Experiments
1. Common laboratory ratio
1.1 Solution inoculum volume (v / v): 1%
1.2 Antibiotic concentration (v / v): 1‰
1.3 Dye ratio: 0.1‰
2. Preparation of Agarose Gel and DNA Electrophoresis
2.1 Agarose gel electrophoresis, to prepare 1% gel.
2.1.1 Measure 1g of agarose, mix it with 100mL of TAE.
2.1.2 Microwave for 1-3 minutes until agarose dissolves.
2.1.3 Pour the mixture into a gel tray with well comb in place.
2.1.4 Let the mixture cool down for 10-15 minutes.
2.1.5 Once solidifyed place the agarose gel into a gel box.
2.1.6 Load samples and marker into the wells of the gel.
2.1.7 Put the gel into the electropheoric chamber.
2.1.8 Start the electropheoric chamber for 20 minutes, 140V.
2.1.9 Remove the gel afterwards and place it in an UV light source.
2.1.10 Compare the sample markings to the marker.
3. Reagents and media configurations need to be sterilized at 121℃ for 20 min
3.1 Commonly used reagent configuration
3.1.1 Reagents used for the yeast validation experiments
| Materials | Dosage |
|---|---|
| 1M Sodium Hydroxide | 4 g+100 mL Purified water |
3.1.2 Antibiotic preparation (Only filtration sterilization is employed in this process)
| Materials | Dosage |
|---|---|
| Carbenicillin（50 mg/ml） | 1 g+20 mL Purified water |
| Kanamycin（50 mg/ml） | 1 g+20 mL Purified water |
3.1.3 50% (V/V) Glycerol (suitable for making frozen stocks)
| Materials | Dosage |
|---|---|
| Glycerol | 50 ml |
| Purified water | 50 ml |
3.2 Culture medium preparation：
3.2.1 Preparing stock solutions of reagents
| Materials | Dosage | 200mL Purified water | Notes |
|---|---|---|---|
| Glucose（20x） | 400 g/L | 80 g | Add to medium at a final concentration of 1x |
| Ura（100x） | 2.24 g/L | 0.448 g | Add to medium at a final concentration of 1x |
3.2.2 YPD medium (Yeast extract peptone dextrose medium)
| Materials | Dosage | 800mL Purified water | Notes |
|---|---|---|---|
| Yeast extract | 10 g/L | 8 g | |
| Peptone | 20 g/L | 16 g | |
| Glucose（20×） | 20 g/L | 40 mL | Add it after sterilization |
| H2O | 1 L | 760 mL | |
| Agar (solid plate) | 30 g/L |
3.2.3 Synthetic complete medium：
| Materials | Dosage | 800mL Purified water | Notes |
|---|---|---|---|
| Ammonium sulphate | 5 g/L | 4 g | |
| YNB (Yeast Nitrogen Base) | 1.7 g/L | 1.36 g | |
| DO Supplement-Ura | 0.78 g/L | 0.624 g | |
| Glucose（20×） | 20 g/L | 40 mL | Add it after sterilization |
| Agar (solid plate) | 30 g/L | 760 mL |
3.2.4 LB medium （suitable for cultivating E.coli and other bacteria）
| Materials | Dosage | 800mL Purified water |
|---|---|---|
| LB Broth | 30 g/L | 20 g |
4. Plasmid Extraction method
4.1.1 Place 1.5 ml of bacterial fluid into a 1.5 ml centrifuge tube and centrifuge at 12,000 rpm for one minute. Repeat step 1 until 3 ml of sample precipitate is collected.
4.1.2 Add 150ul of Buffer P1 and mix with a vortex mixer.
4.1.3 Add 150ul of Buffer P2 and softly invertate for eight times to mix well. (This step should be finished within 2 mins)4.1.4 Add 350ul of Buffer P5 and softly invertate for eight times, until the color of mixture is even.
4.1.5 Centrifuge at 12000 rpm for 7 minutes.
4.1.6 Place the adsorbent column CP3 in the collection tube and pour the supernatant from the
4.1.7 Centrifuged tube (Do not mix in any sediment). After resting for 1 min, centrifuge at 12,000 rpm for 2 minutes and pour off the waste solution.
4.1.8 Add 300 ul of Buffer PWT to the adsorbent column CP3 and let it stand for two minutes.
4.1.9 Centrifuge at 12,000 rpm for one minute and pour off the waste solution.
4.1.10 Put adsorbent column CP3 with the waste solution removed into the collection tube and centrifuged at 12000 rpm for 3 mins.
4.1.11 Remove the adsorbent column CP3 from the collection tube and place it in a 1.5ml.
4.1.12 Centrifuge tube. Place the combined centrifuge tube into a dry bath incubator at 50°C for 2 mins (with lid opened).
