Material: 2×Hieff PCR Master Mix, Primer (10 μM), DNA template, ddH₂O

Equipment: Thermal cycler, microcentrifuge tubes (0.2 mL), micropipettes and sterile tips

Materials:​ plasmid, restriction endonucleases (HindIII, XbaI), 10× CutSmart Buffer, sterile water.

​​Equipment:​​ Microcentrifuge tubes, water bath, micropipettes and tips

Method:

Part A: Digestion of phaC_pRSFDuet-1 / phaP_pRSFDuet-1 (HindIII + XbaI)

Part B: Digestion of phaCAB_pRSFDuet-1 / phaP_pRSFDuet-1 (AflII + XbaI)

1. Add reagents in the order listed above into a clean 0.5 mL microcentrifuge tube.
2. Gently flick the tube or pipette to mix.
3. Spin briefly to collect contents at the bottom.
4. Incubate at 37°C for 30 minutes in a heat block or water bath.

Material: TAE buffer, Agarose gel, DNA stain, DNA ladder.

Equipment:​​ Horizontal gel electrophoresis apparatus, power supply, microwave or hot plate for melting agarose, gel casting tray and combs, UV transilluminator

Method:

1. To prepare the gel: Melt agarose in an appropriate buffer. Add a DNA stain to the melted agarose. Pour the mixture into a casting tray equipped with a comb. Allow the gel to solidify for 20–30 minutes.
2. To load the samples:
• Mix the DNA samples with a loading dye. Carefully pipette each mixture into the wells of the solidified gel.
• Ensure a DNA ladder is loaded into one well as a reference.
• To run the electrophoresis (the main experiment): Submerge the gel in a buffer-filled electrophoresis chamber. Connect the chamber to a power supply.
• Run the gel at a voltage of 80–120 V.
• Continue until the DNA bands have separated adequately.
3. To visualize the results: Place the gel on a transilluminator.
4. Use either UV or blue light to visualize the separated DNA bands.

Materials:​​ Agarose gel slice containing the DNA band of interest, gel extraction kit

​​Equipment:​​ Microcentrifuge tubes (1.5 mL or 2 mL), sterile scalpel or razor blade for cutting gel, UV transilluminator, water bath, microcentrifuge, micropipettes, and tips

Method:

1. First, excise the agarose gel slice containing the DNA fragment. Add buffer B2 to the gel slice at a volume 3 times its weight. Heat the mixture at 50 degrees Celsius until the gel is completely dissolved.
2. Transfer the resulting binding mix to a purification column. Centrifuge the column for 30 seconds.
3. Discard the flow-through filtrate from the collection tube. Place the column back into a new collection tube.
4. Add 500 µL of wash solution to the column. Centrifuge at 9000 x g for 30 seconds. Discard the flow-through filtrate. Return the column to a new collection tube.
5. Repeat the washing step (adding 500 µL wash solution and centrifuging at 9000 x g for 30 seconds) once more.
6. Place the column back into the collection tube and centrifuge for 1 minute to dry the membrane.
7. Transfer the column to a new 1.5 mL Eppendorf tube. Add 15-40 µL of Elution buffer directly to the center of the column membrane. Allow it to stand at room temperature for 1 minute.
8. Centrifuge the column for 1 minute to elute the DNA. Keep and store the eluted DNA solution.

​​Materials:​​ DNA fragments with homologous overlaps, homologous recombination/cloning kit, sterile water.

​​Equipment:​​ Microcentrifuge tubes, Thermal cycler, micropipettes, and tips

Method:

The target gene fragment was directionally inserted into the multiple cloning site (MCS) of the vector using the ClonExpress Ultra One Step Cloning Kit.

