a.Relevant culture media

i．YPD medium
This medium is used for culturing the original strain CENPK113-11C：

Component	Dosage
Tryptone	2g
Yeast extract	1g
Glucose	2g
ddH2O	To 100ml
Agar powder (solid)	1.8-2.0g

ii．SD medium（+Ura/His）
This medium is used for transformation cultivation and fermentation：

Component	Dosage
YNB	0.67g
Glucose	2g
ddH2O	To 100ml
Ura	0.002g
His	0.002g
Agar powder (solid)	1.8-2.0g

iii．Deft-D medium
This medium is used for the fermentation of the transformed bacterial strains,
Step1（Reserve）:

Component	Dosage
(NH4)2SO4	2.5g
KH2PO4	14.4g
MgSO4·7H2O	0.5g
Ura	0.06g
His	0.04g
ddH2O	To 900ml

Step2（Before use）:

Component	Dosage
Step1	900ml
200g/l Glucose	100ml
Vitamin	1ml
Trace Metal Solution	2ml

iv．SD medium+4-Pyrimidinecarboxylic acid
This medium is used for discarding plasmids, such as gRNA plasmids.

Component	Dosage
SD medium (+ His/Ura)	100ml
4-Pyrimidinecarboxylic acid	0.1g

v．LB medium（+Amp）
This medium is used for screening Escherichia coli that carry the Amp resistance marker.

Component	Dosage
Tryptone	2g
Yeast extract	1g
Sodium chloride	2g
Agar powder	3g
Purified water	200ml
Ampicillin	20mg

Strain Preservation for Experimental Samples
Strain preservation is used for storing experimental samples to ensure the continuity of future experiments.
First, add 80% glycerol (note: glycerol is viscous, so precise measurement of volume is required), followed by the addition of the cell suspension.

Preservation Type	80% glycerol	Bacterial culture
Standard Strains	250μL	750μL
Competent cells	65μL	250μL

b.Saccharomyces cerevisiae Transformation Experiment

Objective: Efficiently introduce exogenous DNA (such as plasmids or linear DNA fragments) into Saccharomyces cerevisiae cells to achieve gene editing or functional validation.
Principle: The use of lithium acetate (LiAc) increases the permeability of the yeast cell wall, and polyethylene glycol (PEG) is used to promote the fusion of DNA with the cell membrane. Single-stranded vector DNA (ssDNA) can protect exogenous DNA from being degraded by intracellular nucleases.
1. On the first day evening (16:00-18:00), pick the monoclonal cells and put them into 2mLYPD medium, and cultivate them overnight at 220rpm and 30°C constant temperature for 12-16h;
2. In the morning of the second day, transfer the overnight-cultured seed strain (diluted 20 times, with a spectrophotometer reading of OD600 between 0.2 and 0.8) to 20mLYPD medium (100mL shake flask) to ensure an initial OD600 of 0.1. Cultivate for 6-8 hours until the OD600 reaches 0.6-1.0.
3. Transfer the bacterial suspension to a 50 mL centrifuge tube and centrifuge at 1000g for 5 minutes at 4°C to collect cells. Discard the supernatant, add 1mL0.1M lithium acetate (LiAc) solution to resuspend the cells, then transfer to a 1.5mL centrifuge tube. Centrifuge at 1200g for 2 minutes (small centrifuge) to collect cells, discard the supernatant, and resuspend in 200 μL 0.1M LiAc solution. Distribute 25-35 μL into individual 1.5mL centrifuge tubes. Centrifuge at 4°C for 21 minutes (small centrifuge) to collect cells, discard the supernatant, and directly add the transformation system.
4. Prepare the conversion solution according to the following table (volume: μL)

Number of conversions	1	2	6
PEG350050%w/v	120	240	720
1.0MLiAc	25	50	150
SSDNA（2mg/mL）	120	240	720
Plasmid or fragment DNA	X	2X	6X
Sterilized water	17-X	34-2X	192-6X
competent cell	25	25*2	25*6
total volume	180	360	1080

(When there are multiple conversions in one batch, first prepare the premix solution, then proceed with aliquoting and sample addition; SSDNA should be heated at 99°C for 10 minutes before use, quickly placed on ice and stored in a -20°C refrigerator)

5. Place the transformation system in a 30°C water bath for 30 minutes; then transfer it to a 42°C water bath for heat shock for 25 minutes; immediately place it on ice for 2 minutes;
6. Centrifuge at 1000g for 1 minute at room temperature, remove the supernatant, add 100-150 μL. If it is a nutritional deficiency screening, directly spread on the plate; if it is a resistance marker screening, add 500 μL of LYPD liquid medium for 2 hours to express the resistance gene, then centrifuge at 1200g for 2 minutes to collect the cells, remove 500 μL of the supernatant, resuspend the cells. (For plasmids or plasmid recombination, spread 25-50 μL; for chromosome integration or gene knockout, spread 100-180 μL)
7. Cultivate in a 30°C incubator for 2-4 days until the transformants appear.
Troubleshooting:

