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Main experimental process:

Making of Lysogeny broth & Lysogeny Agar

Materials for Lysogeny broth (1 liter formula):

Tryptone: 10g

Yeast extract: 5g

NaCl: 10g

ddH2O: add up to 1L

*Lysogeny agar: based on the above, add 15g of agar per liter. Additionally, add 50mg/ mL Kanamycin: 5µL.

Glycerol stock inoculation

Samples numbered from C1-C8 (Representative target gene wild type and five mutants and two auxiliary enzyme target genes)

Materials:

Lysogeny broth: 5mL x8

Samples C1-C8: each 50µL

50mg/ mL Kanamycin: 5µL x8

The samples are all inoculated in the Lysogeny broth (with kanamycin added) and kept overnight in an incubator (at 37 °C, 220 rpm).

Alkaline lysis

Materials:

Samples C1-C8

RNase A (Degrades RNA in the sample): 150µL

Anhydrous ethanol (dilute Buffer PW2): 80mL

Buffer P1 (Bacterial resuspension buffer): 30mL

Buffer P2 (Bacterial lysis buffer, contains SDS/NaOH): 30L

Buffer P3 (Neutralization buffer): 40mL

Buffer PW1 (Removes proteins and other impurities from the lysate): 60L

Buffer PW2 (Removes residual salts from the plasmid DNA): 20mL

Elution Buffer (Elutes purified plasmid DNA): 20mL

Columns for plasmid DNA adsorption/binding: x8

2mL Collection Tube: x8

*150µL RNase A needs to be added to 30mL Buffer P1 before the experiment, to degrade the RNA in the sample.

*80mL Anhydrous ethanol needs to be added to 20mL Buffer PW2 before the experiment.

Procedure:

1. Centrifuge C1-C8 at 10,000rpm for 1min. Discard supernatant (lysogeny broth) and keep the cell pellets (C1-C8).

2. Resuspend pellets in 250µL Buffer P1 and mix gently by inversion 8-10 times.

3. Cell lysis: Add 250µL Buffer P2 and mix gently by inversion 8-10 times.

4. Neutralization: Add 350µL Buffer P3 and mix gently by inversion 8-10 times. Centrifuge at 12,000rpm for 10min.

5. Transfer the supernatant to an adsorption column for plasmid DNA adsorption/binding in a 2mL collection tube. Centrifuge at 12,000 rpm for 1min. Discard the waste and return the column to the collection tube.

6. Add 500µL Buffer PW1 to the adsorption column. Centrifuge at 12,000 rpm for 1min. Discard the waste and put the adsorption column back into the collection tube.

7. Add 600µL Buffer PW2 to the adsorption column. Centrifuge at 12,000 rpm for 1min. Discard the waste and put the adsorption column back into the collection tube.

8. Repeat step 7.

9. Centrifuge at 12,000 rpm for 1min to dry the adsorption column.

10. Place the adsorption column in a new sterilized 1.5ml centrifuge tube. Add 80μL Elution Buffer to the center of the membrane of the column adsorption column. Leave at room temperature for 2 min, then centrifuge at 12,000 rpm for 1min to elute DNA.

11. Plasmid DNA is extracted from bacterial cells. Stored at -20°C to prevent degradation.

Fig 1. Plasmid extraction and concentration determination

Polymerase Chain Reaction

Mixture used for PCR (100µL):

2× Phanta Flash Master Mix: 25µL

ddH2O: 19µL

Template DNA (from samples C1-C8): 2µL

Forward primer: 2µL

Reverse primer: 2µL

DNA polymerases used:

Low-fidelity / standard (for C1-C7)

94°C 5min, 94°C 30sec, 65°C 30sec, 72°C 1min, 72°C 5min, 12°C ∞

High fidelity/ high accuracy (for C8&C9)

98°C 5min, 98°C 30sec, 65°C 30sec, 72°C 1min, 72°C 5min, 12°C ∞

Fig 2. PCR reaction time and procedure

Agarose Gel Electrophoresis

Added sample: Vector backbone of pET28a plasmid, numbered as C9

*This sample is used as a negative control for PCR

Making of gel (1%):

Agarose: 1g

TAE: 100mL

10000× YeaRed nucleic acid gel stain: 5µL

Fig 3. Agarose gel electrophoresis process

Procedure:

1. Make agarose gel using the formula from above.

2. Heat until there are no obvious visible particles, and let it cool slightly.

3. Stain the mixture with 5µL of 10000x YeaRed nucleic acid gel stain.

4. Pour the gel onto the electrophoresis tray and put the gel comb in before allowing it to solidify.

5. Remove the comb and load the samples needed to be tested.

6. Place the gel in the electrophoresis tank with the running buffer for about 50min.

7. Then check the results below an ultraviolet light.

Expected band sizes:

C1: pET28a- wildtype, 963BP

C2: pET28a- truncated, 861BP

C3: pET28a- 287, 963BP

C4: pET28a- 288, 963BP

C5: pET28a- 289, 963BP

C6: pET28a- AAA, 963BP

C7: MtnN, 699BP

C8: ATed, 1767BP

C9: Vector backbone, 5341BP

Markers used:

2,000 bp

15,000bp (for C8&C9)

Results: Bands for C3-C6 did not appear. This might have happened because the PCR cycling parameters (denaturation/annealing/extension times) were not optimized for these variants, since C3-C5 are all point mutations and C6 is a triple amino acid substitution. We then re-amplified it and it was successful.

Fig 4. Agarose gel electrophoresis results

DNA Gel Extraction

Material:

Buffer GDP (DNA binding buffer): 80mL

Buffer GW (wash buffer): 20mL

Elution Buffer (elutes purified plasmid DNA): 20mL

Columns for plasmid DNA adsorption/binding: ×8

2mL Collection Tube: ×8

*80mL anhydrous ethanol needs to be added to 20mL Buffer GW before the experiment.

Procedure:

1. After electrophoresis, excise the agarose gel under UV light.

2. Add 700µL Buffer GDP. Incubate in a water bath at 50°C for 10min, mix by inversion twice during the process.

3. Centrifuge at 12,000rpm for 60 sec twice to collect liquid.

4. Add 300 µL Buffer GDP, wait for 1 minute, and centrifuge for 60sec.

5. Add 700 µL Buffer GW (with ethanol), centrifuge at 12,000rpm for 60sec.

6. Repeat step 5.

7. Centrifuge at 12,000 rpm for 2 minutes to dry the column.

8. Add 30 µL Elution Buffer, let sit for 2 minutes, then centrifuge at 12,000rpm for 1 minute.

9. Stored at -20°C to prevent degradation.

Homologous Recombination

Materials (20µL):

DNA fragments (C1-C8): 2µL ×8

pCE-Zero vector, linearized (50 ng/µL): 6µL ×8

2× Clon Express Mix: 10µL ×8

ddH2O: 2µL ×8

Procedure:

1. Prepare 8 systems by combining the above materials: 2µL of DNA fragments (C1-C8), 6µL of pCE-Zero vector, linearized (50 ng/µL), 10µL of 2× Clon Express Mix, 2µL of ddH2O.

2. Insert the PCR tubes into the PCR thermal cycler. Ligate the fragments at 50˚C for 5min.

Transformation of competent cells

Materials:

E.coli TOP10 Competent Cells (stored in –80°C): 100µL ×8

Ligated recombination plasmid (C1-C8): 10µL ×8

Lysogeny Agar (plates, with Kanamycin added): ×8

Lysogeny Broth (antibiotic-free): 700µL ×8

Procedure:

1. Take out the E. coli TOP10 competent cells from the –80 °C freezer and immediately place them on ice to thaw.

2. Add the DNA samples to the competent cells, mix gently, and keep on ice for 30 minutes.

3. Place the cells in a 42 °C water bath for 45 seconds, then immediately return them to ice for about 2 minutes. Do not shake or disturb the tubes.

4. Add 700µL of Lysogeny broth, mix gently, and incubate at 37 °C with shaking (200 rpm) for 1 hour.

5. Spread the culture evenly onto the Lysogeny agar plates. Incubate the plates at 37 °C overnight.

Fig 5. Monoclonal plate after transformation

Polymerase Chain Reaction

Mixture used for PCR (100µL):

2× Phanta Flash Master Mix: 25µL

ddH2O: 19µL

Template: 6 samples per plate, 8 plates (48 in total)

Forward primer: 2µL

Reverse primer: 2µL

*Pick up the samples from the plate using a pipette tip. There are a total of 8 plates, from which 6 samples are chosen from each, resulting in a total of 48 samples.

