Goal: To create a functional and nutritional liquid or solid medium for bacteria growing. Activation, cultivation, and fermentation for

microorganisms.

Materials:

1) Conical flask

2) Autoclave

3) Tryptone

4) Yeast Extract

5) Sodium Chloride

6) Double distilled H2O

7) Agar

Procedures:

1) Add 10 g tryptone, 5 g yeast extract, and 10 g of sodium chloride per liter of the culture medium. (Note: 1.5% agar powder was added to the solid culture medium);

2) Add into 1L conical flask;

3) Maintain at 121 ℃ for 20 minutes in an autoclave for sterilization.

The construction strategy in this chapter takes pETDuet-BGCI-gene123 as an example, and the construction methods of other recombination plasmids are the same as pETDuet-BGCI-gene123.

Ⅰ. Obtain target gene fragments by 1st PCR

Goal: To amplify BGCI-gene1, BGCI-gene2, BGCI-gene3, and backbone middle fragment for 2nd overlap PCR.

Materials:

1) 2x PrimeStarMix

2) Gene template

3) Double distilled water (ddH2O)

4) Primer-F

5) Primer-R

6) PCR tube

7) PCR thermal cycler

* PrimeStarMix includes buffer and dNTPs for PCR. The extention rate for PrimeStarMix (2x) is 1000bp/10 s.

Procedures:

1) Add 2x PrimerStarMix 25µl, gene template 1µl, primer-F 1µl, and primer-R 1µl to centrifuge tube. Add ddH2O to the centrifuge tube up to 50µl. If bubbles are observed in the mixture, centrifuge the mixture for a few seconds to remove the bubbles;

2) Insert PCR tubes into PCR thermal cycler. Pre-denature the fragments at 95˚C for 3 minutes;

3) Perform 35 cycles of 30 seconds 95˚C denaturing, 30 seconds 55˚C annealing, and 2 minutes extension 72˚C extension;

4) Extend the fragments for 1 minutes at 72˚C;

5) If not use immediately, preserve the fragments at 4˚C;

6) Recover PCR fragments by agarose gel electrophoresis.

Ⅱ. Agarose gel construction, deployment and recycle

Goal: To produce a functional and well-shaped gel for electrophoresis.

Materials:

1) Casting Tray

2) Well comb

3) Microwave

4) 1x TAE buffer

5) Agarose

6) 10000x Nucleic acid gel stain

7) Conical flask

Procedures:

1) Dissolve 1.5g Agarose in 200mL Conical flask;

2) Add 100mL TAE buffer;

3) Heat the material with microwave;

4) Melt the mixture at 30 sec interval;

5) See whether bubbles are forming, if did succesfully mixed;

6) Add 3μl nucleic acid gel stain (10000x);

7) Let it cool for 5~10 minutes;

8) Pour the agarose mixture into the casting tray;

9) Place the appropriate well comb to create the wells;

10) Wait to solidify ~30 minutes;

11) Remove the comb and place the gel in the gel box.

Ⅲ. Agarose gel electrophoresis

Goal: To verify the DNA bands' lengths and thus preparing for the next step of gel extraction to recover the bands that are of the correct length.

Procedures:

1) Extract 50µl of every sample; add 5µl of loading buffer (10x) to each sample;

2) Place the gel in the horizontal electrophoresis system;

3) Extract 50µl of the mixed solution containing the sample, mix them till their colors are almost uniform;

4) Add the marker to the first well and load 50µl of each sample into the wells;

5) The procedure runs at 120V for 20min;

6) Take out the gel from the device and proceed to following steps.

Ⅳ. DNA gel extraction

Goal: To recover DNA fragments in agarose gel.

Materials:

1) Agarose gel with PCR products obtaining from 1st PCR;

2) 1.5 mL Eppendorf (EP) tube

3) Agarose Gel Electrophoresis Recovery Kit

Procedures:

1) Cut the slice of gel containing PCR products of BGCI-gene1, BGCI-gene2, BGCI-gene3, and backbone middle fragment obtaining from 1st PCR, cutting off as much unneeded gel as possible, and place it in an 1.5 mL EP tube;

2) Add 500 µl buffer B2 and put the tube into a 50°C hot water bath until gel has completely melted;

3) Transfer the solution containing melted gel into a purification column that's in a collection tube and perform centrifugation at 12000 rpm for 30 seconds;

4) Empty the collection tube, put the purification column back in, and add 500 µl wash solution containing pure ethanol. Perform centrifugation at 12000 rpm for 30 seconds, and empty collection tube again;

5) Repeat steps 4;

6) Perform centrifugation one more time at 12000 rpm for 2 minutes, then open the cap of the tube and let it sit for one minute to allow the ethanol to evaporate;

7) Transfer purification column to a new 1.5 mL EP tube. Add 50 µl double distilled water (ddH2O) at the center of the purification column;

8) Cap the lid and let the tube sit for 1 minute. Perform centrifugation at 12000 rpm for 1 minute;

9) Discard the purification column. Store DNA fragments in the EP tube at 4°C.

