【1】Experimental preparation

[1]Amplify the plasmid of interest
Two of the synthesized expression plasmids (pACT1 and pTDH3) were transformed into DH5α, and the strains containing the target plasmids were screened out. The plasmids were extracted after 18 hours of expansion at 37℃ and 180rpm.

[2]Extracted the target plasmid
* Add 0.2~0.5ml Buffer P1 to RNase A dry powder, beat and mix for 5~10 times to fully dissolve the RNase A, and then all the RNase A is transferred to Buffer P1 and stored at 2-8℃.
* Buffer PW2 was stored at room temperature by adding 4 x volume of absolute ethanol as indicated by the bottle label.
* Sterilized 1.5-m l centrifuge tube and used for DNA collection.
* LB medium and the corresponding culture tubes or culture flasks.
* Below centrifugation was performed at room temperature and column blockage at low temperatures.
1.The plasmid-containing strains were seeded in 50ml tubes containing 10 m l of L A medium and incubated in a 37℃ shaker for 12 to 16 h to amplify the plasmid.
2.The ls were centrifuged at 3,000~5,000 g for 10 min and 10 to 15 m l of broth were collected.
3. Pour down the medium and buckle the absorbent paper and gently beat the residual liquid. Add 500 μl Buffer P1/RNase A mixed liquid and resuspended. Before use, ensure that the RNase A is added to the Buffer P1. Thorough resuspension of bacteria is quite important to achieve high yield, and cell clumps should be invisible after resuspension.
4. The resuspension was transferred to a 2-m l centrifuge tube. Add to the 500 μl Buffer P2. Gently upside down 8 to 10 times. Place at room temperature for 2 minutes, occasionally reverse several minutes. Gently reverse the mixing, do not vortex, otherwise it will cause genomic DNA breakage and contamination. When the amount of bacterial solution exceeds 10ml, the lysate will be very viscous and difficult to mix. After full lysis, the solution becomes viscous and bright. If necessary, slowly reverse the lysate until the lysate becomes clear, but not over 4 minutes.
5. Add 700 μl Buffer P3 and immediately reverse and mix well 15~20 times. After adding Buffer P3, the mixing should be reversed immediately to prevent precipitation agglomeration and affecting the neutralization effect. The mixing process must be gentle. When the amount of bacterial liquid is more, neutralization will be more difficult to proceed, increase the number of inverted to the solution thoroughly neutralization.
6.It was centrifuged at 6. 13,000 ×g for 10 min.
7. Cover the HiPure DNA Mini Column III column in the collection tube. Transfer half the volume of the supernatant to the column. The mixture was centrifuged at 13,000 g for 30~60 seconds.
8. Pour away the effluent and put the column back into the collection tube. Transfer the remaining supernatant to the column. The mixture was centrifuged at 13,000×g for 30~60 seconds.
9. Pour away the effluent and put the column back into the collection tube. Add the 500 μl Buffer PW1 to the post. The mixture was centrifuged at 13,000×g for 30~60 seconds. This step can be omitted in treating nuclease knockout strains (end A-), such as JM109 et al. Treatment with nuclease-rich strains (end A +) such as HB101 cannot be omitted. The Buffer PW1 contains protein denaturants, please wear gloves to treat it. HiPure Plasmid Plus Kits is recommended when treating nuclease-containing strains to improve plasmid stability.
10. Pour the effluent, put the column back into the collection tube and add 600 μl Buffer PW2 to the column.13,000 ×g centrifugation for 30-60 seconds.
11. Pour the effluent, put the column back into the collection tube and add 600 μl Buffer PW2 to the column.13,000 ×g centrifugation for 30~60 seconds.
12. Pour the filtrate and put the column back into the collection tube. The drying column was centrifuged at 13,000 ×g for 2 min. Don't ignore this step. This step was taken to remove the residual ethanol in the column.
13. Cover the column in a sterilized 1.5ml centrifuge tube. Add 75~100 μl Elution Buffer or sterilized water to the center of the membrane of the column. Let for 2 min and centrifuged at 12,000 ×g for 1 min.
14. Leave the column and save the plasmid at-20℃.

