SE

Media was sterilized in an autoclave. SE medium must be used when using G418
 SC

Media was sterilized in an autoclave.
 SD-His

Media was sterilized in an autoclave.
 SD-Ura

Media was sterilized in an autoclave.
 Medium for Escherichia coli

Media was sterilized in an autoclave.
  Medium for Saccharomyces cerevisiae

Plasmid Extraction

1. Collect 1-4 ml of overnight LB culture. Centrifuge at 12,000×g for 1 minute to pellet the bacteria. Decant or pipette off as much of the supernatant as practical.
2. Resuspend the bacterial pellet in 250μl of Buffer S1 by vortexing.
3. Add 250μl of Buffer S2, and mix by gently inverting the tube for 4-6×. Do not vortex.
4. Add 350μl of Buffer S3, and mix by gently inverting 6-8×. Centrifuge at 12,000×g for 10 minutes to clarify the Iysate. Do not vortex.
5. Place a Miniprep column into an uncapped 2 ml Microfuge tube. Transfer the clarified supernatant from Step 4 into the Miniprep column. Transfer the Miniprep column and 2 ml Microfuge tube to microcentrifuge and spin at 12,000 × g for 1 minute.
6. Pipette 500μl of Buffer W1 into each Miniprep column. Centrifuge at 12,000×g for 1 minutes. (Buffer W1 Wash Washing with Buffer W1 is required only in cases where the plasmid has been propagated in an endA+ bacterial strain. )
7. Pipette 700 μl of Buffer W2 into each Miniprep column. Centrifuge at 12,000 ×g for 1 minute.
8. Discard the filtrate from the 2ml Microfuge tube. Place the Miniprep column back into the 2 ml Microfuge tube. Add 700 μl of Buffer W2 to the Miniprep column and centrifuge at 12,000 ×g for 1 minute.
9. Discard filtrate from the 2 ml Microfuge tube. Place the Miniprep column back into the 2 ml Microfuge tube. Centrifuge at 12,000 ×g for 1 minute.
10. Transfer the Miniprep column into a clean 1.5 ml Microfuge tube (provided). To elute the purified plasmid DNA, add 60-80 μl of Eluent(or double distilled water) to the center of the membrane. Let it stand for 1 min at room temperature. Centrifuge at 12,000 ×g for 1 minute.

PCR-Super Fidelity DNA Polymerase
Pipette these reagents in the order listed:

PCR cycling program

PCR-Taq DNA Polymerase
Pipette these reagents in the order listed:

PCR cycling program

Pipette these reagents listed in the table. The reaction mixture is prepared in the room temperature.

Mix gently and spin down. Incubate at 37℃ in a water thermostat for 4 hours.

The reaction mixture in microcentrifuge tubes is as followed:

Vortex and spin briefly to collect the drops.
Incubate the mixture at 25℃ for 1 hour.
Use 5μL of the ligation mixture for transfor mation.

Ligation confirmation

1. Introduce the recombinant plasmid into the bacteria(we used E.coli DH 5α) by chemical transformation or electrotransformation.
2. Spread the broth on LB solid medium and place the plate in the 37℃ incubator overnight.
3. Prepare the reaction system of PCR-Taq DNA polymerase except DNA in eight microtubules.
4. Randomly choose and mark eight bacterial colonies. Pick these colonies and dissolve in the reaction mixture.
5. Perform PCR.
6. Run gel electrophoresis to separate PCR product. Remove the gel and place it over a UV or Blue-light illuminator to visualize the EtBr intercalated DNA for confirmation of experimental results.

Add the agarose and TAE buffer as this table into a flask whose volume is 2-4 times greater than the one of the solution being prepared.

Heat the mixture in the microwave oven for 90 seconds. After heating, remove the flask from the microwave oven with a glove and shake the flask gently to completely blend the liquid mixture. Till the solution cool down to 50-60℃, add 1 μL of DNAgreen(UV) and shake to mix. Pour the gel into a tray and insert the well comb, leave the gel to solidify for 10-15 minutes at room temperature. Once the gel is solidified, gel is ready for use.

