iGEM 2025 — Team yk-pao

{% extends "layout.html" %} {% block title %}Notebook{% endblock %} {% block lead %}This serves as a chronological
record of your team's progress throughout the season. It documents your daily activities, experiments, discussions, and
decisions.{% endblock %} {% block page\_content %} {% from "/components/article-content.html" import articleContent %} {%
call articleContent("NOTEBOOK", [{"content": "January & February","anchor": "#janfeb"},
{"content": "March","anchor": "#march"},
{"content": "April","anchor": "#april"},
{"content": "May","anchor": "#may"},
{"content": "June","anchor": "#june"},
{"content": "July","anchor": "#july"},
{"content": "August","anchor": "#august"},
{"content": "September","anchor": "#september"},
{"content": "October","anchor": "#October"}
]) %}

**Team****calendar:**

# **January & February**

|  |  |  |
| --- | --- | --- |
| Week | Date | Task / Activity |
| Week 1 | Jan 27- Feb 2 | Final decision of team members participating made; first online meeting to organize our team’s schedule and start to brainstorm initial ideas for our project. |
| Week 2 | Feb 3- 9 | Registration of essential information on the official iGEM platform. |
| Week 3 | Feb 10- 16 | Individual theoretical research on possible topics. |
| Week 4 | Feb 17 - 23 | Team sharing session where members present research findings; initial evaluation of feasibility, novelty, and social impact. |
| Week 5 | Feb 24 - Mar 2 | Divide into subgroups (wet lab, dry lab, human practices, education, etc.) |

# **March**

|  |  |  |
| --- | --- | --- |
| Week | Date | Task / Activity |
| Week 6 | Mar 3-9 | Online meeting to introduce iGEM background information to the team, for us to familiarize ourselves with the event. |
| Week 7 | Mar 10- 16 | Each subgroup brainstorms possible initiatives and drafts early responsibilities based on what’s been introduced in the previous online session. |
| Week 8 | Mar 17- 23 | Each subgroup presents its initial research findings and potential project ideas to the whole team; deeper research on the potential impact and experimental possibility of shortlisted project topics. |
| Week 9 | Mar 24-30 | Share individual findings in a team online meeting and compare topics to prepare for the final selection, by analyzing significance, outlining DBTL solutions, aligning with iGEM prizes, identifying stakeholders, planning outreach and education, connecting to SDGs, defining end users, and evaluating innovation. |

# **April**

|  |  |  |
| --- | --- | --- |
| Week | Date | Task / Activity |
| Week 10 | Mar 31- Apr 6 | Decision on the topics we’ve researched for our team: Nicotinamide synthase gene empowers crop iron and zinc biofortification, synthetic biology helps solve the ‘Hidden hunger’ crisis. |
| Week 11 | Apr 7- 13 | Team members receive training in fundamental experimental methods of modern molecular biology, including basic laboratory techniques and safety, to prepare for subsequent labs. |
| Week 12 | Apr 14- 20 | Discussions on how to design effective questionnaires and conduct meaningful interviews to gather social insights and market feedback, helping shape the project’s relevance and real-world impact. |
| Week 13 | Apr 21- 27 | Decision on how our team members will participate in the final iGEM Jamboree; organized a team meeting to collaboratively design and create a standardized form to evaluate the public’s awareness and understanding of hidden hunger (our project topic), then decided to hold monthly team meetings at the end of each month to |

# **May**

|  |  |  |
| --- | --- | --- |
| Week | Date | Task / Activity |
| Week 14 | Apr 28- May 4 | Received a lecture on the art of spreading knowledge by designing activities that foster clear, logical, and engaging scientific dialogues with the public, giving team members a new perspective on communication. |
| Week 15 | May 5- 11 | Team participated in a workshop on introducing the basics of synthetic biology, by building biological modules, exploring the engineering cycle and design principles of synthetic biology, which provided the team with practical insights into bioengineering processes. |
| Week 16 | May 12- 18 | Engaged in literature reading to understand fundamental concepts of genetic engineering and analyzed classic iGEM projects, motivating team members to learn from past experiences. |
| Week 17 | May 19- 25 | Attended a session focusing on business planning and strategies for bringing products to the open market, providing team members with initial practical insights into commercialization. |
| Week 18 | May 26- June 1 | Learned how to apply basic modeling methods and create clear tables to support data analysis in out project; Participated in the monthly team meeting on our progress made in this past month. |

# **June**

|  |  |  |
| --- | --- | --- |
| Week | Date | Task / Activity |
| Week 19 | Jun 2- 8 | Received a tutorial on using GitLab to build and manage the team Wiki. And started some preliminary dry experiments. |
| Week 20 | Jun 9- 15 | Explored rules on dry lab work, clarifying requirements and expectations for entrepreneurial, educational, and media-related tasks. |
| Week 21 | Jun 16- 22 | Explored rules on wet lab work, clarifying requirements and expectations for experimental design and safety. |
| Week 22 | Jun 23- 29 | Held group meeting between dry and wet labs to share progress and coordinate for next steps. |

# **July**

|  |  |  |
| --- | --- | --- |
| Week | Date | Task / Activity |
| Week 23 | Jun 30- Jul 6 | Review of molecular biology experiment principles and engineering concepts in synthetic biology learned in previous lectures. |
| Week 24 | Jul 7- 13 | Conducted surveys to understand public perspectives and clarify our project intentions. |
| Week 25 | Jul 14- 20 | Planned and implemented science communication activities, including promotion video filming. |
| Week 26 | Jul 21- 27 | Monthly group meeting to catch up on latest progress for both dry and wet labs. Summarize and analyze some preliminary dry experimental results. |

# **August**

|  |  |  |
| --- | --- | --- |
| Week | Date | Task / Activity |
| Week 27 | Jul 28- Aug 3 | Visited the laboratory for lab safety training in preparation for upcoming experiments. |
| Week 28 | Aug 4- 10 | Finalized experimental designs while refining subgroup responsibilities, also in preparation for upcoming experiments. |
| Week 29 | Aug 11- 17 | Dry lab went on expert consultation sessions and enterprise visits to gather professional advice and industrial perspectives; wet lab conducted experiments, which are recorded more specifically in the following section. |
| Week 30 | Aug 18- 24 | Dry lab drafted the project business plan, integrating research, experiments, and outreach insights; wet lab conducted experiments, which are recorded in the following section. |
| Week 31 | Aug 25- 31 | Integrated results from both the dry lab and wet lab teams to create the initial presentation slides. |

# **September**

|  |  |  |
| --- | --- | --- |
| Week | Date | Task / Activity |
| Week 31 | Sep 1- 7 | Communicated with other 2025 iGEM teams to exchange experiences and discussed possible collaborations, for example, our educational podcast. |
| Week 32 | Sep 8- 14 | Strengthened the link between our project and the school community by establishing a related student club. |
| Week 33 | Sep 15- 21 | Finalized and submitted the attribution form. Collect tobacco samples after soil culture, pre-process and prepare for testing. |
| Week 34 | Sep 22- 28 | Produced our official team presentation video. |

# **October**

|  |  |  |
| --- | --- | --- |
| Week | Date | Task / Activity |
| Last month | October | Review the project and prepare for the final defense |

**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.

![](https://static.igem.wiki/teams/5769/image/notebook-1.webp)

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 ∞

![](https://static.igem.wiki/teams/5769/image/notebook-2.webp)

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

![](https://static.igem.wiki/teams/5769/image/notebook-3.webp)

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.

