Protocol

Expression of Sweet Protein

E. Coli Trial

    Plasmid construction and cell transformation

    Plasmid transformation

    • 1. Take out BL21 (DE3) receptive E. Coli cells from the -80℃ refrigerator, leave them at room temperature or partially melt them with the palm of your hand, and place them on ice while in the ice-water mixture to continue melting.
    • 2. Take out the plasmids from the refrigerator at -20 ℃, dissolve them with 4 µL ddH2O per tube, centrifuge at 14,000 rpm for 1-3 mins, and add 0.01-1ug plasmids per 100 µL receptive cells after thawing. Tap the bottom of the Eppendorf tube to mix. It is carried out in sequence: ice standing for 5 mins, liquid nitrogen freezing for 5 mins, water bath at 37 ℃ for 5 mins, ice bath for 5 mins.
    • 3. In the super clean table, 700 µL of antibiotic-free LB (or YEB) medium was added to each tube, and expand the culture at 28 ° C for 2-3 hours with shaking.

    Screening and activation

    • 1. Prepare LB solid medium containing kanamycin (1 mg/L), and coat each plate with 60 μL of bacterial solution.
    • 2. Plates were inverted and incubated in an incubator at 37℃ overnight.
    • 3. Monoclonal clones carrying transformation plasmids were selected from BL21 (DE3) competent cells growing on plates with a 200 μL yellow pipette tip and seeded in 3mL LB liquid medium containing kanamycin (50 μg/mL, diluted 1:1000). Stock solution concentration was 50 mg/mL (the tip was left in the culture tube). The cells were incubated overnight at 37℃ with shaking.

    IPTG induction

    • 1. Pick three single, well-isolated colonies and inoculate it into 4 mL LB medium containing 50 μg/mL kanamycin, respectively.
    • 2. Incubate the cells in shaker at 37℃ with shaking at 200rpm.
    • 3. Monoclonal clones carrying transformation plasmids were selected from BL21 (DE3) competent cells growing on plates with a 200 μL yellow pipette tip and seeded in 3mL LB liquid medium containing kanamycin (50 μg/mL, diluted 1:1000). Stock solution concentration was 50 mg/mL (the tip was left in the culture tube). The cells were incubated overnight at 37℃ with shaking.

    Expression assessment

    Protein extraction

    • 1. Sample preparation Harvest cell pellet from 450 μL culture, and lyse with 300 μL lysis Buffer(50 mM Tris-HCl, 500 mM NaCl, 5%glycerol, 0.5%TritonX-100, pH=8.0) using sonicator for 1 minutes.
    • 2. Whole cell lysate: Mix 50 μL 5x loading buffer to 100 μL cell lysate as the sample of whole cell lysate. Heat the sample at 100℃for 10 minutes, and centrifuge at 15, 000 rpm for 2 minutes.
    • 3. Supernatant and pellet of cell lysate: Centrifuge the remained 200 μL cell lysate at 15, 000 rpm for 10 minutes, and collect the supernatant and pellet of cell lysate, respectively Mix 90 μL 5 x loading buffer to 180 μL supernatant as the sample of supernatant of cell lysate. Resuspend all of the precipitation with 130 μL 5 x loading buffer as the sample of pellet of cell lysate. Heat the samples at 100℃for 10 minutes, and centrifuge at 15, 000 rpm for 2 minutes before loading into the gel.

    Western Blot

    • 1. Gel Preparation
    • 2. Sample preparation
    • Take 40 μL of each sample and add 10 μL of 5 x SDS-PAGE loading buffer. Boil the protein samples using a PCR machine at 95-100℃ for 5-10 minutes. Immediately cool the samples on ice for 5 minutes.
    • 3. Addition of samples
    • Add 20 μL of each prepared sample to the wells of the gel. Additionally, add a pre-stained molecular weight marker to one of the wells.
    • 4、Electrophoresis
    • (for Brazzein)120V, 60 mins.
    • (for Thaumatin)150V, 60 mins.
    • 5. Wash the gel
    • Transfer the gel to a clean tray containing EB solution for 3 minutes.
    • 6. Activation of PVDF membrane
    • 7. Soak the filter paper in the EB solution
    • 8. Protein transfer
    • On the semi-dry transfer apparatus, place the components from negative to positive in the following order: Filter Paper, Gel, Membrane, Filter Paper(top to bottom). Be sure to eliminate any air bubbles when placing the components. Close the electrode cover of the transfer apparatus and electrically transfer the components at 15V for 42 mins.
    • 9. Transferring and Blocking
    • After transferring, block the membrane with 5%BSA at room temperature for 2 hours.
    • 10. First Antibody Incubation
    • Dilute the primary antibody with the blocking solution(1:5000). Once the blocking solution is removed, immediately add the diluted primary antibody. Incubate the membrane at room temperature or 4℃ on a shaker for 15 hours(from 7:30 p.m. to 10:30 a.m.). It is preferable to incubate at a lower temperature. If incubating the primary antibody overnight in the blocking solution, ensure it is done at 4℃ to prevent contamination and protein degradation.
    • 11. Washing the First Antibody
    • After incubation with the primary antibody is complete, carefully remove the primary antibody solution. Place the membrane in a square container and add TBST(Tris-buffered saline with Tween) to completely cover the PVDF membrane. Shake the container on a low-speed shaker for 10 minutes. Repeat this washing step three times, each time replacing the TBST solution.
    • 12. Secondary Antibody Incubation and Washing
    • Dilute the secondary antibody with the blocking solution(1:5000). Submerge the PVDF membrane containing the transferred proteins into a square container filled with the diluted secondary antibody. Incubate the membrane while shaking gently for 2 hours. After the incubation is complete, carefully remove the secondary antibody solution. Place the membrane in a square container and add TBST to completely cover the PVDF membrane. Shake the container on a low-speed shaker for 10 minutes. Repeat this washing step three times, each time replacing the TBST solution.
    • 13. Staining
    • Prepare the DAB(3, 3'-Diaminobenzidine) working solution by mixing equal volumes of the DAB solution A and B(Note: handle the solution in a light-protected manner). Using a pipette, carefully apply an appropriate amount of the DAB working solution to cover the PVDF membrane. Allow the reaction to proceed for a suitable duration as per the staining requirements. After the desired staining intensity is achieved, remove the excess DAB solution and rinse the membrane with deionized water. Place the membrane in an ECL(Enhanced Chemiluminescence) imaging system and perform image acquisition according to the instrument's instructions.