Gel electrophoresis

1. Remove the comb from the cast gel.
2. Place the gel into an electrophoresis system and make sure that the volume of 1×TAE buffer is sufficient.
3. After submerging the wells of the gel, and load DNA marker and samples.
4. Load 5μL of DNA size marker (2or 10kDa).
5. Mix 10μL of the samples with 1μL 10×loading buffer and then load the mixture. The volume depends on the purpose of the gel（confirmation or purification）.
6. Place the electrodes with the cover over the gel system and start the electrophoresis.
7. Set voltage to 120 V. Set the appropriate gel run time as 30 minutes. After the run is over, switch off the system.
8. Remove the gel and place it over a UV or Blue-light illuminator to visualize the EtBr intercalated DNA either for confirmation of experimental results or gel excision and DNA purification.

Gel extraction

1. Remove the gel from the electrophoresis system.
2. To visualize the DNA prior to extraction one cannot use UV illuminators due to risk of mutation caused by UV exposure. Instead, a Blue-light illuminator in a dark room can be used, which is sufficient for accurate gel excision. Note: If the possibility of avoiding UV irradiation is out of the question, please try to keep the exposure of the gel to UV under 10 seconds to avoid damaging DNA.
3. Excise the bands with the DNA of interest keeping the excess of gel to a minimum as it decreases the yield of DNA after cleanup.
4. Follow the AxyPrep Plasmid Miniprep Kit, which is listed below as follows:
5. Excise a gel slice of up to 200 mg containing the DNA fragment using a clean scalpel or razor blade. Cut as close to the DNA as possible to minimize gel surplus. Place the gel slice into a 1.5 mL tube.
6. Add 200 μL of Binding Buffer. Mix thoroughly by pipetting.
7. Incubate the gel mixture at 60 °C for 10 minutes or until the gel slice is completely melt. Mix the tube by inversion every few minutes to facilitate the melting process. Ensure that the gel is completely dissolved.
8. Place a Miniprep column into an uncapped 2 ml Microfuge tube. Transfer the clarified supernatant from Step 4 into the Miniprep column. Transfer the Miniprep column and 2 ml Microfuge tube to microcentrifuge and spin at 12,000 × g for 1 minute.
9. Discard the filtrate from the 2ml Microfuge tube.
10. Pipette 500μl of Buffer W1 into each Miniprep column. Centrifuge at 12,000×g for 1 minutes.
11. Place the Miniprep column back into the 2 ml Microfuge tube and centrifuge at 12,000 ×g for 1 minute.
12. Discard the filtrate from the 2ml Microfuge tube.
13. Place the a microtube of water and the Miniprep with cap open in 60℃ oven for 5 minutes. The water can be kept in the oven longer if the temperature isn’t high enough.
14. Pipette proper volume of warm water into the miniprep placed on the 2mL microfuge tube. Centrifuge at 12,000×g for 1 minutes.
15. Store the water containing purified DNA at -20℃.

Chemical transformation

1. Take competent bacteria out of the 80 °C freezer and melt on ice.
2. Turn on the UV sterilization function in the clean bench for about 15 minutes to sterilize the working environment before opening the test tube with the bacteria to avoid any risk of contamination.
3. Take out petri dishes with required medium and antibiotic out of the refrigerator.
4. Put 5 μL of plasmid into 20 μL of competent cells culture. Also, prepare as many of the transformation controls as possible: (a) Transform the bacteria without DNA - contamination control. (b) Transform the bacteria with a linearized digested vector - to check if DNA is properly digested. (c) Transform the bacteria with purified non-digested DNA - positive control, to check transformation efficiency.
5. Hold the samples on ice for 30 minutes.
6. Perform a heat shock for 1.5 min at 42 °C.
7. Incubate samples on ice for 5 minutes.
8. Suspend the bacteria with 200μL of LB (Luria Broth).
9. Incubate samples in the 37 °C heat block for 60 minutes.
10. Plate out this mixture on an LB petri plate with the correct antibiotic.
11. Incubate the plates overnight at an optimal temperature for selected bacteria.

Cell lysis by sonication

1. Resuspend the bacterial cell biomass with 2 mL of resuspension buffer (PBS pH7.2, 50 mM).
2. Sonication was carried out on ice using the Scientz Ultrasonic Homogenizer JY92-ⅡN.
3. Sonication process parameters:
4. 30% power
5. 3s ON / 7s OFF sonication for 20 minutes
6. After sonication, centrifuge the sample at 12 000 RPM at 4 °C for 10 min. Carefully transfer supernatant into another tube for further use.
7. Remaining precipitation is then stored at -20 °C

