on
the plasmid carrying transporter XanQ F94Y88.

*Rationale.* Genomic *asd* deletion makes growth dependent on plasmid *asd*, stabilizing
the plasmid and replacing antibiotic selection. A markerless Cre–loxP strategy via pKD-Cre(ts) is used.

#### Build

1. **pKD-Cre(ts) vector.** On pKD46, PCR the P1 *cre* ORF and Gibson-replace downstream
   of PBAD; amplify/screen/sequence in DH5α.
2. **EcN Δ*asd* chassis.** Replace chromosomal *asd* with kanaR-lox71 by λ-Red;
   transform pKD-Cre, induce at 30 °C (1 mM arabinose) to excise kanaR; passage 42 °C ×6 to cure pKD-Cre →
   non-resistant Δ*asd* chassis.
3. **Integration.** Co-clone optimized XanQ F94Y88 and *asd* into pWT021a, transform into
   Δ*asd* chassis to obtain YES302.

![]({{ uploads_url }}eng/100.avif)

#### Test

1. **Knockout verification.** Sequencing confirms correct *cre* insertion, *asd*
   deletion, kanaR removal.
2. **Auxotrophy.** No growth in LB without DAP; restored with 100 μg/mL DAP.
3. **Xanthine transport.** In M9 + 100 μg/mL xanthine, YES302 transports 90% within 60 min,
   higher than wild-type EcN.
4. **Antibiotic sensitivity.** No growth in LB/M9 containing Amp/Sm/Kan/Apr/Cm (25–100 μg/mL).
5. **Simulated GI survival.** SGF 5 min + SIF 60 min → survival ≈ 50%.

#### Learn

1. Achievements. Resistance marker removed; auxotrophic chassis established;
   high transport maintained.
2. Areas for Optimization. Verify long-term plasmid stability (avoid
   XanQ/*asd* loss) for the next continuous-passage cycle.

### Cycle 2: Continuous Passage of Plasmid

#### Design

1. **Core Objective** : Simulate the growth environment of engineered bacteria in vivo without antibiotic selection pressure, test the genetic stability of the pWT021a-XanQF94Y88-*asd* plasmid, and ensure the strain maintains its function long-term.
2. **Design Logic** : Through "non-selective pressure passage + dual detection (gene presence + functional activity)", eliminate the risk of functional failure caused by plasmid loss.

#### Build

1. **Passage culture** : culture: Inoculate YES302 into LB medium without antibiotics (containing 100 μg/mL DAP), culture at 37°C with 220 rpm shaking; subculture into fresh medium at a 1% inoculation volume every 12 hours for 20 consecutive passages.
2. **Sample collection** : samples at passages 1, 5, 10, 15, and 20; perform gradient dilution and spread on antibiotic-free LB plates; pick 30 single colonies from each passage for subsequent detection.

#### Test

Verification Indicators and Results:

1. **Gene stability (Colony PCR):** Colony PCR of single colonies from each passage successfully amplified the XanQ and *asd* genes; the plasmid retention rate at passage 20 was 100%, and no gene mutations were confirmed by sequencing.
2. **Functional stability (xanthine transport):**YES302 at passage 20 still transported 88% of xanthine within 60 minutes, showing no significant difference from passage 1 (90%), confirming no functional attenuation.
3. **CFU-OD600calibration:**Establish a CFU-OD600 standard curve for YES302; confirm that OD600 = 1 corresponds to approximately 10⁸ CFU/mL, providing a basis for subsequent quantitative experiments.

#### Learn

1. Achievements: The plasmid maintained good stability and functional activity after 20 passages, proving that YES302 can stably carry functional genes under non-selective pressure. Achievements: The plasmid maintained good stability and functional activity after 20 passages, proving that YES302 can stably carry functional genes under non-selective pressure.
2. Areas for Optimization: The issue of "oral strains being easily damaged by gastric acid/digestive enzymes in the gastrointestinal tract" needs to be addressed; thus, the next cycle will focus on constructing a "microcapsule delivery system" to improve intestinal survival rate.

### Cycle 3: Microcapsule Delivery

#### Design

Core Objective. Use pH-sensitive Eudragit® L100-55 (soluble at pH > 5.5) to
construct microcapsules for “stomach protection → intestinal release”.

