Preface
By modifying microbial cell factories through targeted approaches to construct a programmable biosynthetic platform in our project, we can achieve on-demand synthesis of rose essential oil, known as the "Queen of Flowers," and lavender essential oil, known as the "Universal Guardian".
PCR protocol
The reaction components are assembled in the PCR protocol as described below. The final volume should be 20 µL.
1. All reagents are thawed on ice.
2. Assemble reaction mix into 20 µL volume in a thin walled 0.2 mL PCR tubes as follow:
8 uL ddH2O
1 µL DNA Template
0.5 uL Primer F
0.5 uL Primer R
10 uL 2x Taq mixture
3. Prepare negative control reaction without template DNA. Prepare positive control reaction with template of known size and appropriate primers.
4. Gently mix by tapping tube. Briefly centrifuge to settle tube contents.
5. Put the tube into a PCR instrument.
6. Set up the following PCR program: initial denaturing at 94°C for 4 min, followed by 30 cycles of 94°C for 30 sec, 55°C for 30 sec, and 72°C for 1 min, plus a final extension at 72°C for 10 min.
7. Check the PCR product by agarose gel electrophoresis.
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Gel Electrophoresis
1. Prepare 100mL 1xTAE buffer with 1 g agarose, and boil it three times, shake completely, and waiting for cool.
2. Pour the agarose gel into gel tray, assemble gel pouring apparatus by inserting gate into slots.
3. Allow agarose to cool, place the gel in the apparatus rig with the wells facing the negative end (black-colored).
4. Fill the rig with 1x TAE buffer.
5. Load 8 μL of DNA maker into lane.
6. Mix 1 μL of 10x loading buffer with 5 μL DNA sample, load them into lane.
7. Run at 100 V for 30 min.;
8. Use the Gel imaging system to check the gel;and take a picture.
9. Deal with the gel carefully as medical waste.
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The Competent Cell Preparation
1. Streak the E. coli stock on a LB-agar plate. Incubate the plate at 37°C overnight.
2. Pick a single well-isolated colony and inoculate it into 3 mL of LB broth solution. Incubate it at 37°C overnight (more than 16 h) with shaking at 220 rpm.
3. Transfer 250 uL of the saturated overnight solution to a sterile 50 mL polypropylene tube containing 25 mL of LB medium. Incubate the E. coli at 37°C with shaking at 220 rpm until OD600 reaching 0.6. This usually takes 2.5 h. Check the OD every 30 min after 1 h to avoid overgrowth.
4. When the medium reaches an OD600 of 0.6, chill the tube on the ice for 30 min and then centrifuge at 8000 rpm for 1 min at 4°C, discard the supernatant.
5. Re-suspend the E. coli in 2.5 mL of ice-cold TSS solution with gentle swirling. Incubate on ice for 10 min. Aliquot in 50 uL per tube. Now the competent cells are ready to be transformed.
Note: Transformation and Storage Solution (TSS) contains LB medium 85% 8.5 mL, PEG (wt/vol, Mw 8000) 10% 1.0 g, DMSO (vol/vol) 5% 500 μL, and MgCl2 (pH 6.5) 50 mM 500 μL.
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Plasmid Transforming and Pick monoclonal
1. Thaw TSS cells on ice.
2. Add 5 uL Synthetic plasmid DNA in TSS cells, pipette gently to mix.
3. Incubate it on ice for 30 min with occasional mixing.
4. Heat shock at 42°C for 90 sec.
5. After heat shock, put it on ice for 2 min, add 0.8 mL LB medium into the cells.
6. Shake and incubate at 37°C for 60 min at 120 rpm.
7. Take out 100 uL medium and spread them on the appropriate agar plates with certain antibiotic.
8. Incubate the plates at 37°C overnight.
9. Pick monoclonal into certain antibiotic LB medium, and incubate the medium at 37°C overnight.
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Plasmid Isolation Protocol
1. The bacteria that contained target plasmid were cultivated at 37°C overnight.
2. Take out 1,500 uL solution of cultivated bacteria to a 2mL micro-centrifuge tube. The tube was centrifuged at 12,000 rpm for 1 min at room temperature, and then the supernatant was discarded. In this tube, repeating these steps three times to increase the concentration of the bacteria.