Ⅴ. Obtain recombination fragments by 2nd overlap PCR

Goal: To amplify recombination fragments of BGCI-gene1 with BGCI-gene2 and backbone middle fragment with BGCI-gene3 by 2nd overlap PCR for further Infusion assay.

Materials:

1) 2x PrimeStarMix

2) Gene template from 1st PCR products

3) Double distilled water (ddH2O)

4) Primer-F

5) Primer-R

6) PCR tube

7) PCR thermal cycler

Procedures:

1) Take recombination process of BGCI-gene1 and BGCI-gene2 as an example, the same as backbone middle fragment and BGCI-gene3;

2) Add 2x PrimerStarMix 25µl, BGCI-gene1 1st PCR product 1µl, and BGCI-gene2 1st PCR product 1µl to PCR tube. Add ddH2O to the centrifuge tube up to 46µl (Except primer-F and primer-R). If bubbles are observed in the mixture, centrifuge the mixture for a few seconds to remove the bubbles;

3) Insert PCR tubes into PCR thermal cycler. Pre-denature the fragments at 95˚C for 3 minutes;

4) Perform 35 cycles of 30 seconds 95˚C denaturing, 30 seconds 55˚C annealing, and 2 minutes extension 72˚C extension;

5) After performing 5 cycles for self-annealing, then pause the PCR thermal cycler;

6) Add primer-F of BGCI-gene1 and primer-R of BGCI-gene2 each 2µl to PCR reaction solutions;

7) Restart PCR thermal cycler for the rest steps;

8) Extend the fragments for 1 minutes at 72˚C;

9) If not use immediately, preserve the fragments at 4˚C;

10) Recover recombination PCR fragments by agarose gel electrophoresis.

Ⅵ. Linearize pETDuet/pRSFDuet/pACYCDuet backbone by restriction endonucleases

Goal: To linearize pETDuet/pRSFDuet/pACYCDuet backbone for Infusion recombination.

Materials:

1) pETDuet backbone plasmid

2) pRSFDuet backbone plasmid

3) pACYCDuet backbone plasmid

4) 10x Cutting buffer

5) EcoRI endonuclease

6) XhoI endonuclease

7) BamHI endonuclease

8) Double distilled water (ddH2O)

Procedures:

1) Use BamHI and XhoI endonucleases to digest pETDuet backbone, use EcoRI and XhoI endonucleases to digest pRSFDuet backbone, and use BamHI and XhoI endonucleases to digest pACYCDuet backbone;

2) Add 5µg backbone plasmid, 10x Cutting buffer 5µl, endonuclease I 5µl, and endonuclease II 5µl in PCR tube. Add ddH2O to PCR tube up to 50µl;

3) Mix by pipette and remove the bubbles by low-speed centrifuge;

4) Insert PCR tubes into PCR thermal cycler. Digest the plasmid at 37˚C for 30 minutes;

5) Recover linearized plasmid backbone fragments by agarose gel electrophoresis.

Ⅶ. Homologous recombination of linearized plasmid backbone with 2nd overlap PCR products

Goal: Perform homologous recombination between the linearized plasmid backbone and 2nd overlap PCR products to generate the complete plasmid. Take pETDuet-BGCI-gene123 as an example.

Materials:

1) CloneExpress Mix (2x)

2) 2nd overlap PCR products of BGCI-gene1 with BGCI-gene2 and backbone middle fragment with BGCI-gene3

3) Linearized pETDuet backbone

Procedure:

1) Measure the concentration of the gel-recovered fragments;

2) Prepare the system for the construction of pETDuet-BGCI-gene123 in PCR tubes: 5μl CloneExpress Mix, 2μl Linearized pETDuet backbone, 1.5μl 2nd overlap PCR products of BGCI-gene1 with BGCI-gene2, and 1.5μl 2nd overlap PCR products of backbone middle fragment with BGCI-gene3;

3) Insert PCR tubes into PCR thermal cycler. Ligase the fragments at 50˚C for 30 minutes;

Ⅷ. Transformation

Materials:

1) Ligated recombination plasmid

2) Liquid LB solution without antibiotics

3) LB Agar plates with corresponding antibiotics

Procedures:

1) Add recombination plasmid into E.Coli DH5α strain;

2) Placed on ice for 20 minutes;

3) Heat shock at 42˚C for 45 seconds, then immediately put on ice for 2-3 minutes;

4) Add 400μL liquid LB solution without antibiotics, incubate at 37 ˚C for 30 minutes;

5) Centrifuge at 5000 rpm for 3 minutes, extract 300μL of supernatant then discard;

6) Apply bacterial liquid to LB Agar plates with corresponding antibiotics, incubate for 12-16 hours at 37˚C.

