Purpose:

Separate and visualize DNA fragments by size; excise target bands.

Steps:

Agarose Gel Preparation

1. Weigh 0.4g agarose powder in a flask; add 50mL 1×TAE buffer.
2. Heat in microwave with intermittent shaking until fully dissolved and clear.
3. Cool solution to 50-60°C.
4. Add 5μL Ultra GelRed dye; mix gently.
5. Seal gel tray ends; insert comb.
6. Pour solution into tray avoiding bubbles. Solidify at RT for 20-30 min.

Sample Loading Electrophoresis

1. Remove comb and end seals after solidification.
2. Place gel in tank with wells facing cathode.
3. Add 1×TAE buffer to cover gel by 1-2mm.
4. Load 5-10μL DNA samples mixed with loading buffer into wells. Include DNA ladder in one well.
5. Run gel at 100V for 30 min.
6. Stop when bromophenol blue reaches 2/3-3/4 gel length.
7. Transfer gel to UV transilluminator; visualize and photograph bands.
8. Excise target DNA fragments based on marker reference for purification.

Purpose: 

Efficiently and accurately join multiple linear DNA fragments (e.g., target gene and linearized vector) into a complete circular recombinant plasmid in vitro.

Steps:

1. Prior to step 2, perform PCR and DNA purification for the target gene fragment.
2. Measure concentrations of purified linearized vector and insert using a K5600C microspectrophotometer.
3. Calculate molar concentrations based on measured concentrations and fragment lengths (bp).
4. Precisely pipette calculated volumes of linearized vector and insert into a new sterile PCR tube or microcentrifuge tube; mix gently.
5. Add an equal volume of Gibson Assembly Master Mix to the DNA mixture.
6. Incubate at 50°C in a PCR instrument or water bath for 45 minutes.

Purpose: 

Rapidly and efficiently screen for positive E. coli clones containing the target agarase gene insert in the plasmid, rather than empty vector clones.

Steps:

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

   DNA Template (50-200 ng)	5 μL
   Forward Primer (10 μM)	0.2 μL
   Reverse Primer (10 μM)	0.2 μL
   High-Fidelity PCR Buffer (2X)	0.3 μL
   ddH2O	4.3 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

1. CRISPR-Cas9 Plasmid Construction for Targeted Genomic Loci

   Purpose: 

   Construct Cas9 plasmids expressing specific sgRNAs to create DNA double-strand breaks at specific yeast genomic loci, providing targets for subsequent homologous recombination and foreign gene integration.

   Steps:

   1. Use online tool CHOPCHOP (https://chopchop.cbu.uib.no/) to design efficient and low-off-target 20 nt sgRNA sequences for target genomic loci (e.g., GAL80, X-3).
   2. Amplify sgRNA fragment using p426-SNR52p-gRNA.csr-1.Y-SUP4t as template with primers sg-F and sg-R; amplify Cas9 fragment using Addgene SpCas9 plasmid as template with primers Cas9-F and Cas9-R. Mix sgRNA and Cas9 fragments at 1:1 molar ratio to assemble p426-P_TEF1-SpCas9-T_CYC1-P_SNR52-sgRNA-T_SUP4 plasmid.
   3. Use Cas9 empty vector (p426-P_TEF1-SpCas9-T_CYC1-P_SNR52-sgRNA-T_SUP4) as template and designed sgRNA sequence as 5' homology arm in primers for PCR linearization to obtain linearized vector backbone.
   4. Transform 100 ng linearized plasmid into E. coli DH5α competent cells after purifying the target gene fragment.
   5. Pick 3 positive transformants for culture, extract plasmids, and send for sequencing verification (e.g., Sangon Biotech) to obtain validated Cas9-sgRNA plasmids targeting the gene locus.
2. Donor DNA Preparation

   Purpose: 

   Prepare linear DNA fragments containing the target gene and homology arms as repair templates for homologous recombination, guiding precise integration of foreign genes into specific genomic loci.

   Steps:

   1. Amplify target gene, promoter, terminator, etc., by PCR using yeast genomic DNA or plasmid as template.
   2. Assemble all elements using Gibson Assembly.
   3. Transform the assembled product into E. coli DH5α, screen positive clones using ampicillin resistance, and verify by colony PCR and sequencing.
   4. Amplify the correct construct using specific primers and purify the gene fragment with SanPrep Column PCR Product Purification Kit to obtain donor DNA.
3. Preparation of S. cerevisiae CEN.PK2-1D Competent Cells

   Using ZYMO Frozen-EZ Yeast Transformation II Kit

   Purpose:

   Disrupt the cell wall barrier to enable efficient uptake of foreign DNA by S. cerevisiae for genetic modification.

   Steps:

   1. Inoculate CEN.PK2-1D strain in 10 mL YPD liquid medium. Incubate at 30°C with vigorous shaking (200-250 rpm) until mid-log phase (OD600 ≈ 0.8-1.0).
   2. Centrifuge at 500 × g for 4 min, discard supernatant. Resuspend cell pellet in 10 mL Solution 1, centrifuge again at 500 × g for 4 min, and discard supernatant to thoroughly wash cells.
   3. Add 1 mL Solution 2 to the washed cell pellet, resuspend completely by pipetting or vortexing. The resulting cell suspension is competent cells, ready for immediate transformation or freezing.
4. Primary Screening

   Purpose:

   Rapidly screen for positive clones with potential correct homologous recombination from a large number of transformants.