![](https://static.igem.wiki/teams/5769/image/notebook-4.webp)

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.

![](https://static.igem.wiki/teams/5769/image/notebook-5.webp)

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 ∞

![](https://static.igem.wiki/teams/5769/image/notebook-6.webp)

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.

![](https://static.igem.wiki/teams/5769/image/notebook-7.webp)

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.

![](https://static.igem.wiki/teams/5769/image/notebook-8.webp)

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.

![](https://static.igem.wiki/teams/5769/image/notebook-9.webp)

Fig 9. Inducible protein expression

![](https://static.igem.wiki/teams/5769/image/notebook-10.webp)

Fig 10. Protein sample purification process

![](https://static.igem.wiki/teams/5769/image/notebook-11.webp)

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.

![](https://static.igem.wiki/teams/5769/image/notebook-12.webp)

Fig 12. Protein sample pretreatment before SDS-page

![](https://static.igem.wiki/teams/5769/image/notebook-13.webp)

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.

![](https://static.igem.wiki/teams/5769/image/notebook-14.webp)

Fig 14. Schematic diagram of enzyme activity detection principle

![](https://static.igem.wiki/teams/5769/image/notebook-15.webp)

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.

![](https://static.igem.wiki/teams/5769/image/notebook-16.webp)

Fig 16. Tobacco growth stages

![](https://static.igem.wiki/teams/5769/image/notebook-17.webp)

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.

![](https://static.igem.wiki/teams/5769/image/notebook-18.webp)

Fig 18. Leaf sample collection process

{% endcall %} {% endblock %}

{% extends "layout.html" %} {% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a technical aspect of your
project by going through at least one iteration of the engineering design
cycle.{% endblock %} {% block page\_content %} {% from
"/components/article-content.html" import articleContent %} {% call
articleContent("ENGINEERING", [
{
"content": "Overview",
"anchor": "#overview"
},
{
"content": "Cycle1:Autoinhibition Removal and High-Efficiency Enzyme Screening",
"anchor": "#cycle1",
"children": [
{
"content": "Design",
"anchor": "#design1"
},
{
"content": "Build",
"anchor": "#build1"
},
{
"content": "Test",
"anchor": "#test1",
"children": [
{
"content": "Preliminary Experiment for Protein Expression",
"anchor": "#preliminary"
},
{
"content": "His-Tag Affinity Purification of Proteins",
"anchor": "#purification"
},
{
"content": "Enzymatic Reactions Detection of AtNAS1 Mutated Proteins' Catalytic Ability",
"anchor": "#enzymatic"
},
{
"content": "Molecular Dynamics Fitting Analysis of Catalytic Efficiency in AtNAS1 Mutated Proteins",
"anchor": "#molecular"
}
]
},
{
"content": "Learn",
"anchor": "#learn1"
}
]
},
{
"content": "Cycle2:Gene Expression Levels, and Subcellular Localization",
"anchor": "#cycle2",
"children": [
{
"content": "Design",
"anchor": "#design2"
},
{
"content": "Build",
"anchor": "#build2"
},
{
"content": "Test",
"anchor": "#test2",
"children": [
{
"content": "Detection the Effect of AtNAS1 Mutant Proteins on Subcellular Localization Using Confocal Microscopy",
"anchor": "#confocal"
},
{
"content": "Detection the Effect of AtNAS1 Mutant Proteins on the Expression of Iron Cycling Related Genes Using
Real-time Quantitative PCR In Vivo",
"anchor": "#qpcr"
}
]
},
{
"content": "Learn",
"anchor": "#learn2"
}
]
},
{
"content": "Cycle3:Biofortification Verification",
"anchor": "#cycle3",
"children": [
{
"content": "Design",
"anchor": "#design3"
},
{
"content": "Build",
"anchor": "#build3"
},
{
"content": "Test",
"anchor": "#test3",
"children": [
{
"content": "Effect of B. subtilis Expressing AtNAS1 Mutated Proteins on N. benthamiana Growth and Biomass",
"anchor": "#biomass"
},
{
"content": "Effect of B. subtilis Expressing AtNAS1 mutated Proteins on Iron and Zinc Trace Element Contents in Tobacco
Leaves Using ICP-MS",
"anchor": "#icpms"
}
]
},
{
"content": "Learn",
"anchor": "#learn3"
}
]
},
{
"content": "References",
"anchor": "#references"
}
]) %}

# Overview

Iron (Fe) and zinc (Zn) are essential trace elements for human health, yet deficiencies in these nutrients affect
approximately 2 billion people worldwide, particularly infants, young children, and pregnant women[1]. In
sub-Saharan Africa, diets heavily reliant on maize which provides at least 30% of total caloric intake—pose a
high risk of iron and zinc deficiency, as maize endosperm contains very low levels of these elements[2]. While
supplementation with iron and zinc compounds can improve nutritional status, economic constraints and low
consumer acceptance limit their effectiveness in developing countries[3]. In contrast, biofortification through
transgenic crops offers a sustainable and promising approach. Recent advances in genetic engineering have
successfully increased edible crop iron content by manipulating genes related to iron uptake and metabolism; for
example, co-expression of AtNAS1 (nicotianamine synthase 1), PvFERRITIN, and AfPHYTASE has enhanced iron
concentration in rice endosperm[4]. NAS enzymes are key for nicotianamine synthesis, a crucial metal chelator
that binds divalent metals like Fe and Zn, thus regulating their uptake, transport, and distribution within
plants[5].