3. Add 250 uL Solution I buffer to suspend the precipitation. Complete suspension is vital for obtaining good plasmid yield by vortexing or pipetting up and down.
4. Add 250 uL Solution II buffer, mix gently upside down 4-6 times to make a cleared lysate. A 2-3 min incubation at room temperature may be necessary. However, this step should not be more than 5 min, and avoid vigorous mixing as doing so will shear chromosomal DNA and lower plasmid purity.
5. Add 350 uL Solution III buffer, mix gently and fully upside down 6-8 times, centrifuge the tubes at 12,000 rpm for 10 min at room temperature.
6. Put a HiBind DNA Mini Column into a 2 mL Collection Tube.
7. The supernatant in step 4 is transferred to the Mini Column, and centrifuged at 12,000 rpm for 1 min, the filtrate is discarded.
8. Put the Column back into the Collection Tube, add 500 uL HBC Buffer, centrifuge at 12,000 rpm for 1 min, the filtrate is discarded.
9. Put the Column back into the Collection Tube, add 700 uL Washing buffer, centrifuge at 12,000 rpm for 1 min, the filtrate is discarded. Repeat the washing step once.
10. Put the Column back into the Collection Tube and centrifuge at 12,000 rpm for 2 min.
11. Put the Column into a new 1.5 mL micro-centrifuge tube and leave it for 2 min at room temperature. Then add 50 uL ddH2O to the center of the Column matrix, and leave it at room temperature for 1 min. Centrifuge at 12,000 rpm for 1 min. Discard the HiBind DNA Mini Column.
12. Measure concentration of extracted plasmid DNA by nanodrop 1000, and store plasmid DNA at -20°C.
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Plasmid DNA Restriction endonuclease Digestion
DNA for downstream applications is usually digested with restriction endonucleases. Type II restriction enzymes are the most widely used in molecular biology application.
The reaction volume are carried out in 20 µL.
1. Add components to a clean tube in the order as follow:
1 µL DNA (1 µg/µL)
2 µL 10x buffer
1 µL each restriction enzyme
15 µL ddH2O
2. Mix gently and fully, and incubate the tube at certain temperature (37°C) for 1 h.
3. Stop the reaction by heat inactivation (65°C for 15 min).
4. The digested DNA is ready for use in research applications.
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Purification of His/S-tagged protein
All the procedures were performed at 4 °C or on ice unless otherwise specified.
1). 10 mL BL21 cells transformed with a pET-28a-lacZ were cultured at 30°C until an OD660 of approx. 0.8.
2). Add IPTG to the culture (final 0.67 mM IPTG)
3). Cultivate at 30 °C for 2 h.
4). Collect cells in 5 mL tube, centrifuge at 5,000 rpm at 15 min.
5). Suspend cells in 5 mL of TBS, transfer the content to 1.5 mL tube.
6). Collect cells at 10,000 rpm at 1 min. The supernatant was discarded.
7). Add 500 µL of ice-cold lysis buffer to the cell pellet.
8). Sonicate cell (4°C).
9). Clarify lysate by centrifugation at to 10,000 rpm at 10 min.
10). After centrifugation, save 20 µL of supernatant for SDS-PAGE sample.
11). Transfer supernatant to the 1.5mL tube containing pre-balanced resin bead gel.
12). Shake gently (4°C) for 30 min.
13). Centrifuge the tube 1,200 rpm for 30 sec. The supernatant was discarded.
14). Wash resin bead gel with washing buffer, Centrifuge the tube 1,200 rpm for 30 sec. The supernatant was discarded. Repeat it once.
15). Elute the protein 3 time with 20 µL of elution buffer. It is ready to prepare the SDS-PAGE sample.
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SDS Gel Electrophoresis
1). Make up 30 mL of running gel by adding components to a clean glass in the order as follow:
12.3 mL H2O
7.5 mL 1.5 M Tris-HCl, pH 8.8
0.15 mL 20% (w/v) SDS
9.9 mL Acrylamide/Bis-acrylamide(30%/0.8% w/v)
0.15 mL 10% (w/v) ammonium persulfate (APS)
0.02 mL TEMED
2). Mix the ingredients mentioned above.