*Advantages.* High biocompatibility, food/drug-safe; solubility matches GI pH (stomach 1.5–3.0;
small intestine 5.5–7.0).

#### Build

1. **Bacterial suspension.** YES302 to OD600=1.0; 4000 rcf 5 min; resuspend in
   ice-cold Ca phosphate (12.5 mM CaCl2).
2. **Capsule formation.** Mix 1:1 with 1 mg/mL L100-55; shake 5 min; adjust pH 5.0 (0.1 M
   HCl); centrifuge to collect.
3. **Wash & store.** PBS (pH<5.0) ×2; resuspend; −20 °C.

#### Test

1. **Survival.** Encapsulated YES302 survives SGF/SIF significantly better than control.
2. **Transport.** Encapsulated YES302 shows no xanthine transport activity.

#### Learn

1. Achievements. GI survival markedly improved; enables oral route.
2. Areas for Optimization. Coating may mask transporters or hinder release;
   need dynamic verification under peristalsis → next: peristaltic-pump culture.

### Cycle 4: Peristaltic Pump Culture

#### Design

Core Objective. Simulate intestinal hydrodynamics with a peristaltic pump to
evaluate YES302 transport more realistically.

*Parameters.* Flow 1 mL/min; circulating fluid SIF + 1 mM xanthine.

#### Build

1. **Immobilization.** Mix encapsulated YES302 with 0.2% alginate; inject into silicone hose;
   cross-link with 0.3% CaCl2 to form hydrogel beads.
2. **System assembly.** Connect pump → hose → reservoir (180 mL SIF + 20 mL 1 mM xanthine) as
   a closed loop.
3. **Culture.** 37 °C; circulate 2 h; sample reservoir.

#### Test

1. **Initial result.** 2 h transport ≈10% (vs. 85% in static).
2. **Analysis.** (i) Flow too high → short contact time; (ii) Bacterial quantity in beads
   107 CFU/mL < static 108; (iii) Only surface cells contact flow.

#### Learn

1. Achievements. Dynamic system established; identified “flow & quantity” as
   key factors.
2. Optimization. Tune flow, raise OD in beads (OD600=2.0), optimize
   cross-linking/release, co-test with uric-acid strains, and adjust hydrogel geometry to enlarge contact
   area.

## Uric Acid Transport

### Cycle 1: Construction of UacT Chassis

#### Design

Core Objective. Overcome “xanthine-only” by screening direct uric-acid
transporters (UacT, PucJ, PucK) and enabling uric-acid transport.

#### Build

1. **Gene amplification.** UacT from *E. coli* K-12; PucJ/PucK codon-optimized from
   *B. subtilis* 168.
2. **Vector construction.** Recombine into pWT021a (J23100 + B0034) and transform DH5α.
3. **Chassis transformation.** Extract plasmids; electrotransform into EcN to obtain YES303
   series (1917-UacT/PucJ/PucK).

![]({{ uploads_url }}eng/33.avif)

#### Test

1. **Method.** Strains to OD600=1.2; mix with 100 μM UA; 37 °C shaking; assay
   supernatant by Elabscience® UA kit.
2. **Results.** UacT: 45 μM in 60 min (45%); PucJ/PucK: 22 μM/18 μM; WT EcN < 5 μM →
   specificity confirmed.
3. **Sequencing.** No mutations/insertions.

#### Learn

1. Achievements. UacT identified as optimal; YES303 (1917-UacT) enables direct
   UA transport.
2. Optimization. Active-site residues affect binding; plan site-directed
   evolution.

### Cycle 2: Directed Evolution by Point Mutations

#### Design

We aim to improve the transport efficiency of UacT through site-directed mutagenesis, and plan to identify key sites and mutation directions using molecular docking technology.

Based on the structure-activity relationship between protein structure and function, the binding ability of UacT to uric acid directly determines its transport efficiency: the tighter the binding (i.e., the smaller the molecular distance) between the two, the higher the efficiency of substrate capture and transport.

Therefore, we plan to use molecular docking technology to accurately locate the key amino acid residues involved in the interaction between UacT and uric acid, determine potential mutation directions, and then screen for the optimal amino acid substitution scheme through saturation mutagenesis.