Problem	Possible cause	Solution
No transformant colonies	1. Low efficiency of receptive state cells 2. Poor or insufficient DNA quality 3. Screening for tablet failure	1. Strictly control the OD600 between 0.6 and 1.0 when collecting cells 2. Use NanoDrop to detect DNA concentration and purity 3. Prepare fresh screening plates
Slow colony growth rate	1. The integration fragments impose a burden on cell growth 2. Overly high screening pressure	1. Extend the culture time or enrich in liquid medium before plate drawing 2. Check whether the concentration of screening reagent is correct

c．Gene editing

Objective: Utilize the CRISPR-Cas9 system and the homologous recombination repair mechanism to achieve precise gene knockout or insertion at specific loci in the yeast genome.
Principle: The gRNA plasmid guides the Cas9 protein to cause DNA double-strand breaks (DSB) at specific loci in the genome. The cell repairs this break through the homologous recombination (HR) mechanism, using a linear repair template co-transformed and having both ends with homologous arms, to integrate the sequence on the template into the genome.

i．Fragment amplification and purification with homologous arms

1.Using primers with specific overlapping sequences, all the basic fragments for splicing are amplified from the corresponding DNA templates (such as plasmids or genomes) through PCR reactions. Among them, the fragments at both ends of the final fusion product are the upstream and downstream homologous sequences of the cutting sites for the target gRNA-guided cas9 protein.
2.All PCR products are detected by agarose gel electrophoresis to confirm the correct and single band size. Then, they are cut and purified using a gel recovery kit.
3.Precisely determine and record the DNA concentration of each purified fragment to prepare for the subsequent multi-fragment fusion.

ii．The first round of OE-PCR (multi-fragment fusion)

1. Based on the size, concentration and location of the genes to be integrated, calculate the required template volume X.
2. Use this mixed DNA as the template and perform the first round of overlapping extension PCR without adding any primers. This will gradually concatenate multiple independent short fragments into a complete, linear long-chain DNA molecule.

iii．The second round of PCR (amplification of the fusion product)

1. Use the products from the first round of OE-PCR as the template, and add a pair of external primers specifically targeting the sequences at both ends of the entire fusion fragment to conduct a second round of PCR amplification, selectively and massively amplifying the full-length target product.
2. After the amplification is completed, re-identify through agarose gel electrophoresis, cut out the correct-sized target band and perform gel recovery.

iv．Yeast co-transformation and gene editing

The repaired template DNA and corresponding gRNA plasmids were co-transformed into yeast recipient cells according to the method of yeast transformation experiment. Gene insertion/ knockout was achieved by homologous recombination repair.

v．Screening and validation

1. Select several independent yeast single colonies from the transformation plates and perform transfer culture.
2. Extract the genomic DNA of the bacterial culture using the quick genomic extraction method. Use specific verification primers and a verification PCR system and procedure to test the genome of each colony.
3. Analyze the band size of the PCR products through agarose gel electrophoresis, and compare and identify the positive clones that have the correct gene insertion or knockout.
Troubleshooting:

Problem	Possible cause	Optimization plan
No target bands or diffuse bands in OEC-PCR	1. The molar ratio of each fragment template is not good 2. Unreasonable design of overlapping region sequences (low Tm value or secondary structure)	1. Optimize the pyramid-shaped molar ratio with the highest amount of central fragment 2. Redesign the primers to ensure that the Tm value of the overlapping region is 55-65°C
No positive clones were verified by colony PCR	1. Low efficiency of gRNA 2. The homologous arms of the repair template are too short 3. Cells tend to repair through non-homologous end joining (NHEJ)	1. Redesign or replace with a more efficient gRNA 2. Extend the length of homologous arms to 40-60bp 3. Consider knocking out key genes of NHEJ pathway to improve HR efficiency

d．Microencapsulation

Objective: To build a physical protective barrier for yeast cells using sodium alginate and chitosan to enhance their survival in harsh environments such as simulated gastric fluid.
Principle: The method utilizes ion gelation, where divalent cations (Ca²+) cross-link with carboxyl groups on sodium alginate molecular chains to form a stable gel network. The positively charged chitosan coating and negatively charged sodium alginate combine through electrostatic interactions, creating a more compact composite membrane.

i．Microcapsule preparation

1. Mixing: Add the prepared yeast cell suspension to the sodium alginate solution (mix 5 mL of yeast suspension into 50 mL of 3% (w/v) sodium alginate solution) and mix thoroughly. A study indicates that adding yeast suspension to a sodium alginate solution with a concentration of 3 g/100cm³ proves effective.
2. Gelation: Using a syringe, drop the above mixture into a pre-prepared 2g/100cm³ calcium chloride solution. This step should ensure that the droplets solidify quickly into uniform gel beads.
3. Primary curing: The gel beads are soaked in calcium chloride solution for about 30 minutes to ensure that the calcium alginate gel network is fully crosslinked and stable primary microcapsule particles are formed.
4. Secondary Encapsulation: After removing the solidified calcium alginate gel beads from the calcium chloride solution, they are rinsed with sterile water and immediately transferred to a chitosan solution at 0.38g/100cm³. The gel beads are then immersed in the chitosan solution for 60 minutes to complete the secondary encapsulation process.
5. Cleaning and drying: The microencapsulated particles after secondary coating are washed with sterile water to remove excess chitosan on the surface. Then, other mild drying methods such as freeze-drying can be selected according to application requirements.

