Purpose: 

Amplify target gene fragments using specific primers.

steps:

1. In a sterile PCR tube, add the following reagents in order:

   DNA Template (50-200 ng)	0.5 μL
   Forward Primer (10 μM)	0.4 μL
   Reverse Primer (10 μM)	0.4 μL
   High-Fidelity PCR Buffer (2X)	10 μL
   ddH2O	9.1 μL

2. Place the tube in the PCR instrument for PCR amplification.

   PCR thermal cycling conditions:

   Step	Time(s)	Temperature(℃)	Cycle
   Initial Denaturation	180	98	1
   Denaturation	10	98	30
   Annealing	5	58	30
   Extension	30-180	72	30
   Final Extension	600	72	1

Using SanPrep Column PCR Product Purification Kit (Cat. B518141-0100)

Purpose: 

Remove enzymes, primers, and other impurities while concentrating target DNA to obtain high-purity templates for downstream experiments.

steps:

1. Preparation: Ensure ethanol has been added to Wash Solution, and isopropanol to Buffer B3. Check for precipitation in Buffer B3.
2. Add 5 volumes of Buffer B3 to the PCR reaction mixture and mix thoroughly.
3. Centrifuge at 8,000 × g for 30 seconds. Discard the flow-through.
4. Add 500 μL of Wash Solution, centrifuge at 9,000 × g for 30 seconds, and discard the flow-through.
5. Repeat step 4 once.
6. Centrifuge the empty column at 9,000 × g for 1 minute.
7. Transfer the column to a clean 1.5 mL tube. Add 15–40 μL of Elution Buffer to the center of the membrane. Incubate at room temperature for 1 minute, then centrifuge for 1 minute. Collect and store the DNA solution.

Purpose: 

Amplify plasmids efficiently using E. coli DH5α transformation and rapidly obtain positive clones via antibiotic selection.

steps:

1. Thaw 50 μL of DH5α competent cells slowly on ice.
2. Add 1–5 μL of ligation product or plasmid DNA to the cells; mix gently and incubate on ice for 5 minutes.
3. Heat-shock at 42°C for 1 minute, then return to ice for 3 minutes.
4. Under sterile conditions, spread the mixture directly onto an LB agar plate containing 150 μg/mL ampicillin.
5. Incubate at 37°C for 12–15 hours (no satellite colonies observed). Pick colonies for verification by colony PCR.

1. Lithium Acetate Transformation Method

   Purpose: 

   Efficiently introduce in vitro constructed recombinant plasmid DNA into engineered Saccharomyces cerevisiae cells.

   steps:

   1. Pick a single colony from a solid plate and inoculate into YPD liquid medium; incubate at 30°C, 220 rpm for 16 h.
   2. Take 500 μL culture, centrifuge at 8000 rpm, and discard supernatant.
   3. Add sequentially: 3 μL of 10 mg/mL salmon sperm ssDNA (denatured at 95°C for 5 min and chilled on ice), 100 μL transformation mix (800 μL 50% PEG3350, 200 μL 2 mol/L LiAc, 7.5 μL β-mercaptoethanol, balance ddH₂O per 1 mL), and 0.1–1 μg plasmid; mix well.
   4. Incubate at 37°C for 30 min, centrifuge at 8000 rpm for 3 min, discard supernatant.
   5. Resuspend cells in 500 μL sterile water, plate 80 μL on uracil-deficient YNB plates, incubate at 30°C for 3–5 days.
2. Preliminary Verification by Lugol’s Iodine Plate Assay

   Purpose: 

   Qualitatively and rapidly determine whether engineered yeast successfully secretes active extracellular enzymes for degrading red algal polysaccharides, based on clear hydrolysis zones around colonies.

   steps:

   1. Inoculate a single colony of engineered strain into 5 mL uracil-deficient YNB liquid medium; culture at 30°C, 220 rpm for 16–24 h.
   2. Spot 2 μL culture onto Lugol’s iodine plates, incubate for 48 h, and observe clear zones. Undegraded areas appear dark blue.
3. Pre-treatment for Enzyme Activity Assay
   1. From a fresh plate (≤1 month old), pick a single S. cerevisiae colony and inoculate into 3 mL uracil-deficient YNB liquid medium; culture overnight at 30°C, 220 rpm to obtain primary seed culture in log phase.
   2. Inoculate 1% activated seed culture of 6 engineered strains into YPDA medium; after 96 h fermentation, collect broth for agarase and neoagarobiose hydrolase activity assays.
4. Agarase Activity Assay

   Purpose: 

   Quantitatively evaluate the overall ability of extracellular agarase secreted by engineered strains to degrade agar polysaccharides into reducing sugars, for screening the most efficient degraders.

   steps:

   1. Precisely weigh dried glucose standard and prepare a 1.0 mg/mL stock solution with deionized water.
   2. Prepare standard series in test tubes as follows:

      Tube No.	Glucose Stock (mL)	Deionized Water (mL)	Glucose Content (μg)
      0	0.0	1.0	0
      1	0.2	0.8	200
      2	0.4	0.6	400
      3	0.6	0.4	600

   3. Add 1.5 mL DNS reagent to each tube, heat in boiling water bath for exactly 5 min, then immediately cool.
   4. Add 8.5 mL deionized water to each tube, mix well.
   5. Measure absorbance at 540 nm using Tube 0 as blank.
   6. Plot standard curve with glucose content (μg) as X and OD540 as Y. Obtain regression equation: Y = 1.391X - 0.07451 (R² = 0.9974).

