LB medium preparation：Yeast extract 5 g/L，Tryptone 10 g/L，NaCl 10 g/L. For solid, add 20 g/L agar.
Yeast medium preparation：Yeast extract 10 g/L，Tryptone 20 g/L，D-glucose 30 g/L(separate)，Lactose 2 g/L. For solid, add 20g/L agar.
YPD30L2 preparation：Yeast extract 10 g/L, Tryptone 20 g/L, D-glucose 30 g/L, Lactose 2 g/L.

The medium, as mentioned above, are put into autoclave at 120 °C for 20 mins.

Primer pairs h-bb-x-3d-f1 and h-bb-x-3u-r1 amplify vector using pHCas9-Nours plasmid as DNA template to obtain the integration backbone plasmid. Size: 3776bp.
Primer pairs h-x-3u-bb-f1 and h-x-3d-bb-r1 amplify the flanking region of homologous arms using genomic CCTCC M94055 as DNA template. Size 444bp.

Primer pairs h-bb-xi-2d-f1 and H-bb-xi-2u-r1 amplify vector using pHCas9-Nours plasmid as DNA template to obtain the integration backbone plasmid. Size 3776bp.
Primer pairs h-xi-2u-bb-f1 and h-xi-2d-bb-r1 amplify the flanking region of homologous arms using genomic CCTCC M94055 as DNA template. Size 444 bp.

PCR reaction setup

Component	Volume (μL)
2× Mix	25
Primer F (10 μM)	2.5
Primer R (10 μM)	2.5
DNA template	2
ddH2O	18
Total	50

PCR instrument set

Steps	Temperature(℃)	Time
1	98	3min
2	98	30sec
3	55	30sec
4	72	2min
5	72	10min
6	4	Forever

*Repeat step2 to 4 for 30 times.

1. Agarose gel preparation: weight out 0.5 g Agarose in a conical flask. Add 50 mL 1×TAE buffer to the conical flask. Melt the conical flask using a microwave oven until the solution become clear. While heating, swirl the flask occasionally. Add 5 μL nucleic acid staining dye (Gel Red) to Agarose gel when this solution cooled down to ~55℃. Pour the molten gel to into casting tray. And, insert the comb at the end of the casting tray. Avoid air bubbles. When it completely solidified, remove the comb to form the wells. Put the gel into the electrophoresis bath, add 1×TAE until it covered the gel.
2. Each sample was mixed the loading dye. And then the samples were added sequentially to the wells of the gel (avoid to break the gel).
3. Electrophoresis: cover the electrophoresis bath, samples had a net negative charge, and an electrical current was applied in 180 V for 15 min.
4. Turn off the power, and observe gel when it exposed to a short wave ultraviolet light source. The size of DNA fragments was determined according to the position of DNA ladder.

1. Cut the band that contains the DNA fragment.
2. Weight the gel, then add appropriate amount of Buffer B2.
3. Heat in a water bath at about 50℃ until the gel is completely dissolved.
4. DNA binding on membrane on adsorption column and centrifuge at 8000 xg for 30sec. Discard the filtrate.
5. Add 500 l wash buffer for cleaning to column and centrifuge at 9000 xg. Discard the filtrate. Repeat this step.
6. Adding DNA eluent for elution. And purified DNA (30μL) was obtained.
7. inoculate X-3 XI-2 gRNA/DH5α（Amp）, X-3（Amp）, XI-2（Amp）, pHCas9-Nourse-1（Amp）, AQ yeast (no antibiotic）add 5 μL ampicillin (working concentration: 100 μg/mL）and 100-200μL inoculant to 5mL LB medium, Incubator shaker foster at 220 rpm (30℃) overnight.