[3]Quantitative plasmids were detected by electrophoresis
1. Take 2.5 μl Loading Buffer, mix 1 μl plasmid and drop into the sample hole.
2.10,000 bp Marker was added for control.
3. Agarose gel was put into electrophoresis and run at 180v for 25min.
4. Plasmid was extracted using automatic gel image system.The target plasmid was verified by enzyme digestion

[4]The target plasmid was verified by enzyme digestion
1) pACT1p enzyme digestion and verification:
Xho I: 9149 bp
Hind Ⅲ: 2991 bp+6158 bp
Sal I :3031 bp+6118 bp
2) pTDH3p enzyme digestion and verification:
Xho I: 12534 bp
EcoR I :3970 bp+8564 bp
EcoR V: 7367 bp+5167 bp
*System configuration:
The dna sample was used in 10 μl
Cutting buffer was prepared at 1.5 μl
Enzyme:1 μl
Sterile water was in 2.5 μl
1.Plasmid samples were taken in a 0.5-m l centrifuge tube
2. Mix well; centrifuge at 4000rpm for 30s and swing to the bottom of the tube
3.37℃, and digested for 2h
4.65℃, insulated for 20min
5. Electrophoresis verification: take 1μl amplification product, mix with 2.5 μl 10 × loading buffer, add to the point sample hole with a pipette, electrophoresis at 180v for 25min, and remove the gel. In control with Marker, the band size was determined with the correct transformants

[5]Medium preparation:
SD medium (200 ml)
YNB:10 ml
Glucose: 4 g
Agar: 4 g
Methionine: 0.004 g
Histidine: 0.004 g
Leucine: 0.012 g
Uracil: 0.008 g
dH2O: 200 ml
1. Glucose, add 4 g of agar each to a tapered flask, 185m l of distilled water and autoclaved at 115 ℃ for 15 min
2. Ultra-clean table disinfection and ultraviolet test extinguishing tube (set 10ml scale line with water in advance)
3. Corresponding corresponding amino acids and uracil, loaded into 5 ml tube and added 5ml water to dissolve.
4. Filter the amino acids and uracil with a sterile filter membrane and connect them to a sterile tube
5. Take 10 ml of YNB from the ultra-clean table to the test tube, add YNB solution and amino acid uracil solution to the autoclaved tapered bottle, and shake at 50 ℃ for half an hour to dissolve

【2】Yeast transformation


[1]Single plasmid yeast transformation
(1)Take 100μl For the BY4741 competent cells melted on the ice, pre cooled target plasmid 2g and Carrier DNA 10 μl, PEG / LiAc 500μl .Suck and beat for several times, mix well, and take a 30℃ water bath for 30 min (turn over for 6-8 times when 15 min)
Note: Carrier DNA 96 ℃ water bath for 3 min, fast ice bath for 3 min, repeat once
(2) Move the competent state to 42 ℃ water bath for 15 min (turn it over for 6-8 times and mix well at 7.5 min).
(3) Centrifuge the supernatant at 5000 rpm for 40s, ddH2O 400μl Suspend again, centrifuge for 30s and discard the supernatant.
(4) ddH2O 50μl Heavy suspension, coated plate, culture at 29 ℃ for 48-96 h.

[2]Yeast transformant colony PCR and electrophoresis verification
• two × Fast PCR Mix 10 ul
• Primer F:1 ul
• Primer R:1 ul
• Bacterial liquid: 2 ul
• ddH2O:6 ul
(1) Add 6μl ddH2O to the PCR tube , take the convertor with uniform size from the plate and add it.
(2) Take 2μl The bacterial solution was cultured in defective SD medium at 29℃ for 180rpm.
(3) Take 4μl for PCR validation, 2μl Bacterial liquid test and the rest for standby.
(4) PCR amplification was carried out according to the system.
(5) Take 1μl Expansion products and 2.5μl10 * loading buffer, mix well, add it into the sample hole with a pipette, conduct 180v electrophoresis for 25min, and take out the gel.
(6) Compare with Marker to determine the stripe size and correct transformant.


