(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.