(1) Prepare for the reaction system shown in Table 1(a). The plasmid template content in the system should be within the range of 10 to 30 ng.

(2) The PCR procedure is shown in Table 2.

Figure 1 Colony PCR reaction system

Figure 2 The program of PCR

DH5α Escherichia coli Transformation Protocol

(1) Pre‑chill components: ~5 minutes before the ligation ends, place one sterile 1.5 mL microcentrifuge (EP) tube on ice. Retrieve competent DH5α E. coli cells from −80 °C and immediately put on ice to thaw. Use pre‑chilled yellow tips. After thawing, gently aliquot competent cells into pre‑chilled tubes as needed.

(2) Add ligation product: Add 5–10 μL of the ligation mixture to the competent cells. Mix gently by pipetting up and down twice (do not vortex). Incubate on ice for 30 minutes. Meanwhile pre‑equilibrate a 42 °C water bath.

(3) Heat shock: Heat shock at 42 °C for 45 seconds, then immediately place tubes back on ice for 2 minutes without shaking.

(4) Recovery: Add 1 mL antibiotic‑free LB medium. Place tubes on a floating rack (or shaker) and incubate at 37 °C, 180 rpm for 60 minutes.

(5) Plating: Centrifuge at 4000 rpm for 5 minutes, remove 900 μL supernatant leaving ~100 μL. Gently resuspend (brief vortex or pipette), then spread onto pre‑warmed LB agar plates containing the appropriate antibiotic using sterile glass beads. Incubate plates overnight at 37 °C.

In this experiment, a fragment or multiple fragments or plasmids with homologous sequences are transferred into S. cerevisiae cells by lithium acetate method. The specific operational methods are as follows:

(1) Inoculate a colony from a plate into 5 mL YPD liquid culture. Incubate at 30 °C.

(2) Next day, inoculate 500 μL of the overnight culture into 5 mL fresh YPD. Incubate at 30 °C, 200 r/min for about 4–6 h.

(3) Take 1 mL culture; centrifuge at 3800 rpm for 2 min; discard supernatant.

(4) Resuspend the pellet in 1 mL sterile water; centrifuge 3800 rpm 2 min; discard supernatant.

(5) Resuspend cells in 1 mL 0.1 M LiOAc. Place on ice; standby.

(6) Boil ssDNA (10 mg/mL) in a PCR tube at 99.9 °C for 20–30 min; then place on ice.

(7) Prepare the transformation system :

Figure 3 transformation system

(8) Add 3 μL per plasmid and ~20 μL ddH₂O to the cells. Add the competent cells into the transformation system; mix gently by flicking.

(9) Incubate 30 min at 30 °C.

(10) Heat shock at 42 °C for 18 min.

(11) Centrifuge at 3800 rpm for 2 min; discard supernatant.

(12) Add 400 μL 5 mM CaCl₂; let stand 5 min.

(13) Add 700 μL YPD; incubate 5 h at 30 °C, 200 rpm.

(14) Centrifuge 3800 rpm for 2 min; discard most supernatant. Resuspend in 200 μL ddH₂O; plate 100 μL on selective medium.

(1) Use a commercial TIANprep Mini Plasmid Kit (or equivalent) to extract the plasmid from E. coli cultures as per manufacturer instructions.

(2) After extraction, perform restriction enzyme digestion with the appropriate enzymes, then analyze by agarose gel electrophoresis to verify the band size.

(1) Harvest 1–5 mL yeast culture (≤ 5 × 10⁷ cells) by centrifugation at 13,000 g for 1 min; discard supernatant.

(2) Resuspend pellet in 300 μL P1 solution by pipetting; add 20 μL Zymolyase, mix, and incubate 1 h.

(3) Pre‑warm P2 solution at 42 °C for 5 min; add to sample and mix gently by inversion.

(4) Pre‑cool centrifuge to 4 °C for 5 min; add pre‑chilled P3; centrifuge at 17,000 g for 20 min.

(5) Transfer supernatant to a fresh 2 mL tube; add 700 μL isopropanol, mix, and centrifuge 17,000 g for 30 min.

(6) Discard supernatant; add 700 μL PW along pellet side; centrifuge 17,000 g 10 min; repeat wash once.

(7) Pre‑heat heat block to 55 °C; dry the white pellet at 55 °C for 10 min.

(8) Pre‑heat sterile water; add 20 μL to pellet, mix, and incubate 55 °C for 10 min to elute plasmid.

(1) Perform seamless cloning using the pEASY®-Basic Seamless Cloning and Assembly Kit (or equivalent) following supplier instructions.

