1.1 LB Medium

Materials:

• 1% (w/v) tryptone
• 1% (w/v) NaCl
• 0.5% (w/v) yeast extract
• 1.5% (w/v) agar
• Ampicillin (100 μg/mL)

Procedure:

(1) Weigh 1% (w/v) tryptone, 1% (w/v) NaCl, 0.5% (w/v) yeast extract, and 1.5% (w/v) agar in a 250 mL conical flask and dissolve in deionized ddH2O.

(2) Autoclave the mixture at 121°C for 20 min.

(3) Allow the solution to cool to 50-60°C.

(4) Add ampicillin to a final concentration of 100 μg/mL. Gently mix the contents.

(5) Aseptically pour the medium into culture plates and allow to solidify.

(6) Store the plates at 4°C overnight.

1.2 YPD Medium

Materials:

• 2% (w/v) tryptone
• 2% (w/v) yeast extract
• 2% (w/v) glucose
• 1.5% (w/v) agar
• Kanamycin (50 μg/mL)
• Ampicillin (100 μg/mL)

Procedure:

(1) Weigh 2% (w/v) tryptone, 2% (w/v) yeast extract, 2% (w/v)

glucose, and 1.5% (w/v) agar in a 250 mL conical flask and dissolve in deionized ddH2O.

(2) Autoclave the glucose solution at 116°C for 15 min.

(3) Allow the solution to cool to 50-60°C.

(4) Add kanamycin and ampicillin to a final concentration of 50 μg/mL and 100 μg/mL, respectively. Gently mix the contents.

(5) Aseptically pour the medium into culture plates and allow to solidify.

(6) Store the plates at 4°C overnight.

Inoculate strains containing the following plasmids into 5 mL of liquid LB medium supplemented with 100 μg/mL ampicillin:

Materials and Equipment:

• RNase A
• Buffer SP1, SP2, and SP3
• Wash Solution
• 100% ethanol
• Miniprep columns
• Collection tubes
• LB bacterial culture
• Centrifuge
• Vortex mixer

Procedure:

(1) Prepare buffers:

a) Add RNase A to Buffer SP1.

b) Add indicated volume of 100% ethanol to Wash Solution.

c) Check Buffer SP2 and SP3 for precipitation.

(2) Equilibrate column:

a) Place Miniprep column in collection tube.

b) Add 500 μL Buffer S, centrifuge at 12,000 ×g for 1 min.

c) Discard filtrate, return column to collection tube.

(3) Harvest bacterial cells:

a) Centrifuge 1.5-5 mL overnight LB culture at 8,000 ×g for 2 min.

b) Discard supernatant.

(4) Resuspend pellet in 250 μL Buffer SP1, vortex thoroughly.

(5) Add 250 μL Buffer SP2, mix by inverting 10 times.

(6) Add 350 μL Buffer SP3, mix immediately by inverting 10 times.

(7) Centrifuge at 12,000 ×g for 10 min.

(8) Transfer supernatant to column, centrifuge at 8,000 ×g for 30 sec.

(9) Discard filtrate, return column to collection tube.

(10) Add 500 μL Wash Solution, centrifuge at 9,000 ×g for 30 sec.

(11) Repeat step 10.

(12) Centrifuge empty column at 9,000 ×g for 1 min.

(13) Transfer column to clean 1.5 mL microcentrifuge tube.

(14) Add 50 μL ddH2O to center of membrane.

(15) Incubate for 1 min at room temperature.

(16) Centrifuge at 9,000 ×g for 1 min to elute DNA.

Materials and Equipment:

• PrimeStar Max Premix (2×)
• Forward and reverse primers (10 μM each)
• DNA template
• Nuclease-free water
• Thermal cycler
• PCR tubes

Procedure:

Materials and Equipment:

• Electrophoresis equipment
• 1×TAE buffer
• Agarose (0.8% w/v)
• YeaRed™ nucleic acid dye
• DNA loading buffer
• DNA marker
• UV transilluminator

Procedure:

(1) Preparation of 1×TAE solution

Dilute 50×TAE concentrates to a working concentration (1×), i.e., add 20 mL of the 50× TAE concentrates mix to 980 mL of ddH2O.