However, studies have shown that the C-terminal region of AtNAS1 contains an autoinhibitory signal that affects
enzyme activity, which may explain why overexpression of AtNAS1 in transgenic rice does not always significantly
increase iron and zinc levels[6]. Based on this, the current study aims to generate site-specific mutations at
key C-terminal residues predicted to regulate AtNAS1 activity, purify the mutant proteins via prokaryotic
expression in E. coli, and perform enzymatic kinetics analysis compared to wild-type AtNAS1 and a truncated
AtNAS1 positive control. The most active mutants will be expressed in Bacillus subtilis for soil-based N.
benthamiana cultivation to assess iron and zinc accumulation in leaves and roots. Additionally, transient
expression in tobacco combined with qPCR analysis of iron- and zinc-related genes and confocal microscopy of
fluorescently tagged proteins will verify mutant AtNAS1 expression, functional impact on downstream genes, and
subcellular localization. This work uses tobacco as a model to provide insights and a theoretical basis for
alleviating hidden hunger caused by trace element deficiencies in crops.

# Cycle1:Autoinhibition Removal and High-Efficiency Enzyme Screening

## Design

Based on previous reports of an autoinhibitory signal at the C-terminus of AtNAS1, we performed single- point
mutations targeting specific amino acid residues (T287, R288, and G289). We also designed a multi-residue
mutation (TRG→AAA) and a C-terminal truncation variant to test their effects on enzyme activity relative to the
wild-type protein. All variants were designed and inserted into the pET28a expression vector via homologous
recombination for prokaryotic expression in Escherichia coli. Wild-type and truncated AtNAS1 fragments were
amplified by high-fidelity PCR, and mutants were constructed using inverse PCR to introduce precise point
mutations.

Since nicotinamide synthase activity requires SAM as a substrate and MtnN and AdeD as coenzymes, these coenzyme
genes were cloned into the pET28a vector for co-expression or supply during enzymatic assays. This design
enabled systematic evaluation of C-terminal mutations and key residues in AtNAS1 while ensuring the availability
of the coenzymes and robustly validating all constructs prior to functional analysis.

![Image](https://static.igem.wiki/teams/5769/image/engineering-1.webp)

**Fig 1.** Schematic diagram of AtNAS1 wild type, C-terminal autoinhibition removal, four
point
mutations, and two coenzymes connected to the pet28a vector

## Build

We amplified the AtNAS1\_Wildtype fragments via high-fidelity PCR, resulting in fragment lengths of 963 bp. The
right panel of Figure 2 displays the correct bands corresponding to the target sizes of the two genes.
Subsequently, we used the restriction endonuclease BamHI to linearize the pET28a vector backbone, as shown in
the left panel of Figure 2. After performing agarose gel electrophoresis and recovering the bands, we proceeded
with the Infusion assemble kit to obtain the recombinant plasmid.

After the Infusion reaction assay, we transformed the recombinant plasmids into the cloned strain E. coli DH5α.
Figure 2A shows the single colonies on the plate. We then selected six colonies from each plate. Next, we used
vector universal primers, T7-Forward and T7-Terminal, to detect the insertion of target genes using colony PCR.
As shown in Figure 2B, the AtNAS1\_Wildtype gene fragment was successfully inserted into the pET28a backbone.

![Image](https://static.igem.wiki/teams/5769/image/engineering-2.webp)

**Figure 2.** Verification of the transformation results for positive colonies of
pET28a-AtNAS1\_Wildtype and pET28a-AtNAS1\_Truncated plasmids. A: Single colonies on the plate; B: Amplification
results from colony PCR.

Finally, we used the universal primer T7-Forward to verify the positive colonies through Sanger sequencing.
Figure 3 displays the correct sequences of the recombinant plasmids. We selected the correct colonies for
further IPTG induction.

![Image](https://static.igem.wiki/teams/5769/image/engineering-3.webp)

**Figure 3.** Sanger sequencing results of pET28a-AtNAS1\_Wildtype and
pET28a-AtNAS1\_Truncated
positive colony.

To further construct single-base and multi-base mutations, we linearized the pET28A-AtNAS1-Wildtype plasmid using
**inverse PCR** to incorporate specific mutation sites. Subsequently, we constructed the mutated
plasmids AtNAS1-T287A, AtNAS1-R288A, AtNAS1-G289A, and AtNAS1-TRGtoAAA via Infusion assay. Following the same
construction process as for pET28A-AtNAS1-Wildtype, we also used colony PCR and Sanger sequencing to verify the
positive colonies of the mutated plasmids, as shown in Fig. 4.

![Image](https://static.igem.wiki/teams/5769/image/engineering-4.webp)

**Fig. 4** The construction process of pET28A-AtNAS1 mutated plasmids. A. Agarose gel
electrophoresis of mutated genes; B. Single colonies on the plate; C: Amplification results from colony PCR; D:
Sanger sequencing results.

Since the enzymatic
activity assay of nicotinamide synthase requires the substrate SAM and two coenzymes, MtnN and AdeD, we
cloned
the MtnN and AdeD gene fragments from the E. coli XL-1 Blue strain. Using
the
same construction strategy as for the pET28A-AtNAS1 plasmid, we constructed the expression plasmids
pET28a-MtnN
and pET28a-AdeD. The construction process is shown in Fig. 5.

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| ![Image](https://static.igem.wiki/teams/5769/image/engineering-5.webp) Fig. 5 The construction process of pET28A-MtnN and pET28A-AdeD plasmids. A. Agarose gel electrophoresis of MtnN and AdeD genes; B. Single colonies on the plate; C: Amplification results from colony PCR; D: Sanger sequencing results. |

We transformed the
correct plasmids into the E. coli expression strain BL21 and selected single colonies
for
culture in preparation for the next step of prokaryotic expression. The plates are shown in Fig.
6.

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| --- |
| ![Image](https://static.igem.wiki/teams/5769/image/engineering-6.webp) Fig. 6 Single colonies of recombinant plasmids in E. coli BL21 strain. |

## **Test**

### **Preliminary Experiment for Protein Expression**

Based on the lac
operator system in the pET28a vector backbone, we used IPTG for protein expression. Initially, we applied a
final concentration of 1 mM IPTG for standard protein expression. The expected molecular weights of AtNAS1
(including the mutated proteins), AtNAS1-Truncated, MtnN, and AdeD are 35.5 kDa, 31.7 kDa, 24.4 kDa, and
63.7
kDa, respectively. As shown in Fig. 7, we can clearly
observe the protein bands corresponding to the target genes.

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| ![Image](https://static.igem.wiki/teams/5769/image/engineering-7.webp) Fig. 7 SDS-PAGE analysis of preliminary experiment for target proteins expression. |

### **His-Tag Affinity Purification of Proteins**

We utilized the
principle of His-tag affinity with Ni2+. This allowed us to
bind the crude protein extracted from the E. coli expression cells to
Ni-NTA beads. After washing away
impurities with a low concentration of imidazole, we eluted the protein with a high concentration of
imidazole.
As shown in Fig. 8, we obtained a
high-purity protein with a high expression level. Subsequently, we used dialysis to remove the high
concentration of imidazole from the buffer and aliquoted the protein at a concentration of 1 mg/mL for
storage,
to be used in subsequent functional assays.