Ⅸ. Colony PCR verification

Goal: E.Coli DH5α strain containing constructed plasmids has multiplied. We aimed to identify mono-colony E.coli DH5α containing correct recombination plasmids by colony PCR.

Materials:

1) Taq Master Mix (2x)

2) Double distilled water (ddH2O)

3) Culture plates post-transformation

Procedures:

1) Add 10μl Taq Master Mix, 1μl primer-F, 1μl primer-R, and 8μl ddH2O into PCR tubes;

2) Pick a single colony with a 10μl sterile pipette tip and perform aspiration and dispensing in PCR tubes;

3) Insert mixture into a PCR thermal cycler, design the PCR program according to the manufacturer's instructions.

4) Perform agarose gel electrophoresis, and the presence of the correct bands indicates the successful construction of recombination plasmids.

Ⅹ. Plasmid extraction and sequencing

Goal: To prepare the recombination plasmids and then to verify the correctness of sequences.

Materials:

1) Bacterial fluid of pETDuet-BGCI-gene123

2) Centrifuge

3) 1.5mL centrifuge tubes and absorption columns

4) Sangon small-scale plasmid extraction kit

Procedures:

1) Take all bacterial fluid into centrifuge for 2 minutes at 12000 rpm and discard all supernatant;

2) Add 250μl Buffer P1 to the bacteria and let it resuspend;

3) Add 250μl Buffer P2 and gently turn the centrifuge tube upside down for 5-10 times to let it mix. Leave the centrifuge tube at room temperature for 2-4min;

4) Add 350μl Buffer P3, immediately and gently invert the centrifuge tube for 5-10 times to fully mix;

5) Centrifuge the tube at 12000 rpm for 15min, and carefully move all the supernatant into the adsorption column and centrifuge at 12000 rpm for 30 sec. Drain the liquid in the collection tube;

6) Add 700μl Wash Solution into the adsorption column, centrifuge at 12000 rpm for 30 sec. Drain the liquid in the collection tube.;

7) Repeat step 6 once;

8) Put empty adsorption column and the collection tube together and centrifuge them at 12000 rpm for 1 min;

9) Add 50μl double distilled water to the center of the adsorption membrane. Leave the tube at room temperature for 1-2min.

10) Transfer the absorption membrane to a new EP tube and then centrifuge it at 12000 rpm for 1min;

11) Store plasmid in the EP tube at 4°C;

12) Sequence the plasmid by specific primers in Sangon Biotech Co., Ltd.

The transformation strategy in this chapter takes Strain-BGCI as an example, and the transformation methods of Strain-BGGII are the same as Strain-BGCI. The only exception difference between Strain-BGCI and Strain-BGCII is the types of antibiotics using for selecting positive colony.

Goal: To co-transform correct combination plasmid into E. coli BL21 for further protein expression. For strain-BGCI, verified pETDuet-BGCI-gene123 and pRSFDuet-BGCI-gene456 plasmids are co-transformed into E. coli BL21. For strain-BGCII, verified pACYCDuet-BGCII-gene143, pETDuet-BGCII-gene792, AND pRSFDuet-BGCII-gene685 are co-transformed into E. coli BL21.

Ⅰ. Co-transformation of E.coli BL21

Materials:

1) Verified pETDuet-BGCI-gene123 and pRSFDuet-BGCI-gene456 plasmids

2) Liquid LB solution without antibiotics

3) LB Agar plates with corresponding double antibiotics

Procedures:

1) Add each 1μl verified pETDuet-BGCI-gene123 and pRSFDuet-BGCI-gene456 plasmids into E.Coli BL21 strain;

2) Placed on ice for 20 minutes;

3) Heat shock at 42˚C for 45 seconds, then immediately put on ice for 2-3 minutes;

4) Add 400μL liquid LB solution without antibiotics, incubate at 37 ˚C for 30 minutes;

5) Centrifuge at 5000 rpm for 3 minutes, extract 300μL of supernatant then discard;

6) Apply bacterial liquid to LB Agar plates with corresponding antibiotics, incubate for 12-16 hours at 37˚C.