(2) Preparation of 0.8% DNA agarose gel

a) Weigh 0.64 g agarose powder using a balance.

b) Pour 80 mL 1× TAE solution using a measuring cylinder.

c) Add the 1× TAE solution and agarose powder into a conical flask and mix them well.

d) Cover the conical flask and heat it in a microwave until all powder is dissolved completely.

e) Cool agarose solution to about 60 °C and add YeaRed™ nucleic acid gel stain to a final concentration of 0.1 μL/mL.

f) Pour gel solution into gel tray and insert well comb. Wait for 20-30 min until the gel is solidified.

(3) Electrophoresis conditions

a) Remove the comb and place the solidified agarose gel into the horizontal electrophoresis bath.

b) Pour the TAE solution into the bath, and make sure the solution cover the surface of the agarose gel

c) Prepare DNA samples by mixing the 6× DNA loading buffer with PCR products at a ratio of 1: 5.

d) Load 5 μL DNA marker and 3.5 μL PCR products into wells of solidified agarose gel.

e) Run gel electrophoresis at 150 V in 1× TAE buffer until sufficient migration (at about 25-30 min).

f) Visualize DNA bands under UV light.

Materials and Equipment:

• Gel extraction kit
• Buffer B2
• Wash Solution
• Elution Buffer
• Heating block (50°C)
• Microcentrifuge

Procedure:

(1) Excise DNA band from agarose gel.

(2) Add three volume Buffer B2 to gel slice.

(3) Incubate at 50°C for 5-10 min until gel is dissolved.

(4) Transfer mixture to spin column, centrifuge at 8,000 ×g for 30 sec.

(5) Discard flow-through.

(6) Add 500 μL Wash Solution, centrifuge at 8,000 ×g for 30 sec.

(7) Repeat step 6.

(8) Centrifuge empty column at 8,000 ×g for 2 min.

(9) Place column in new 1.5 mL microcentrifuge tube.

(10) Add 30 μL ddH2O to column membrane.

(11) Incubate at room temperature for 1 min.

(12) Centrifuge at 8,000 ×g for 1 min to elute DNA.

Materials and Equipment:

• AscI and PmeI restriction enzymes
• 10×CutSmart buffer
• Plasmid DNA and target fragment
• Nuclease-free water
• Thermal cycler
• Gel extraction kit

Procedure:

Materials:

• T4 DNA Ligase
• 10×T4 DNA Ligase buffer
• Vector and insert DNA
• Nuclease-free water

Procedure:

Materials and Equipment:

• Plasmid DNA
• Competent E. coli DH5α cells
• Ice bath
• 42°C water bath
• Antibiotic-free liquid medium
• Solid medium with antibiotics
• L-shaped spreader
• Incubator (37°C)

Procedure:

(1) Add 2.5 μL plasmid DNA to 50 μL competent E. coli DH5α cells.

(2) Incubate on ice for 30 min.

(3) Heat shock at 42°C for 90 sec.

(4) Incubate on ice for 2 min.

(5) Add 900 μL antibiotic-free liquid medium.

(6) Incubate at 37°C, 200 rpm for 1 h.

(7) Centrifuge at 3,000 rpm for 5 min.

(8) Resuspend pellet in 100 μL medium.

(9) Spread on solid medium with appropriate antibiotics.

(10) Incubate plates at 37°C overnight.

Materials and Equipment:

• 2× Hieff Ultra-Rapid II HotStart PCR Master Mix
• Forward and reverse primers (10 μM each)
• Bacterial colonies
• Nuclease-free water
• Thermal cycler

Procedure:

(1) Pick 8 well-isolated colonies from the transformation plate onto a new LB agar plate, and incubate at 37 ℃ for 6 h.

