Goal: Amplify the complete coding genes of PDI and its isoforms to obtain target fragments with homologous recombination sites.

Materials:

50 μL PCR amplification reaction system:

PCR amplification reaction procedure:

Procedure:

Mix all components in labeled PCR tubes, followed by brief centrifugation. Load samples into a pre-programmed thermal cycler running through the PCR amplification reaction procedure above.

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

Materials:

Procedure:

Dissolve 0.3 g Agarose in 30 mL erlenmeyer flask. Add 1× TAE buffer to reach the target volume. Heat the material with microwave at short intervals until the agarose dissolves completely. Add 3μl nucleic acid gel stain when it cool. Pour the mixture into a casting tray, insert a well comb, wait 7-10 minutes for the gel to solidify. Remove the comb and place the gel in the gel box.

Goal: To verify the DNA bands’ lengths and thus preparing for the next step of gel extraction to extract the bands that are of the correct length, eliminating the genetic fragments that didn’t successfully undergo PCR.

Materials:

Procedure:

Mix 5 μl of PCR product with 1μl of loading buffer, followed by loading into the wells alongside the DNA marker. Perform electrophoresis at a constant voltage of 180V for 20 minutes in 1×TAE buffer. After electrophoresis, place the gel in a UV gel imaging system for observation.

Goal: To abtain a sufficient quantity of engineered strains with stable activity for subsequent experiments.

Materials:

Procedure:

Add 5 mL of LB medium, 5 μL of pRSFDuet glycerol stock, and 5 μL of kanamycin (Kan⁺) antibiotic to labeled tubes. Place the tubes in a shaking incubator at 37°C and 220 rpm. Allow the bacteria to grow overnight (12-16 hours).

Goal: To obtain high-purity plasmid DNA from host cells, including empty vectors or recombinant vectors.

Materials:

Procedure:

1. Transfer 2 mL of the overnight culture into a 1.5 mL microcentrifuge tube. Centrifuge at 12000 rpm for 1 min, and drop the solution.
2. Add 250 μL Buffer P1 containing RNase A, and mix thoroughly by vortexing.
3. Add 250 μL Buffer P2, and gently invert the tube up and down 10 times to fully lyse the bacterial cells.
4. Add 350 μL Buffer P3, immediately invert gently up and down 10 times to mix thoroughly, then centrifuge at 12,000 rpm for 2 minutes.
5. Place the FastPure DNA Mini Columns spin column into the Collection Tubes, carefully pipette the supernatant into the adsorption columns, centrifuge at 12,000 rpm for 1 minute, discard the waste liquid, and put the adsorption column back into the collection tube.
6. Add 500 μL of Buffer PW1, centrifuge at 12,000 rpm for 1 minute, and discard the waste liquid.
7. Add 600 μL of Buffer PW2, centrifuge at 12,000 rpm for 1 minute, and discard the waste liquid.
8. Repeat step 7.
9. Put the spin column back into the collection tube, and centrifuge at 12,000 rpm for 1 minute.
10. Place the spin column into a new clean 1.5 mL centrifuge tube. Add 40 μL of ddH₂O to the middle part of the adsorption membrane, let it stand at room temperature for 1 minute, then centrifuge at 12,000 rpm for 1 minute to obtain the plasmid DNA solution.

Goal：Double-digest the plasmid vector to linearize it, providing a backbone for recombination and preventing self-ligation.

Materials:

20 μL double digestion reaction system:

Procedure:

Add all the components to PCR tubes, followed by brief centrifugation at 12,000 rpm for 10 seconds. Incubate the reaction system in a 37 ℃ water bath for 20 minutes to ensure complete digestion of the vector plasmid into linearized fragments.

Goal: To isolate and purify linearized vector DNA fragments from agarose gel, while removing impurities such as uncut circular vectors, other restriction-generated fragments, proteins, and agarose.

Materials:

Procedure:

1. After completing agarose gel electrophoresis, precisely excise the gel slice containing linearized vector DNA under UV illumination. Transfer the excised gel slice to a 1.5 mL centrifuge tube.
2. Add an equal volume of Buffer GDP. Incubate at 65°C in a metal bath until the gel becomes completely transparent.
3. Transfer the solution to the spin column and centrifuge at 12000 rpm for 60 seconds.
4. Discard the filtrate. Place the spin column in the collection tube. Add 300 μL Buffer GDP to the spin column, and let it stand for 1 minute. Centrifuge at 12000 rpm for 60 seconds.
5. Discard the filtrate. Place the spin column in the collection tube. Add 700 μL Buffer GW to the spin column. Centrifuge at 12000 rpm for 60 seconds.
6. Repeat Step 5.
7. Discard the filtrate. Place the spin column in the collection tube. Centrifuge at 12000 rpm for 2 minutes.
8. Place the spin column into a new clean 1.5 mL centrifuge tube. Add 30 μL of ddH₂O to the middle part of the adsorption membrane, let it stand for 2 minute. Centrifuge at 12,000 rpm for 1 minute. Store the DNA solution at -20℃.