4.1.13 Drop 50ul of ddH2O in the middle of the adsorption column CP3. Place in a dry bath. incubator at 50°C for 5 minutes (with lid closed). Then centrifuge at 12,000rpm for 2 mins.
4.1.14 The DNA plasmid solution is collected into the 1.5 ml centrifuge tube, then we could assay the concentration with qubit 4.
5. Golden-gate assembly and bacterial transformation
5.1 Connect the synthesized plasmid containing the target gene to HcKan-O (universal carrier) (enzymes: BSA1, T4 DNA ligase)
5.1.1 Mix all the materials as the table mentioned below in the PCR tube.
| mixture | input |
|---|---|
| HCKAN_O | 4 μL |
| Gene | 4 μL |
| 10×T4DNA ligase buffer | 1 μL |
| T4 DNA ligase | 0.5 μL |
| BsaI | 0.5 μL |
| ddH2O | 0 μL |
5.1.2 Put the tube with mixture into the thermal cycler and react according to the following procedure.
| Temperature | Time |
|---|---|
| 37℃ | 1 h |
| 25℃ | 1 h |
| 55℃ | 15 min |
| 80℃ | 15 min |
| 12℃ | - |
5.2 Connect the promoter+Gene+terminator to the POT plasmid (enzymes: ESP3I, T4 DNA ligase)
5.2.1 Mix all the materials as the table mentioned below in the PCR tube.
| mixture | input |
|---|---|
| POT | 1 μL |
| HCKAN_P-X | 2 μL |
| HCKAN_O-Gene | 3 μL |
| HCKAN_T-X | 2 μL |
| 10×T4 DNA ligase buffer | 1 μL |
| T4 DNA ligase | 0.5 μL |
| ESP3I(BSMBI) | 0.5 μL |
| ddH2O | 0 μL |
5.2.2 Put the tube with mixture into the thermal cycler and react according to the following procedure.
| Temperature | Time |
|---|---|
| 37℃ | 1 h |
| 25℃ | 1 h |
| 55℃ | 15 min |
| 80℃ | 15 min |
| 12℃ | - |
5.3 DH5 α conversion operation
5.3.1 Thaw the competent cells DH5 α on ice 10 minutes in advance.
5.3.2 Take the PCR tube out of thermal cycler after the reaction is completed. Gently mix all the assembly system with 100 ul competent cell, and put it on ice bath for 20 minutes without shaking
5.3.3 After the ice bath is completed, water bath heat shock at 42 ℃ for 45 s without shaking.
5.3.4 Take a sample ice bath for 2 min.
5.3.5 Add 500 μL of non-resistant sterile LB liquid medium.
5.3.6 Place in a shaker at 37 ℃ and shake at 250 rpm for 45-60 minutes to revive and culture.
5.3.7 Take 100 μL of the bacterial solution and evenly spread it onto LB agar plates containing the appropriate antibiotic. Incubate the plates at 37℃ overnight to observe the growth of bacterial colonies.
6. Verification Operation
6.1 Colony PCR
6.1.1 Pick and incubate single colony bacteria
- In the laminar flow hood, add 1 ml of LB liquid medium containing the appropriate antibiotics into a 1.5 ml EP tube, and use the pipette tip to pick (white) single colonies and discard them into the centrifuge tube. (Pick 10-15 single colonies from each plate.)
6.1.2 Incubate at 37°C with 220 rpm shaking for 3 hours on a shaker. Proceed with single clony PCR validation.
6.1.3 Mix all the materials as the table mentioned below in the PCR tube.
| Materials | Dosage |
|---|---|
| 2×Taq PCR mix | 5 μL |
| Primer F | 0.4 μL |
| Primer R | 0.4 μL |
| ddH2O | 3.2 μL |
| Template DNA | 1 μL |
6.1.4 Mix the PCR system using a vortex mixer, and centrifuge shortly. Then place the PCR tube in the thermal cycler, follow the steps below to set up the program, and close the cover to run the program.
| Temperature | Time |
|---|---|
| 94℃ | 3 min |
| 94℃ 20s | 30 cycle |
| 56℃ 30s | |
| 72℃ 90s | |
| 72℃ | 5 min |
| 12℃ | - |
6.1.5 After PCR, perform electrophoresis
6.2 Enzyme restriction validation operation.
6.2.1 Mix all the materials as the table mentioned below in the PCR tube.
| Materials | Dosage |
|---|---|
| Vector | 5 μL |
| 10x cut smart buffer | 1 μL |
| BSAI(POT)/ESP3I(HCKAN-O) | 1 μL |
| ddH2O | 3 μL |
6.2.2 Put the tube with mixture into the thermal cycler and react according to the following procedure.
| Temperature | Time |
|---|---|
| 37℃ | 30 min |
| 80℃ | 15 min |
| 12℃ | - |
6.2.3 After PCR, perform electrophoresis.
7. Yeast Experiment Operation Procedure
7.1 Yeast Competent Cell Preparation Protocol (Strain: BY4741)
7.1.1 Inoculate the BY4741 yeast strain into 5 mL of YPD medium and incubate overnight to prepare the seed culture.
7.1.2 The next day, dilute the seed culture 10-fold and measure its optical density at 600 nm (OD600).
7.1.3 Using the diluted culture, inoculate 20 mL of YPD medium to achieve an initial OD600 of 0.1. Incubate at 30°C in a shaker until the OD600 reaches 0.6 to 1 (growth rate approximately doubles every 1.5 hours).