ii．Micro capsule validation

1. Set up four groups:
Group 1: Take 100 microliters of yeast solution and place it in 5 ml of gastric juice for two hours.
Group 2: Take 100 microliters of yeast solution and place it in 5 ml of ddH2O for two hours.
Group 3: 100 microliters of yeast solution was prepared according to the above microcapsule preparation steps and placed in 5ml gastric juice for two hours.
Group 4: Take 100 microliters of yeast solution and prepare it according to the above microcapsule preparation steps and place it in 5ml ddH2O for two hours.
For Group 1 and Group 2, the microcapsules were directly coated after two hours. For Groups 3 and 4, the microcapsules were removed after two hours and transferred to a buffer solution containing 2 mL of sterile 5% sodium citrate for dissolution. The mixture was vigorously vortex-oscillated for 10-15 minutes to ensure complete dissolution and uniform release of yeast. The resulting bacterial suspension was then coated with the disintegrated microcapsules.
Troubleshooting:

Problem	Possible cause	Optimization plan
The gel beads are not formed or have a severe streaking effect	1. Low concentration of sodium alginate 2. Insufficient calcium chloride concentration	1. Properly increase the concentration of sodium alginate (e.g., to 4%) 2. Ensure that the concentration of calcium chloride solution is not less than 2%
The size of microcapsules is not uniform	1. Manual drip is unstable 2. Improper choice of syringe needle	1. Use an injection pump to drip at a constant rate 2. Replace different types of needles to control droplet size
The survival rate of yeast is low after being wrapped	1. Contamination caused by non-sterile operation 2. Too long soaking time or toxic reagent	1. Sterile operation is performed in the laminar flow unit throughout 2. Optimize the curing and coating time to ensure that the reagents used are at the cell culture level

a．Rapid genomic extraction

1. Resuspend the overnight culture uniformly by vortex or gentle inversion. Aspirate 100μL of the culture into a clean 2.0mL microcentrifuge tube.
2. At room temperature, use a microcentrifuge at 13,500 x g relative centrifugal force for 1 minute to precipitate the bacterial cells at the bottom of the tube.
3. Aspirate the supernatant with a pipette, and pay attention not to touch or suck the cell precipitate at the bottom of the tube.
4. Add 100μL of rapid lysis buffer to the centrifuge tube containing the cell precipitate. Vortex 10-15 seconds until the precipitate is completely resuspended and the solution becomes homogeneous.
5. Place the centrifuge tube in a constant temperature homogenizer or water bath at 70℃ and incubate for 7 minutes.
6. Add 300μL anhydrous ethanol to the lysis solution and vortex for 1min. Centrifuge at 13,500×g for 3 minutes. Carefully pour off the ethanol supernatant.
7. Gently add 300μL of 70% ethanol to the tube and vortex for 1 min. Centrifuge at 13,500×g for 3 minutes to resuspend the DNA precipitate, then pour off the ethanol wash.
8. Open the centrifuge tube and place it on a constant temperature homogenizer or water bath at 60℃ until no visible liquid residue is left. Add 80μL ddH2O.
9. Centrifuge at 13,500 ×g for 1 minute. At this time, the supernatant contains dissolved DNA, which is the final genomic DNA sample.

b．Plasmids extraction

1. Take 2mL of the bacterial solution and add 2mL to a centrifuge tube. Centrifuge at 10,000×g for 1min. Discard the supernatant as thoroughly as possible.
2. Add 250μL Solution I (resuspension) to the centrifuge tube. Vortex until the bacteria are completely resuspended and no visible clumps are visible.
3. Add 250μL Solution II (lysis buffer) to the tube. Immediately gently invert the tube 10 times to ensure complete mixing of contents. Let it stand at room temperature for 2 minutes.
4. Add 350μL Solution III (neutralizing solution) to the tube. Immediately gently invert the centrifuge tube up and down 10 times to mix thoroughly. Let it stand at room temperature for 2 minutes.
5. Centrifuge at 13,500 × g for 10 minutes.
6. Mount the adsorption column (mounting tube) onto a clean 2mL collection tube. Carefully transfer the centrifuged supernatant to the column using a pipette, ensuring each transfer does not exceed 700 μL. Centrifuge at 13,500×g for 1 minute. Discard the filtrate from the collection tube.
7. Add 500μL HBC Buffer to the adsorption column. 13,500×g centrifuge for 1min and discard the filtrate.
8. Add 700μL DNA Wash Buffer to the adsorption column. Centrifuge at 13,500×g for 1min and discard the filtrate.
9. Place the empty adsorption column into the collection tube and leave it empty at 13,500×g for 2min.
10. Place the adsorption column into a new, clean 1.5mL centrifuge tube. Open the column lid and dry it for 5-6 minutes at 60℃.
11. Carefully add 30-100μL Elution Buffer to the center of the adsorption film and place it for 2min at 60℃.
12.13500g Centrifuge for 1min. The liquid at the bottom of the centrifuge tube is the extracted plasmid DNA solution.