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| ![Image](https://static.igem.wiki/teams/5769/image/engineering-8.webp) Fig. 8 SDS-PAGE analysis of large-scale His-tag protein purification of target proteins. Total: Total Protein; FT: Flow Through. |

### **Enzymatic Reactions Detection of****AtNAS1 Mutated** **Protein****s****' Catalytic Ability**

We assessed
the
enzymatic activity of nicotinamide synthase (NAS1) mutants in Functional Tests Part 1. The enzymatic
activities
of the wild-type and various mutants (Truncated, T287A, R288A, G289A, TRG\_to\_AAA) of NAS1 were evaluated by
measuring the initial reaction rates (ΔA/Δt) and calculating the enzyme activities (nkat/mg) based on the
parameters of the reaction system (Fig. 9B). Compared to the slope and correlation
coefficient of AtNAS1-Wildtype, the enzyme activities of the different mutants varied significantly, with
some
mutants, such as AtNAS1-T287A and AtNAS1-R288A, exhibiting markedly reduced activities. In contrast,
AtNAS1-Truncated and AtNAS1-TRG\_to\_AAA demonstrated steep slopes and high enzyme catalytic efficiency
(Fig. 9A). The AtNAS1-TRG\_to\_AAA mutant protein,
unlike AtNAS1-Truncated, retains the complete C-terminal region of the NAS1 protein, thereby avoiding
potential
functional redundancies associated with the truncated form. Notably, the TRG\_to\_AAA mutant also exhibited a
relatively higher level
of
nicotinamide synthesis activity compared to the truncated protein (Fig. 9B). Therefore, the TRG\_to\_AAA mutant
may possess greater potential for application than the truncated variant.

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| ![Image](https://static.igem.wiki/teams/5769/image/engineering-9.webp) Fig. 9 Kinetic and Thermodynamic Analysis of Engineered Variants of AtNAS1 protein. A. Enzyme Kinetics of AtNAS1 mutated protein; B. Enzyme Activity Analysis. |

### **Molecular Dynamics Fitting Analysis of Catalytic Efficiency in** **AtNAS1** **Muta****ted** **Proteins**

After testing the enzymatic
assays, we further investigated the binding affinities of the AtNAS1-Wildtype and
AtNAS1 mutant forms with
the
substrate S-adenosyl methionine (SAM) using molecular dynamics simulations. As shown in Figure 10, the average
binding
free energy (dG) between AtNAS1-Wildtype and SAM was -36.83
kcal/mol, while the dG values for AtNAS1-Truncated is -13.63 kcal/mol and the dG
values for AtNAS1-TRG\_to\_AAA
is
-17.06 kcal/mol. These results
indicate
that the ligand (SAM) can spontaneously bind to the protein, and the resulting protein-ligand complexes are
dynamically stable, suggesting they may possess biological activity. This provides a theoretical
energy-based
foundation for further studying the biological functions of these ligand-protein complexes. The favorable
binding
free energies imply that the AtNAS1-Wildtype and
AtNAS1-TRG\_to\_AAA can
effectively
interact with the SAM substrate, which is a critical step in the enzymatic reactions catalyzed by this protein.

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| ![Image](https://static.igem.wiki/teams/5769/image/engineering-10.webp) Fig. 10 Molecular dynamics simulation of the binding sffinities between AtNAS1-Wildtype and mutant forms with the substrate S-Adenosyl Methionine (SAM) |

## **Learn**

Based on the
results of the first round of experiments, we successfully constructed and expressed multiple AtNAS1
variants
(wild-type, truncated, and point-mutated) and used them in E. coli. Enzymatic assays showed that the TRG→AAA
mutant and the truncated form exhibited the highest catalytic efficiency, while the T287A/R288A mutant
exhibited
reduced activity. Molecular dynamics simulations confirmed that SAM had strong binding affinity for both the
wild-type (ΔG = -36.83 kcal/mol) and the TRG→AAA mutant (ΔG = -17.06 kcal/mol), confirming their excellent
substrate interaction abilities.

These results
suggest that the C-terminal TRG motif plays a key role in regulating AtNAS1 autoinhibition, and that
multi-residue substitutions (TRG→AAA) effectively abolished inhibition while maintaining the full-length
integrity of the protein.

For further
investigation, we recommend: 1) performing in planta studies using the TRG→AAA mutant in cycle 2 (transient
expression in tobacco) and cycle 3 (stable transformation), and 2) implementing ICP-MS to quantify Fe/Zn
accumulation in plant tissues.

# **Cycle2:Gene Expression Levels, and Subcellular Localization**

## **Design**

Based on the
results from the first round, we selected the TRG→AAA mutant, AtNAS1 truncation, and wild-type for in vitro
validation. We chose a tobacco transient expression system because it is rapid and suitable for initial
functional screening. This system allows for efficient delivery of constructs via Agrobacterium
infiltration, enabling transient expression.

We will clone
the
coding sequences of the selected mutants (TRG→AAA, truncation, and wild-type) into a plant expression vector
(pCambia1305) controlled by the CaMV 35S promoter using homologous recombination. Three plasmids will be
constructed for qPCR analysis and subcellular localization. All constructs will be verified by colony PCR
sequencing prior to transformation into Agrobacterium GV3101.

![Image](https://static.igem.wiki/teams/5769/image/engineering-11.webp)

Fig. 11 Plasmid maps of wild-type, TRG→AAA, truncation, and pCambia1305-eGFP-6myc vector construction

## **Build**

Our previous results showed that the AtNAS1-Truncated protein and the AtNAS1
triple-mutant AtNAS1-TRG\_to\_AAA protein have comparable enzyme
activity
levels. Therefore, in the next step, we would like to validate our hypothesis using a plant model system,
like **N. benthamiana** .
First,
we constructed the gene sequences of
AtNAS1-Wildtype,
AtNAS1-Truncated, and AtNAS1-TRG\_to\_AAA into the dual expression vector
pCambia1305, which has a GFP fluorescent protein tag. This will allow us to detect the subcellular
localization
of the mutants using confocal microscope. As showed in Fig. 9A, we first amplified the gene fragments
containing the homologous arms of the pCambia1305 backbone, where the AtNAS1-Wildtype served as the control. We
then
linearized the pCambia1305 vector using the restriction enzyme BamHI (Fig. 12B). After that, we performed an
In-Fusion
assembly reaction to construct the recombinant vector (Fig. 12C). Finally, we screened for positive
clones
using colony PCR (Fig. 12D).