7) For strain-BGCI, use double antibiotics of kanamycin and ampicillin to select positive colonies. For strain-BGCII, use triple antibiotics of kanamycin, ampicillin, and chloramphenicol to select positive colonies.

Ⅱ. Colony PCR verification for multi plasmids

Materials:

1) Taq Master Mix (2x)

2) Double distilled water (ddH2O)

3) Culture plates co-transformation

Procedures:

1) For verification of multi plasmids in E.coli BL21 colonies, primers for each plasmids are used to amplify target bands, respectively;

2) Add 10μl Taq Master Mix, 1μl primer-F, 1μl primer-R, and 8μl ddH2O into PCR tubes;

3) Pick a single colony with a 10μl sterile pipette tip and perform aspiration and dispensing in PCR tubes;

4) Insert mixture into a PCR thermal cycler, design the PCR program according to the manufacturer's instructions.

5) Perform agarose gel electrophoresis, and the presence of the correct bands indicates the successful construction of plasmids.

Induction of protein expression for strain-BGCI and strain-BGCII

Ⅰ. Amplification culture for correct colony

Goal: To acquire large amounts of target proteins for SDS-PAGE

Materials:

1) Liquid LB solution

2) E.coli BL21 of strain-BGCI and strain-BGCII bacterial fluid

3) Antibiotics: kanamycin, ampicillin, and chloramphenicol

4) Inducer: IPTG

5) Iso-thermic shaker

Procedures:

1) Add 100μl of strain-BGCI and strain-BGCII bacterial fluid and 100μl of corresponding multi antibiotics into 100mL of liquid LB solutions, respectively;

2) Culture bacterial liquid in iso-thermic shaker for 3 hours until absorbance OD600nm to 0.4-0.6;

3) Add IPTG into bacterial liquid with 1mM final concentration;

4) Sample for each bacterial liquid cultured at 25°C and 37°C for 0.5h, 1h, 3h, and 6h, respectively.

Ⅱ: Protein crude extraction and SDS-PAGE

Goal: To verify the expression of desired proteins.

Materials for protein crude extraction:

1) 500μl bacterial liquid for different induced conditions

2) Centrifuge

3) Double distilled water (ddH2O)

4) 6x Protein loading buffer

Procedures for protein crude extraction:

1) Centrifuge samples at 12000 rpm for 1 minute;

2) Discard supernatant and resuspend precipitation with 50μl 1x Protein loading buffer diluted by ddH2O;

3) Boiled samples at 95°C for 15 minutes in PCR thermal cycler.

Materials for SDS-PAGE:

1) Sangon 12.5% SDS-PAGE Color Preparation kit

2) Electrophoresis buffer (Tris-Glycine)

3) Protein ladder

4) 6x Protein loading buffer

5) Vertical electrophoresis system

6) Coomassie blue staining solution

Procedures for SDS-PAGE：

1) Add and mix 2.2mL 2x separating gel solution, 2.2mL 2X separating gel buffer, and 44μL catalyst in centrifugation tubes for separating gel;

2) Slowly pipetting mixture into casting stand and frame to avoid bubbles;

3) Add 1 mL ddH2O at the top of separating gel;

4) Wait the separating gel to solidify for 8 minutes;

5) Discard the water that was added previously;

6) Add and mix 825μL 2x stacking gel solution, 825μL 2X stacking gel buffer, and 11μL catalyst in centrifugation tubes for stacking gel;

7) Add stacking gel mixture until the cast is completely filled, then slowly insert comb without producing any air bubbles;

8) Wait the stacking gel to solidify for 12 minutes, then carefully remove the comb, and wash the wells with electrophoresis buffer;

9) Add 10μL protein ladder into the first well, then load samples in each successive well;

10) Transfer gel into vertical electrophoresis system;

11) Run at 120V for 90 minutes;

12) Stain the gel with Coomassie blue staining solution for 10 minutes, then destain the gel using destaining solution repeatedly until gel background become transparent.

1 The test results for Total Antioxidant Capacity (T-AOC)

Goal: Total antioxidant capacity represents the levels of antioxidant substances and antioxidant enzymes in sample. We used bacterial fluids expressing strain-BGCI and strain-BGCII to test the total antioxidant capacity via DPPH method. DPPH is a stable substance that is soluble in ethanol and has a maximum absorption at 515 nm. When antioxidants are added to DPPH solutions, the antioxidant capacity of antioxidant substances can be quantified by changes in absorbance value.