c．PCR technology

Objective: Amplify specific DNA fragments and separate and detect DNA by size by agarose gel electrophoresis.
Principle: PCR amplifies target DNA exponentially through cycles of denaturation, primer annealing, and extension guided by DNA polymerase. Gel electrophoresis utilizes the negative charge characteristic of DNA molecules, which migrate toward the positive electrode under an electric field. The migration rate is inversely proportional to the logarithm of molecular size, enabling effective separation.

i．PCR verification —— Taq enzyme PCR system

Component	volume
2XTaq MasterMix	5μL
Forward Primer	0.25μL
Reverse Primer	0.25μL
Template DNA	2μL
ddH2O	2.5μL
Total	10μL

Turn on the PCR instrument and select the "YZ" item. Modify the extension time according to the template length of the target fragment (1min/1000bp)

ii．PCR amplification ——PrimerStar enzyme system

Component	volume
5×Primer Star Buffer	10μL
dNTP Mix	4μl
Primer1	2μl
Primer2	2μL
Template	2μL
Primer Star Enzyme	0.5μL
ddH2O	29.5μL
Total	50μL

Open the PCR instrument and select the "PS" item. Modify the extension time according to the template length of the target fragment (1min/1000bp)

iii．High efficiency PCR verification, amplification ——Super-Fidelity enzyme system

The reaction system was configured according to the requirements of Super-FidelityPCR.
SF system for PCR verification:

Component	volume
5×SF Buffer	2μL
dNTP Mix	0.2μl
Primer1	0.4μl
Primer2	0.4μL
Template	2μL
Super-Fidelity Enzyme	0.2μL
ddH2O	4.8μL
Total	10μL

The time program uses the "YZ" project, and note that the extension time is 30s/1000bp.
SF system for PCR amplification:

Component	volume
5×SF Buffer	10μL
dNTP Mix	4μl
Primer1	2μl
Primer2	2μL
Template	2μL
Super-Fidelity Enzyme	0.5μL
ddH2O	29.5μL
Total	50μL

Turn on the PCR instrument and select the "SF" item. Modify the extension time according to the template length of the target fragment (1min/1000bp)

iv．Gene splicing by overlap extension PCR，OE PCR

Based on the length and concentration of the genes to be fused and their location in the target fusion gene, calculate the required template volume X according to the "pyramid" molar ratio (for example, for five fragments, use a ratio of 1:3:5:3:1).
Prepare the reaction system according to the requirements of OE-PCR. Note the amount of template used for each fragment (volume is determined by concentration)

Component	volume
5×PS Buffer	5μL
dNTP Mix	3μl
Total Template	Xμl
Primer Star Enzyme	0.5μL
ddH2O	(16.5-X)μL
Total	25μL

d．DNA agarose gel electrophoresis verification

i．Gelatinous agar preparation

1. Weigh 0.35g agarose (Agarose) and place it in a 250mL conical flask.
2. Add 35mL of 1× TAE buffer (Tris-acetate-EDTA buffer) to the bottle.
3. Heat in the microwave oven for about 20s until the agarose is completely dissolved and the solution is clear.
4. Cool at room temperature to about 50-60°C, add 10000:1 nucleic acid dye (GelRed) according to the volume of the gel, and mix thoroughly.
5. Insert the hole comb, pour the solution into the electrophoretic gel plate mold, and leave it at room temperature until it solidifies into the gel. (It is necessary to avoid the gel being too dry. After solidification, soak it in 1× TAE)

ii．Sample preparation and electrophoresis

1. Mix 50μL reaction system with 5μL6×DNALoadingBuffer (if TaqDNAPolymerase is used for rapid extraction of products, some systems can be directly loaded without adding loading buffer).
2. Place the solidified agarose gel in the electrophoresis tank and add 1× TAE buffer until it completely covers the surface of the gel.
3. Sample addition to Marker well: 8/10 μL 2/10kb DNA Marker
4. Slowly add the sample to the corresponding gel hole.
5. Cover the electrophoresis tank and connect the power supply:
Gel extraction experiment: 25min; band verification experiment: 18-20min; voltage current about 138V,400mA
After electrophoresis, the gel was taken out and observed under a UVtransilluminator or gel imaging system to record the results of the bands.

e.Gel extraction and DNA concentration determination

i．DNA gel extraction

1. Add the cut agarose gel block to 1×XP2Buffer (volume 1000μL/g gel) and heat it in a constant temperature water bath or metal bath for 5-10min at 60°C. During this period, gently invert and mix the gel block every 2min until the gel block is completely dissolved.
2. Place the DNA recovery column (silicaspincolumn) in a 2mL collection tube, add 700μL of the dissolved gel solution, and place it in a high-speed refrigerated centrifuge at 10000×g for 1min.
3. Pour out the filtrate in the collection pipe.
4. Add 300μL XP2Buffer to the column, put it in the centrifuge, 13000×g, 1min, and discard the filtrate.
5. Add 700μL SPWBuffer (washing buffer) to the column, centrifuge at 10000×g for 1min, and discard the filtrate; repeat this step twice.
6. Centrifuge the empty column at 13000×g for 2min to remove residual eluate.
7. Transfer the recycling column to a new 1.5mL RNAse/DNAse-free centrifuge tube, open the lid and place it in a constant temperature metal bath of 60°C for 6min to dry and remove residual ethanol.
8. Slowly add 16-30μL nuclease-free water (ddH2O or ElutionBuffer) to the center of the column membrane, and incubate with the lid closed at 60°C for 2min in a constant temperature metal bath.
9. Centrifuge 13000×g for 2min, collect the eluate, and the recovered DNA solution is obtained.