(2) Prepare the assembly reaction (see Table 8); use an optimal molar ratio of vector : each insert = 1 : 2.

(3) Mix linearized vector fragment(s) with Basic Assembly Mix; incubate at 50 °C for 20 min.

(1) Incubate transformation plates at 37 °C (generally colonies are ready within 12–16 h; pick ~12 colonies).

(2) Add 10 μL ddH₂O into a PCR tube.

(3) Touch a sterile toothpick to a single colony; swirl into the water (solution becomes slightly cloudy).

(4) Use 1 μL of the suspension as PCR template.

(5) Run PCR (reaction composition & cycling per standard colony PCR or as described for yeast colony PCR).

Single colonies from transformation plates (typically 12) are first streaked for purity and incubated at 30 °C. Because yeast has a cell wall, lysis/alkaline treatment is required before PCR. Steps:

(1) Add 30 μL of 20 mM NaOH into each PCR tube.

(2) Pick a small amount of cells with a toothpick into the tube; solution becomes slightly cloudy.

(3) Perform 3 cycles: 99 °C 5 min then 4 °C 1 min; hold at 4 °C (this is the template).

(4) Use lysate as template for colony PCR (reaction mixture & cycling per Tables 1 & 2).

(1) Inoculation: Pick a single colony into 5 mL SC‑Trp (glucose) medium; incubate 30 °C shaking until OD₆₀₀ ≈ 1.6 (~36 h).

(2) Induction setup: Harvest 2 mL (3000 g, 3 min), wash once with sterile water, resuspend in SC‑Trp medium lacking glucose; adjust OD₆₀₀ to 0.6–0.8; add 150 μL 40× raffinose + 300 μL 20× galactose.

(3) Incubation: Continue at 30 °C. Incubate 24 h for live cell counting; 36 h for IRI activity measurement.

(4) Cell density adjustment: Dilute culture to OD₆₀₀ = 0.5 (≈ 1 × 10⁷ cells/mL) for downstream assays.

(1) Cold‑shock treatment: Aliquot 1 mL per tube. Treat at −24 °C for 1.5 / 2 / 2.5 / 3 h or at −80 °C for 5 / 6 / 7 / 8 min; include an untreated control.

(2) Serial dilutions: Prepare 90 μL sterile water in each well (96‑well plate). Add 10 μL culture to first well (1:10). Change tip, serially transfer 10 μL into next wells for further 10× dilutions. For control, perform single 1:10 then plate.

(3) Plating: Plate suitable dilutions (e.g., 10⁻⁴, 10⁻⁵) onto SD‑CAA; 0.1 mL per plate; spread evenly; dry 15–20 min.

(4) Incubation: Invert plates; incubate 37 °C 24–48 h.

(5) Counting & calculation: Select plates with 30–300 colonies; compute mean of triplicates. Viable cell concentration (CFU/mL) = (Average colony count ÷ plated volume (mL)) × dilution factor.

The IRI activity of peptides is measured by optical microscopy on a cooling stage. 20 μL of sample is dropped from ~1 m onto a pre‑chilled thin aluminum block over liquid nitrogen to vitrify.

The vitrified sample is transferred to the cooling stage at −60 °C for 1 min, then warmed to −9 °C at 5 °C·min⁻¹ and annealed at −9 °C for 30 min.

Images are captured every 5 min during annealing. NIS‑Elements D software is used to measure mean grain areas (MGAs) of ice crystals: five random regions, all crystal areas quantified; three biological replicates per sample.

Mean MGA values are computed (e.g., with Prism 5.0) and compared to controls to infer IRI performance.

AKTA FPLC system used unless otherwise stated.

(1) Sample loading: Filter clarified sample (0.22 μm) and load 50 mL onto affinity column at 2 mL/min (autosampler optional).

(2) Washing: Wash with Binding Buffer (10 mM Na₂HPO₄, 10 mM NaH₂PO₄, 500 mM NaCl, 20 mM imidazole) for 8 CV at 4 mL/min.

(3) Elution: Elute with Elution Buffer (same salts + 500 mM imidazole) for 3–5 CV at 2 mL/min; collect 1 mL fractions.

(4) Buffer exchange/desalting: Direct pooled peak onto equilibrated desalting column with Formulation Buffer (10 mM sodium acetate, 150 mM NaCl, pH 5.0) at 2 mL/min; monitor A₂₈₀; collect fractions ≥10 mAU.

(1) Run instrument pre‑heating program.

(2) Pipette 20 μL sample into crucible; weigh and record mass.

(3) Place crucible in crimping tool; position lid (convex side up) and seal.

(4) Insert sealed pan into DSC cell; close cover; start measurement program.