[3]Determine the growth curve of single plasmid yeast, and verify that single plasmid has no significant effect on yeast growth

(1) Take single colonies of BY4741, pTDH3p and pACT1p into the liquid SD medium, and conduct amplification and culture at 29 ℃ at 180rpm until the OD660 value is 0.2.
(2) Take 2% as the inoculum quantity to connect the bacteria to the liquid SD culture medium that has been repackaged. One kind of bacteria is connected to three bottles of culture medium to take the average value of multiple experiments, and then expand and culture at 29℃ at 180rpm.
(3) The OD660 value shall be measured by spectrophotometer at 0h, 4h, 8h, 12h, 24h, 36h, 48h, 60h and 72h after inoculation, and the average value shall be taken for three times of each bacterial test.
(4) Summarize the data and draw a curve.

[4]Fluorescence observation of single plasmid yeast
Fluorescence observation of transformants: 100μl was taken from the 5ml EP tube shaken for 3~4 days, washed with 1ml PBS, 4000rpm, 1min, discarded supernatant, resuspended with a new 50~100μl PBS, and 10μl of the resuspended transformants solution was prepared for observation. Then, turn on the microscope and software ZEN3. 4. The corresponding GFP and DAPI channel were used for fluorescence observation.

[5]Glycerol tube strain preservation
1、Preservation of BY4741 and BY4741-DmIav, BY4741-ACT1
(1)50ml of bacterial solution was separately packed into 2ml finger tubes, centrifuged at 5000g for 5-10min, and the supernatant was discarded.
(2) Add 500ml of fresh SD culture medium into each finger tube, resuspension and merge into one tube.
(3) After centrifugation, discard the supernatant and add 1ml fresh SD medium to resuspension.
(4) Under aseptic operation conditions, add 800mL of bacterial solution and 1200mL of 50% glycerin solution into the sterilized 2mL cryopreservation tube, mix gently and fully;
(5) Store in - 80 ℃ refrigerator.
2、Preservation of pTDH3p and pACT1p plasmids
(1) 50ml of bacterial solution was separately packed into 2ml finger tubes, centrifuged at 5000g for 5-10min, and the supernatant was discarded.
(2) Add 500ml fresh LB culture medium into each finger tube, resuspension the cell and merge it into one tube.
(3) After centrifugation, discard the supernatant and add 1ml fresh LB medium to resuspension.
(4) Under aseptic operation conditions, add 1000mL bacterial solution and 1000mL 50% glycerol solution into the sterilized 2mL cryopreservation tube respectively, mix gently and fully;
(5) Store in - 80 ℃ refrigerator.

[6] Oil mirror making
For 30 ml of BY4741-ACT1 transformants, BY4741-DmIav transformants and BY4741 cultured at 29 °C, 180 rpm for 72 h, we made temporary mounts for observation.
1. Wipe the slides. Mix 200 μl of the bacterial solution with 200 μl of ink and drop it onto the slide with a dropper.
2. After the low magnification field of view is adjusted accurately, the temporary film is adjusted from the low magnification mirror to the 100 times oil mirror.
3. Turn the rough adjuster to make the oil lens drop and immerse in the glycerol, gently contact the slide.
4. Adjust the lens to make the field of view clear.
5. Move the thruster and turn the micro-adjuster to observe the specimen. Look for areas with dense yeast and take pictures with your phone.
6. After observation, turn the rough adjuster to raise the lens barrel and remove the slide. Immediately wipe the glycerin off the lens with a mirror polishing paper.

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