Engineering

Cycle 1: Knockout of Resistance Genes

Design

Core Objective. Address Sm^r-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 Δasd 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 Δasd chassis.
3. Integration. Co-clone optimized XanQ F94Y88 and asd into pWT021a, transform into Δasd chassis to obtain YES302.

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.

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).

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.

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.

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 (V_max > 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); K_m=163.7 μM; V_max=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.