ii．DNA concentration determination

Procedure (NanoDrop)
1. Turn on the machine for preheating and calibrate/blank according to the instrument SOP.
2. Make a blank on the sample table with 2µL ddH2O and follow the instrument prompts to perform baseline/blank.
3. Take 2µLDNA samples and drop them on the measuring position to measure and record the concentration.
4. Wash the measuring position with ddH2O immediately after measurement and gently dry it with clean paper towels.
Troubleshooting:

Problem	Possible Cause	Solution
No target bands	1. Poor quality or improper concentration of template DNA 2. The annealing temperature is not appropriate 3. Primer design issues	1. Extract high quality templates again and quantify them accurately 2. Set the annealing temperature for gradient PCR optimization 3. Check for hairpin structures or dimerization of primers
The electrophoretic bands are blurred or diffuse	1. Too much sample on the plate 2. Aging of electrophoresis buffer 3. Poor gel quality	1. Reduction of DNA loading 2. Use newly configured 1× TAE or TBE buffer 3. Ensure that the agar is completely dissolved and used immediately after solidification

The plasmid was constructed by one-step cloning

1. Linearization of the carrier is prepared by double enzymatic method to make the carrier completely linearized, or directly use the linearized skeleton (such as 6005 skeleton)
2. Insertion fragments are obtained by designing forward and reverse primers based on the homologous sequences upstream and downstream and the restriction sites. PCR amplification is carried out, and the products are detected and confirmed by agarose gel electrophoresis. Then, they are purified using a gel recovery kit and their concentration is measured.
3. Calculation of carrier and fragment usage The optimal DNA usage in the recombinant reaction system can be estimated as [0.02× fragment base logarithm]ng, and the corresponding volume can be calculated according to the measured concentration.
4. Reconstitute the reaction mixture on ice. After mixing thoroughly, briefly centrifuge to collect the reaction solution at the bottom of the tube. React for 30 minutes at 37℃; cool to 4℃ or immediately place on ice for cooling.

Component	Reorganization response	Negative control-1	Negative control-2	positive control
Linearization vector	Xμl	Xμl	0μl	1μl
insert fragments	Y1-Ynμl	0μl	Y1-Ynμll	1μl
5×CE MultiS Buffer	2μl	0μl	0μl	2μl
Exnase MultiS	1μl	0μl	0μl	1μl
ddH2O	To 10μl	To 10l	To 10μl	To 10μl

5. Transformation of recombinant products:
① Chemical competent cells (e.g., DH5α Competent cell) used for ice thawing and cloning should be at least 5 min.
② Take 10μl of the recombinant product and add it to 100μl of the receptive cells. Shake the tube wall to mix and let it stand on ice for 30min.
③ After incubation at 42℃ for 60 seconds in a water bath, immediately place it on ice for 2 minutes for cooling.
④ Add 800μl LB medium (without antibiotics) and shake the bacteria for 1h at 37℃ rpm.
⑤ The corresponding resistant LB plate solid culture medium was preheated in a 37℃ incubator.
⑥ 2,400g centrifuge for 5min and discard 750μl of the supernatant. Resuspend the bacteria in the remaining medium and gently spread it on the plate with the correct resistance using a sterile spreader.
⑦ 37℃ incubate inverted in the incubator for 12-16 h.
6.Recombinant product identification
① Pick several independent single colonies from the transformed plate and carry out transfer culture.
② Use specific verification primers and use E. coli as the template to carry out PCR verification. The PCR products are analyzed by agarose gel electrophoresis to determine the size of the bands and identify them by comparison.
③ Select strains according to the results of agarose gel electrophoresis, extract plasmids, and send plasmids for sequencing to verify the successful construction of plasmids.

a．Use of the enzyme analyzer

Objective: To monitor yeast growth curve (OD600) and fluorescent protein expression level in real time with high throughput in 96-well plate.
Principle: The assay measures cell density by detecting the absorption of light at a specific wavelength (600nm). For fluorescence, it uses a specific excitation light to illuminate the sample and detects the intensity of the emitted light, thus quantifying the concentration of fluorescent molecules

i．Preparation of yeast initial liquid culture
1. Cool the sterilized liquid medium (Deft D+Ura, 121℃,25 minutes) to room temperature.
2. Inoculate a single yeast colony from the petri dish into 2mL of selective pressure liquid medium.
3. Place the test tube in a 30℃ shaker overnight until the yeast cells reach the logarithmic growth phase.
4. Transfer yeast to 20ml medium and cultivate under different conditions.