Materials:

1) Bacterial fluids expressing strain-BGCI or strain-BGCII

2) Extraction buffer

3) DPPH solution

4) Centrifuge

5) 96-wells plate

6) Microplate reader

7) Double distilled water(ddH2O)

Procedures:

1) Add 2mL bacterial fluids to centrifuge tubes, then centrifuge at 12000 rpm for 2 minutes;

2) Resuspend precipitation with 1mL extraction buffer;

3) Put on ice for 30 minutes;

4) Use ultrasonic apparatus to pre-treat samples: power set at 200W, ultrasound 3s, interval 10s, repeated 30 times;

5) Centrifuge at 10000 rpm for 10 minutes;

6) Transfer supernatant to a new tube for measurement;

7) Prepare tubes for blank and test. Add 20μL extraction buffer and 380μL DPPH solution to blank tube. At the same time, add 20μL bacterial crude extraction and 380μL DPPH solution to test tube;

8) React in dark at room temperature for 20 minutes;

9) Add 200μL reaction solutions to 96-well plate;

10) Use microplate reader to measure the absorption value of samples at 515nm;

11) Calculate the total antioxidant capacity: DPPH free radical clearance rate (%) = (Ablank-Atest)÷Ablank×100%

2 The test of the fermentation product antibacterial experiment

Goal: Use the double-layer agar plate to test the antibacterial activity of fermentation products expressing strain-BGCI or strain-BGCII via observe antibacterial plaque.

Materials:

1) Bacterial fluids expressing strain-BGCI or strain-BGCII

2) Test strain K-12

3) LB solid medium plate without antibiotics

4) Positive control: 1g/L Ciprofloxacin

5) Standard sample: 200mg/L Squalene

Procedures:

) Prepare the double-layer agar plate, with the bottom layer containing 1.5% LB solid medium and the top layer containing 0.8% LB solid medium poured after the bottom layer had cooled.

2) Mix and culture strain K-12 bacterial fluids in double-layer agar plate. Streak the swab evenly across the surface of the nutrient agar plate to create a bacterial lawn.

3) Put the double-layer agar plate in the incubator at 37℃ for 24 hours;

4) Add 4μL drops of each antibacterial agent onto the surface of the inoculated agar plates;

5) At the same time, add 4μL drops of Ciprofloxacin and Squalene onto the surface of the inoculated agar plates;

6) Cover the plates with lids and incubate them upside down at 37°C for 24 hours.

7) After incubation, observe the plates for zones of inhibition (clear areas where bacteria did not grow).

3 Determination of squalene in the fermentation broth by HPLC

Goal: To determine if our target terpenoid compound is squalene, we conducted testing on the fermentation broth of the bacterial strains.

Materials:

1) Bacterial fluids expressing strain-BGCI or strain-BGCII

2) LBG medium

3) Sonicator

4) Centrifuge

5) Double distilled water(ddH2O)

6) 0.22μm filter membrane

7) Methanol

8) Ethyl acetate

9) Pure acetonitrile

10) Waters HPLC system

11) Waters XBridgeTM C18 HPLC column

Procedures:

1) Fermentation was carried out using a biphasic fermentation method, with 10% volume of normal heptane added on top of the LBG medium;

2) After fermentation, 1 mL of the 24-hour whole-cell catalytic liquid was taken, centrifuged at 13,000×g for 10 minutes, and the supernatant was discarded;

3) 400μL of saline solution was added to wash the fermentation cells, centrifuged at 13,000×g for 10 minutes, and the supernatant was discarded;

4) ddH2O was added, thoroughly mixed, and brought to a volume of 400μL;

5) The cells were disrupted by ultrasonication at a working power of 20%, for 2 minutes with 3-second on and 5-second off cycles;

6) Subsequently, 600μL of ethyl acetate was added, mixed well, and subjected to ultrasonic cleaning twice for 15 minutes each;

7) The mixture was then centrifuged, and 400μL of the extract phase was obtained;

8) The extract was concentrated using a vacuum centrifuge to evaporate the solvent, then re-dissolved in 200μL of methanol, filtered through a 0.22μm filter membrane, and ready for analysis;

9) Squalene yield detection was performed using high-performance liquid chromatography (HPLC) under the following conditions: Column: Waters XBridgeTM C18 (3.5μm 4.6mm×150mm); Column temperature: 35℃; Mobile phase: 100% pure acetonitrile; Flow rate: 1 mL/min; Detector: Photodiode array detector at 196 nm wavelength.