TRV verification

    Plasmid construction and cell transformation

    Plasmid transformation

    • 1. Take out GV3101 receptive Agrobacterium cells from the -80℃ refrigerator, leave them at room temperature or partially melt them with the palm of your hand, and place them on ice while in the ice-water mixture to continue melting.
    • 2. Take out the plasmids from the refrigerator at -20 ℃, dissolve them with 4 µL ddH20 per tube, centrifuge at 14,000 rpm for 1-3 min, and add 0.01-1ug plasmids per 100 µL receptive cells after thawing. Tap the bottom of the Eppendorf tube to mix. It is carried out in sequence: ice standing for 5 min, liquid nitrogen freezing for 5 min, water bath at 37 ℃ for 5 min, ice bath for 5 mins.
    • 3. In the super clean table, 700 µL of antibiotic-free LB (or YEB) medium was added to each tube, and expand the culture at 28 ° C for 2-3 hours with shaking.

    Screening and activation

    • 1. Prepare LB solid medium containing kanamycin and rifampicin, and spread 60 μL of bacterial suspension on each plate.
    • 2. Invert the plates and incubate at 28℃ in an incubator for 2-3 days.
    • 3. For each shake culture, take 3 mL of LB liquid medium (containing 50 mg/mL Kan and 50 mg/mL Rif) in a 15 mL centrifuge tube, gently pick a single colony from the solid medium, insert the pipette tip below the liquid surface and gently blow to make the bacteria inside the tip enter the medium, then push the tip into the tube, and activate by shaking at 28℃ for 24-36 hours.
    • 4. Strain preservation: 200 μL of 50% glycerol (ddH2O : glycerol=1:1 diluted) + 600 μL of bacterial suspension, mix well by gently flicking with a finger. Store at -80℃.
    • 5. Strain activation: Take 100 μL of activated bacterial suspension and inoculate it into 10 mL of LB liquid medium (containing 50 mg/mL Kan and 50 mg/mL Rif) at a ratio of 1:100, and shake culture overnight at 28℃.

    Agrobacterium detection

    • a. Synthesize corresponding detection primers.
    • b. Prepare the PCR amplification system, mix thoroughly after preparation, and use the PCR instrument for amplification, with the amplification program set according to the primer information, etc.

    • Reaction system:
      • Group name Volume
      Plasmid 1.0 μL
      Primer 1(10μM) 1.0 μL
      Primer 2(10μM) 1.0 μL
      2x Taq Mix 10.0 μL
      ddH2O Up to 20 μL
      Total 20 μL

    • Reaction process:
      • step 1 98℃ 3 mins
      step 2 (34 cycles) 98℃ 20s
      step 3 (34 cycles) 56℃ 20s
      step 4 (34 cycles) 72℃ 40s
      step 5 72℃ 5 mins
      step 6 12℃ ∞

    • c. Gel electrophoresis detection, prepare a 1% agarose gel, weigh 0.5 g agarose powder and dissolve in 50 mL 1xTAE buffer, heat until boiling in a microwave, until the solution becomes transparent, cool slightly and add 5 μL SuperRed (10000x). Pour the gel into a gel mold tool with a comb inserted, and wait for it to solidify. Add the mixed samples and markers to the gel wells. The specific experiment determines the sample loading amount. The volume of marker loading is based on the following standards: 11-well gels correspond to 2 μL, 8-well correspond to 5 μL, and 6-well correspond to 10 μL. Electrophoresis is carried out at 150V until the loading buffer's bands reach the middle or two-thirds of the gel, then stop the electrophoresis;
    • d. View the results of PCR amplification, if the electrophoresis bands of the positive control and samples are clear and the size is correct, and the negative control has no bands, it indicates that the sample can proceed to the next step.

    Plant inoculation

    Inoculum resuspension before infection

    • Centrifuge at 4℃, 4000rpm for 10 mins to collect the bacteria, discard the culture medium, and resuspend in the resuspension liquid MMA (10 mmol/L MgCl2, 10 mmol/L MES, 100 mmol/L AS). Measure the OD600 with a UV spectrophotometer, and dilute with the resuspension liquid to the appropriate multiple (adjusted according to the initially measured OD value) until OD600 = 0.6, and let it stand at room temperature to induce for at least 3h.

    Inoculation

    • a. Inoculate when the seedlings have developed four cotyledons.
    • b. Use a syringe to inoculate the abaxial side of the cotyledons or true leaves of the seedlings. Remove the needle from the syringe, and gently scratch the abaxial side of the cotyledon with the needle to damage it without puncturing through. After drawing the bacterial liquid into the syringe, press it against the puncture hole to inject. Observing the color on the abaxial side of the leaf becoming darker indicates successful injection. Repeat this process until the entire leaf is soaked. It is necessary to inject the top 4 leaves from the bottom up of the seedling (when there are branches, only inject the top leaf). The remaining bacterial liquid can be injected into the root system.

    Expression assessment

    RNA extraction

    • 1. Sample collection and preparation: Take approximately 0.2g of fresh sample and place it into a 2 mL centrifuge tube pre-equipped with 2 steel balls. Immediately immerse the tube in liquid nitrogen, then disrupt the sample by vortexing at 42 Hz for 60 seconds. After vortexing, place the tube back into liquid nitrogen and freeze for later use.
    • 2. To each tube, add 1 mL of Trizol and vortex until the sample thaws from solid to liquid. After a 5-minute incubation in a fume hood, add 200 µL of chloroform to each tube, vortex for 15 seconds, and then let it stand for 3 minutes.
    • 3. Centrifuge at 4℃ at 12,000 rpm for 15 minutes. Take 500 µL of the supernatant and mix it 1:1 with 500 µL of isopropanol. Let it stand for 20 minutes, then centrifuge at 12,000 rpm for 10 minutes. Decant the supernatant.
    • 4. To each tube, add 1 mL of 75% ethanol (nuclease-free water: anhydrous ethanol = 5:15), and mix quickly by inverting the tube. Centrifuge again at 7,000 rpm for 3 minutes, then decant the supernatant.
    • 5. Centrifuge once more at 6,800 rpm for 1 minute. Aspirate the supernatant and air-dry the pellet with the lid open.
    • 6. Add 30-50 µL of DEPC water to each tube and store at -20℃.