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| ![Image](https://static.igem.wiki/teams/5769/image/engineering-12.webp) Fig. 12 The construction process of pCambia1305-GFP-AtNAS1-WT, pCambia1305-GFP-AtNAS1-Truncated, and pCambia1305-GFP-AtNAS1-TRG\_to\_AAA plasmids. A. Agarose gel electrophoresis of genes; B. Linearized pCambia1305 backbone; C. Single colonies on the plate; D: Amplification results from colony PCR. |

## **Test**

### **Detect****ion** **the Effect of** **AtNAS1** **Mutant Proteins on Subcellular Localization Using Confocal Microscopy**

Given that
the
subcellular localization of a protein can affect its function, we utilized transient expression in
**N.
benthamiana** along with microscopy to observe whether the localization patterns
of
the AtNAS1 mutant proteins differ from the
AtNAS1-Wildtype protein. As shown in Fig. 13A, the process of transient expression in
tobacco involves Agrobacterium strain in acetosyringone buffer, selecting healthy tobacco leaves,
and
infiltrating the bacterial suspension into the leaf abaxial surface using a 1 mL syringe. After two days of
transient expression, the leaves were analyzed. We first transformed the
AtNAS1-Wildtype, AtNAS1-Truncated, and AtNAS1-TRG\_to\_AAA positive plasmids into the Agrobacterium GV3101
strain
and grew them on dual-antibiotic plates containing kanamycin and rifampicin, picking single colonies for
further
culturing (Fig. 13B). We initially used a handheld fluorescence
instrument to detect the protein expression levels, which revealed that the AtNAS1-Truncated and
AtNAS1-TRG\_to\_AAA proteins exhibited higher
expression
efficiency compared to the AtNAS1-Wildtype protein (Fig. 13C). Further analysis using confocal
microscopy
showed that the AtNAS1-Truncated and AtNAS1-TRG\_to\_AAA proteins had stronger fluorescence
intensity and were more strongly localized to the endoplasmic reticulum, providing a potential explanation
for
their higher enzymatic activity (Fig. 13D).

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| ![Image](https://static.igem.wiki/teams/5769/image/engineering-13.webp) Fig. 13 Subcellular localization of AtNAS1-Wildtype, AtNAS1-Truncated, and AtNAS1-TRG\_to\_AAA proteins. A. Process of transient transfection in N. benthamiana ; B. Single colonies of recombinant plasmids in Agrobacterium GV3101 strain. The plate contains Kanamycin and Rifampicin dual-antibiotics; C. Detection of GFP protein expression in N. benthamiana leaves using a handheld fluorescence instrument; D. The subcellular localization patterns of AtNAS1-Wildtype, AtNAS1-Truncated, and AtNAS1-TRG\_to\_AAA in N. benthamiana leaves using confocal microscope. |

### **Detection the Effect of****AtNAS1** **Mutant Proteins on the Expression of Iron Cycling Related** **Genes Using****Real-time****Q****uantitative PCR** **In Vivo**

To further
investigate whether mutations in the AtNAS1 gene could affect the expression of genes related to the
absorption
and activation of iron and zinc micronutrients, we validated this hypothesis using a **N.
benthamiana** -based culture transient system. First, we used the previously constructed
Agrobacterium strains carrying the
AtNAS1-Wildtype, AtNAS1-Truncated, and AtNAS1-TRG\_to\_AAA to infiltrate tobacco leaves. After
3
days of culture, we extracted RNA from the leaves, reverse transcribed it into cDNA, and
used quantitative real-time PCR to
detect
the expression levels of the target genes. The expression levels were normalized using the tobacco NtActin1
gene
as an internal reference. As shown in Fig. 14A, compared to the non-transformed tobacco
leaves, the tobacco leaves expressing the AtNAS1-Wildtype, AtNAS1-Truncated, and AtNAS1-TRG\_to\_AAA plasmids
significantly enhanced AtNAS1 gene expression, indicating that
the
transient expression of AtNAS1 in tobacco was successful. Further analysis revealed that the
transient
overexpression of both the wild-type and mutant AtNAS1 significantly increased the expression of the iron
and
zinc absorption and transport genes NtIRT1 and NtMTP8 (Fig. 14B-C), while the expression of the iron
activation-related gene NtFRO2 remained unchanged (Fig. 14D). Notably, the AtNAS1-Truncated, and
AtNAS1-TRG\_to\_AAA exhibited even higher expression
levels
of the iron and zinc absorption and transport genes NtIRT1 and NtMTP8, suggesting their greater potential
for
iron biofortification (Fig. 14B-C).

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| ![Image](https://static.igem.wiki/teams/5769/image/engineering-14.webp) Fig. 14 Overexpression of wild-type and mutant AtNAS1 affects the expression of genes related to iron and zinc homeostasis. A: Relative expression levels of the AtNAS1 gene in tobacco leaves; B: Relative expression levels of the iron transporter gene NtIRT1 in tobacco leaves; C: Relative expression levels of the zinc transporter gene NtMTP8; D: Relative expression levels of the iron activation gene NtFRO2 in tobacco leaves. |

## **Learn**

Based
on
the combined results from cycles 1 and 2, we demonstrate that TRG→AAA effectively eliminates AtNAS1
autoinhibition while maintaining structural integrity. It also exhibits increased catalytic efficiency
(cycle 1
enzymatic analysis) and enhanced in-plant function: confocal microscopy revealed enhanced ER localization,
while
qPCR confirmed significant upregulation of the metal transporters NtIRT1 and NtMTP8 (14B-C). Molecular
dynamics
simulations further validated its robust SAM binding affinity (ΔG: wild-type -36.83 kcal/mol vs. TRG→AAA
-17.06
kcal/mol), consistent with its enhanced performance. Crucially, the TRG→AAA mutant retains the full-length
protein structure while achieving activity comparable to the truncated variant, minimizing potential
functional disruption.

These
findings provide a mechanistic basis for the observed physiological effects: the ER-localized AtNAS1 mutant
enhances nicotinamide synthesis, thereby mobilizing iron/zinc and activating endogenous transport systems,
while maintaining metal activation pathways.

In
the third cycle, we will:

1) express wild-type and TRG→AAA mutants in Bacillus subtilis for soil cultivation in tobacco;

2)
quantify iron and zinc accumulation in stems and leaves using inductively coupled plasma mass spectrometry
(ICP-MS) to assess biofortification efficacy under realistic growth conditions;

The
transition from controlled models (E. coli, transient tobacco experiments) to soil-based validation
addresses
environmental relevance and scalability, bridging molecular design with agricultural applications. The
TRG→AAA
mutant's balanced combination of high activity, intact structure, and minimal ecological risk makes it an
ideal candidate for future field-based biofortification solutions.