7.1.4 Measure the OD600 again. Transfer 5 mL of this culture to a centrifuge tube and centrifuge at 3000 rpm for 5 minutes to collect the yeast cells.
7.1.5 Resuspend the yeast pellet in 4 mL of Solution A and wash the cells. Centrifuge again at 3000g for 5 minutes and carefully remove the supernatant.
7.1.6 Resuspend the yeast pellet in 0.5 mL of Solution B to create competent cells.
7.1.7 Portion the competent cells into 50 μL aliquots in sterile 1.5 mL cryogenic vials. Slowly freeze these vials and store at -80°C for future use.
7.1.8 When needed, remove the vials from the -80°C freezer, allow them to thaw at room temperature, and use the competent cells directly for transformation procedures.
7.2 Transformation
7.2.1 Prepare the premix for each plasmid transformation, requiring a total of 360 μL premix.
| Solution C | 350 μL |
| Plasmid (approximately 200 ng/μL) | 10 μL (Adjust the volume according to the plasmid concentration) |
| Total Volume | 360 μL (Top up any shortfall with ddH2O) |
7.2.2 Aspirate 360 μL of the premix and add it to resuspend the yeast competent cells. Use the pipette tip to repeatedly blow and draw the precipitate, thoroughly suspending the yeast cells in the premix at the bottom of the tube.
7.2.3 Place it in a 30°C water bath for heat shock for 45 min, shake to mix thoroughly every ten minutes. After the water bath, centrifuge at 3000g for 3 min.
7.2.4 Resuspend the precipitate in 1 mL of sterile water.
7.2.5 Spread 100 μL of the yeast suspension on SC-URA tablet.
7.2.6 Put the tablet after spreading into 30 ℃ celcius incubator for 2 days to gain successfully transformed single-cloned yeast.
7.3 Colony PCR of S.cerevisiae
7.3.1 Plate streaking for bacterial enrichment
- mark and pick six single colonies to perform streaking for isolation. After incubating at 30°C for one day, colonies of an appropriate size (rice grain size) can be selected for clone PCR verification. Then the plate should be placed back into the incubator for further incubation for 2 days.
7.3.2 lyse yeast cells
- Dispense 50 μl of freshly prepared 20 mM Sodium Hydroxide (NaOH) solution into a PCR tube. Add yeast cells and thoroughly mix.
- Place the tube in the PCR machine and run the following program.
- Upon completion, chill the tube on ice. Perform a quick spin, and use 1 μl of the supernatant as the template for colony PCR.
| Temperature | Time |
|---|---|
| 99℃ 5 min | 3 cycle |
| 12℃ 5 min | |
| 12℃ | - |
8. Fermentation and detection of Limonene/Ocimene/Myrcene/borneol/Cineole/Nerol/Nerolidol
8.1 Fermentation conditions
8.1.1 Inoculate A single colony of S.cerevisiae within YPD medium and cultivate overnight, then the seed solution would be obtained.
8.1.2 Measure the OD600.
8.1.3 The seed culture was inoculated to an initial OD600 of 0.1 into a fertilized 250 mL flask containing 50 mL SC-URA medium and cultivated at 30 ℃ and 250 rpm.
8.1.4 After fermentation for 12 h, 5 ml dodecane (10%) overlay culture was added.
8.1.5 The fermentation process was ended at 120 h.
8.1.6 Transfer the fermentation solution from the flask into a 5 mL centrifuge tube and centrifuge to obtain the organic phase.
8.1.7 Prepare a sample for GC-MS analysis by combining equal volumes (500 μL) of the dodecane extract and ethyl acetate.
8.2 GC-MS analysis
8.2.1 1μL samples were injected into a GC-MS [7890A (GC) + 5975C (MS), Agilent] equipped with a HP5MS column (30 m × 0.25 mm × 0.25 μm) and operated with He carrier gas at 1.0 mL/min.
8.2.2 Temperature and voltage were set as follows: ionization voltage 70 eV, split ratio of 30:1; transfer line temperature 250°C; ion source temperature 230°C.
8.2.3 The oven program was as follows: 80°C (held for 1 min), 80–90°C (2°C min–1), 90–270°C (20°C min–1), and held for 5 min, with a total program of 21 min.
8.2.4 Analysis is carried out by SIM (Single Ion Monitoring) mode: m/z = 50–250.