1. Forward and reverse primers were designed based on the restriction enzyme sites within the MCS of the vector. The vector was linearized via PCR using these primers.
2. Primers for amplifying the gene fragment were designed to include terminal sequences homologous to the ends of the linearized vector. This ensured that the PCR-amplified vector and the gene fragment possessed homologous ends (15-25 bp in length).
3. The homologous recombination between the linearized vector and the gene fragment was performed using the ClonExpress Ultra One Step Cloning Kit (Vazyme). The reaction mixture, prepared with a specific molar ratio of the components, was incubated at 50°C in a metal bath for 15 minutes.

​​Materials:​​ Digested vector and insert DNA, T4 DNA Ligase, ligation buffer, sterile water.

​​Equipment:​​ Microcentrifuge tubes, incubator, micropipettes, and tips.

​​Materials:​​ Competent E. coli cells, plasmid DNA or ligation/assembly product, LB recovery medium (sterile), selection agar plates

​​Equipment:​​ Sterile microcentrifuge tubes (1.5 mL), water bath, shaking incubator, spreaders or glass beads, micropipettes and tips

Method:

All recombinant products/enzyme-linked products need to be first transferred to E. coli DH5α, subjected to Colony PCR and sequencing, and then the plasmid extracted from the sequenced E. coli DH5α without errors; Transfer the plasmid into E. coli BL21 (DE3).

1. Add 1 μL of recombinant products or plasmids to 100 μL of competent cells.
2. Let it stand on ice for 10 minutes, heat shock at 42 °C for 90 seconds, and let it stand on ice for 2 minutes.
3. Add 1000 μL of LB medium (without antibiotics) and resuscitate for 30 minutes
4. Mix the competent cells evenly and spread them uniformly onto an LB solid agar plate containing the antibiotic corresponding to the vector's resistance. Place the plate inoculated with the bacterial solution upside down in a 37°C constant temperature incubator and incubate for 16 hours.

Materials: 2×Hieff® Ultra-Rapid II HotStart PCR Master Mix, primer, ddH₂O, single colony

Equipment: Thermal cycler, microcentrifuge tubes (0.2 mL), micropipettes and sterile tips

Method:

Under aseptic conditions, prepare LB liquid medium containing the appropriate antibiotic, then use a sterile toothpick or pipette tip to pick the target colony and inoculate it into 5 mL of LB medium, mixing gently. Place the culture tube on a shaker at 37 °C and 200 rpm for 8–16 hours until the culture becomes turbid, indicating that the bacteria have grown extensively.

Material: Plasmid extraction kit

Equipment: Microcentrifuge, microcentrifuge tubes (1.5 mL), micropipettes and tips

Method：

1. Equilibrate column: Place a Miniprep column into an collection tube. Add 500 μl Buffer S to column, centrifuge at 12,000 × g for 1 min. Discard the filtrate in collection tube. Return the column to the collection tube.
2. Collect 1.5-5 ml of overnight LB bacteria culture. Centrifuge at 8,000 × g for 2 min to pellet the bacteria. Discard as much of the supernatant.
3. Resuspend the bacterial pellet in 250 μl of Buffer SP1 by vortexing. Make the pellet completely resuspended.
4. Add 250 μl of Buffer SP2, and mix by gently inverting the tube for 5-10 times. And incubate at room temperature (15–25°C) for 2-4 min.
5. Add 350 μl of Buffer SP3, and mix immediately by gently inverting the tube for 5-10 times.
6. Centrifuge at 12,000 × g for 5-10 min. Transfer the supernatant to column, centrifuge at 8,000 × g for 30 sec, discard the filtrate in the collection tube.
7. (Optional) Add 500 μl Buffer DW1, Centrifuge at 9,000 × g for 30 sec, discard the filtrate in the collection tube. Return the column to the collection tube.
8. Add 500 μl Wash Solution, centrifuge at 9,000 × g for 30 sec, discard the filtrate in the collection tube. Return the column to the collection tube.
9. Repeat step 9.
10. Place the column back into the collection tube. Centrifuge at 9,000 × g for 1 min.
11. Transfer the column into a clean 1.5 ml Eppendorf tube. To elute plasmid, add 50~100 μl of Eluent buffer to the centre of the membrane. Incubate for 1 min at room temperature. Centrifuge at 9,000 × g for 1 min. Keep the DNA solution.