# **Cycle3:Biofortification Verification**

## **Design**

Based
on
the results from rounds 1 and 2, the TRG→AAA mutant demonstrated excellent enzyme activity and enhanced
upregulation of metal transporters in tobacco. The third round transitioned to soil functional verification
under relevant environmental conditions. The wild type, truncated mutant, and TRG→AAA were cloned into the
pHT43
vector via homologous recombination to ensure compatibility with Bacillus subtilis. Plasmid integrity was
initially verified in Escherichia coli DH5α by colony PCR and Sanger sequencing. Positive clones were
transformed into Bacillus subtilis WB800N, screened on resistance plates, and verified by PCR according to
established protocols.

Soil
Cultivation, Bacillus strains were inoculated
at
varying concentrations in sterilized soil at a uniform volume. Nicotiana benthamiana seedlings were
transplanted
into the soil inoculated with Bacillus, and a control group was included. After 2 weeks, stems and leaves
were
harvested and Fe/Zn concentrations were analyzed by ICP-MS. This design ensured that protein engineering
(cycle
1) and gene expression and subcellular localization (cycle 2) were tested under actual agricultural
constraints.
The expression of Bacillus subtilis reduced the risk of genetic modification while providing a scalable path
for future field trials in major crops.

![Image](https://static.igem.wiki/teams/5769/image/engineering-15.webp)

Fig. 15 Plasmid maps of wild-type, TRG→AAA, truncation, and pHT43 vector construction

## **Build**

To further
evaluate the application potential of AtNAS1 mutants in plants, we also used **N.
benthamiana** as a model system and used
**B** **.**  **subtilis** harboring
expression plasmids as a bioinoculant for soil-grown tobacco. Using the same construction strategy as
for
the pET28A-AtNAS1 plasmid, we constructed the B . subtilis expression
plasmids pHT43-AtNAS1-Wildtype, pHT43-AtNAS1-Truncated, and pHT43-AtNAS1-TRG\_to\_AAA. The construction process is
shown in
Fig.
16.

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| ![Image](https://static.igem.wiki/teams/5769/image/engineering-16.webp) Fig. 16 The construction process of pHT43-AtNAS1-WT, pHT43-AtNAS1-Truncated, and p pHT43-AtNAS1-TRG\_to\_AAA plasmids. A. Agarose gel electrophoresis of genes; B. Linearized p pHT43 backbone; C. Single colonies of DH5alpha on the plate; D: Single colonies of B. subtilis on the plate. |

## **Test**

### **Effect of** **B** **.** **subtilis** **Expressing AtNAS1** **Mutated** **Protein****s** **on** **N. benthamiana** **Growth and Biomass**

The previous
research results have shown that compared to the AtNAS1-Wildtype, both the AtNAS1-Truncated and the
AtNAS1-TRG\_to\_AAA mutants have higher enzyme activity and a
high efficiency to activate genes related to
iron
cycling. Since the AtNAS1-Truncated removes the C-terminal domain of
the
protein, it may lead to the partial loss of other biological functions, so we are more focused on the
application of the AtNAS1-TRG\_to\_AAA mutant. In our tobacco pot culture experiment, we
used **B** **.**  **subtilis** as
a probiotic. When the tobacco plants grew
at the 4-leaf stage, we
applied 10 mL of B . subtilis at different
concentrations to the root system for root colonization. After two weeks of growth, we measured the plant
fresh biomass and then dried and ground the
shoot to determine the iron and zinc
micronutrient contents using ICP-MS. As shown in Figure 17, we found that after applying
B . subtilis , the fresh biomass of the tobacco plants
actually
showed a decreasing trend, and the leaves exhibited partial curling, which may be related to the applied
concentration and still requires further optimization.

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|  |
| ![Image](https://static.igem.wiki/teams/5769/image/engineering-17.webp) Fig. 17 Phenotypic analysis of AtNAS1 transient expressing tobacco plants. A: Images showing the growth of pHT43-AtNAS1-Wildtype and pHT43-AtNAS1-TRG\_to\_AAA transient expressing tobacco plants under different B . subtilis optical densities (PBS Control, OD=0.5, OD=1.0); B: The fresh weight of tobacco plants. |

### **Effect of** **B** **.** **subtilis** **Expressing AtNAS1** **mutated** **Protein****s** **on Iron and Zinc Trace Element Content****s** **in Tobacco Leaves** **Using ICP-MS**

After
harvesting
the tobacco samples with **B** **.**  **subtilis** root
application, as shown in Figure 18A, we dried the shoot of the tobacco plants at 60°C and
ground them into powder. We then performed microwave digestion of the samples using nitric acid and hydrogen
peroxide, and used ICP-MS to measure the micronutrient contents. The raw data from the instrument was
converted
to mg/kg units using standard calibration curves. As shown in Figure 18B, compared to the PBS control, the
tobacco
plants inoculated with B . subtilis carrying the
pHT43-AtNAS1-Wildtype and pHT43-AtNAS1-TRG\_to\_AAA plasmids showed increased iron (Fe) and zinc (Zn)
micronutrient contents in the shoot at different B . subtilis concentrations.
However, at the OD=0.5 concentration, there was no significant difference in either Fe or Zn content between
the
AtNAS1-Wildtype and AtNAS1-TRG\_to\_AAA treatments. At the OD=1 concentration, the tobacco
plants
inoculated with the B . subtilis carrying the
pHT43-AtNAS1-TRG\_to\_AAA plasmid had significantly higher Fe content in the shoot compared to the
pHT43-AtNAS1-Wildtype,
while the Zn content did not differ. This suggests the potential application of the B .
subtilis strain
carrying
the pHT43-AtNAS1-TRG\_to\_AAA plasmid for iron biofortification. Overall, our experiment has achieved
preliminary success, but further optimization is still needed to fully understand the effects of the
different
B . subtilis treatments
on
plant biomass and micronutrient accumulation.

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| ![Image](https://static.igem.wiki/teams/5769/image/engineering-18.webp) Fig. 18 A: Sample processing process, B: Fe element determination results, C: Zn element determination results |

## **Learn**

In the third round, we
transitioned
to soil-based validation, using Bacillus subtilis as a delivery vehicle for AtNAS1 variants in tobacco. The
TRG→AAA mutant consistently demonstrated superior performance: ICP-MS analysis revealed a significant
increase
in iron (Fe) accumulation in tobacco spores at high bacterial density (OD = 1), a 1.5-fold increase compared
to
the wild type, while zinc (Zn) levels remained stable. This confirmed that the mutant enhanced metal
bioavailability without disrupting overall homeostasis. However, the reduction in plant biomass at higher OD
concentrations highlighted the need for dose optimization. The successful integration of Bacillus subtilis
as a
bioinoculant underscores the potential of non-GMO soil amendment strategies for enhancing micronutrient
uptake, consistent with real-world agricultural constraints.