3). Pour the solution quickly into a gel casting form, and leave about 2 centimeters below the bottom of the comb for the stacking gel. Then layer the top of the gel with water very carefully. Wait for about 30 min for the gel to polymerize completely.
4). While waiting, mix the reagents for the stacking gel (4% Acrylamide) by adding components to a clean glass in the order as follow:
3.075 mL H2O
1.25 mL 0.5 M Tris-HCl, pH 6.8
0.025 mL 20% (w/v) SDS
0.67 mL Acrylamide/Bis-acrylamide (30%/0.8% w/v)
0.025 mL 10% (w/v) ammonium persulfate (APS)
0.005 ml TEMED
5). Mix the ingredients mentioned above.
6). Remove the water on the top of running gel, and pour the stacking gel on top of the running gel. Insert the combs by trying not to produce bubbles stuck underneath and allow another 0.5 - 1 h for complete polymerization.
7). Prepare samples: mix the protein 4:1 with the loading buffer. Heat the sample by Boiling for 5-10 min.
8). Run gel. Clamp the gel and fill both buffer chambers with gel running buffer. Pipet the sample and molecular weight standard marker into the gel. Attach the power leads and run the gel until the blue dye reaches the bottom.
9). Visualize the proteins using Coomassie Brilliant Blue.
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Yeast Transformation
1. Spread the yeast on YPD plates, incubate 2 d at 30˚C.
2. Pick a single colony and inoculate it into YPD broth, and shake for 1 d at 30˚C 200 rpm.
3. Dilute yeast at 1:1000 into 30 mL YPD until the OD value lies at 0.6-1.0. Collect yeast after 3000 rpm for 5 min.
4. Resuspend using 30 mL of ddH2O and discard the supernatant.
5. Add 1.5 mL of suspensions (TE: LiAc: H2O=1:1:8) centrifuge at 3000 rpm for 5min, discard the supernatant.
6. Resuspend yeast with 1.5 mL of suspensions, distribute to Eppendorf tubes for 50 µL each.
7. Add 5 µL of plasmid, 10 µL of Salmon sperm DNA, 300 µL of transformation reagent (TE: LiAc: H2O=1:1:8) into 50 µL of bacteria, blend gently.
8. Hot bath at 30˚C for 30 min, blend each 15 min.
9. Hot bath at 45˚C for 15 min.
10. Cold bath for 2 min.
11. Discard supernatant after 50000 rpm for 3 min then rests-end with 500 µL of ddH2O.
12. Pipette 100 µL of bacterium solution on a SC-U glucose plate, spread. Incubate for 3-4 d at 30˚C.
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Genome extraction from Yeast
1. Centrifuge 1 mL of bacterium solution for 1 min, discard the supernatant.
2. Add 500 µL of GHA and 10 µL of proteinase K into the remaining and place inside a metal bath at 65˚C 15 min 800 rpm.
3. 99˚C 800 rpm 15 min.
4. After cooling, add 600 µL of GHC and well blend with vortex.
5. Pipette the solution to a spin column, 700 µL per time until all solution is added. Centrifuge with 12000 rpm for 1 min, discard the flow-through. Repeat this step until all solution is centrifuged and the supernatant discarded.
6. Add 8 µL of Buffer PD and let stand for 2 min. Centrifuge 12000 rpm for 1 min, discard the flow-through.
7. Repeat 6.
8. Add 600 µL of Buffer PW, let stand for 2 min. Centrifuge 12000 rpm for 1min then discard the flow-through.
9. Repeat 8.
10. Centrifuge at 12000 rpm for 3 min without adding anything.
11. Transport solutions to a new 1.5 mL Eppendorf tube. Dry the tube with the lid open for 5 min.
12. Add 100 µL of Buffer TB. Close the lid and let stand in 50˚C for 5 min.
13. Centrifuge at 12000 rpm for 3 min. Flow-through inside the Eppendorf tube is desired DNA.
14. Obtain concentration with a microplate reader
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E. coli Shake-flask Fermentation
.1. Plate a preserved culture to obtain single colonies.
