ZYMOLYASE Experimental Protocol

ZYMOLYASE Experimental Protocol

1. Culture cells overnight in 3 mL selective medium.

2. Collect cell pellets by centrifuging briefly twice in a microcentrifuge.

3. Resuspend the cells in 200 μL of the following solution:

• ​5 mL SCE*
• ​60 μL 10 mg/mL 100T Zymolyase
• ​10 μL 2-ME (M755744)

4. Vortex to mix, and incubate at 37 °C for 30–60 minutes.

5. Add 400 μL 0.2 N NaOH/1% SDS (S767322), mix by gently inverting, and incubate on ice for 5 minutes.

6. Add 300 μL 3 M K/5 M potassium acetate (P108329), pH 4.5, mix by gently inverting, and incubate on ice for 5 minutes.

7. Centrifuge in a microcentrifuge for 2 minutes, pour the supernatant into a new centrifuge tube, and centrifuge again. Transfer 500 μL of the supernatant to a new centrifuge tube.

8. Add 300 μL isopropanol (I112021), vortex to mix, and let stand at room temperature for 5 minutes.

9. Centrifuge in a microcentrifuge for 5 minutes to collect the DNA precipitate, wash the precipitate with 70% ethanol and centrifuge again.

10. Dry the precipitate in a vacuum concentrator and resuspend it in 25 μL TE buffer (T476214).

11. Transfer 2 μL of the sample into electrocompetent Escherichia coli, and after recovery, culture all on plates.

*SCE buffer formulation:

1 M sorbitol (D755727)

0.1 M sodium citrate (S189183), pH 7.6

0.06 M EDTA (E278775)

Yeast Transformation Methods and Reagents

Introduction:

Yeast transformation can be achieved through various methods, among which the lithium ion treatment method and protoplast fusion method are the most widely used. These methods differ significantly in transformation efficiency, and the use of different reagents can also affect the results. For example, in protoplast transformation, the quality of polyethylene glycol (PEG) is crucial for DNA uptake efficiency, and its batch testing method is similar to the evaluation of serum quality in cell culture. This study compared the transformation effects of the lithium ion method and the protoplast fusion method, and evaluated the differences between Zymolyase and Lyticase as cell wall-degrading enzymes in protoplast transformation.

Materials and Methods:

• Yeast Strains

All transformation experiments used Saccharomyces cerevisiae INVSc1 from Invitrogen. The phenotype of this yeast is MAT his3(Δ)1 leu2 trp1-289 ura3-52. The transformation experiment utilized its leu phenotype, and transformants were selected through LEU2 complementation.

• Plasmid DNA

Plasmid YEp351/A/M was used as control DNA in the transformation experiment. This plasmid contains LEU2, which can achieve leucine complementation to select transformants. Meanwhile, the plasmid carries an easily detectable marker gene for verification. The plasmid contains a fusion of the ADC1 promoter and the MEL1 coding region, which allows MEL1 to be expressed in a glucose environment and synthesize β-galactosidase. β-galactosidase can be detected by various chromogenic substrates, such as using the enzymatic cleavage reaction of X-β-Gal.

1.Lithium Cation Method for Transforming Saccharomyces cerevisiae

Materials

• YPD liquid medium (Y778413)
• TE buffer (T476214)
• 100 mM lithium acetate (L102778, dissolved in TE buffer, sterilized)
• Plasmid DNA
• 50% PEG 4000 (P615468, aqueous solution, sterilized)
• 1 M sorbitol (D755727, dissolved in TE buffer)
• Leu selective agar plates: 1% glucose (D755713), 0.67% yeast nitrogen source (Y110517), 1 M sorbitol (D755727)

Protocol

12. Culture overnight in YPD liquid medium (2% glucose, 2% peptone, 1% yeast extract) at 30°C with shaking (aerated). The cell density of the culture should be approximately 1×10^7 cells/mL.

13. For each transformation, take 4.5 mL of the culture, centrifuge at 3000 rpm for 5 minutes to collect the yeast pellet.