1. Harvest E. coli by centrifugation at 12000 rpm for 1 minute, and then remove the supernatant.
2. Pipette 250 μL Buffer S1 into the tubes (cool down at 4 degrees) to suspend the precipitation.
3. Pipette 250 μL Buffer S2 into the tubes with gently mixing to break the bacteria (this step must be done in 5 min)
4. Pipette 350μL Buffer S3 into the tubes with gently mixing and centrifuge at 12000rpm for 10 min, and then the supernatant can be purified using spin filter (neutralization reaction, remove impurity like protein) .
5. Extract plasmids DNA using the column extraction method.

    All the PCR products were treated with the restriction enzymes NotI and XhoI, including the cassettes of gmd-wcaG and wbgL-lac12, as well as X-3 and XI-2 plasmid.
DNA fragment digestion

Component	Volume (μL)
PCR fragments (Homologous recombination templates)	25
Not1	1
Xho1	1
CutSmart	5
ddH2O	18
Total	50

Plasmid digestion for vector preparation

Component	Volume (μL)
Plasmid vector	30
Not1	1
Xho1	1
CutSmart	5
ddH2O	13
Total	50

*Gel extraction (same as before) for DNA fragment
*Gel electrophoresis for vector

The system: Room temperature/30 minutes

Component	Volume (μL)
Cut Vector	10
PCR fragments after digestion (Homologous recombination templates)	7
T4 ligase	1
T4 ligase buffer	2
Total	20

1. Add 10 μL recombination products to 2 tubes of 100 μL E. coli DH5α competent cells and incubate cells on ice for 30 minutes;
2. Heat-shock cells for 90 seconds in a 42 degrees water bath and cool down on ice for 3 minutes immediately;
3. Add 900 μL LB medium to the tubes and put it on the shaker at 37 degrees for 1 hour;
4. Centrifuge at 5,000 rpm for 5 minutes and remove 900 μL supernatant;
5. Suspend the precipitation and pipette the solution into the LB culture plate with ampicillin. For cultivation of stains, the plate was put on the incubator at 37 degrees overnight.

1. Culture yeast Y2H in YPDA medium at 30 degrees;
2. Pick single yeast into 2 ml tubes and add 1 ml YPD medium, and shake to grow the cells;
3. Pipette the solution into YPD medium at the ratio of 1:100 and incubate at 30 degrees for 18-24 hours until the OD600 reaches 1.5-2；
4. Pipette the culture at the ratio of 1:4 into YPD medium at 30 degrees for 3 hours until the OD600 reaches 0.2;
5. Pipette the yeast culture into 1.5 ml tube and centrifuge at 12,000 rpm for 1 minute;
6. Remove the supernatant and add 1ml TE buffer and suspend the precipitation;
7. Remove the supernatant and add 0.1ml TE-LiAc solution, and suspend the precipitation.

1 boil the salmon sperm DNA (10μL/μg) for 10-20 minutes and put it in an ice bath for 10 minutes;
2 put 10μL salmon sperm DNA in 100μL yeast competent cells and 5μL plasmids and shake it;
3 add 600μL TE-LiAc-PEG solution and mix it through vortex;
4 incubate on the shaker at 200rpm and 20 degrees for 30 minutes;
5 add 70μL DMSO and mix;
6 heat shock for 15 minutes at 15 minutes;
7 put it in an ice bath for 2-10 minutes;
8 centrifuges at 12000rpm for 1 minutes and remove the supernatant;
9 add 100μL 1*TE and suspend the precipitation.

place the validated strains in glycerol tubes to obtain E. coli with the target plasmid and stored at -80 °C until use. The samples were identified by colony PCR, NotI and XhoI double enzyme digestion, and then sent for Sanger sequencing. Positive colonies which were as X-3-gmd wcaG and XI-2-wbgL-lac12 were picked and inoculated in 3 mL LB supplemented 3 μL Amp overnight.

0.4 ml of S. cerevisiae culture were inoculated 40 ml YPD20 medium for 8h. Collect bacteria after several period of growth. Then, the culture transferred to 20 ml YPD30L2 medium to produce 2'-FL. After initial growth, OD600 reached 10 and regularly taking samples to measure.