DNA polymerase used: High fidelity/ high accuracy

98°C 5min, 98°C 30sec, 65°C 30sec, 72°C 1min, 72°C 5min, 12°C ∞

Fig 6. Colony PCR process

Agarose Gel Electrophoresis

Making of gel (1%):

Agarose: 1g

TAE: 100mL

10000× YeaRed nucleic acid gel stain: 5µL

* Due to the large number of samples, the experiment will require 2 gels.

Marker used: 2,000 bp

Procedure:

1. Make agarose gels using the formula from above.

2. Heat until there are no obvious visible particles and let it cool slightly.

3. Stain the mixtures each with 5µL of 10000x YeaRed nucleic acid gel stain.

4. Pour the 2 gels onto 2 electrophoresis trays, and on each tray, put 4 combs in before allowing them to solidify.

5. Remove the comb and load the samples needed to be tested along with the 2 markers.

6. Place the gel in the electrophoresis tank with the running buffer for about 50 minutes.

7. Then check the results below an ultraviolet light.

*After making sure the results of the transformed E. coli colonies are acceptable, pick one single colony to inoculate.

Fig 7. Results of colony PCR

Inoculate into Lysogeny Broth

Materials:

Lysogeny broth: 5mL x8

Template: 1 colony per plate, 8 plates

50mg/ mL Kanamycin: 5µL x8

The samples are all inoculated in the Lysogeny broth (with kanamycin added) and kept overnight in an incubator (at 37 °C, 220 rpm).

Scale-Up into Larger Culture

Materials:

Lysogeny Broth: 100mL x8

Overnight bacterial culture: 2 mL

Kanamycin: 100 µL

Place in a shaking incubator at 37 °C, 220 rpm, and grow for 4 hours.

Induction with IPTG

Materials:

Large-scale fermented bacterial culture: 100mL x8

IPTG: added to a final concentration of 0.5mM

Transfer culture to 16 °C, 220 rpm for 12 hours (overnight) to induce protein expression.

Fig 8. Small amount of protein induced expression

His-Tag Purification

Materials:

PBS: 5mL x8

50% BeyoGold™ His-tag Purification Resin (reduction-resistant chelating type): 1mL ×8 (to obtain 0.5mL of settled resin)

20 mM imidazole: 0.5 mL ×16

250 mM Imidazole: 0.5 mL ×32

Procedure:

1. Centrifuge bacterial culture at 15,000g for 10 minutes at 4 °C.

2. Discard the supernatant and keep the cell pellet.

3. Add an appropriate volume of PBS, around 5 mL.  

4. Sonicate the suspension on ice (to ensure the solution does not overheat).

(at 200-300W, depending on effects, in 3-second intervals for 10 minutes each sample)

5. Centrifuge at 10000g for 20 minutes at 4 °C.

6. Centrifuge 50% BeyoGold™ His-tag Purification Resin at 4 °C (1000g × 10 s) and discard the storage solution. Add one column volume of non-denaturing lysis buffer to the resin, mix to equilibrate, centrifuge again at 4 °C (1000g × 10 s), and discard the liquid. Repeat the equilibration 1–2 more times, discarding the liquid each time.

7. Mix 0.5mL of BeyoGold™ His-tag Purification Resin with 4mL of bacterial lysate supernatant we obtained in step.

8. Incubate the mixture at 4 °C on a shaker for 60 min.

9. Load this mixture of lysate and BeyoGold™ His-tag Purification Resin into an empty column tube with a collection tube at the bottom. Open the bottom cap to let the liquid flow out naturally by gravity.

10. Use 20 mM imidazole to wash twice, then use 250 mM Imidazole to elute four times.

Fig 9. Inducible protein expression

Fig 10. Protein sample purification process

Fig 11. Protein sample purification process

SDS-PAGE

Materials:

2× lower gel solution

2× lower gel buffer

modified catalyst

Anhydrous ethanol

2× upper gel solution

2× upper gel buffer

Making of 1.50 mm-thick gel:

1. Mix equal volumes of 2× lower gel solution and 2× lower gel buffer (4 mL each) in a mixing cup. Additionally, add 80 μL of modified catalyst.

2. Pour the mixed lower gel solution into the gel mold until the liquid level is about 1.5 cm below the top edge of the gel mold. Then pour Anhydrous ethanol till full to keep the surface flat.