14. Resuspend the yeast in 4 mL TE buffer, and centrifuge again at 3000 rpm for 5 minutes.

15. Carefully resuspend the yeast in 3 mL 100 mM lithium acetate/TE buffer, and shake gently at room temperature for 30 minutes.

16. Centrifuge at 2000 rpm for 5 minutes, carefully remove the supernatant, resuspend the yeast in 100 μL lithium acetate/TE solution, and transfer to a 1.5 mL microcentrifuge tube. The final density of the yeast at this point is approximately 5×10^8 cells/mL.

17. Add no more than 10 μg of plasmid DNA (total volume no more than 10 μL) to the yeast suspension, and incubate at 30°C for 30 minutes without shaking.

18. Add 300 μL 50% PEG solution, mix, and incubate at 30°C for 1 hour without shaking.

19. Heat shock at 42°C for 5 minutes.

20. Immediately centrifuge for 20 seconds, carefully remove the PEG solution, and resuspend the cells in 1 mL sorbitol/TE solution.

21. Spread the cells on selective medium plates, and incubate in an inverted position after the surface of the plates is dry.

2.Protoplast Method for Transforming Saccharomyces cerevisiae

Materials

• 1 M sorbitol (D755727), 25 mM EDTA (E278775), pH 8 50 mM DTT (D104859). The sorbitol/EDTA solution is prepared and sterilized separately. Molecular biology-grade 1 M DTT stock solution (sterile filtered) is aliquoted into 500 μL portions, frozen and stored. Before use, add DTT to 9.5 mL sorbitol/EDTA solution.
• 1 M sorbitol (autoclaved)
• 1 M sorbitol, 1 mM EDTA, 10 mM sodium citrate buffer (S189183), pH 5.8 (for Zymolyase)
• 1 M sorbitol, 1 mM EDTA, 10 mM Tris buffer (T755536), pH 7.5 (for Lyticase)
• Zymolyase (dissolved in water), 60 U/mL
• Lyticase (L303583, dissolved in water), 1200 U/mL
• 1 M sorbitol, 10 mM Tris, pH 7.5, 10 mM CaCl₂ (C431202)
• 20% PEG (P615468)/10 mM Tris, pH 7.5, 10 mM CaCl₂ (prepared fresh)
• Leu selective agar plates: 1% glucose (D755713), 0.67% yeast nitrogen source (Y110517), 1 M sorbitol (D755727)
• Leu selective top agar tubes: 10 mL 1% glucose, 0.67% yeast nitrogen source, 1 M sorbitol, contained in large test tubes with foam stoppers; melted and maintained at 47°C when in use
• Sterile molecular biology-grade water
• 5% SDS (S767322, aqueous solution)

Protocol

1. Streak the frozen preserved yeast onto YPD agar plates (1% yeast extract, 2% peptone, 2% glucose, 2% agar), and culture at 28–30°C for 24–48 hours.

2. Inoculate a single colony into 100 mL YPD liquid medium (1% yeast extract, 2% peptone, 2% glucose) in a 500 mL Erlenmeyer flask, and culture overnight with shaking at 28°C, 250–300 rpm.

3. Measure the OD₅₂₀ of the culture the next day (zero the spectrophotometer with sterile YPD). If necessary, dilute to a readable OD within the range of 0.1–1.0.

• ​If the OD is between 0.2–0.3, collect the cells directly.
• ​Or dilute to OD 0.2 and continue culturing until OD 0.3, then collect the cell pellet by centrifugation at 1500×g for 10 minutes at room temperature.

4. Thoroughly resuspend the cell pellet in 10 mL sterile water, and transfer to a sterile 15 mL screw-cap tube.

5. Centrifuge at 1500×g for 5 minutes at room temperature, discard the supernatant, and retain the cell pellet.

6. Resuspend the yeast in 10 mL freshly prepared 1 M sorbitol, 25 mM EDTA, pH 8, 50 mM DTT, centrifuge at 1500×g for 5 minutes at room temperature.