![]({{ uploads_url }}eng/55.avif)

#### Build

We imported the three-dimensional structure of the UacT protein and the standard structure of the uric acid molecule into AutoDock Vina software for molecular docking. We analyzed the interaction modes between UacT and uric acid (e.g., hydrogen bonds, hydrophobic interactions, van der Waals forces) to identify the amino acid residues that are likely the most critical for this interaction.

#### Test

We initially identified M274 and V320 as key interaction sites. These two residues stabilize the conformation of uric acid through hydrophobic interactions and van der Waals forces, respectively, and are core sites that affect the tightness of binding between UacT and uric acid.

#### Learn

Due to time constraints, in this cycle, we have not yet constructed a saturation mutagenesis library for UacT at these two sites or conducted further screening to determine which amino acid mutations are most beneficial for improving transport efficiency.

In subsequent studies, we will continue to conduct in-depth research: we will adopt high-throughput screening methods combined with functional verification experiments to evaluate the impact of each mutant on uric acid transport efficiency. Meanwhile, we plan to use molecular dynamics simulation technology to observe changes in protein conformation after mutation and their effects on substrate binding ability. To ensure the reliability of screening results, strict positive and negative controls will be set up, and statistical analysis will be used to determine the optimal mutation scheme.

## Uric Acid Metabolism

### Cycle 1: Cloning & Expression of URI + Enzyme Activity

#### Design

Core Objective. Introduce uricase (URI) into YES301 to prepare for “transport +
degradation”. Selected *B. cereus* SKIII enzyme (Vmax > 2000
μM·min−1·mg−1; stable at 37 °C).

*Strategy.* Express/purify in pET-28a/BL21 for parameters → clone into pWT021a and transform EcN to
check intra/extra-cellular distribution.

#### Build

1. **Optimization & cloning.** Codon-optimize URI (1482 bp) → pET-28a (6×His) → BL21 (DE3).
2. **Expression & purification.** IPTG 1 mM at 37 °C 4 h; sonication (3 s on/5 s off, 40%);
   Ni-NTA; 10 kDa ultrafiltration; BCA quant.
3. **Secretion check.** Clone into pWT021a; transform EcN; WB on lysate & supernatant.

#### Test

1. *SDS-PAGE.* Single band at 56.3 kDa; purity > 90%.
2. *Activity.* UA standard at 293 nm (0–200 μM, R²=0.9976); Km=163.7 μM;
   Vmax=2577 μM·min−1·mg−1; 0.2 mg/mL degrades 450 μM UA ≈80 min.
3. *Secretion.* URI only in lysate; extra ~40 kDa band in supernatant; modeling shows 1–169 aa
   non-catalytic, 170–494 catalytic.

#### Learn

1. Achievements. High-purity, high-activity URI obtained.
2. Findings. URI mainly intracellular; N-terminal 1–169 aa affects secretion →
   truncate to UricaseT.

### Cycle 2: URIT Expression & Activity after Optimization

#### Design

Core Objective. Remove N-terminal 1–169 aa to ~36.7 kDa (UricaseT) to improve
secretion while retaining activity. Design D214V & Q438R mutations.

#### Build

1. **Truncation/mutation.** Amplify 170–494 aa → pET-28a; obtain UricaseT, D214V, Q438R.
2. **Host.** Clone UricaseT into pWT021a (J23100) → EcN for secretion tests.

#### Test

1. *SDS-PAGE.* Clear ~40 kDa bands.
2. *In-vitro activity.* 450 μM UA almost fully degraded ≈60 min; UricaseT ≈55 min; mutants ~similar.
3. *WB secretion.* Distinct ~36.7 kDa band in whole protein; faint band in supernatant → partial
   natural secretion.
4. *M9 secretion.* Intracellular in LB & M9; supernatant band weaker in M9 → medium affects
   secretion but detectable.

#### Learn

1. Achievements. Truncation improves secretion with high activity and lower
   burden.
2. Insights. Supernatant protein < 10 μg/mL; next add signal peptides or
   co-express with transporters.

### Cycle 3: Co-expression of URIT and XanQ

#### Design

Core Objective. Introduce UricaseT into YES301 (XanQ) to build dual-function
strain YES301-UricaseT (YES304).