    RNA reverse transcription

    • Reaction system:
      • Group name Volume
      Random Primer 1.0 μL
      2x R-mix 10.0 μL
      E-mix 1.0 μL
      gDNA remover 1.0 μL
      DEPCH2O Up to 20 μL
      Total 20 μL

    • Reaction process:
      • step 1 25℃ 10 mins
      step 2 42℃ 15 mins
      step 3 4℃ ∞


    PCR

    • Reaction system:
      • Group name Volume
      Plasmid 1.0 μL
      Primer 1(10μM) 1.0 μL
      Primer 2(10μM) 1.0 μL
      2x Taq Mix 10.0 μL
      ddH2O Up to 20 μL
      Total 20 μL

    • Reaction process:
      • step 1 98℃ 3 mins
      step 2 (35 cycles) 98℃ 20s
      step 3 (35 cycles) 56℃ 20s
      step 4 (35 cycles) 72℃ 40s
      step 5 72℃ 5 mins
      step 6 16℃ ∞


    Agarose gel electrophoresis

    • 1. Dissolve 0.5g agarose in 50mL 1xTAE solution, heat until boiling, until the solution becomes transparent, cool slightly and add 5 μL SuperRed (10000x).
    • 2. Pour the gel into a gel mold tool with a comb inserted, and wait for it to solidify.
    • 3. Place the gel into the electrophoresis tank, add 1xTAE buffer until the gel is fully covered, and expel the bubbles from the wells.
    • 4. Add the mixed samples and markers to the wells. The specific experiment determines the sample loading amount. The volume of marker loading is based on the following standards: 11-well gels correspond to 2 μL, 8-well correspond to 5 μL, 6-well correspond to 10 μL.
    • 5. Perform electrophoresis at 150V until the dye front of the loading buffer reaches the middle or two-thirds of the gel, then stop the electrophoresis.
    • 6. Imagine the gel using ultraviolet light.

    Protein extraction

    • 1. Sample preparation Harvest cell pellet from 450 μL culture, and lyse with 300 μL lysis Buffer(50 mM Tris-HCl, 500 mM NaCl, 5%glycerol, 0.5%TritonX-100, pH=8.0) using sonicator for 1 minutes.
    • 2. Whole cell lysate: Mix 50 μL 5x loading buffer to 100 μL cell lysate as the sample of whole cell lysate. Heat the sample at 100℃for 10 minutes, and centrifuge at 15, 000 rpm for 2 minutes.
    • 3. Supernatant and pellet of cell lysate: Centrifuge the remained 200 μL cell lysate at 15, 000 rpm for 10 minutes, and collect the supernatant and pellet of cell lysate, respectively Mix 90 μL 5 x loading buffer to 180 μL supernatant as the sample of supernatant of cell lysate. Resuspend all of the precipitation with 130 μL 5 x loading buffer as the sample of pellet of cell lysate. Heat the samples at 100℃for 10 minutes, and centrifuge at 15, 000 rpm for 2 minutes before loading into the gel.

    Western Blot

    • 1. Gel Preparation
    • 2. Sample preparation
    • Take 40 μL of each sample and add 10 μL of 5 x SDS-PAGE loading buffer. Boil the protein samples using a PCR machine at 95-100℃ for 5-10 minutes. Immediately cool the samples on ice for 5 minutes.
    • 3. Addition of samples
    • Add 20 μL of each prepared sample to the wells of the gel. Additionally, add a pre-stained molecular weight marker to one of the wells.
    • 4、Electrophoresis
    • (for Brazzein, Tricine system)120V, 60 mins.
    • (for Thaumatin, Tris system)150V, 60 mins.
    • 5. Wash the gel
    • Transfer the gel to a clean tray containing EB solution for 3 minutes.
    • 6. Activation of PVDF membrane
    • 7. Soak the filter paper in the EB solution
    • 8. Protein transfer
    • On the semi-dry transfer apparatus, place the components from negative to positive in the following order: Filter Paper, Gel, Membrane, Filter Paper(top to bottom). Be sure to eliminate any air bubbles when placing the components. Close the electrode cover of the transfer apparatus and electrically transfer the components at 15V for 42 mins.
    • 9. Transferring and Blocking
    • After transferring, block the membrane with 5%BSA at room temperature for 2 hours.
    • 10. First Antibody Incubation
    • Dilute the primary antibody with the blocking solution(1:5000). Once the blocking solution is removed, immediately add the diluted primary antibody. Incubate the membrane at room temperature or 4℃ on a shaker for 15 hours(from 7:30 p.m. to 10:30 a.m.). It is preferable to incubate at a lower temperature. If incubating the primary antibody overnight in the blocking solution, ensure it is done at 4℃ to prevent contamination and protein degradation.
    • 11. Washing the First Antibody
    • After incubation with the primary antibody is complete, carefully remove the primary antibody solution. Place the membrane in a square container and add TBST(Tris-buffered saline with Tween) to completely cover the PVDF membrane. Shake the container on a low-speed shaker for 10 minutes. Repeat this washing step three times, each time replacing the TBST solution.
    • 12. Secondary Antibody Incubation and Washing
    • Dilute the secondary antibody with the blocking solution(1:5000). Submerge the PVDF membrane containing the transferred proteins into a square container filled with the diluted secondary antibody. Incubate the membrane while shaking gently for 2 hours. After the incubation is complete, carefully remove the secondary antibody solution. Place the membrane in a square container and add TBST to completely cover the PVDF membrane. Shake the container on a low-speed shaker for 10 minutes. Repeat this washing step three times, each time replacing the TBST solution.
    • 13. Staining
    • Prepare the DAB(3, 3'-Diaminobenzidine) working solution by mixing equal volumes of the DAB solution A and B(Note: handle the solution in a light-protected manner). Using a pipette, carefully apply an appropriate amount of the DAB working solution to cover the PVDF membrane. Allow the reaction to proceed for a suitable duration as per the staining requirements. After the desired staining intensity is achieved, remove the excess DAB solution and rinse the membrane with deionized water. Place the membrane in an ECL(Enhanced Chemiluminescence) imaging system and perform image acquisition according to the instrument's instructions.