7. Resuspend the cells in 10 mL 1 M sorbitol, centrifuge at 1500×g for 5 minutes at room temperature.

8. Resuspend the cells in 10 mL sorbitol/citrate buffer (1 M sorbitol, 1 mM EDTA, 10 mM sodium citrate buffer, pH 5.8), take 1.6 mL of the cell suspension and add 2.5 mL Zymolyase to detect the time and efficiency of protoplast formation; retain the remaining cells.

9. Resuspend the cells in 10 mL sorbitol/Tris buffer (1 M sorbitol, 1 mM EDTA, 10 mM Tris, pH 7.5), take 1.6 mL of the cell suspension and add 2.5 mL Lyticase to detect the time and efficiency of protoplast formation; retain the remaining cells.

10. After adding cell wall lytic enzymes (Zymolyase or Lyticase), incubate at 30°C, take 200 μL samples at 0, 5, 10, 20, 30, 40, 50, and 60 minutes respectively into 2 mL 1 M sorbitol, then add 800 μL 5% SDS, mix, and measure OD₈₀₀ to evaluate cell lysis (using 1 M sorbitol as blank control).

11. Plot the OD₈₀₀–time curve, and calculate the protoplast formation efficiency: Efficiency = (OD₈₀₀ at each time point / initial OD₈₀₀) × 100; transformation can be performed when the protoplast efficiency reaches 70%.

12. Add 13 mL Lyticase or Zymolyase to the remaining cells, and incubate for the measured time until the protoplast efficiency reaches 70%. Note to operate gently to avoid protoplast rupture.

13. Centrifuge at 750×g for 10 minutes at room temperature, discard the supernatant, and retain the pellet.

14. Gently resuspend the protoplasts in 1 mL 1 M sorbitol, centrifuge at 750×g at room temperature.

15. Resuspend the protoplasts in 5 mL 1 M sorbitol, 10 mM Tris (pH 7.5), 10 mM CaCl₂, centrifuge and resuspend in 300 μL of the same solution.

16. For each transformation, take 100 μL of protoplasts into a sterile 15 mL centrifuge tube, add 10 μg of YEp351/A/M control plasmid, and incubate at room temperature for 10 minutes.

17. Add 1.0 mL of freshly prepared 20% PEG / 10 mM Tris (pH 7.5), 10 mM CaCl₂, mix gently, and incubate at room temperature for 10 minutes.

18. Centrifuge at 750×g for 10 minutes at room temperature, carefully discard the PEG solution, and invert the tube to drain excess liquid.

19. Gently resuspend the protoplasts in 1 mL 1 M sorbitol.

20. Take 100 μL of protoplasts and add to 10 mL of melted top agar, pour onto selective plates, and let solidify.

21. Invert the plates and culture at 28–30°C; transformed colonies can be seen in 4–6 days.

Results and Discussion

Successful transformation of Saccharomyces cerevisiae was achieved using YEp351/A/M. Transformation efficiency was significantly affected by the method and the type of lyase. The efficiency of the protoplast transformation method (Zymolyase: 652 transformants/10 µg plasmid; Lyticase: 53 transformants/10 µg) was much higher than that of the lithium ion transformation method (6 transformants/10 µg). Although protoplast transformation has high efficiency, it requires removal and regeneration of the cell wall, with higher operational difficulty and cost; the lithium acetate method is simple but has lower efficiency.

The difference in lyases is the main reason for the variation in protoplast transformation efficiency. Zymolyase is composed of various carbohydrate hydrolases (such as β-1,3-glucan pentasaccharide hydrolase, β-1,3-glucanase, protease, and mannosidase, etc.), which can synergistically degrade the cell wall, retaining sufficient cell wall structure for regeneration while forming approximately 70% protoplasts. In contrast, Lyticase has limited degradation specificity, which may lead to insufficient or excessive removal of the cell wall, thereby reducing fusion and regeneration efficiency.

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