*Architecture.* Bicistronic J23100 + B0034 drives XanQ & UricaseT on pWT021a-*asd*.

#### Build

1. Amplify pWT021a-XanQ backbone + UricaseT; assemble XanQ-B0034-UricaseT; verify by colony PCR &
   sequencing.
2. Electrotransform EcN → YES301-UricaseT (YES304).

#### Test

1. *Xanthine transport.* Near-complete in 60 min; early rate slightly lower vs YES301 but final
   amount similar.
2. *UA degradation.* 100 μM UA → ~50% in 60 min, ≫ YES301 (no UricaseT).
3. *Crude enzyme.* Lysate degrades 450 μM UA ≈70 min → sufficient intracellular activity.
4. *Medium effect.* Secretion lower in M9 but detectable.

#### Learn

1. Achievements. Dual-function probiotic YES301-UricaseT built.
2. Next. Optimize secretion (signal peptides) and verify in-vivo efficacy.

### Cycle 1: Initial Version — Construction and Verification of the Basic Dynamic Model

#### Design

Designs the framework for “intestinal purine absorption – serum uric acid – engineered
bacteria action”.

1. Intestinal purines: I0 input; bacterial transport k1, conversion k2.
2. Serum uric acid: conversion coefficient (intestinal→serum), renal clearance β.
3. Bacterial quantity: Logistic growth (r, Bmax, death), consistent with YES301 transport data
   (83%/60 min at 150 μM xanthine).

#### Build

1. Dynamic equation for intestinal purines with B(t)/Bref coupling.
2. Serum uric acid dynamics with conversion & renal clearance.
3. Bacterial logistic equation (growth → stabilization).

#### Test

1. Parameter fitting: residual after 60 min error < 10% vs experiment.
2. Serum UA: normal diet I0=200 mg/day → normal range; 400 mg/day → >420 μmol/L.
3. Bacteria growth: matches shake-flask trend; B approaches Bmax at 24 h.

#### Learn

1. Fixed I0 oversimplifies real meal pulses.
2. Misses renal tubular reabsorption → deviation for some patients.
3. Reproduction assumption invalid under GI simulation → needs revision.

### Cycle 2: Final Version — Dynamic Model of Intestinal Purine & Serum Uric Acid

#### Design

1. I(t) as Gaussian meal pulses.
2. Add intestinal uric acid Ugut with reabsorption Kreabs and bacterial degradation.
3. Rebuild bacterial quantity as exponential decay B(t)=B0e−kt.
4. Link Ugut and serum UA with reabsorption term.

#### Build

1. Replace I0 by I(t) in intestinal equation.
2. Introduce Ugut equation and reabsorption coupling.
3. Update serum UA equation with reabsorption; add γ for bacterial effect.
4. Adopt exponential decay with initial condition B(0+) = B(0−)+B0.

#### Test

1. I(t) shows three peaks (100/150/120 mg) → postprandial peaks 1–2 h later.
2. Reabsorption case: error reduced from 25% to < 8%; more bacteria → lower serum UA.
3. Decay verified with YES302 (24 h residual 30%); curves overlap across B0=10⁶/10⁷ CFU.

#### Learn

1. Need low-threshold user tool.
2. Parameter input should leverage voice.
3. Improve real-time & portability → hardware carrier.

### Cycle 3: Interactive Voice Assistant

#### Design

1. Use optimized model as dosage core; voice to capture diet/personal traits.
2. Hardware: ESP32-S3-N16R8 + INMP441 + IPS screen + ML307R 4G.
3. Modules: dosage guidance, science content, multilingual, reminders.

#### Build

1. Embed equations; connect to China Food Network; NLP maps food→purine in real time.
2. Assemble core board + mic/amp/screen + 4G; avg response < 3 s without Wi-Fi.
3. Implement multilingual, reminders, and science entries.

#### Test

1. 10 datasets: UriGuard vs manual model error < 5%; voice food recognition 92%.
2. Outdoor 4G query “100 g beef” 2.8 s ~ indoor 2.5 s.
3. Multilingual TTS 95%+; reminders 100% trigger; 80% users think language easy.

