1. Plasmid extraction

Use AxyPrep Plasmid Miniprep Kit for rapid purification and extraction of plasmid DNA.

(1) Aseptically label and take 2 mL of bacterial solution into a centrifuge tube, mass balance, arrange the centrifuge tube symmetrically into the centrifuge, check the closure condition, set the speed at 12000 rpm for 1 min, and retain the precipitate.

(2) Resuspend the bacterial pellet in 250 μl of Buffer S1 by vortexing.

(3) Add 250 µl of Buffer S2, and mix by gently inverting the tube 4-6×within 5 min.

(4) Add 350 µl of Buffer S3, and mix by gently inverting 6-8×. Centrifuge at 12,000rmp for 10 minutes to clarify the lysate.

(5) Pipette the supernatant from the centrifuge tube and transfer it to the preparation tube, which is placed in a 2 mL centrifuge tube and centrifuged at 12000 rpm for 1 min, and discard the filtrate.

(6) Put the preparation tube back into a 2 mL centrifuge tube, add 500 μL of Buffer W1, centrifuge at 12000 rpm for 1 min, and discard the filtrate.

(7) Place the preparation tube in a centrifuge tube, add Buffer W2 700 μL, set the speed to 12000 rpm and centrifuge for 1 min, discard the filtrate. Repeat the procedure in (7).

(8) Centrifuge in vacuum for 2 min at 12000 rpm.

(9) Transfer the preparation tube to a sterilized 1.5 mL centrifuge tube, add 30 μL of ddH2O to the membrane of the preparation tube after a 65°C water bath, and let it stand at room temperature for about 1 min at 25°C until ethanol could not be detected by light sniffing, then centrifuge the tube at 12000 rpm for 1 min, and then dispose of the tube.

2、Agarose gel electrophoresis

(1) Prepare a clean and dry 250 mL conical flask, weigh 0.25 g of agarose on a weighing balance, and add 25 mL of 1×TAE.

(2) Put the conical flask into a microwave oven, and set the microwave oven to high heat mode for 1 min to fully dissolve the agarose, and then after it cools a little, add 2.5 µL of 10000×ExGreen, shake gently, and then pour it onto the gelatin plate inserted with a comb and wait for solidification. Pour it onto the plate with the comb inserted and wait for solidification.

(3) Pipette a certain amount of reaction solution.

(4) Turn on the electrophoresis instrument. Set the voltage at 125 V for 25 min.

(5) Observe under the gel imager and compare the size of the bands.

3、PCR method

(1) Acquisition of target fragments

The PCR reaction system and conditions were as shown in Table 1-1. The PCR reaction system and conditions are shown in Table 1-1.

Table 1-1 PCR reaction system

* PCR cycling program: 95°C, 5 min; 32 cycles × (95°C, 15 s; 56°C~72°C, 15 s; 72°C, 1 min). The annealing temperature should be adjusted according to the Tm value of the primer, and generally set to 3 ~ 5 ℃ lower than the Tm value of the primer; for complex templates, it is necessary to achieve effective amplification by adjusting the annealing temperature and extending the extension time.

(2) DNA digestion

Follow the instructions of the reaction system of the relevant enzyme, and the enzymatic system is shown in Table 1-2.

Table 1-2 Enzyme cleavage reaction system

*Configuration of 20 µL system: incubate at 37°C for 5 minutes in a thermostatic water bath and store at 4°C.

4. PCR products purification

PCR products can be rapidly purified using the AxyPrep PCR Clean-up Kit.

(1) Measure the volume of PCR reaction solution (X mL) with a pipette gun and addat least 100 µL of Buffer A (three times the volume of X mL).

(2) Blow the solution with a pipette gun, transfer the solution to a preparation tube (the preparation tube is placed in a 2 mL centrifuge tube), adjust the rotational speed to 12000 rpm, centrifuge for 1 min, and discard the filtrate.

(3) Place the preparation tube back into the 2 mL centrifuge tube, add 700 µL of Buffer W2 to the preparation tube, adjust the speed to 12000 rpm, centrifuge for 1 min, and discard the filtrate.

(4) Place the preparation tube back into the 2 mL centrifuge tube, add 400 µL of Buffer W2 to the preparation tube, adjust the speed to 12000 rpm, centrifuge for 1 min, and discard the filtrate.