Transgenic tomato 1.0

    Agrobacterium preparation

    Plasmid construction and cell transformation

    • Take 1 μL of plasmid and add it to 50 μL of GV3101 Agrobacterium competent cells, mix thoroughly, then transfer to an electroporation cuvette. After electroporation, add 1 mL of LB liquid medium, mix well, and transfer to a 1.5 mL centrifuge tube. Incubate at 30℃ and 180 rpm on a shaker for 30 minutes. Take 50 μL of the activated Agrobacterium culture and inoculate onto LB solid medium, and incubate at 30℃ in the dark for 48 hours.

    Agrobacterium detection

    • a. Synthesize corresponding detection primers;
    • b. Prepare the PCR amplification system, mix thoroughly after preparation, and use the PCR instrument for amplification, with the amplification program set according to the primer information, etc.;

    • Reaction system:
      • Group name Volume
      Plasmid 1.0 μL
      Primer 1(10μM) 1.0 μL
      Primer 2(10μM) 1.0 μL
      2x Taq Mix 10.0 μL
      ddH2O Up to 20 μL
      Total 20 μL

    • Reaction process:
      • step 1 98℃ 3 mins
      step 2 (34 cycles) 98℃ 20s
      step 3 (34 cycles) 56℃ 20s
      step 4 (34 cycles) 72℃ 40s
      step 5 72℃ 5 mins
      step 6 12℃ ∞

    • c. Gel electrophoresis detection, prepare a 1% agarose gel, weigh 0.5g agarose powder and dissolve in 50 mL 1xTAE buffer, heat until boiling in a microwave, until the solution becomes transparent, cool slightly and add 5 μL SuperRed (10000x). Pour the gel into a gel mold tool with a comb inserted, and wait for it to solidify. Add the mixed samples and markers to the gel wells. The specific experiment determines the sample loading amount. The volume of marker loading is based on the following standards: 11-well gels correspond to 2 μL, 8-well correspond to 5 μL, and 6-well correspond to 10 μL. Electrophoresis is carried out at 150V until the loading buffer's bands reach the middle or two-thirds of the gel, then stop the electrophoresis;
    • d. View the results of PCR amplification, if the electrophoresis bands of the positive control and samples are clear and the size is correct, and the negative control has no bands, it indicates that the sample can proceed to the next step.

    Tomato Genetic Transformation

    Seed Disinfection

    • Rinse with sterile water for 2 minutes, disinfect with 75% alcohol for 40 seconds, wash with 84 disinfectant for 7 minutes, rinse with sterile water three times, and soak in sterile water for 1 hour.

    Seed Sowing

    • Sow the disinfected tomato seeds on germination medium, and culture in the dark for 3-4 days. After the seeds have germinated and the radicle is visible, place them in a lighted tissue culture chamber for growth for 4-5 days.

    Preparation and Pre-culture of Explants

    • When the tomato seedlings have fully developed cotyledons, use a scalpel to remove the cotyledon petioles and tips, leaving the middle part which is then cut into 2-3 segments and inoculated onto the pre-culture medium. Pre-culture at 23±2℃ for 2-3 days.

    Agrobacterium Infection and Co-cultivation

    • Pick the Agrobacterium and prepare a resuspended solution with an OD(600) of 0.1; infect for 10-15 minutes. Place the dried explants onto the co-cultivation medium and culture in the dark at 23±2℃ for 2 days.

    Selection and Differentiation Rooting

    • Transfer the recovered callus to the selection medium and culture at 23℃ with a photoperiod of 16 hours light/8 hours darkness for 15-30 days. The selected callus is then inoculated onto the differentiation medium and cultured under the same conditions for 30-40 days. When the differentiated seedlings grow to about 2-3 cm in height, excise them from the callus and inoculate onto the rooting medium. Culture under the same light and temperature conditions for 10-15 days.

    Transgenic detection of tomato

    DNA extraction by CTAB method

    • 1. Add β-mercaptoethanol to the CTAB buffer (final concentration of 0.2%) and preheat at 65℃ before use.
    • 2. Weigh 100 mg of strawberry fruit tissue/50 mg of leaf tissue, grind into a powder in liquid nitrogen, and transfer to a pre-chilled 2 mL centrifuge tube.
    • 3. Add 500 μL of the preheated CTAB, mix well, and incubate at 65℃ for 1 hour, mixing every 15 minutes by inversion.
    • 4. Allow the mixture to cool to room temperature, add 500 μL of chloroform: isoamyl alcohol (24:1) for extraction, centrifuge at 10,000x g for 10 minutes, and collect the supernatant.
    • 5. Add an equal volume (500 μL) of chloroform: isoamyl alcohol (24:1) to the supernatant for a second extraction, centrifuge at 10,000x g for 10 minutes, and collect the supernatant again
    • 6. Add 2/3 volume of pre-chilled at -20℃ isopropanol and 1/10 volume of 3M NaAc (pH=5.4), mix gently, and precipitate at -20℃ for 1 hour.
    • 7. Centrifuge at 10,000x g for 10 minutes, and discard the supernatant.
    • 8. Wash the pellet with 500 μL of 70% ethanol, centrifuge at 10,000x g for 10 minutes, and discard the supernatant.
    • 9. Repeat step 8.
    • 10. Leave the tube open for 10 minutes to allow any remaining ethanol to evaporate, then add 50 μL of TE solution containing RNase (30 μL/mL), and incubate at 37℃ for 30 minutes.
    • 11. Use a NanoDrop ultra-micro nucleic acid protein analyzer to measure the absorbance of the DNA solution at wavelengths of 230nm, 260nm, and 280nm to obtain the DNA concentration. Take 2 μL of the DNA solution for agarose gel electrophoresis to check the integrity of the DNA, and store at -20℃.