(2) Pick the colonies, resuspend them in 10 μL of sterile water, and the resuspension is lysed at 98 °C for 10 min.

(3) Centrifuge the tubes for 5 min to pellet the cell debris.

(4) Add 1 μL of the supernatant as a DNA template and followed by PCR amplification according to the following table:

(5) After amplification, analyze 3.5 μL of the PCR reaction by agarose gel electrophoresis. The procedure is as previously described.

(6) Inoculate the positive transformants in an LB culture medium containing antibiotics at 200 rpm, 37 ℃ overnight.

(7) Extracted the integrated plasmids as described above.

Materials and Equipment:

• NotI restriction enzyme
• 10× CutSmart buffer
• Plasmid DNA
• Nuclease-free water
• Thermal cycler
• Agarose gel electrophoresis equipment

Procedure:

Materials:

• S. cerevisiae strain (1974-Cas)
• YPD and 2×YPD media
• Sterile water
• Integrated fragment
• gRNA plasmid
• Salmon sperm ssDNA (10 mg/mL)
• LiAc, PEG, and other transformation reagents
• Selection plates (Amp+Kan)

Procedure:

(1) Preparation of Yeast Culture

a. Pick a single colony and inoculate it into a test tube containing 3 mL of YPD liquid medium.

b. Incubate overnight at 30°C with shaking at 240 rpm.

c.Dilute the overnight culture 10-fold and measure OD600. The OD600 should be greater than 0.2.

d. Inoculate a 250 mL Erlenmeyer flask containing 25 mL of 2×YPD medium with the overnight culture to an initial OD600 of 0.2.

e. Incubate at 30°C with shaking at 240 rpm until the OD600 reaches 0.8-1.0.

(2) Preparation of Competent Cells

a. Centrifuge the culture at 3,000 rpm for 5 min at 20°C to collect the cells.

b. Resuspend the cell pellet in 25 mL of sterile water.

c.Centrifuge at 3,000 rpm for 5 min at 20°C. Repeat this washing step twice.

d. Resuspend the cell pellet in 1 mL of sterile water and transfer to a 1.5 mL microcentrifuge tube.

e. Centrifuge at 12,000 rpm for 30 sec and discard the supernatant to obtain competent yeast cells.

f. Preheat a water bath to 42°C and a heat block to 100°C.

(3) Transformation Procedure

a. Prepare carrier DNA by heating 2 mg/L salmon sperm ssDNA at 100°C for 5 min, then immediately place on ice.

b. Add the following components to microcentrifuge tubes containing competent cells in the order and amount specified in the table below.

c. Heat-shock the mixture at 42°C for 15 min.

d. Centrifuge at 12,000 rpm for 30 sec and discard the supernatant.

e. Add 1 mL of YPD liquid medium and resuspend the cell pellet.

f. Seal the tube with parafilm.

g. Incubate in a shaking incubator at 30°C and 240 rpm for 2 h to allow cell recovery.

h. Centrifuge at 3,000 rpm for 4 min.

i. Discard 600 μL of the supernatant and resuspend the cells in the remaining liquid.

j. Spread 200 μL of the cell suspension evenly onto a selective solid medium plate.

k. Incubate the plates inverted at 30°C for two days.

Materials and Equipment:

• Yeast Lysis Buffer
• Neutralization Buffer
• 2×Yeast Colony PCR Mix
• Thermal cycler

Procedure:

(1) Add 10 µL of Yeast Lysis Buffer to a PCR tube.

(2) Transfer 0.2-1 mm of yeast monoclone from a sterile suction head into the PCR tube containing Yeast Lysis Buffer.

(3) Mix thoroughly by gentle pipetting or vortexing, then centrifuge briefly to collect the liquid at the bottom of the tube.

(4) Incubate at 95°C in a thermal cycler for 5 min to fully release yeast DNA.

(5) Add 10 µL of Neutralization Buffer and mix well. Use this mixture as the DNA template for PCR detection and followed by PCR amplification according to the following table:

(8) Inoculate the positive transformants in an YPD culture medium containing antibiotics at 220 rpm, 30 ℃.