(5) Adjust the speed to 12000 rpm and centrifuge in vacuo for 1 min. Transfer the preparation tube to a sterilized 1.5 mL centrifuge tube, add 30 μL of ddH2O from a 65°C water bath to the preparation tube membrane, and allow to stand at room temperature for approximately 1 min. After centrifugation at 12000 rpm for 1 min, add 30 µL of the filtrate back into the preparation tube and let it stand for 1 min (until ethanol is not detected by sniffing), then centrifuge at 12000 rpm for 1 min, and store at -20 °C.

5. One-step cloning and ligated fragment method Escherichia coli transformation

Follow One-step cloning- ClonExpress® MultiSOne Step Cloning Kit (C113) (Vazyme) instructions for operation.

(1) Calculate the amount of DNA required for the recombination reaction according to the formula: Optimum number of fragments per fragment = [0.02 x number of base pairs in the fragment] ng.

(2) Configure the following reaction system on ice. (20 μL reaction system).

Table 2 One-step cloning ligation system

(3) Use a pipette to gently mix (do not shake or jar) and briefly centrifuge to collect the reaction solution to the bottom of the tube.

(4) Incubate at 37℃ (PCR instrument) for 30 min and immediately lower to 4℃ or immediately cool on ice. (The reconstituted product can be stored at -20℃ for up to one week and can be thawed for transformation)

6、E.coli transformation

(1) Take 50 µL of sensory cells out of the -80℃ultra-low temperature refrigerator, quickly put them on ice to melt for about 2 min, and put the plasmid DNA on ice to melt as well.

(2) Add 1 µL of plasmid to 50 µL of sensory cells, flick the wall of the tube to mix well, and leave it on ice for 30 min, avoiding heating with fingers.

(3) Heat-excite the cells in a metal bath at 42℃ for 90 s, and immediately put them on ice for 2 min. Add 900 µL of pure LB liquid medium into the centrifuge tube, mix well, and then put them on a shaker for 1-2 hours, at 37℃, and adjust the rotational speed to 200 rpm.

(4) Adjust the speed to 6000 rpm, centrifuge for 1 min, discard the supernatant (about 200 µL remaining), resuspend the organisms with sterilized ddH2O, and take 160 µL of the bacterial solution and spread it on the plate. Add 120 µL of ddH2O to the centrifuge tube to dilute the remaining bacterial solution, and spread all of it on the plate.

(5) Incubate at 37℃ for 12-16 h in a constant temperature incubator.

7. Yeast competent cell preparation

(1) Inoculate a single colony into 7 mL SC liquid medium, incubate in 220 rpm shaker at 30℃ for 18-24 h. Dilute in 50 mL SC liquid medium to OD600=0.1, and continue to incubate for about 6 h, until OD600= 0.6.

(2) Transfer 50 mL of bacterial solution to a 50 mL centrifuge tube, and let it stand on ice for 15 min. At 4℃, centrifuge at 4000 rpm for 10 min and discard the supernatant.

(3) Resuspend the organisms by adding 40 mL of ddH2O pre-cooled at 4°C to a 50 mL centrifuge tube. Centrifuge at 4000 rpm at 4℃for 10 min, discard the supernatant.

(4) Add 25 mL of pre-cooled ddH2O at 4°C to a 50 mL centrifuge tube to resuspend the organisms. Centrifuge at 4000 rpm at 4°C,for 10 min, discard the supernatant.

(5) Add 2 mL of 1 M sorbitol pre-cooled at 4°C to a 50 mL centrifuge tube to resuspend the organisms. 4°C, 4000 rpm centrifuge for 10 min, discard the supernatant. Centrifuge at 4000 rpm for 10 min and discard the supernatant.

(6) Resuspend the organisms with 300 μL of pre-cooled 1 M sorbitol at 4℃.

8. Yeast Electrotransformation

(1) Add 1-5 μL of plasmid DNA to 50 μL of yeast receptor cells, transfer into a pre-cooled electroshocking cup at 4℃, and leave on ice for 5 min.

(2) Dry the water droplets on the outer wall of the shock cup, set the program of the electrotransfer device to "Sc2", and shock.

(3) Add 1 mL of SC liquid medium immediately after shocking, transfer to a 1.5 mL centrifuge tube and incubate for 1 h at 30℃ in a constant temperature incubator.

(4) Adjust the speed of centrifugation to 4000 rpm, centrifuge for 5 min and discard the supernatant. Pipette 100 μL of 1 M sorbitol to resuspend the bacteria and spread on solid medium.

9. Brewer's yeast genome extraction

(1) Take 1 mL of overnight cultured yeast liquid into a 1.5 mL centrifuge tube, centrifuge at 12000 rpm for 1 min, collect the bacterial body and discard the supernatant.