(5) After run, open data; select appropriate curve, set onset, and record freezing point from software output.

1. Cytotoxicity Test with L929 Cells

(1) Culture L929 mouse fibroblast-like cells in RPMI-1640 complete medium until log-phase growth.

(2) Trypsinize for 2 min, collect by centrifugation at 1000 rpm for 5 min.

(3) Prepare an antifreeze protein solution at 0.25 mg/mL in RPMI-1640 complete medium.

(4) Seed 2×10⁴ cells per well in a 96-well plate in 200 µL protein solution.

(5) Incubate for 48 h at 37℃, 5% CO₂; use blank medium as control.

(6) After incubation, trypsinize, collect, wash twice with sterile PBS, and resuspend in DPBS.

(7) Prepare stain mix: DPBS:PI:AM = 1000:2:0.5, vortex to mix.

(8) Combine 50 µL stain mix with 50 µL cell suspension per well, mix gently, and incubate for 30 min in the dark.

(9) Observe under an inverted fluorescence microscope, image, count live/dead cells, and calculate viability.

(1) Cultivate yeast cells until the culture OD600 reaches 3.0–5.0.

(2) Centrifuge at 1000 × g 5 °C for 5 min and discard the supernatant.

(3) Resuspend the pellet in 30 mL sterile water centrifuge at 3000 × g 5 °C for 5 min and discard the supernatant.

(4) Resuspend the cells in 20 mL of 1 mol/L sorbitol by vortexing.

(5) Let stand overnight then centrifuge at 3000 × g 5 °C for 5 min and discard the supernatant.

(6) Resuspend the cells in 20 mL SPE solution (1 mol/L sorbitol, 10 mmol/L Na-EDTA, 10 mmol/L Na-phosphate, pH 7.5), add 40 μL Zymolyase solution (10 mg/mL in SPE) and 30 μL 1 mol/L β-ME solution and incubate with gentle shaking at 30 °C for 20 min.

The preparation and extraction of metabolomic samples were performed following standard protocols modified from. Briefly, the yeast strain was cultured in 5mL of SC medium and incubated with shaking at 30 °C for 24 h.

(1) 5mL of culture sample was collected and centrifuged at 1200 × g for 5 min at 4 °C. The cell pellet was washed twice with Milli-Q water and immediately submerged in a prechilled solution of 60% (v/v) methanol/water to quench the reaction rapidly. After a 30-s incubation at 40 °C, the samples were centrifuged at 4000 × g for 5 min at 4 °C to collect the cell pellets.

(2) The cells were then washed twice with phosphate-buffered saline at 4 °C, followed by a final wash with Milli-Q water to remove any residual culture medium. The cell pellets were collected by centrifugation at 4000 × g for 5 min at 4 °C.

(3) To extract the metabolites, 700 µL of an extraction solvent containing an internal standard (methanol：acetonitrile：water = 4:2:1, v/v/v) was added to the cell pellets. The mixture was vigorously shaken for 1 min and placed in a –20 °C freezer for 2 h. Subsequently, the samples were centrifuged at 25,000 × g and 4 °C for 15 min.

(4) The supernatant (600 µL) was carefully transferred to a new EP tube, followed by freeze-drying. The dried samples were then reconstituted in 180 µL of a methanol：water solution (1:1, v/v) and vortexed for 10 min until complete dissolution.

(5) The reconstituted samples were centrifuged at 25,000 × g and 4 °C for 15 min. The supernatant was transferred to a new EP tube and stored at –80 °C until further analysis.

Targeted metabolite quantification was performed on a Waters ACQUITY UPLC I-Class system coupled to a Waters TQ-S triple quadrupole mass spectrometer (Waters, USA) operated in MRM mode. Chromatographic separation was achieved on a Waters ACQUITY UPLC BEH C18 column (1.7 μm, 2.1 × 100 mm) maintained at 40 °C. The mobile phase consisted of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). The gradient elution was as follows:0–0.5 min, 2% B; 0.5–1.5 min, 2–30% B; 1.5–3.0 min, 30–95% B; 3.0–4.0 min, 95% B; 4.0–4.1 min, 95–2% B; 4.1–6.0 min, 2% B.

The flow rate was set to 0.4 mL/min, and the injection volume was 2 μL.

The MS parameters were optimized for each MRM transition. The two monitored transitions for the target analyte were m/z 360 > 85 and m/z 360 > 163, with dwell times of 20 ms each. The capillary voltage was set to 3.0 kV, the desolvation temperature to 500 °C, and the cone voltage to 30 V. Collision energies were optimized at 20 eV and 25 eV for the two transitions, respectively.