Over three cycles, we
systematically validated protein engineering solutions for micronutrient biofortification:

• Cycle 1 (in vitro enzymology):
TRG→AAA was identified as a highly active mutant (kinetics: ≈ wild-type efficiency; ΔG = -17.06 kcal/mol SAM
bound).

• Cycle 2 (In-plant Transient
Assays): We confirmed endoplasmic reticulum-localized expression and upregulation of metal transporters
(NtIRT1/NtMTP8), linking mutant activity to physiological gene activation.

• Cycle 3 (Soil-Tobacco System):
We
used ICP-MS to confirm stem iron accumulation and determined functional efficacy under relevant
environmental conditions.

The TRG→AAA mutant was the
optimal
variant, balancing catalytic efficiency, structural integrity, and scalability for agricultural use.
Methodologically, we integrated in silico design (molecular dynamics), in vitro validation (enzyme assays),
and
in-plant testing (transient/soil systems), providing a reproducible workflow for synthetic biology projects.

Contributions to iGEM and Future Teams

We have contributed identified
genetic components (e.g., the AtNAS1-TRG→AAA mutant and the pHT43-Bacillus vector) to a registry, enabling
future teams to bypass initial screening and directly deploy high-performing variants. Our multi-cycle
approach integrating enzyme kinetics,
confocal
microscopy, qPCR, and ICP-MS provides a template for
validating
biofortification strategies, highlighting the need to cross-validate molecular data with phenotypic results.

Using Bacillus subtilis (a GRAS
organism) for soil delivery mitigates concerns about the release of genetically modified organisms, but
further
safety considerations are needed, and future efforts, such as the addition of a suicide gene switch, are
planned. Our project demonstrates how to
integrate protein engineering with agricultural applications, highlighting the steps from rational design to
field-ready solutions. Future teams could apply this process to other nutrients/crops or optimize
Bacillus-plant synergies to achieve broader sustainability goals.

# References

1. Kumar S, Palve A, Joshi C, et al.
Crop biofortification for iron (Fe), zinc (Zn) and vitamin A with transgenic approaches[J]. Heliyon, 2019, 5(6).

2. Ekpa O, Palacios-Rojas N,
Kruseman G,
et al. Sub-Saharan African maize-based foods-processing practices, challenges and opportunities[J]. Food
Reviews International, 2019, 35(7): 609-639.

3. Gibson R S, Hotz C, Temple L, et
al.
Dietary strategies to combat deficiencies of iron, zinc, and vitamin A in developing countries: development,
implementation, monitoring, and evaluation[J]. Food and Nutrition Bulletin, 2000, 21(2): 219-231.

4. Narayanan N, Cueto-Reaño M F,
Eroğlu
S, et al. Iron biofortification through genetic modification in rice, wheat, and cassava and its potential
contribution to nutritional security[J]. CABI Reviews, 2022.

5. Seregin I V, Kozhevnikova A D.
Nicotianamine: a key player in metal homeostasis and hyperaccumulation in plants[J]. International journal
of molecular sciences, 2023, 24(13): 10822.

6. Seebach H, Radow G, Brunek M, et
al.
Arabidopsis nicotianamine synthases comprise a common core-NAS domain fused to a variable autoinhibitory C
terminus[J]. Journal of Biological Chemistry, 2023, 299(6).

{% endcall %} {% endblock %}

{% extends "layout.html" %} {% block title %}Experiments{% endblock %} {% block
lead %}Describe the research, experiments, and protocols you used in your
project. It is designed to provide sufficient information for other teams to
replicate our work.{% endblock %} {% block page\_content %} {% from
"/components/article-content.html" import articleContent %} {% call
articleContent("EXPERIMENTS", [
{"content": "Preparation of LB culture medium", "anchor": "#lb-prep", "children": [
{"content": "Preparation of Kanamycin", "anchor": "#kanamycin-prep"},
{"content": "Inoculation of glycerol bacteria", "anchor": "#glycerol-inoculation"},
{"content": "Plasmid extraction", "anchor": "#plasmid-extraction"}
]},
{"content": "Construction of E. coli Expression Plasmids", "anchor": "#ecoli-construction", "children": [
{"content": "PCR amplification of target genes", "anchor": "#pcr-amplification"},
{"content": "Agarose gel electrophoresis AGE", "anchor": "#age-electrophoresis"},
{"content": "Preparing the Gel", "anchor": "#gel-preparation"},
{"content": "Setting Up and Running the Gel", "anchor": "#gel-setup"},
{"content": "Visualize the DNA", "anchor": "#dna-visualization"},
{"content": "Enzyme-digested vector", "anchor": "#enzyme-digestion"},
{"content": "Homologous recombination", "anchor": "#homologous-recombination"},
{"content": "Transformation into competent E. coli cells", "anchor": "#transformation"},
{"content": "Colony PCR", "anchor": "#colony-pcr"},
{"content": "Seed Culture", "anchor": "#seed-culture"},
{"content": "Scale-Up Culture", "anchor": "#scale-up-culture"}
]},
{"content": "Protein Expression and Purification", "anchor": "#protein-expression", "children": [
{"content": "Preliminary Experiment for Protein Expression", "anchor": "#preliminary-experiment"},
{"content": "His-Tag Affinity Purification of Proteins", "anchor": "#his-tag-purification"}
]},
{"content": "Functional Tests Part1: Mutation Sites Selection", "anchor": "#mutation-selection", "children": [
{"content": "Enzymatic Reactions Detection of AtNAS1 Mutated Proteins' Catalytic Ability", "anchor":
"#enzymatic-reactions"},
{"content": "Molecular Docking and Prediction of AtNAS1 Mutated Proteins with Substrates", "anchor":
"#molecular-docking"},
{"content": "Molecular Dynamics Fitting Analysis of Catalytic Efficiency in AtNAS1 Mutated Proteins", "anchor":
"#molecular-dynamics"}
]},
{"content": "Functional Tests Part2: Plant Model Validation", "anchor": "#plant-validation", "children": [
{"content": "Construction of N. benthamiana Transient Expression Plasmids", "anchor": "#benthamiana-construction"},
{"content": "Transient N. benthamiana Expression Assay", "anchor": "#benthamiana-expression", "children": [
{"content": "Detection the Effect of AtNAS1 Mutant Proteins on Subcellular Localization Using Confocal Microscopy",
"anchor": "#subcellular-localization"},
{"content": "Detection the Effect of AtNAS1 Mutant Proteins on the Expression of Iron Cycling Related Genes Using
Real-time Quantitative PCR In Vivo", "anchor": "#iron-cycling-genes"}
]},
{"content": "Construction of B. subtilis Expression Plasmids", "anchor": "#subtilis-construction"},
{"content": "Biofortification Experiment of Soil-Based N. benthamiana Cultivation Using B. subtilis Stably Expressing
AtNAS1 Mutated Proteins", "anchor": "#biofortification-experiment", "children": [
{"content": "Effect of B. subtilis Expressing AtNAS1 Mutated Proteins on N. benthamiana Growth and Biomass", "anchor":
"#growth-biomass"},
{"content": "Effect of B. subtilis Expressing AtNAS1 mutated Proteins on Iron and Zinc Trace Element Contents in Tobacco
Leaves Using ICP-MS", "anchor": "#trace-elements"}
]}
]}
]) %}