   Enzyme Activity Assay:

   1. Add 200 μL fermentation broth to 800 μL Tris-HCl buffer (pH 8.0) containing 0.3% w/v agar in a colorimetric tube. React with shaking at 40°C for 20 min.
   2. Add 1 mL DNS reagent, heat in boiling water bath for 5 min, cool, and dilute to 10 mL with distilled water. Mix well.
   3. Measure absorbance at 540 nm using inactivated enzyme as control. Calculate reducing sugar content using standard curve (Y=1.391X-0.07451, R²=0.9974).

   Unit definition (U): Enzyme amount required to produce 1 μg reducing sugar per minute under above conditions.

5. Neoagarobiose Hydrolase Activity Assay

   Purpose: 

   Specifically detect the efficiency of engineered strains hydrolyzing neoagarobiose (agar degradation product) into galactose, ensuring complete degradation of algal polysaccharides into utilizable monosaccharides.

   steps:

   Galactose Standard Curve (Using Abcam Galactose Assay Kit):

   1. Dilute kit-provided galactose standard (100 mmol/μL) to 1 mmol/μL working solution using Assay Buffer.
   2. Take 10 μL working solution, add 990 μL Assay Buffer, mix thoroughly to obtain 10 nmol/μL diluted standard.
   3. Add reagents to wells as per table:

      Tube No.	Glucose Stock (mL)	Deionized Water (mL)	Glucose Content (μg)
      0	0.0	1.0	0
      1	0.2	0.8	200
      2	0.4	0.6	400
      3	0.6	0.4	600
      4	0.8	0.2	800
      5	1.0	0.0	1000

   4. Then add sequentially to each well: 2 μL Galactose Probe, 2 μL Galactose Enzyme Mix, and 2 μL HRP.
   5. Incubate the reaction system at 37°C in the dark for 40 min.
   6. Measure the absorbance (OD₅₇₀) of each well at 570 nm.
   7. Plot the galactose standard curve with galactose content (nmol) as X and OD₅₇₀ as Y, obtaining the regression equation: Y = 0.07252X + 0.03044 (R² = 0.9945).

   Enzyme Activity Assay:

   1. Dilute the galactose standard (Abcam kit) to 1 nmol/μL: Add 10 μL of 100 nmol/μL galactose standard to 990 μL galactose assay buffer and mix thoroughly.
   2. Add 0, 2, 4, 6, 8, and 10 μL of the diluted standard to a series of wells. Adjust each well to 50 μL with galactose assay buffer, resulting in 0, 2, 4, 6, 8, and 10 nmol galactose standards per well.
   3. Plot the standard curve with galactose concentration (nmol) as X and OD570 as Y.
   4. Calculate galactose content in the fermentation broth using the standard curve (Y=0.07252X+0.03044, R²=0.9945).
   5. To 25 μL fermentation broth (using inactivated enzyme as control), add 25 μL of 2 mM neoagarobiose, 44 μL galactose assay buffer, 2 μL galactose probe, 2 μL galactose enzyme mix, and 2 μL HRP. Total reaction volume is 100 μL. Incubate at 37°C for 40 min.
   6. Measure absorbance at 570 nm.

   Unit definition (U):Enzyme amount required to convert 1 μmol galactose per minute.

Purpose: 

Mild hydrolysis of red algal powder using dilute HCl to convert hard-to-utilize polysaccharides into fermentable reducing sugars, providing a carbon source for yeast.

steps:

1. Mix 5g algal powder with 0.1 mM HCl solution at 1:10 ratio (total volume 50 mL); react at 115°C for 15 min.
2. After reaction, adjust hydrolysate pH to 7.0 using NaOH solution to suit yeast growth.
3. Dilute the neutralized hydrolysate 1:1 with deionized water, use as YP fermentation medium in shake flasks.
4. Measure initial reducing sugar concentration of diluted medium to assess carbon source content.

Purpose:

High concentrations of red algal polysaccharides increase medium viscosity, inhibiting strain growth. Determine the specific conditions for liquefaction using low HCl concentrations.

Steps:

1. Treat 25 mL YPDA medium with HCl gradients (0.001-0.01 M) at 121°C for 20 min.
2. Analyze by HPLC to detect neoagarobiose and monosaccharides.