#### Learn

1. Add GI efficiency/comorbidities for special populations.
2. Iterate hardware (ESP32-C6; 12 h+ battery; ≤100 g).
3. Database weekly updates via user feedback + manual review.

![]({{ uploads_url }}eng/ihp-first-slogan.webp)

### Cycle 1: Project Promotion & Popularization of Synthetic Biology

#### Design

Define initial probiotic plan after clinical interviews; address GM safety concerns via
science communication.

#### Build

1. Illustrated brochure (hyperuricemia basics, project intro, GM benefits & regulation).
2. Lectures: kindergarten animations; middle-school textbook-aligned sessions; cultural-creative products。

![]({{ uploads_url }}eng/ihp-cycle1-build1.webp)
![]({{ uploads_url }}eng/ihp-cycle1-bulid2.webp)

#### Test

Warm responses; pre/post questionnaires show GM acceptance ↑ in students.

![]({{ uploads_url }}eng/ihp-cycle1-test1.webp)

#### Learn

Offline activities effective but limited by manpower/time → next: expand with online
platforms.

### Cycle 2: Expanding the Audience

#### Design

Add social media + platform modules to scale impact.

#### Build

1. WeChat/Xiaohongshu/Instagram accounts for realtime updates & Q&A.
2. Build “Suprobiotics” platform for public + clinician/researcher collaboration.

![]({{ uploads_url }}eng/ihp-cycle2-test1.webp)
![]({{ uploads_url }}eng/ihp-cycle2-bulid2.webp)

#### Test

Reads and follower diversity ↑; Suprobiotics visitors and sign-ups ↑; trust in GM ↑.

#### Learn

Still need accessibility for disabled/remote/low-education groups → next: inclusivity.

### Cycle 3: Inclusivity

#### Design

Identify barriers and design targeted inclusive measures.

#### Build

1. Kindergarten/middle-school/college tiered teaching；quiz games with cultural-creative prizes。
2. Lectures in Yuexi County (resource-limited); encourage perseverance in science.
3. Children’s picture book (with audio) to lower concept threshold; braille brochure.
4. Activities with autistic children; culture-integrated Tai-Chi sessions; “Light of Life” art contest.

![]({{ uploads_url }}eng/ihp-cycle3-bulid3.webp)
![]({{ uploads_url }}eng/ihp-cycle3-bulid2.webp)
![]({{ uploads_url }}eng/ihp-cycle3-bulid4.webp)
![]({{ uploads_url }}eng/ihp-cycle3-bulid5.webp)
![]({{ uploads_url }}eng/ihp-cycle3-bulid6.webp)

#### Test 3

Students and autistic children showed strong engagement; art contest broadened public
expression.

![]({{ uploads_url }}eng/ihp-cycle3-test1.webp)

#### Learn 3

1. Braille brochure needs direct feedback loop with visually-impaired users; explore tactile/voice tools.
2. Work with special-education orgs for more targeted tools.
3. Adopt standardized feedback scales + long-term tracking to evaluate sustainability and impact.

### Cycle 4: Commercial Exploration

#### Design 4

Advance lab strain to clinical application; build “safe probiotic therapy” product line.

![]({{ uploads_url }}eng/ihp-cycle4-build4.webp)

#### Build 4

1. **Compliance & Production.** Preclinical + Ph I/II; GMP workshops; partner for scale-up;
   stabilize strain efficiency >80%.
2. **Channels & Pricing.** B-end: hospitals & pharmacies; C-end: Tmall/JD; tiered pricing
   (custom vs basic).
3. **Brand & Ops.** Suprobiotics “Product Q&A”; physician KOLs; public-welfare program with free
   trials.
4. **Finance & Risk.** Budget (RMB: 4M R&D / 3M clinical / 3M production / 2M promotion);
   patents to protect core tech.