ii．Preparation of 96-well plate and lid
1. Ensure that the 96-well plate and lid are sterile.
2. Place the sterile 96-well plate in the laminar flow unit.
3. According to the experimental design, the corresponding volume of medium, inducer and yeast suspension was added to each hole.
4. Cover the 96-well plate or seal it with a breathable membrane to prevent evaporation and contamination.

iii．Sample testing

See the experimental results section for details

iv．Enzyme spectrometer and software setting
1. Turn on the enzyme labeler and start the Gen5 software.
2. Adjust the parameters and create a new experimental protocol to measure both fluorescence values and growth curves.
3. Plate type: 96 hole plate.
4. Mode: dynamic measurement, continuous track vibration.
5. temperature ：30℃.
6. Track vibration: intensity 0, duration 10 seconds.
7. Total running time: 40 minutes, measured every 4 minutes.
8. Growth curve: absorbance at 600nm (OD value).
9. Fluorescence value: the excitation wavelength is 488nm and the emission wavelength is 525nm.

v．Results and conclusions
1. After completing the data collection, export the original data of the 96-well plate from the microplate reader.
2. Export the data corresponding to the fluorescence and growth curves to an Excel spreadsheet file.
3. Save the results in the supporting software and export the data as an Excel spreadsheet file.
4. Close the software and then close the microplate reader.
Troubleshooting:

Possible Cause	Optimization solution
1. Inaccurate sampling volume 2. Cell sedimentation or uneven distribution	1. Use a multi-channel pipette and calibrate it regularly 2. Set the track oscillation before each reading to ensure uniform cell suspension
1. The culture medium itself produces fluorescence 2. Yeast autofluorescence	1. The well containing only the culture medium was set as a blank control for deduction 2. A wild-type strain without fluorescent protein was set as a negative control

b．IF immunofluorescence

Objective: To use fluorescently labeled antibodies to specifically label and observe target proteins displayed on the surface of yeast cells.
Principle: The primary antibody specifically binds to the target protein (e.g., V5 tag) on the cell surface, and then the secondary antibody with fluorescent groups specifically binds to the primary antibody. The fluorescent signal can be observed by fluorescence microscopy to locate the target protein.

i．Cell culture and preparation
1. Harvest appropriate amount of cells: centrifuge at 3000g for 10 minutes on a desktop centrifuge to collect the cell precipitate.
2. Washing: Discard the supernatant, resuspend the cell precipitate with 1mL× PBS (pH7.0), and wash twice.

ii．Fixation and translocation
1. Fixation of cells: Discard the supernatant from the last wash and resuspend the cell precipitate in 1 mL of 4% polyformaldehyde solution. Incubate at room temperature for 15 to 20 minutes.
Important note: Avoid fixation for more than 30 minutes, and do not fix overnight, because the spontaneous fluorescence of yeast cells will be significantly increased by prolonged fixation, which seriously interferes with the detection of target signals.
2. Washing fixative: After centrifugation for 3000g for 2min, the polyformaldehyde solution was discarded and washed the cells twice with 1mL 1× PBS to completely remove the residual fixative.
3. To achieve cellular permeabilization (i.e., target localization within cells), completely cover bacterial colonies with permeabilizing solution (0.1% Triton X-100) and incubate at room temperature for 10-20 minutes.
*Note: This step is of vital importance and is the key to replacing the wall-dissolving enzyme. Triton X-100, a detergent, permeabilizes the plasma membrane and partially softens the cell wall to facilitate antibody entry. Thoroughly rinse the slides three times with 1× PBS for 5 minutes each to remove the permeabilizing agent.

iii．Antibody staining
1. Sealing: Centrifuge the cells and discard the supernatant. Resuspend the cell precipitate in 1mL of 1× PBS solution containing 5% skim milk, and seal it at room temperature for 30 minutes. This step is the key to reduce non-specific binding.
2. Primary Antibody Incubation: After centrifugation and discarding the blocking buffer, cells are washed with 1×PBS. The cell precipitate is resuspended in 300μL 1×PBS containing a rabbit polyclonal anti-V5 antibody. The dilution ratio should start at 1:200 and be adjusted according to the antibody data sheet. Incubation is performed at 25°C for 1 hour or overnight (4℃).
3. Washing: After centrifugation, wash twice with 1mL 1×PBS to remove all unbound primary antibodies. This step is crucial to prevent the subsequent binding of secondary antibodies with unbound primary antibodies, thereby reducing background fluorescence.
4. Secondary Antibody Incubation: After centrifugation and discarding the supernatant, resuspend the cell precipitate in 300 μL of 1× PBS buffer containing goat anti-rabbit IgG and Coralite® Plus555 fluorescently labeled secondary antibody. Incubate for 1 hour. According to manufacturer guidelines, dilution should start at 1:500. All incubation steps must be conducted under light protection to prevent fluorescence quenching.
5. Final washing: Centrifuge the cells and wash them thoroughly with 1mL 1×PBS at least twice. This step is crucial to eliminate unbound secondary antibodies, which are the main source of background fluorescence from indirect staining.