    Western Blot

    • 1. Gel Preparation
    • 2. Sample preparation
    • Take 40 μL of each sample and add 10 μL of 5 x SDS-PAGE loading buffer. Boil the protein samples using a PCR machine at 95-100℃ for 5-10 minutes. Immediately cool the samples on ice for 5 minutes.
    • 3. Addition of samples
    • Add 20 μL of each prepared sample to the wells of the gel. Additionally, add a pre-stained molecular weight marker to one of the wells.
    • 4、Electrophoresis
    • (for Brazzein, Tricine system)120V, 60 mins.
    • (for Thaumatin, Tris system)150V, 60 mins.
    • 5. Wash the gel
    • Transfer the gel to a clean tray containing EB solution for 3 minutes.
    • 6. Activation of PVDF membrane
    • 7. Soak the filter paper in the EB solution
    • 8. Protein transfer
    • On the semi-dry transfer apparatus, place the components from negative to positive in the following order: Filter Paper, Gel, Membrane, Filter Paper(top to bottom). Be sure to eliminate any air bubbles when placing the components. Close the electrode cover of the transfer apparatus and electrically transfer the components at 15V for 42 mins.
    • 9. Transferring and Blocking
    • After transferring, block the membrane with 5%BSA at room temperature for 2 hours.
    • 10. First Antibody Incubation
    • Dilute the primary antibody with the blocking solution(1:5000). Once the blocking solution is removed, immediately add the diluted primary antibody. Incubate the membrane at room temperature or 4℃ on a shaker for 15 hours(from 7:30 p.m. to 10:30 a.m.). It is preferable to incubate at a lower temperature. If incubating the primary antibody overnight in the blocking solution, ensure it is done at 4℃ to prevent contamination and protein degradation.
    • 11. Washing the First Antibody
    • After incubation with the primary antibody is complete, carefully remove the primary antibody solution. Place the membrane in a square container and add TBST(Tris-buffered saline with Tween) to completely cover the PVDF membrane. Shake the container on a low-speed shaker for 10 minutes. Repeat this washing step three times, each time replacing the TBST solution.
    • 12. Secondary Antibody Incubation and Washing
    • Dilute the secondary antibody with the blocking solution(1:5000). Submerge the PVDF membrane containing the transferred proteins into a square container filled with the diluted secondary antibody. Incubate the membrane while shaking gently for 2 hours. After the incubation is complete, carefully remove the secondary antibody solution. Place the membrane in a square container and add TBST to completely cover the PVDF membrane. Shake the container on a low-speed shaker for 10 minutes. Repeat this washing step three times, each time replacing the TBST solution.
    • 13. Staining
    • Prepare the DAB(3, 3'-Diaminobenzidine) working solution by mixing equal volumes of the DAB solution A and B(Note: handle the solution in a light-protected manner). Using a pipette, carefully apply an appropriate amount of the DAB working solution to cover the PVDF membrane. Allow the reaction to proceed for a suitable duration as per the staining requirements. After the desired staining intensity is achieved, remove the excess DAB solution and rinse the membrane with deionized water. Place the membrane in an ECL(Enhanced Chemiluminescence) imaging system and perform image acquisition according to the instrument's instructions.


Transgenic tomato 2.0

    Agrobacterium preparation

    Plasmid construction and cell transformation

    • Take 1 μL of plasmid and add it to 50 μL of GV3101 Agrobacterium competent cells, mix thoroughly, then transfer to an electroporation cuvette. After electroporation, add 1 mL of LB liquid medium, mix well, and transfer to a 1.5 mL centrifuge tube. Incubate at 30℃ and 180 rpm on a shaker for 30 minutes. Take 50 μL of the activated Agrobacterium culture and inoculate onto LB solid medium, and incubate at 30℃ in the dark for 48 hours.

    Agrobacterium detection

    • a. Synthesize corresponding detection primers;
    • b. Prepare the PCR amplification system, mix thoroughly after preparation, and use the PCR instrument for amplification, with the amplification program set according to the primer information, etc.;

    • Reaction system:
      • Group name Volume
      Plasmid 1.0 μL
      Primer 1(10μM) 1.0 μL
      Primer 2(10μM) 1.0 μL
      2x Taq Mix 10.0 μL
      ddH2O Up to 20 μL
      Total 20 μL

    • Reaction process:
      • step 1 98℃ 3 mins
      step 2 (34 cycles) 98℃ 20s
      step 3 (34 cycles) 56℃ 20s
      step 4 (34 cycles) 72℃ 40s
      step 5 72℃ 5 mins
      step 6 12℃ ∞

    • c. Gel electrophoresis detection, prepare a 1% agarose gel, weigh 0.5g agarose powder and dissolve in 50 mL 1xTAE buffer, heat until boiling in a microwave, until the solution becomes transparent, cool slightly and add 5 μL SuperRed (10000x). Pour the gel into a gel mold tool with a comb inserted, and wait for it to solidify. Add the mixed samples and markers to the gel wells. The specific experiment determines the sample loading amount. The volume of marker loading is based on the following standards: 11-well gels correspond to 2 μL, 8-well correspond to 5 μL, and 6-well correspond to 10 μL. Electrophoresis is carried out at 150V until the loading buffer's bands reach the middle or two-thirds of the gel, then stop the electrophoresis;
    • d. View the results of PCR amplification, if the electrophoresis bands of the positive control and samples are clear and the size is correct, and the negative control has no bands, it indicates that the sample can proceed to the next step.

    Tomato Genetic Transformation

    Seed Disinfection

    • Rinse with sterile water for 2 minutes, disinfect with 75% alcohol for 40 seconds, wash with 84 disinfectant for 7 minutes, rinse with sterile water three times, and soak in sterile water for 1 hour.

    Seed Sowing

    • Sow the disinfected tomato seeds on germination medium, and culture in the dark for 3-4 days. After the seeds have germinated and the radicle is visible, place them in a lighted tissue culture chamber for growth for 4-5 days.

    Preparation and Pre-culture of Explants

    • When the tomato seedlings have fully developed cotyledons, use a scalpel to remove the cotyledon petioles and tips, leaving the middle part which is then cut into 2-3 segments and inoculated onto the pre-culture medium. Pre-culture at 23±2℃ for 2-3 days.