{% endblock %}


{% extends "layout.html" %}
{% block page\_content %}

![]({{ uploads_url }}hero/p-1.webp)
![]({{ uploads_url }}hero/p-2.webp)
![]({{ uploads_url }}hero/p-3.webp)

# RESULTS

Results

* [Construction of Engineered Probiotics](#inc-content-1)
* [Experiment on Antibiotic-sensitive
  Engineered Probiotics](#inc-content-2)
* [Design of Uric Acid Transporter UacT](#inc-content-3)
* [Development of Engineered Probiotics with
  Uric Acid Metabolism Ability](#inc-content-4)
* [In-depth Dialogue with Stakeholders](#inc-content-5)
* [Math Model & Suprobiotics](#inc-content-6)
* [Inclusivity Success](#inc-content-7)
* [Future Plans](#inc-content-9)
* [Wet Lab: Further Optimization](#inc-content-10)
* [Human Practice: Continuously
  Evolving](#inc-content-11)
* [Commercialization Blueprint](#inc-content-12)

## Construction of Engineered Probiotics

With the prevalence of high-purine diets, hyperuricemia has gradually become younger and more widespread, with
its incidence increasing year by year. Currently, most drugs for treating hyperuricemia have toxic and side
effects, and their therapeutic effects are highly limited. Wild strains capable of degrading purine substances
have been isolated and screened from the human body, food, and environment, and their application in
hyperuricemic mouse models has shown a certain therapeutic effect. This further indicates that the direction of
treating hyperuricemia through probiotics is feasible and has promising application prospects.

In recent years, the use of genetically engineered bacteria based on synthetic biology to reduce intestinal
uric acid has become a major research focus, as they can be more specifically and effectively customized to
address hyperuricemia and related diseases. This approach enhances the degradation capacity of strains by
introducing specific metabolic pathways or genes, thereby more effectively reducing uric acid production and
minimizing uric acid accumulation in the body.

Therefore, our team has conducted research on constructing engineered probiotics for the prevention and
treatment of hyperuricemia. In the preliminary foundational work, the team used *E. coli* Nissle 1917 (EcN)
as the chassis bacterium, overexpressed the xanthine transporter XanQ, enhanced its activity through rational
design, and optimized its expression level in the cytoplasmic membrane—enabling it to efficiently transport
xanthine and hypoxanthine.

The construction of the engineered probiotic chassis mainly includes the following two aspects:

(1) Double-Site Mutation of XanQ

Mutating the 88th serine to threonine and the 94th phenylalanine to tyrosine in XanQ significantly improved its
xanthine transport capacity. The transport efficiency of XanQ F88T/F94Y was 213.19% that of wild-type XanQ.

The determination of xanthine transport ability for the XanQ double-point mutant compared with the wild-type
XanQ

![]({{ uploads_url }}results/res-wet1.webp)

(2) Promoter and RBS Engineering

By selecting the promoter J23100 and RBS B0034, the xanthine transport level of the strain with EcN as the
chassis was successfully increased by 8.6-fold, and its recognition ability for hypoxanthine was significantly
enhanced (4.0-fold that of the original EcN). This optimized strain was named YES301.

![]({{ uploads_url }}results/res-wet2.webp)

Evaluation of the xanthine transport capacity of XanQF94Y/S88T after the expression level was regulated by
different strength promoters (A) and RBS (B)

### Experiment on Antibiotic-sensitive Engineered Probiotics

We successfully constructed the antibiotic-sensitive engineered probiotic YES302 by scarless knockout of the
*asd* gene in the EcN1917 genome and introducing a recombinant plasmid carrying both the *asd* gene
and the xanthine transporter XanQ mutant.

![]({{ uploads_url }}results/res-wet3.webp)

Performance Tests of YES302

(A) Plasmid passage experiment; (B) Xanthine transport capacity of YES302 and its 20th passage derivative;
(C) Survival rate test under simulated gastrointestinal fluid conditions; (D) Xanthine transport capacity
after simulation of gastrointestinal fluids.

![]({{ uploads_url }}results/res-wet4.webp)

Antibiotic Sensitivity of YES302

YES302 exhibited efficient extracellular xanthine transport capacity, with a transport efficiency of
approximately 90%. The strain remained sensitive to multiple antibiotics in LB and M9 media and showed high
plasmid stability—retaining over 95% of plasmid function even after 20 consecutive passages. In addition,
YES302 had a significant survival rate under simulated gastrointestinal fluid conditions.

These results indicate that YES302 can reduce the xanthine concentration in the reaction system through
efficient extracellular xanthine transport, demonstrating its potential as a safe and sustainable microbial
therapy for hyperuricemia. This strain does not rely on traditional antibiotic selection markers, reducing
biosafety risks and laying a foundation for its future clinical application.