(9) The positive yeast cells were expanded in culture, prepared again as a sensory state using the method described above, and transformed by LiAc method

(1) RNA Extraction from Yeast Cells

Culture yeast strains in YPD medium and harvest cells at the logarithmic growth phase. Extract total RNA using the S. cerevisiae RNA extraction kit (TRNzol Universal Total RNA Extraction Reagent, DP424) following the manufacturer's protocol:

a. Resuspend 5×10⁶ - 1×10⁷ cells in 1 mL TRNzol Universal reagent.

b. Incubate the homogenized sample at room temperature for 5 min to ensure complete dissociation of nucleoprotein complexes.

c. Add 0.2 mL chloroform per 1 mL of TRNzol Universal reagent used. Cap the tube securely and shake vigorously for 15 sec. Incubate at room temperature for 3 min.

d. Centrifuge at 12,000 rpm for 15 min at 4°C. The mixture will separate into three phases: a lower red organic phase, an interphase, and a colorless upper aqueous phase containing RNA.

e. Transfer the aqueous phase (approximately 500 µL) to a new microcentrifuge tube.

f. Add an equal volume of isopropanol to the aqueous phase, mix well, and incubate at room temperature for 10 min.

g. Centrifuge at 12,000 rpm for 10 min at 4°C. Discard the supernatant.

h. Wash the RNA pellet with 1 mL of 75% ethanol (prepared with RNase-free water).

i. Centrifuge at 10,000 rpm for 5 min at 4°C. Carefully remove the ethanol without disturbing the pellet.

j. Air-dry the RNA pellet at room temperature for 2-3 min.

k. Resuspend the RNA in 50 µL of RNase-free water, depending on the experimental requirements.

(2) Quantitative PCR

Materials and Equipment:

• RNA template
• Primers specific to the target gene
• SYBR Green
• qPCR Master Mix
• Nuclease-free water
• 96-well PCR plate
• qPCR machine

Procedure:

a. Add the reaction reagents listed below to a sterile nuclease-free tube placed on ice.

b. Mix gently and incubate at 25°C for 10 min.

c. Incubate at 50°C for 30 min.

d. Stop the reaction at 85°C for 5 min and immediately cool the sample on ice.

e. Analyze the amplification plots to determine the Ct (cycle threshold) values for each sample.

f. Use the Ct values to quantify gene expression or DNA copy number.

g. Normalize the target gene expression to a reference gene (ACT1) and use the 2^-ΔΔCt method for analysis.

(1) Preparation of β-Carotene Standard Curve

a. Prepare a 50 mg/L β-carotene stock solution by dissolving 1.25 mg of β-carotene standard in 10 mL of n-hexane and diluting to 25 mL in a volumetric flask.

b. Prepare a dilution series of 2.5, 5, 7.5, 10, 15, 20, and 25 mg/L from the stock solution.

c. Use n-hexane as a blank control.

d. Add 200 µL of each standard (in triplicate) to wells of a microplate.

e. Measure absorbance at 450 nm using a plate reader.

f. Plot the standard curve with absorbance values on the y-axis and β-carotene concentration (mg/L) on the x-axis.

(2) β-Carotene Extraction and Quantification

a. To the 0.2 mL cell suspension, add 200 µL of sterile water, 0.5 mm glass beads, and 1 mL of chloroform.

b. Disrupt cells by vortexing for 3 min, followed by a 1-min ice bath. Repeat this cycle 3-4 times.

c. Centrifuge at 4°C for 1 min and collect the supernatant in a 5 mL centrifuge tube.

d. Repeat the extraction process 3-4 times until the cell pellet is colorless.

e. Evaporate the collected supernatant under a stream of nitrogen.

f. Resuspend the dried extract in 1 mL of hexane.

g. Measure the β-carotene concentration using the prepared standard curve and calculate the yield.