    Agrobacterium Infection and Co-cultivation

    • Pick the Agrobacterium and prepare a resuspended solution with an OD(600) of 0.1; infect for 10-15 minutes. Place the dried explants onto the co-cultivation medium and culture in the dark at 23±2℃ for 2 days.

    Selection and Differentiation Rooting

    • Transfer the recovered callus to the selection medium and culture at 23℃ with a photoperiod of 16 hours light/8 hours darkness for 15-30 days. The selected callus is then inoculated onto the differentiation medium and cultured under the same conditions for 30-40 days. When the differentiated seedlings grow to about 2-3 cm in height, excise them from the callus and inoculate onto the rooting medium. Culture under the same light and temperature conditions for 10-15 days.

    Transgenic detection of tomato

    DNA extraction by CTAB method

    • 1. Add β-mercaptoethanol to the CTAB buffer (final concentration of 0.2%) and preheat at 65℃ before use.
    • 2. Weigh 100 mg of strawberry fruit tissue/50 mg of leaf tissue, grind into a powder in liquid nitrogen, and transfer to a pre-chilled 2 mL centrifuge tube.
    • 3. Add 500 μL of the preheated CTAB, mix well, and incubate at 65℃ for 1 hour, mixing every 15 minutes by inversion.
    • 4. Allow the mixture to cool to room temperature, add 500 μL of chloroform: isoamyl alcohol (24:1) for extraction, centrifuge at 10,000x g for 10 minutes, and collect the supernatant.
    • 5. Add an equal volume (500 μL) of chloroform: isoamyl alcohol (24:1) to the supernatant for a second extraction, centrifuge at 10,000x g for 10 minutes, and collect the supernatant again
    • 6. Add 2/3 volume of pre-chilled at -20℃ isopropanol and 1/10 volume of 3M NaAc (pH=5.4), mix gently, and precipitate at -20℃ for 1 hour.
    • 7. Centrifuge at 10,000x g for 10 minutes, and discard the supernatant.
    • 8. Wash the pellet with 500 μL of 70% ethanol, centrifuge at 10,000x g for 10 minutes, and discard the supernatant.
    • 9. Repeat step 8.
    • 10. Leave the tube open for 10 minutes to allow any remaining ethanol to evaporate, then add 50 μL of TE solution containing RNase (30 μL/mL), and incubate at 37℃ for 30 minutes.
    • 11. Use a NanoDrop ultra-micro nucleic acid protein analyzer to measure the absorbance of the DNA solution at wavelengths of 230nm, 260nm, and 280nm to obtain the DNA concentration. Take 2 μL of the DNA solution for agarose gel electrophoresis to check the integrity of the DNA, and store at -20℃.

    Western Blot

    • 1. Gel Preparation
    • 2. Sample preparation
    • Take 40 μL of each sample and add 10 μL of 5 x SDS-PAGE loading buffer. Boil the protein samples using a PCR machine at 95-100℃ for 5-10 minutes. Immediately cool the samples on ice for 5 minutes.
    • 3. Addition of samples
    • Add 20 μL of each prepared sample to the wells of the gel. Additionally, add a pre-stained molecular weight marker to one of the wells.
    • 4、Electrophoresis
    • (for Brazzein)120V, 60 mins.
    • (for Thaumatin)150V, 60 mins.
    • 5. Wash the gel
    • Transfer the gel to a clean tray containing EB solution for 3 minutes.
    • 6. Activation of PVDF membrane
    • 7. Soak the filter paper in the EB solution
    • 8. Protein transfer
    • On the semi-dry transfer apparatus, place the components from negative to positive in the following order: Filter Paper, Gel, Membrane, Filter Paper(top to bottom). Be sure to eliminate any air bubbles when placing the components. Close the electrode cover of the transfer apparatus and electrically transfer the components at 15V for 42 mins.
    • 9. Transferring and Blocking
    • After transferring, block the membrane with 5%BSA at room temperature for 2 hours.
    • 10. First Antibody Incubation
    • Dilute the primary antibody with the blocking solution(1:5000). Once the blocking solution is removed, immediately add the diluted primary antibody. Incubate the membrane at room temperature or 4℃ on a shaker for 15 hours(from 7:30 p.m. to 10:30 a.m.). It is preferable to incubate at a lower temperature. If incubating the primary antibody overnight in the blocking solution, ensure it is done at 4℃ to prevent contamination and protein degradation.
    • 11. Washing the First Antibody
    • After incubation with the primary antibody is complete, carefully remove the primary antibody solution. Place the membrane in a square container and add TBST(Tris-buffered saline with Tween) to completely cover the PVDF membrane. Shake the container on a low-speed shaker for 10 minutes. Repeat this washing step three times, each time replacing the TBST solution.
    • 12. Secondary Antibody Incubation and Washing
    • Dilute the secondary antibody with the blocking solution(1:5000). Submerge the PVDF membrane containing the transferred proteins into a square container filled with the diluted secondary antibody. Incubate the membrane while shaking gently for 2 hours. After the incubation is complete, carefully remove the secondary antibody solution. Place the membrane in a square container and add TBST to completely cover the PVDF membrane. Shake the container on a low-speed shaker for 10 minutes. Repeat this washing step three times, each time replacing the TBST solution.
    • 13. Staining
    • Prepare the DAB(3, 3'-Diaminobenzidine) working solution by mixing equal volumes of the DAB solution A and B(Note: handle the solution in a light-protected manner). Using a pipette, carefully apply an appropriate amount of the DAB working solution to cover the PVDF membrane. Allow the reaction to proceed for a suitable duration as per the staining requirements. After the desired staining intensity is achieved, remove the excess DAB solution and rinse the membrane with deionized water. Place the membrane in an ECL(Enhanced Chemiluminescence) imaging system and perform image acquisition according to the instrument's instructions.


Sweetness Detection

Metabolism test

    Glucose Colorimetric Assay

    See the kit: Glucose (Glu) Colorimetric Assay Kit (GOD-POD Method) (Elabscience).


Expression assessment

    Thaumatin ELISA Detection

    See the kit: Thaumatin ELISA Kit(hnybio).