   Steps:

   1. Verify by colony PCR: Pick single colonies from uracil-deficient YNB plates, transfer to tubes containing PCR mix, and use GAL80 locus verification primers F and R to check target gene integration in the yeast genome.
5. Counter-Selection

   Purpose:

   Eliminate the URA3 selection marker gene from positive clones to obtain engineered strains containing only the target gene without exogenous markers.

   Steps:

   1. Inoculate transformants with correct primary screening bands into YPD liquid medium; incubate at 30°C, 220 rpm for 16 h.
   2. Spread onto solid YPD plates containing 1 mg/mL 5-fluoroorotic acid (5-FOA) for counter-selection. Perform PCR verification again on colonies growing on the plates; strains verified correctly indicate successful marker loss.

1. Quantification of Squalene Accumulation

   Steps:

   Squalene Standard Curve Preparation:

   1. Add 0.5 mL grinding beads to a disruption tube, then add 0.5 mL fermentation broth and 1 mL ethyl acetate.
   2. Disrupt cells using a Bioprep-24R instrument, then centrifuge at 12,000 × g for 1 min.
   3. Use a needle to aspirate the supernatant (ethyl acetate layer), attach a 0.22 μm organic solvent filter, discard the first few drops (~3 drops) to prime the filter and remove dead volume. Collect subsequent filtrate into clear HPLC vials for analysis.
   4. Precisely weigh 10.0 mg squalene standard (purity ≥95%), dissolve in ethyl acetate, and dilute to 10 mL in a volumetric flask to prepare 1.0 mg/mL stock solution.
   5. Perform gradient dilution of the stock solution with ethyl acetate to prepare a series of standard working solutions:

      Tube No.	100 μg/mL Stock (μL)	Ethyl Acetate (μL)	Squalene Content (μg)	Final Concentration (μg/mL)
      0	0	1000	0	0
      1	50	950	50	50
      2	100	900	100	100
      3	200	800	200	200
      4	300	700	300	300
      5	400	600	400	400
      6	500	500	500	500
      7	1000	0	1000	1000

   6. Filter the upper ethyl acetate layer through a 0.22 μm membrane for HPLC analysis. Record the squalene chromatographic peak area for each concentration.
   7. Plot squalene concentration (μg/mL) as X and the measured average peak area as Y. Perform linear regression to obtain the equation: Y = 14498.99X + 387171.62, R² = 0.9992.

   Squalene Quantification:

   1. Add 0.5 mL fermentation broth to a disruption tube containing 0.5 g of 0.5 mm glass beads and 1 mL ethyl acetate.
   2. Disrupt cells using a Bioprep-24R instrument, then centrifuge at 10,000 × g for 1 min.
   3. Filter the upper ethyl acetate layer through a 0.22 μm membrane for HPLC analysis.

   HPLC Conditions:

   System: Shimadzu LC-16 with SPD-16 UV detector

   Column: Agilent Poroshell 120 EC-C18 (2.1 × 100 mm)

   Eluent: 100% acetonitrile

   Flow rate: 0.5 mL/min

   Injection volume: 2 μL

   Detection wavelength: 210 nm

2. Detection of Rare Ginsenoside Rh1 Production

   Steps:

   Rh1 Standard Curve Preparation:

   1. Precisely weigh 3.0 mg of 20(S)-Rh1 standard, dissolve in 1000 μL methanol to prepare a 3 mg/mL stock solution.
   2. Label 7 clean HPLC vials as 1-7.
   3. Pipette corresponding volumes of 3 mg/mL stock into each vial and add HPLC-grade methanol to adjust the total volume to 400 μL:

      Tube No.	Stock (mL)	Methanol (μL)	Concentration (mg/mL)
      1	4	396	0.01
      2	10	390	0.025
      3	20	380	0.05
      4	40	360	0.10
      5	100	300	0.25
      6	200	200	0.50
      7	400	0	1.00

   4. Filter the methanol layer through a 0.22 μm membrane for HPLC analysis. Record the Rh1 peak area for each concentration.
   5. Plot Rh1 concentration (g/L) as X and average peak area as Y. Perform linear regression to obtain the equation: Y = 3878X + 38658, R² = 0.9990.

   Rh1 Quantification:

   1. Add 0.5 mL fermentation broth to a disruption tube containing 0.5 g of 0.5 mm glass beads and 1 mL n-butanol.
   2. Disrupt cells using a Bioprep-24R instrument, then centrifuge at 10,000 × g for 1 min.
   3. Filter the upper n-butanol layer through a 0.22 μm membrane for HPLC analysis.

   HPLC Conditions:

   System: Shimadzu LC-16 with SPD-16 dual-wavelength UV detector

   Column: Neptune 5u C18 (250 × 4.6 mm)

   Mobile phase: Water/acetonitrile

   Detection wavelength: 203 nm

   Column temperature: 35°C

   Gradient elution: 0-6 min (40-100% B), 6-18 min (100% B), 18-25 min (100-40% B), 25-35 min (40% B)

Purpose:

Determine the optimal ratio of red algal polysaccharides to glucose in the medium for squalene production by engineered strain Sq-Ag5.

Steps:

1. Prepare YPDA liquid media with glucose concentrations (0, 2.5, 5, 7.5, 10 g/L) and red algal polysaccharide concentrations (10, 15, 20, 25, 30, 40 g/L).
2. Inoculate 1% (v/v) primary seed culture into 250 mL flasks containing 25 mL YPDA medium. Ferment at 30°C, 220 rpm for 96 h.
3. Measure squalene content in fermentation broth by HPLC to determine the optimal carbon source composition.