3. Let rest at room temperature (25 °C) for 10 minutes.

4. Mix equal volumes of 2× upper gel solution and 2× upper gel buffer (1.0 mL each) in another mixing cup. Additionally, add 20 μL of modified catalyst.

5. Discard the Anhydrous ethanol carefully.

6. Pour the upper gel solution on top of the lower gel until the solution reaches the top of the glass plate. Slowly insert the comb into the gel, avoiding bubbles.

7. Let the gel rest at room temperature (25 °C) for about 15 minutes until the upper gel solidifies.

8. Place the gel in an electrophoresis tank and carefully remove the comb.

9. Repeat 8 times to make 8 gels.

Electrophoresis procedure:

1. Load the protein marker first, then the samples obtained from the last step by washing and eluting.

2. Run at 120V for around 90 minutes (time can be adjusted freely depending on the results).

3. Stain gel with Coomassie blue staining solution, and observe under ultraviolet lights.

Fig 12. Protein sample pretreatment before SDS-page

Fig 13. Protein gel staining and destaining

Enzyme Activity Assay

1. Purchase substrates and express the enzymes required for the remaining catalytic reactions.

Sulfoadenosylmethionine (SAM) substrate

2. Determine NAS1 enzyme activity using a microplate spectrophotometer:

a. First, set the microplate reader to continuous sampling mode at 37℃ and 265 nm. Perform five technical replicates for the same AtNAS1 mutant protein, with each reaction volume of 100 μL.

b. Each test reaction contained a final concentration of 1 mg/ml mtnN, 1 mg/ml adaD, 50 mM Tris-HCl (pH 8.7), and 0.15 mg/ml NAS1 protein. The mixture was preincubated at 37℃ for 3 min.

c. The enzymatic reaction was initiated by adding 0.125 mM SAM and mixing five times. The initial rate of change in absorbance over time was measured using the Lambert-Beer law and the extinction coefficient of adenine (λ = 265 nm) of 6700 M/s. Enzyme activity was calculated as nkat/mg protein.

d. Using wild-type AtNAS1 protein and C-terminal truncated AtNAS1 protein as controls, the optimal AtNAS1 protein point mutation was screened.

Fig 14. Schematic diagram of enzyme activity detection principle

Fig 15. Enzyme activity detection process

Agrobacterium Injection into Tobacco

1. Select healthy, pest-free tobacco plants (usually with 4-6 expanded leaves).

2. Select competent strain GV3101.

3. Prepare in LB or YEB medium until the logarithmic growth phase.

4. Prepare injection buffer.

5. Inoculate Agrobacterium into liquid culture and incubate at 28℃ with shaking at 200 rpm until the OD600 reaches 0.8-1.0.

6. Collect the cells by centrifugation, discard the supernatant, and resuspend in injection buffer to adjust the OD600 to 0.3-0.5.

7. Add acetylsalicylic acid and let stand for 1-3 hours to enhance T-DNA transfer efficiency.

8. Place the Agrobacterium suspension into a syringe (e.g., a 1 mL syringe without a needle or with a microneedle).

9. Gently inject the solution into the interveinal tissue on the underside of the tobacco leaf, distributing it evenly throughout the intercellular spaces.

10. Avoid penetrating the leaf or causing extensive damage to ensure leaf health. After injection, place the plant under suitable growth conditions.

11. Expression can usually be measured after 2 days.

12. qPCR and fluorescence observation are performed.

Fig 16. Tobacco growth stages

Fig 17. Agrobacterium injection into tobacco leaves

Soil-grown Tobacco

1) Healthy tobacco seedlings were selected for soil cultivation.

2) Experimental treatments were performed when the plants reached the four-leaf stage, a stage when their root systems are well-developed and suitable for inoculation with exogenous microorganisms.

3) Bacillus subtilis was used as a probiotic for root symbiosis and colonization.

4) According to the experimental design, different concentrations of B. subtilis suspension (OD600 values of 0.5 and 1.0) (10 mL/plant) were applied directly to the tobacco roots to promote rhizosphere microbial colonization.

5) After probiotic administration, the tobacco plants were allowed to continue growing under suitable growth conditions for approximately two weeks.

6) During this period, plant growth was observed.

7) After two weeks, plant fresh weight was measured as an indicator of growth performance.

8) Samples were dried and ground, and trace element contents such as iron and zinc were determined by ICP-MS to analyze the effects of probiotic treatment on nutrient absorption.

Fig 18. Leaf sample collection process