Sweetness detection

    TAS1R2 protein interaction detection

    Material Preparation

    • Human Taste Receptor Type 1 Member 2 (TAS1R2) ELISA Kit (hnybio);
    • Thaumatin ELISA Kit(hnybio);
    • Mouse HA Tag ELISA Kit (hnybio);
    • Solid-phase antibody coated with human receptor protein T1R2 antibody on a microtiter plate;
    • TAS1R2 Standard;
    • Standard dilution solution;
    • Thaumatin enzyme-labeled antigen;
    • Samples of 35S-Thaumatin red ripe fruit (11 samples);
    • Samples of E8-Thaumatin red ripe fruit (11 samples);
    • Sample dilution solution;
    • Wash solution;
    • HA tag enzyme-labeled antibody;
    • Tetramethylbenzidine (TMB);
    • Double-distilled water (ddH2O);
    • Color developing solution A;
    • Color developing solution B;
    • Stop solution;

    Procedure

    • 1. Retrieve the T1R2 solid-phase carrier from the 4℃ refrigerator and allow it to equilibrate at room temperature for 10 minutes.
    • 2. Add 40 μL of standard dilution solution to the sample wells.
    • 3. Add 10 μL of T1R2 standard stock solution to the sample wells, seal with plastic film, and incubate at 37℃ for 30 minutes.
    • 4. Remove the plate, tear off the plastic film, gently tap the plate to dislodge any liquid, and pat dry on paper towels.
    • 5. Fill the sample wells with wash solution, after 30 seconds gently tap the plate to dislodge any liquid, and pat dry on paper towels, repeat this step five times.
    • 6. Add 50 μL of Thaumatin enzyme-labeled antigen to the positive control wells, and to the other sample wells add 10 μL of 35S-Thaumatin red ripe fruit protein samples and E8-Thaumatin red ripe fruit protein samples respectively, along with 40 μL of sample dilution solution, seal with plastic film, and incubate at 37℃ for 30 minutes.
    • 7. Remove the plate, tear off the plastic film, gently tap the plate to dislodge any liquid, and pat dry on paper towels.
    • 8. Fill the sample wells with wash solution, after 30 seconds gently tap the plate to dislodge any liquid, and pat dry on paper towels, repeat this step five times, retain the last wash solution in the positive control wells.
    • 9. Add 50 μL of HA tag enzyme-labeled antibody to the experimental wells, seal with plastic film, and incubate at 37℃ for 30 minutes.
    • 10. Remove the plate, tear off the plastic film, gently tap the plate to dislodge any liquid, and pat dry on paper towels, then add 50 μL of developing solution A and B sequentially to the sample wells. Seal with plastic film and develop the color at 37℃ for 15 minutes.
    • 11. Remove the plate, tear off the plastic film, and add 50 μL of stop solution to each well.
    • 12. Measure the absorbance values at a wavelength of 450 nm using a microplate reader.
    • 13. Organize and analyze the data.



        Electronic Tongue Detection

        Establishing a Database

        • 1. Pick fresh and ripe Micro-Tom tomatoes, wash them clean, and then put them into a high-speed blender to juice for 5 minutes. After that, put the juice into a centrifuge at 7000rpm for 5 minutes, collect the supernatant, dilute it 10 times, and store it on ice.
        • 2. Weigh 1 mg of standard Thaumatin solid into a 2 mL centrifuge tube, prepare a 1 mg/mL Thaumatin standard solution, and store it on ice.
        • 3. According to the sweetness agent curve, select different concentration gradients of Thaumatin to make standard solutions. Add 0 μL, 15 μL, 30 μL, 45 μL, 60 μL of 1 mg/mL Thaumatin standard solution into 5 separate 5 mL small beakers, and then make up to 3 mL with diluted 10 times tomato juice.
        • 4. Collect data with the electronic tongue sensor in a room temperature environment (ensure that the bottom of the sensor is fully immersed during collection). Set the multiplier for each sensor to 100 times, repeat the collection for each sample 5 times, and after testing each sample, rinse the electronic tongue sensor part with ultrapure water.
        • 5. Establish PCA model, LDA-KNN model, and SVM model for the data collected by the electronic tongue.

        Sweetness Detection

        • Control Group:
        • 1. Pick fresh and ripe wild group Micro-Tom tomatoes, wash them thoroughly, and then put them into a high-speed blender to juice for 5 minutes. After that, put the juice into a centrifuge at 7000rpm for 5 minutes, collect the supernatant, dilute it 10 times, and store it on ice.
        • 2. Divide the diluted 10 times tomato juice from the control group into 5 mL small beakers, with 3 mL in each tube.
        • 3. Collect data with the electronic tongue sensor in a room temperature environment (ensure that the bottom of the sensor is fully immersed during collection). Set the multiplier for each sensor to 100, repeat the collection for each sample 5 times, and after testing each sample, rinse the electronic tongue sensor part with ultrapure water.
        • 4. Use the previously established LDA-KNN model and SVM model for prediction, and record the experimental data.

        • Experimental Group:
        • Mature tomatoes containing the 35S promoter TRV vector
        • 1. Pick fresh and ripe Micro-Tom tomatoes containing the 35S promoter TRV vector, divide them into four groups (labeled 1, 2, 3, 4), wash them clean, and then put them into a high-speed blender to juice for 5 minutes. After that, put the juice into a centrifuge at 7000rpm for 5 minutes, collect the supernatant, dilute it 10 times, and store it on ice.
        • 2. Divide the diluted 10 times tomato juice from each group into 5 mL small beakers, with 3 mL in each tube.
        • 3. Collect data with the electronic tongue sensor in a room temperature environment (ensure that the bottom of the sensor is fully immersed during collection). Set the multiplier for each sensor to 100, repeat the collection for each sample 5 times, and after testing each sample, rinse the electronic tongue sensor part with ultrapure water.
        • 4. Use the previously established LDA-KNN model and SVM model for prediction, and record the experimental data.