iv．Prepare observation samples
1.Cover slip preparation: Place a clean cover slip in a sterile petri dish and add 1 mg/mL ConA solution/poly-L-lysine. Let it sit for 15-30 minutes. Rinse thoroughly with distilled water 2-3 times and air-dry. For untreated specimens, add a 1× PBS suspension of yeast mixed with 80% glycerol at a 1:1 ratio.
2.Mounting: Drop a small amount of stained cell suspension onto a pre-coated cover slip. Allow several minutes for cell sedimentation and adhesion. Add a tiny drop of anti-fluorescence quenching mountant, then carefully place the slide on top. Wipe off excess liquid with lens paper, and consider sealing the edges with nail polish for a polished finish.
*Postscript: Anti-fluorescence quenching slides may be used at this stage. When capturing images, fluorescence excitation should be performed only when capturing images after a suitable field of view has been found to maximize protection of the fluorescence signal.

v．Microscopic operation and image acquisition
Use CLSM600 confocal scanning microscope to observe the sample according to the instructions.

See the experimental results section for details

vi．Data analysis and interpretation of results
1. Qualitative analysis: The successful experimental results will be observed with a clear green fluorescent signal in the periphery of the cell, forming a "fluorescent halo", indicating that the V5-marked protein has been successfully displayed on the surface of the cell.
2. Quantitative analysis: Protein display efficiency can be quantitatively compared by measuring the average fluorescence intensity of each cell in image analysis software (e.g., ImageJ). This can be used to evaluate differences in protein display levels between different strains or induction conditions.
Troubleshooting:

Problem	Possible Cause	Solution
No fluorescent signal or weak signal	1. The target protein was not successfully displayed 2. The concentration of primary or secondary antibody is too low or has failed	1. Confirm protein expression by Western blot 2. Gradient optimization of antibody working concentrations; use fresh antibodies
Background fluorescence intensity (non-specific staining)	1. Inadequate closure 2. High antibody concentration 3. Incomplete washing	1. Extend the sealing time or increase the concentration of BSA/saliva in the sealing fluid 2. Increase antibody dilution 3. Increase the number of washes and prolong the washing time

c.The use of HPLC

The enzymatic degradation products (TPA, MHET, BHET) of PET were quantified by HPLC.

i. ample treatment
1. Take 50 µL of the reaction mixture.
2. Add 50 µL acetonitrile (MeCN) to quench the reaction.
3. Centrifugation (8000 rpm, 4 minutes, about 6200 x g).
4. Filter through 0.2 µm nylon filter membrane to obtain clear solution.
5. When measuring enzyme activity, the phosphate buffer can be diluted to a suitable volume.

ii. Flow phase configuration
Phase A: aqueous phase (often with 0.1% formic acid or buffer salts).
Phase B: Acetonitrile (MeCN).
Gradient elution: 0% B → 27.5% B (the specific procedure can be set).
The mobile phase must be filtered (0.22 µm) and degassed before use.

iii. Instrumentation
Detection wavelength: 254 nm or 240 nm (depending on the product).
Chromatography column temperature: 30-40 °C.
Volume: 1-10 µL.
Automated sampler: set the needle washing program and sample bottle position.
Column balance: the column is balanced with mobile phase before running.

iv. Operating procedure
1.Turn on the HPLC host, computer and control software (e.g. Empower, LabSolutions).
2.Connect the system module and perform Purge (high flow rate exhaust for 1-2 minutes).Balanced chromatography column.
Place the sample bottle and set the sequence (sample number, name, method, data file name).
Confirm that the baseline is stable and the pressure is stable, then click run.

v. Calibration and quantification
1.Five concentration gradients were prepared using TPA, MHET, and BHET standards, and calibration curves were drawn.
2.The peak areas of the samples were measured, and the concentrations were calculated based on the calibration curves.
3.Absorbance measurement: If it exceeds 2.5 AU, the sample needs to be diluted before measurement.
4.Blank control: Use the same buffer solution.
See the experimental results section for details

d．Transmission electron microscopy used

Objective: To precisely separate and quantitatively analyze specific chemical components in a sample mixture (e.g., PET degradation products TPA, MHET).
Principle: Using an reversed-phase chromatography column, the separation is achieved based on the differential partition coefficients of components between the stationary phase (non-polar) and mobile phase (polar). Different components elute at different times (retention time), with peak areas proportional to concentrations, enabling both qualitative and quantitative analysis.

i．Step 1: Cell culture and collection
1. Liquid culture: Pick a single colony and inoculate it into a conical flask containing liquid SD+ura medium. The culture is shaken at 30℃,220rpm overnight until the late logarithmic growth (OD600≈1.0-1.5), which usually takes 16-24 hours.
2. Transfection culture: The bacterial suspension with an initial OD of 0.1 was transferred to 20 mL Deft-D+ura medium and incubated overnight for 12-24 hours until the OD value reached above 10. (Two parallel transfection experiments using Cenpk, three empty control groups, and three experimental groups each) After PCR validation of the colonies confirmed their accuracy
3. Collection of bacterial body: Take 15mL of bacterial liquid on the second day, and centrifuge at 3000-4000rpm for 5-10 minutes. Carefully discard the upper clear culture medium.
4. Cleaning: Resuspend the precipitate with pre-cold 0.1M phosphate buffer (1× PBS, pH7.4) or sterile deionized water, and centrifuge to wash twice to completely remove proteins, sugars and other impurities in the culture medium, which may cause interference in subsequent processes.
5. Add microplastics: Resuspend yeast (50ml centrifuge tube) in 5ml of 0.1% Tween-801×PBS solution and add 0.02/0.015g (for ease of weighing, or 0.01, depending on how much you can weigh) microplastic powder.
6. Incubate at 30°C and 800rpm for 4-5 hours.
7. After incubation, centrifuge at 4000rpm for 10 minutes to collect the bottom sediment.