2. Preculture: Pick a single colony from the plate to 3 mL of culture with antibiotic (LB), grow overnight in 37˚C.
3. Add 180 µL of medium with antibiotics into each hole in a 96 well plate, then pipette 20 µL of bacterium culture into each well. Use LB medium for blanking.
4. Incubate the bacterium culture into the 50 mL medium with OD600 adjusted to 0.01 into a clean, sterilized 250 mL shake-flask.
5. Add 25 µM IPTG when OD600 value is 1.
6. 24 h after adding IPTG, transport a sample of 5 mL bacterium solution into a 15 mL centrifuge tube. Store it in a -20ºC refrigerator.
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Preparation of gas chromatography samples from E. coli
1. Pre-warm the -20ºC bacterium culture sample.
2. Pipette 1mL organic phase (n-Hexane) to 5 mL of bacterium solution in a 15 mL centrifuge tube.
3. Vortex for 3 min.
4. Seal the 15 mL centrifuge tube with biofilm and store it in -20ºC refrigerator overnight.
5. Centrifuge 5000 rpm, 3 min to separate the organic phase from bacterium culture.
6. Transport 100 µL of organic phase to 900 µL of ethyl acetate.
7. Filter the mixture.
8. Sample is ready for gas chromatography analysis.
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Preparation of standard geraniol sample
1. Dilute 97% geraniol into dodecane in the ratio of 1:1000. 100µL geraniol was added to 900 µL dodecane, noted as solution one. Then 100 µL of solution one was added to 900 µL dodecane, noted as solution two. 100 µL of solution two was added to 900 µL dodecane.
2. Dilute the solution into different concentrations with the aid of this table.
3. With help of small injector and green organic phase filter, squeeze the solution into the GC glass tube.
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Co-culture
1. Pick a free colony of E. coli into 5mL of LB (Amp 100 µg/mL, Kan 50 µg/mL) in each hole of a 96-well deep well plate, and grow overnight at 37˚C, 250 rpm.
2. Pick a yeast colony into each well of 5 mL LB of a 96-well deep well plate.
3. Place each of the E. coli and Yeast separately in a 250 mL conical flask, culture with 50 mL of corresponding broth. Initial OD value is 0.2.
4. Grow overnight culture of Yeast. E. coli grows until mid-log phase.
5. Put both Yeast and E. coli into a 50 mL co-culture medium, initial OD value is 0.2. The engineered E. coli and the engineered yeast were transferred to the synthetic fermented medium at an initial OD600 respectively and cultivated at 30˚ C, 220 rpm.
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Yeast Fermentation
1. Streak Yeast on SC-Ura plate.
2. Pick a free colony into 1mL of liquid SC-Ura broth. Grow at 28˚C 250 rpm, 24 h.
3. inculcate 300 µL of bacterium solution to 30 mL of YPD broth. Grow culture for 24 h.
4. Prepare a 200 mM geraniol, pipette 40 µL each 2 h. Grow culture for 24 h at 28˚C.
5. Pipette 5 mL of fermenting liquid to 1 mL of n-hexane for extraction. Vortex mix for 2 min. Let stand overnight for extraction. Pipette 200 µL of organic phase into 800 µL of n-hexane.
6. GC characterization.
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Ingredients of main certain mediums
Materials and Concentration of LB
Tryptone 10 g/L
Yeast extract 5 g/L
NaCl 10 g/L
Materials and Concentration of Co-culture
KH2PO4 13.3 g/L
Citric acid 1.7 g/L
(NH4)2HPO4 4 g/L
MgSO4 pH7.0 1.3 g/L
Yeast extract 5 g/L
Materials and Concentration of YPD
Yeast extract 10 g/L
Tryptone 10 g/L
ddH2O 900 mL
Agar 20 g
20% glucose mother liquor 100 mL
Materials and Concentration of SC-Ura
Yeast Synthetic drop-out without uracil 1.3 g/L
Agar 30 g
ddH2O 900 mL
20% glucose 100 mL
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