### Design of Uric Acid Transporter UacT

To enhance the uric acid clearance capacity of engineered probiotics, we expressed three membrane proteins
with potential uric acid transport functions in the chassis strain *E. coli Nissle* 1917 (EcN 1917) and
conducted in vitro uric acid transport experiments to compare their transport efficiencies.

The results showed that the strain harboring UacT (1917-UacT) reduced the initial uric acid concentration
(100 μM) in the reaction system by more than 40 μM within 1 hour, exhibiting the strongest uric acid transport
activity (Figure 1). Therefore, we selected UacT as the core uric acid transport module of the engineered
bacterium YES303. Potential optimization targets of UacT were predicted through molecular docking analysis,
laying a foundation for subsequent site-directed mutagenesis and directed evolution. In the future, we will
further optimize the expression level of UacT through promoter/RBS engineering.

![]({{ uploads_url }}results/res-wet5.webp)

Results of Uric Acid Transport Experiments

(Note: The ordinate represents "Uric Acid Uptake (μM)", and the abscissa represents different strains: 1917,
1917-UacT, 1917-PucK, 1917-PucJ; the culture condition is "LB+UA".)

### Development of Engineered Probiotics with Uric Acid Metabolism Ability

To reduce the amount of uric acid already produced in the body, we further superimposed the expression of
uricase on the basis of the engineered probiotics, endowing the strain with uric acid degradation capacity.
This provides a comprehensive and effective approach for the strain to prevent and treat hyperuricemia.

![]({{ uploads_url }}results/res-wet6.webp)

(A) Xanthine Transport and (B) Uric Acid Degradation of YES301-UricaseT Strain

After coupling with uricase, 1x10⁹ CFU/mL of the engineered bacterium could degrade 48.01% of uric acid when
co-incubated with 100 μM uric acid for 60 minutes.

## In-depth Dialogue with Stakeholders

### Math Model & Suprobiotics

Based on experimental data and the Monod equation, we constructed the "Intestinal Purine-Serum Uric Acid
Dynamic Model" and optimized it through three rounds of the Design-Build-Test-Learn (DBTL) cycle: In Cycle 1,
we established the basic dynamic relationship among "intestinal purines, serum uric acid, and bacterial
quantity"; in Cycle 2, we introduced a Gaussian pulse to fit dietary purine fluctuations and added an
intestinal uric acid reabsorption module, reducing the simulation error of serum uric acid from 25% to less
than 8%; in Cycle 3, we embedded the intelligent voice assistant UriGuard to enable automatic input of
"personal characteristics + dietary data", with the error of personalized dosage calculation being less than
5%.

This work breaks the traditional "one-size-fits-all" medication model, provides a quantitative tool for
clinical personalized probiotic dosage guidance, and promotes the transformation of hyperuricemia management
from "empirical medication" to "precision calculation". The combination of the model and the voice assistant
solves the cumbersome problem of manual input for users, enhancing the convenience and accessibility of
medication guidance.

We innovatively built Suprobiotics, a comprehensive science popularization and communication platform in the
probiotic field. For the general public, this platform can eliminate barriers to accessing health information
and help them identify reliable probiotic products; for researchers, it can facilitate the sharing of
synthetic biology technologies and academic cooperation; for industrial development, it provides a "scientific
and transparent" development paradigm, promoting the transition of engineered bacteria from the laboratory
stage to the market, which is in line with the requirements of "Healthy China 2030" for the transformation of
innovative medical resources.

### Inclusivity Success

We extended popular science lectures from Shanghai to remote mountainous schools; created Braille and audio
brochures for the visually impaired, and developed a multilingual interactive assistant for the hearing
impaired. For children with autism, we designed calm tactile-experience lessons, and for young learners, we
crafted creative tools such as an intestinal whiteboard and children’s picture books.

Meanwhile, we integrated cultural creativity—combining probiotic knowledge with Tai Chi health classes and
co-hosting the national "Light of Life" painting competition. We adopted color-blind-friendly color schemes on
our popular science website to ensure a smooth browsing experience for those with visual impairments. Finally,
we collaborated with 33 iGEM teams to publish the Smashing Synthetic Biology Myths handbook, addressing public
concerns with clear scientific evidence.