(2) Pipette 200 μL of STES buffer into the above centrifuge tube, blow with a pipette gun to resuspend the bacteria, and add tinfoil parcels into the centrifuge tube respectively. Add 200 μL of autoclaved quartz sand wrapped in tinfoil and 200 μL of phenol chloroform into the centrifuge tube.

(3) After vortexing for 10 min with a vortex oscillator, add 200 μL of TE buffer to the centrifuge tube and vortex for 1 min.

(4) Adjust the speed of the centrifuge to 12000 rpm and centrifuge for 5 min. Take 300 μL of supernatant into a clean 1.5 mL centrifuge tube.

(5) Add 30 μL of 3M NaAc and 1 mL of anhydrous ethanol to the centrifuge tube, mix well, and then place it in an ultra-low temperature refrigerator at -80℃ for 1 h or overnight.

(6) Remove the centrifuge tube from the -80℃ ultra-low temperature refrigerator, adjust the speed to 12000 rpm, centrifuge for 15 min at 4℃ to precipitate DNA, discard the supernatant and wash twice with 1 mL of 75% ethanol.

(7) Aspirate appropriate amount of 75% ethanol to wash the precipitate, adjust the speed to 12000 rpm, centrifuge for 1 min, discard the supernatant. Repeat once.

(8) Let it stand at room temperature for 5 min, and after the ethanol is evaporated to dryness, add 100 μL of TE or ddH2O after 65℃ water bath.

10. Validation of recombinant strains

Select a number of single colonies on the yeast electrotransfer plate for trans-spotting, and then perform colony PCR. Or pick colonies and connect them to 5 ml of SC
leucine-deficient medium to extract the genome and then carry out genomic PCR, use M13 as primers. The specific methods of colony PCR were as follows:

(1) Add 20 μl of 20 mmol/L NaOH solution to the PCR tube;
(2) Pick the colony with the tip of the gun and put it into the PCR tube;
(3) Cook the bacteria using PCR instrument, 99℃ for 5min, 4℃ for 1min, and cycle five times;
(4) Centrifuge, take 1 μl of supernatant and add to the PCR reaction system;
(5) Agarose gel electrophoresis verification.

Pick a single colony of yeast into SC(-leu) liquid medium, incubated for 20 h, and then diluted to an OD600 of 0.1. After incubation at 30℃ for 3 h, add different pheromone concentrations (0 μM, 1 μM, 5 μM, 10 μM, 25 μM, respectively). After induction treatment for 1 h, take 1.5 mL of bacterial liquid, centrifuged at 12000 rpm for 2 min, and the supernatant was discarded, wash with 1 mL of 1×PBS buffer and take appropriate amount of cells to make a microscope observation slide, and Observe the morphology of the yeast under the 40X objective lens. Observe the intensity of GFP under 475 nm excitation light.

Pick a single colony of yeast into SC(-leu) liquid medium, incubated for 20 h, and then diluted to an OD600 of 0.1. After incubation at 30℃ for 3 h, add different pheromone concentrations (0 μM, 1 μM, 5 μM, 10 μM, 25 μM, respectively). After induction treatment for 1 h, detect the fluorescence intensity of GFP by flow cytometry.

Use UV spectrophotometer to determine the OD600 value of the pre-culture bacterial solution, add 100ml SC leucine-deficient medium to the sterilized 250ml conical flask. Add the bacterial solution, so that the starting OD600 value of the final volume of the culture solution is 0.1. Then add a certain amount of 25mM CuSO4, so that the final culture solution has a copper ion concentration of 10μM, the conical flask will be placed into a constant temperature shaker to cultivate at 30°C and 200 rpm. After that, samples were taken every 2 h, measure the OD600 by UV spectrophotometer, and measure the GFP expression by enzyme marker until 24 h.

Prepare 25 mM of ZnSO4, MgSO4, MnSO4 solution respectively, use UV spectrophotometer to determine the OD600 value of the pre-culture bacterial solution, add a certain amount of bacterial solution in 2 ml of SC leucine-deficient liquid medium, so that the final volume of the culture of the OD600 value of 0.1. Then dilute 25 mM of CuSO4, ZnSO4, MgSO4, MnSO4 solution according to the Ten-fold gradient dilution. add a certain amount of CuSO4, ZnSO4, MgSO4, MnSO4 solution respectively to the 2ml of bacterial solution with OD600 value of 0.1, so that the concentration of metal ions in the final culture volume was 10 μM. Put the inoculated test tubes into a constant temperature shaker for 24h, 30°C, 200 rpm. The samples were processed for 24h, and were subjected to flow cytometric assay.