Goal：To construct a recombinant vector by ligating the target gene to the linearized vector via homologous recombination.

Materials:

Procedure:

1. Prepare the system:

1. Place the system in 50°C water bath for 15 minutes, then cool to 4℃ immediately

Goal：To introduce the recombinant vector into DH5α for amplification, followed by verification of the target gene sequence, insertion orientation, and vector integrity via sequencing.

Materials:

Procedure:

1. Put 10 μL pRSFDuet-PDI plasmid into 100 μL E. coli DH5α competent cells, then place on ice for 20 minutes.
2. Heat-shock at 42℃ for accurate 45 seconds, then immediately put on ice for 2 minutes.
3. Add 800 μL of LB liquid medium, then incubate at 37℃ for an hour.
4. Centrifugate at 12000 rpm for 1 minute.
5. Remain 100 μL supernatant, spreading on kan+ plate, then incubate at 37℃ for 12-16 hours.

Goal: To introduce the target plasmid into the expression strain for protein production.

Materials:

Procedure:

1. Thaw competent cells on ice for 10 minutes.
2. Add 1-5 µL plasmid DNA to 50 µL cells, mix gently, then incubate on ice for 30 min.
3. Heat-shock at 42°C for accurate 45 seconds, then immediately put on ice for 2 minutes.
4. Add 500 µL LB medium (no antibiotic), incubate at 37°C for 1 hour.
5. Plate 100 µL of the transformed cells on LB agar plates (antibiotic), and incubate at 37℃ for 12-16 hours.

Goal: To optimize protein expression under inducible conditions.

Materials:

Procedure:

1. Inoculate 5 mL LB (antibiotic) with single colony, incubate at 37°C, 220 rpm for 12–16 hours.
2. Transfer 1 mL culture to 100 mL TB/autoinduction medium. Incubate at 37°C until OD₆₀₀ ≈ 0.6 (3–4 h).
3. Add IPTG to final concentration (e.g., 0.5 mM).
4. Induce at:
   1. 37°C, 4–6 h for soluble proteins
   2. 16–18°C, 16–20 h for complex proteins

Goal: To lyse cells to extract soluble proteins.

Materials:

Procedure:

1. Resuspend the pellet in 5 mL lysis buffer.
2. Sonicate on ice for 3 cycles: 30 seconds pulse, 30 seconds rest at 40% amplitude.
3. Centrifuge at 15,000 ×g, for 30 minutes at 4°C.
4. Collect the supernatant as the crude extract.

Goal: To isolate His-tagged protein via immobilized metal affinity.

Materials:

Procedure:

1. Equilibrate 1 mL of Ni-NTA resin with lysis buffer.
2. Load the crude extract onto the column and collect the flow-through.
3. Wash the column with 10 column volumes (CV) of wash buffer.
4. Elute the target protein with 5 CV of elution buffer, collecting 0.5 mL fractions.
5. Analyze the collected fractions using SDS-PAGE.

Goal: To confirm protein size and purity.

Materials:

Procedure:

1. Mix 10 µL protein sample with 2× Laemmli buffer in clean centrifuge tubes, Vortex for 10 seconds. Boil for 5 minutes.
2. Load the ladder and samples into the lanes.Run at 120 V for 90 min.
3. Stain with Coomassie Blue stain for 30 minutes. Incubate the gel in destaining solution overnight (12-16 hours).

Goal: To screen monosaccharide expression profiles.

Materials:

Procedure:

1. Spot samples and standards 1 cm from the bottom of the TLC plate.
2. Develop the plate in the solvent system until the solvent front reaches 8 cm from the starting line.
3. Air-dry the plate completely. Spray with staining reagent, and heat at 105°C for 10 minutes to visualize the spots.
4. Calculate and compare the Rf values of sample spots with those of standard sugars for identification.

Goal: To measure reducing sugar content via colorimetry.

Materials:

Procedure:

1. Mix 50 µL of sample with 100 µL of DNS reagent in centrifuge tubes.
2. Incubate in a boiling water bath for 5 minutes, then immediately cool the mixture on ice to stop the reaction.
3. Add 100 µL of ddH₂O to the tube and mix thoroughly.
4. Measure OD₅₄₀, calculate concentration using standard curve.

Goal: To assess sweetness profile of expressed proteins.

Materials:

Procedure:

1. Dialyze samples into buffer.
2. Calibrate sensors with reference solutions.
3. Measure samples in triplicate.
4. Analyze data via PCA (Principal Component Analysis).

Goal: To investigate the binding mode between sweet proteins and sweet taste receptor proteins.

Procedure:

1. Download the structural file of the sweet taste receptor protein from the Protein Data Bank (PDB), and use AutoDock Tools software to preprocess the structure.
2. Use the AlphaFold tool to predict the three-dimensional structures of the sweet proteins TLP-A and TLP-B.
3. Perform molecular docking between the sweet protein TLP and the sweet taste receptor using the Z-dock online tool.
4. Analyze the docking results by combining visualization tools and interaction analysis methods.