ii．Step 2: Electron microscopy
1. Dehydration and sample preparation
2. Resuspend the precipitate with an equal volume of pre-cold 2.5% glutaraldehyde and fix it overnight for 4℃ hours.
3. Low speed centrifugation (800xg, 5 minutes) to collect the sample, carefully remove the supernatant, and thoroughly wash with 0.1M1×PBS buffer for 3 times, each time 10 minutes, to remove the excess fixative.
4. Post-fixation (optional, but strongly recommended): Add 1% osmium acid solution and fix for 2 hours at 4℃ in the dark.
*Note: Operate in a fume hood
5. Rinse again: rinse with deionized water for 3 times, each time for 10 minutes, to completely remove the osmium acid.
6. The samples were immersed in the following ethanol solutions successively for 10 minutes each:30% ethanol, 50% ethanol, 70% ethanol, 90% ethanol, 90% acetone, 100% acetone three times
7. Infiltration embedding pretreatment
8. Replace the original acetone with 1:1 embedding agent acetone solution, and soak it in a shaking bed of 120rpm for one hour;
9. Then replace the original solution with a 3:1 embedding agent acetone solution and soak overnight on a shaker at 120rpm.
10. Infiltration and embedding:
11. Infiltration: First prepare a bottle of embedding agent in proportion, replace yesterday's 3:1 (embedding agent to acetone) with pure embedding agent, and put it in a 37 degree oven for pure infiltration for one hour.
12. Encapsulation: First, a drop of embedding agent is dropped in the embedding plate. The sample is picked up by a bamboo stick at both ends of the sample groove of the embedding plate, and a written label is placed in the middle.
13. Polymerization: Place in a mini polymerization box at 37 degrees for 12 hours according to the preset program, and automatically heat to 65 degrees for 48 hours.
14. Section: ultra-thin section machine cuts 60-80nm ultra-thin sections.
15. Staining: uranium lead double staining (uranium 2% acetic acid saturated alcohol solution, lead citrate, each staining 15min), section was dried at room temperature overnight.
16. Results: Transmission electron microscopy was observed and images were collected for analysis.
Troubleshooting:

Problem	Possible Cause	Optimization plan
The cell structure is blurred and has vacuoles	1. Fixed not in time or adequately 2. Incomplete dehydration	1. Add the fixative immediately after centrifugation and ensure that the fixation time is sufficient 2. Strictly implement the gradient dehydration steps to ensure sufficient time for each gradient
Slicing crease or rupture	1. Incomplete encapsulation of blocks 2. The blade is notched	1. Strictly control the temperature and time of polymerization 2. Replace with a new glass or diamond knife

Supplementary Table A: Key Reagents & Consumables (KR/C)

Product Name	Supplier	Catalog No.
Phanta Super-Fidelity DNA Polymerase	Vazyme	P501-d3
PrimeSTAR® HS DNA Polymerase	TaKaRa	R010A
Artificial Gastric Juice (pH 1.3)	Yuanye	R41110-100ml
DL2,000 DNA Marker	TaKaRa	3427A
Yeast Genomic DNA Extraction Kit	TIANGEN	DP307-02
E.Z.N.A.® Gel Extraction Kit	Omega Bio-tek	D2500-02
ClonExpress MultiS One Step Cloning Kit	Vazyme	C113-02
E.Z.N.A.® Cycle-Pure Kit	Omega Bio-tek	D6492-02
E.Z.N.A.® Plasmid Mini Kit I	Omega Bio-tek	D6943-02
DL10,000 DNA Marker	TaKaRa	3584A
4S Green Plus Nucleic Acid Stain	Sangon Biotech	A616656
2 x Taq Plus Master Mix (Dye Plus)	Vazyme	P212-03
10 x DNA Loading Buffer	Vazyme	P022
V5-tag Polyclonal antibody	Proteintech	14440-1-AP
Multi-rAb™ CoraLite® Plus 555-Goat Anti-Mouse Recombinant Secondary Antibody (H+L)	Proteintech	RGAR003
Sodium alginate	Sangon Biotech	A602116

Supplementary Table B: Major equipment (ME)

Instrument Name	Manufacturer	Model No.
T100TM Thermal Cycler	BIO-RAD	T100
Multi-Mode Microplate Reader	BioTek (Agilent)	Synergy H1
Centrifuge	HITACHI	CT15RE
Transmission Electron Microscope	HITACHI	H-7650
Essentia Series Liquid Chromatograph	SHIMADZU	LC-16
Laser Confocal Microscope	SOPTOP	CLSM610