        • Transgenic mature tomatoes containing the 35S promoter
        • 1. Pick fresh and ripe transgenic Micro-Tom tomatoes containing the 35S promoter, divide them into four groups (labeled 1, 2, 3, 4), wash them clean, and then put them into a high-speed blender to juice for 5 minutes. After that, put the juice into a centrifuge at 7000rpm for 5 minutes, collect the supernatant, dilute it 10 times, and store it on ice.
        • 2. Divide the diluted 10 times tomato juice from each group into 5 mL small beakers, with 3 mL in each tube.
        • 3. Collect data with the electronic tongue sensor in a room temperature environment (ensure that the bottom of the sensor is fully immersed during collection). Set the multiplier for each sensor to 100, repeat the collection for each sample 5 times, and after testing each sample, rinse the electronic tongue sensor part with ultrapure water.
        • 4. Use the previously established LDA-KNN model and SVM model for prediction, and record the experimental data.

        • Transgenic mature tomatoes containing the E8 promoter
        • 1. Pick fresh and ripe transgenic Micro-Tom tomatoes containing the E8 promoter, divide them into four groups (labeled 1, 2, 3, 4), wash them clean, and then put them into a high-speed blender to juice for 5 minutes. After that, put the juice into a centrifuge at 7000rpm for 5 minutes, collect the supernatant, dilute it 10 times, and store it on ice.
        • 2. Divide the diluted 10 times tomato juice from each group into 5 mL small beakers, with 3 mL in each tube.
        • 3. Collect data with the electronic tongue sensor in a room temperature environment (ensure that the bottom of the sensor is fully immersed during collection). Set the multiplier for each sensor to 100, repeat the collection for each sample 5 times, and after testing each sample, rinse the electronic tongue sensor part with ultrapure water.
        • 4. Use the previously established LDA-KNN model and SVM model for prediction, and record the experimental data.



      Vacuole Integrated Positioning

      Rapid Testing on Tobacco

        Agrobacterium Preparation

        Plasmid Transformation

        • 1. Take the competent GV3101 Agrobacterium cells from the -80℃ freezer, place them at room temperature or use the palm of your hand to partially thaw them, and when they are in a slush state, place them on ice to continue thawing.
        • 2. Take the plasmid from the -20℃ freezer, add 4 μL of ddH2O to each tube to dissolve it, centrifuge at 14,000 rpm for 1-3 minutes. When the cell pellet has thawed, add 0.01-1 ug of plasmid per 100 μL of competent cells, gently tap the bottom of the Eppendorf tube to mix, and then proceed as follows: incubate on ice for 5 minutes, freeze in liquid nitrogen for 5 minutes, incubate in a 37℃ water bath for 5 minutes, and chill on ice for 5 minutes.
        • 3. Under sterile conditions, add 700 μL of antibiotic-free LB (or YEB) medium to each tube, and culture at 28℃ with shaking for 2-3 hours.
        • 4. Prepare LB solid medium containing kanamycin (1 mg/L), and spread 60 μL of bacterial suspension on each plate.
        • 5. Invert the plates and incubate at 28℃ for 2-3 days.

        Bacterial Strain Activation

        • 1. For each culture, take 3 mL of LB liquid medium (containing 50 mg/mL Kan and 50 mg/mL Rif) in a 15 mL centrifuge tube, gently pick a single colony from the solid medium, submerge the pipette tip below the liquid surface and gently blow to ensure the bacteria enter the medium, then inoculate the tube, and activate with shaking at 28℃ for 24-36 hours.
        • 2. Take 100 μL of the activated bacterial culture and inoculate it into 10 mL of LB liquid medium (containing 50 mg/mL Kan and 50 mg/mL Rif), and culture overnight at 28℃ with shaking.

        Strain Preservation

        • Take an EP tube, add 200 μL of 50% glycerol (ddH2O : glycerol=1:1 diluted) + 600 μL of bacterial culture, mix well by gently flicking with a finger. Store at -80℃.

        Tobacco Plant Inoculation

        Inoculum resuspension before infection

        • Centrifuge at 4℃, 4000rpm for 10 mins to collect the bacteria, discard the culture medium, and resuspend in the resuspension liquid MMA (10 mmol/L MgCl2, 10 mmol/L MES, 100 mmol/L AS). Measure the OD600 with a UV spectrophotometer, and dilute with the resuspension liquid to the appropriate multiple (adjusted according to the initially measured OD value) until OD600 = 0.6, and let it stand at room temperature to induce for at least 3h.

        Tobacco Cultivation Conditions

        • Plant 5 seeds per pot, place the small pots in large pots, add a spoonful of fertilizer (such as flower fertilizer) to the large pots, and fill with about 2 cm of tap water. Place in a growth chamber and regularly observe the growth condition. After germination, transplant and continue to cultivate in separate pots.

        Agrobacterium Inoculation of Tobacco

        • When the seedlings have developed four cotyledons, use a 1 mL syringe without a needle to scratch the abaxial side of the cotyledons. Draw the bacterial suspension into the syringe and inject it into the scratched area until the entire cotyledon is soaked.

        Verification and Selection

        • Verification and screening using laser scanning confocal microscopy technology.


      Tomato location verification

        Tobacco Plant Inoculation

          Inoculum resuspension before infection

          • Centrifuge at 4℃, 4000rpm for 10 mins to collect the bacteria, discard the culture medium, and resuspend in the resuspension liquid MMA (10 mmol/L MgCl2, 10 mmol/L MES, 100 mmol/L AS). Measure the OD600 with a UV spectrophotometer, and dilute with the resuspension liquid to the appropriate multiple (adjusted according to the initially measured OD value) until OD600 = 0.6, and let it stand at room temperature to induce for at least 3h.

          Tobacco Cultivation Conditions

          • Plant 5 seeds per pot, place the small pots in large pots, add a spoonful of fertilizer (such as flower fertilizer) to the large pots, and fill with about 2 cm of tap water. Place in a growth chamber and regularly observe the growth condition. After germination, transplant and continue to cultivate in separate pots.

          Agrobacterium Inoculation of Tobacco

          • When the seedlings have developed four cotyledons, use a 1 mL syringe without a needle to scratch the abaxial side of the cotyledons. Draw the bacterial suspension into the syringe and inject it into the scratched area until the entire cotyledon is soaked.

          Verification and Selection

          • Verification and screening using laser scanning confocal microscopy technology.

        • Learn more in Proof of concept