Nepetalactol detection

1. Take the target strain out of the -80°C refrigerator and put it in a foam ice box, take out 3 ml of the sterilized YPD+Glu medium into a test tube, inoculate 1% bacterial solution into the test tube, and put it in 30 Incubate in a shaker for 24h.
2. Remove the test tube. Add 30 ml of sterilized YPD+Glu medium to each of the three sterilized shake flasks. Transfer the bacterial solution (300ul) in a 1% test tube to a shaker flask, and place it in a shaker at 30°C for 24h.
3. Remove the shake flask. Add 30 ml of sterilized YPD+Gal medium to each of the three sterilized shake flasks.
4. Transfer the liquid in the shake flask to a large 50 ml centrifuge tube, centrifuge at 4000r for 5 min, and discard the supernatant.
5. Add 25-30 ml sterile water to a large centrifuge tube, centrifuge at 4000r for 5 min, and discard the supernatant.
6. Repeat step 5.
7. Take a tube of YPD+Gal medium in a shake flask and add it to a large centrifuge tube, blow and mix the precipitate, then transfer it back to the shake flask, and put it into a shaker at 30°C for 48 hours.
8. take out the shake flask and the fermentation is complete.
9.Take out 1ml of the bacterial liquid after the fermentation is completed, and add ethyl acetate solution mixed with one thousandth pure formic acid. Extraction is carried out, the organic phase is removed, and the aqueous phase is discarded.
10.Extracts were analyzed by GC EI-MS using an Agilent 6890N GC system coupled to an Agilent 5875 mass selective detector. Three μL of extracts were injected in pulsed splitless mode at 50 PSI for 0.5 min with an injection temperature of 250 °C. Hydrogen was used as a carrier gas at a constant flow of 1 mL/min. A 10 min temperature gradient ranging from 80 to 300 °C was used to separate analytes over a DB-5 ms column (25 m × 0.20 mm × 0.33 μm film thickness).Analytes were identified and quantitated using linear calibration curves with authentic standards in concentrations ranging up to100 mg/L. Nepetalactol was measured by following ions 135 and 168 m/z.

Yeast transformation (integrated into the genome)

Bacterial culture
1.Inoculate 5 mL of liquid YPD (or 10 mL of SC), and incubate overnight at 30°C with shaking at 250 r/min (test tube);
2.Count the cell density, inoculate the final density of 5 × 106 cells/mL to 50 mL of LYPD solution at 30 °C (shaking flask), and culture with shaking at 250 r/min for 3-5 h;
3.Culture to at least 2×107 cells/mL, which can be used for transformation.
Note: The dosage and steps in the above steps can be flexibly changed according to the needs. The key is to obtain a suitable yeast solution for the competent operation.

Obtain competent cells
1.Use a 50mL sterile centrifuge tube, centrifuge at 4000g for 5min, discard the culture medium, and collect the cells;
2.Washing: Suspend the cells in 25mL sterile water, centrifuge at 4000g for 5min, and discard the liquid phase (this step is repeated twice);
Suspend the cells in 1mL of 0.1M lithium acetate (pipette suction), which is the yeast competent, and can be stored in a refrigerator at 4°C for a week.

Yeast transformation and inoculation
1. Transfer a small amount of suspension (between 50μl-200μl according to the concentration of the suspension) to a sterile 1.5mL (or 2mL) centrifuge tube, centrifuge the cells at 12000r/min for 3s (or 4000g for 30s), and use a micropipette to aspirate the acetic acid lithium;
Note: This step needs to control the amount of competent cells in a single transformation process (if the yeast pellet obtained by centrifugation is too thick, the success rate of yeast transformation may not be high). thin thickness. In order to control this number, it can be flexible. For example, a small amount of suspension can be aspirated before centrifugation for control.
2. Boil 1 mL of ssDNA sample for 5 min in advance, and then quickly cool in ice water;
3. Add the following substances in sequence: 240μL PEG (because PEG is relatively viscous, after adding PEG, blow off the precipitate and mix with a vortexer), 36μL 1.0M lithium acetate, 50μL ssDNA, 34μL sterile water and plasmid DNA (about 700-1000ng), blowing and sucking, mixing and shaking evenly;
4. Heat shock in a water bath at 42°C for 60min;
5.Precipitate cells at 12000r/min for 3s, and remove the transformation mixture with a micropipette (pay attention to removing as thoroughly as possible);
6. Suck 0.5-1.0mL of sterile water into the reaction tube, and blow with a pipette to suspend the precipitate;
7. Coat the plate with a suitable volume of suspension (100-200μl) and incubate for 2-3 days