These initiatives not only broke down barriers for different groups to access scientific knowledge but also
enabled diverse populations to actively engage in the understanding and discussion of synthetic biology. They
provide a replicable practical model for global research teams to conduct inclusive science popularization,
and also promote the transformation of synthetic biology from a "professional field" to "shared by all",
practicing the concept of "science knows no boundaries".

## Future Plans

### Wet Lab: Further Optimization

For YES302 – Advanced Functional Testing
We will expand the performance evaluation of YES302 by testing
its transport efficiency for mixed purine substrates, simulating the complex purine composition found in real
diets. This will help assess the strain’s effectiveness in physiologically relevant conditions.

For YES303 – Safety and Transport Optimization
To enhance biosafety, we will remove the antibiotic
resistance marker in YES303 using the same auxotrophy-based strategy applied in YES302. Functionally, we plan
to carry out site-directed mutagenesis of UacT combined with promoter/RBS engineering to further improve the
transporter's activity and expression strength, aiming for higher uptake rates of uric acid.

For YES304 – Safety and Degradation Optimization
We will also apply the antibiotic-free safety design to
YES304 by deleting its resistance marker. Meanwhile, we will optimize URIT expression through promoter and RBS
engineering to enhance its secretion and uric acid degradation capacity in vivo.

Toward an Integrated Therapeutic Strain
Finally, we aim to combine the optimized modules into a single
next-generation strain that integrates: Front-end xanthine transport to reduce precursors of uric acid in the
gut, and Back-end uric acid transport and degradation to remove residual uric acid. This integrated engineered
probiotic is expected to provide a comprehensive and efficient microbial therapy for hyperuricemia.

### Human Practice: Continuously Evolving

We plan to update the *Smashing Synthetic Biology Myths* Handbook, adding new topics such as "Safety of
Interaction Between Engineered Bacteria and Intestinal Flora" and "Verification of No Side Effects from
Long-Term Use"; develop an animated short film on "Intestinal Purine Metabolism" (in multiple languages) and a
"Probiotic Puzzle Game" for kindergartens; and collaborate with iGEM teams to conduct global popular science
lectures. These efforts aim to specifically address the public's concerns about transgenic technology, lower
the cognitive threshold for synthetic biology, achieve "full-age-group" coverage of popular science through
diverse educational tools, and enhance the public's awareness of hyperuricemia prevention and trust in
engineered bacteria therapy.

We will also establish a "quarterly interview + real-time feedback" mechanism, regularly inviting clinicians,
enterprise representatives, and users from special groups to participate in seminars to collect suggestions on
strain optimization and platform functions; additionally, a "demand feedback channel" will be added to the
Suprobiotics platform to promptly respond to users' needs regarding popular science content and product
functions.

### Commercialization Blueprint

In accordance with the *Classification of Biological Products and Requirements for Application Data*, we
will compile the safety data (such as antibiotic sensitivity and gene transfer risk tests) and efficacy data
of the YES302/YES304 strains, and plan to submit an Investigational New Drug (IND) application within one
year. We will collaborate with hospitals to conduct Phase I clinical trials (focused on the tolerance of
healthy volunteers) and Phase II clinical trials (focused on verifying efficacy in patients). These efforts
aim to promote the transformation of engineered bacteria from "laboratory achievements" to "clinical drugs".
Through compliance procedures and clinical data verification, we will ensure the safety and efficacy of the
products, providing legal and scientific basis for their market launch.

We also plan to cooperate with enterprises to optimize industrial-scale fermentation processes (expanding
fermenter scale and reducing costs) and develop freeze-dried microcapsule formulations that can be stored at
room temperature. In the initial stage, the products will be promoted in China through the "hospital
prescription + online platform" model, with differentiated pricing (high-end customized version and basic
version). This will address the issues of "high cost and difficult transportation" in the industrialization of
engineered bacteria and expand the product coverage. The differentiated pricing and regional adaptation design
will meet the needs of different populations, promoting engineered bacteria therapy to become a safe treatment
option for hyperuricemia patients worldwide, and creating both economic value and public health value.

{% endblock %}