​​Materials:​​ Bacterial culture containing the expression plasmid, IPTG for lac/T7 systems, and arabinose for the araBAD promoter.

​​Equipment:​​ Shaking incubator at appropriate temperature, flasks

Method:

1. Culture the bacteria at 37°C in the temperature shaker overnight
2. Expand the bacterial fluid at a ratio of 1:50 and shake it for 2h
3. Until the log phase, add 0.1mM IPTG to the liquid
4. Measure the OD₆₀₀ at different time intervals by using the microplate reader
5. Record the OD₆₀₀ value by computer and draw the curve of the growth using the data
6. Replace medium (for cycle 2 and cycle 3)

• For cycle 2:

1. Centrifuge the bacterial fluid, remove the supernatant, and keep the cells at the bottom.
2. Add new LB broth, resuspend the cell in the fluid
3. Put them into the temperature shaker and induce the protein to express (24h)

• For cycle 3:

1. Centrifuge the bacterial fluid, remove the supernatant, and keep the cells at the bottom
2. Add new LB broth, which contains 0.2% L-arabinose to the cells, resuspend the cell in the fluid
3. Put them into the temperature shaker and induce the protein to express (24h)

​​Materials:​​ Protein sample, 5× sample loading buffer, SDS-PAGE Rapid Preparation Kit, SDS-PAGE running buffer (1X Tris-Glycine-SDS), protein molecular weight marker, SDS-PAGE rapid staining solution

Equipment:​​ Vertical gel electrophoresis apparatus, power supply, gel casting system (glass plates, spacers, combs), heating block, gel haker 

Method:

1. Assemble the molding mold properly. First, prepare the lower layer (1.00mm).
2. Take 2.7 mL of the lower gel solution (2X) and 2.7 mL of the lower gel buffer (2X) of equal volume each, and mix them in the gel preparation cup.
3. Add 55 µL improved coagulant to the mixed solution in step 2, and gently stir to mix evenly, avoiding the formation of bubbles.
4. Add the lower gel solution, which has been thoroughly mixed in Step 3, to the gel mold, ensuring that the distance from the liquid surface to the top edge of the glass plate is 1.5 cm. Then, cover the surface of the lower gel solution with a layer of water or alcohol to maintain a smooth gel surface and prevent the formation of bubbles.
5. Leave the mixture at room temperature (25°C) for 6 to 10 minutes, until a distinct boundary forms between the lower gel and the covering layer.
6. Prepare the upper gel solution(1.00mm)
7. Slowly pour off the covering layer, then take equal volumes of the 2X upper gel solution and 2X colored upper gel buffer solution, each 0.75 mL, and mix them in a new gel preparation cup.
8. Add 15 µL of the improved coagulant to the mixed solution from Step 7, and gently stir to mix, avoiding the formation of bubbles.
9. Add the upper gel solution to the top of the lower gel layer until the gel solution reaches the top of the glass plate. Slowly insert the comb into the gel, taking care to avoid the formation of bubbles.
10. Leave the gel to set for 10–15 minutes, allowing the upper gel to solidify. Carefully remove the comb, and use a pipette tip or syringe to wash the wells with electrophoresis buffer to ensure they are clean before proceeding with the SDS-PAGE electrophoresis.

1. Extract 1 mL of the target bacterial suspension, centrifuge, and collect the pellet.
2. Mix the pellet with 100 µL of loading buffer.
3. Place the mixed solution in a 100°C water bath and boil for 10-15 minutes.

1. Load 10 μL per well.
2. Run at 80 V (stacking gel) → 120 V (separating gel, ~1.5 h)
3. Stain with SDS-PAGE rapid staining solution