**Outline**

**1. Preparation of LB (Luria-Bertani) culture medium**

**2. Construction of E. coli Expression Plasmids**

pET28a-AtNAS1\_Wildtype

pET28a-AtNAS1\_Truncated

pET28a-AtNAS1\_T287A

pET28a-AtNAS1\_R288A

pET28a-AtNAS1\_G289A

pET28a-AtNAS1\_TRGtoAAA

pET28a-MtnN

pET28a-AdeD

**3. Protein Expression and Purification**

**3.1 Preliminary Experiment for Protein Expression**

**3.2 His-Tag Affinity Purification of Proteins**

**4. Functional Tests Part1: Mutation Sites Selection**

**4.1 Enzymatic Reactions Detection of AtNAS1 Mutated Proteins' Catalytic Ability**

**4.2 Molecular Docking and Prediction of AtNAS1 Mutated Proteins with Substrates**

**4.3 Molecular Dynamics Fitting Analysis of Catalytic Efficiency in AtNAS1 Mutated Proteins**

**5. Functional Tests Part2: Plant Model Validation**

**5.1 Construction of N. benthamiana Transient Expression Plasmids**

pCambia1305-AtNAS1wt-eGFP-6myc

pCambia1305-AtNAS1truncated-eGFP-6myc

pCambia1305-AtNAS1\_TRG\_to\_AAA-eGFP-6myc

**5.2 Transient N. benthamiana Expression Assay**

**5.2.1 Detection the Effect of AtNAS1 Mutant Proteins on Subcellular Localization Using Confocal
Microscopy**

**5.2.2 Detection the Effect of AtNAS1 Mutant Proteins on the Expression of Iron Cycling Related Genes
Using Real-time Quantitative PCR In Vivo**

**5.3 Construction of B. subtilis Expression Plasmids**

pHT43-AtNAS1wt

pHT43-AtNAS1truncated

pHT43-AtNAS1\_TRG\_to\_AAA

**5.4 Biofortification Experiment of Soil-Based N. benthamiana Cultivation Using B. subtilis Stably
Expressing AtNAS1 Mutated Proteins**

**5.4.1 Effect of B. subtilis Expressing AtNAS1 Mutated Proteins on N. benthamiana Growth and
Biomass**

**5.4.2 Effect of B. subtilis Expressing AtNAS1 mutated Proteins on Iron and Zinc Trace Element Contents in
Tobacco Leaves Using ICP-MS**

# 1. Preparation of LB (Luria-Bertani) culture medium

Materials:

1. Sodium Chloride 5g
2. Conical flasks
3. Distilled water 500ml
4. Yeast Extract 2.5g
5. Tryptone 5g

Procedures:

Liquid medium:

1. Add 5 grams of Tryptone, 2.5 grams of Yeast extract, and 5 grams of sodium chloride into the conical
   flask.
2. Add 500 mL of distilled water to the flask
3. Sterilization at 121℃ for 10-15 minutes

## **1.1 Preparation of Kanamycin**

Materials:

1. 0.5 mg
   kanamycin sulfate
2. 10 ml
   distilled water
3. 15 ml
   Centrifuge Tube

Procedures:

1. Add 0.5 mg
   kanamycin sulfate to the centrifuge tube
2. Add 10 mL
   of distilled water to the tube
3. Use a
   syringe and a sterile syringe filter for filtration. 1 ml I each centrifuge tube.

## **1.2 Inoculation of glycerol bacteria**

Procedure:

1. Add 100 μL kanamycin to 100 mL LB culture medium
2. Pour 5 mL of the mixture of LB culture medium and kanamycin into the tube
3. Add 50 μL glycerol bacteria to each 8 separate centrifuge tube.
4. Repeat twice for each of the glycerol bacteria.
5. Pour 5 mL of the mixture of LB culture medium and kanamycin into the tube
6. Put the tubes into the shaking incubator at 37°C and 220 RPM, set for 12-16 hours

## **1.3 Plasmid extraction**

Material:

1. Buffer
   P1
2. Buffer P2
3. Buffer
   P3
4. Buffer
   PW
5. Buffer PW
   2
6. Elution
   Buffer

Procedure:

1. Separate
   the inoculated glycerol bacteria into centrifuge tubes, each 1.5 ml.
2. Centrifuge
   the tubes at 10000 rpm for 1 minute
3. Pour out
   the liquid and centrifuge at 10000 rpm for 10 seconds to make sure the LB culture medium is
   poured out thoroughly
4. Add 250 μL
   of Buffer P1 to one centrifuge tube of each type of glycerol bacteria. Stir the bacteria with
   Buffer P1.
5. Add the
   mixture to the other 5 centrifuge tubes and mix.
6. Add 250 μL
   Buffer P2. Stir 8-10 times
7. Add 350
   Buffer P3. Shake 8-10 times.
8. Centrifuge
   the tubes at 12000 rpm for 10 minutes.
9. Use a
   pipette to transfer liquid to the DNA collection tubes
10. Centrifuge
    at 12000 rpm, 10minutes.
11. Add 600 μL
    of Buffer PW2to each tube
12. Centrifuge
    at 12000 rpm for 1 minute.
13. Repeat
    steps 11 and 12.
14. Add the
    mixture to a new 1.5 ml DNA collection tube.
15. Still 2
    minutes. Centrifuge at 12000 rpm for 1 minute for elution of DNA

# **2.** **Construction of E. coli Expression Plasmids**

## **2.1 PCR amplification of target genes**

Material:

1. Te
2. DNA Primer
   1
3. DNA Primer
   2
4. 2×Hieff PCR
   Master Mix
5. Water
6. PCR
   machine
7. Procedure:

1. Add 2 μL plasmid
2. Add 2 μL
   DNA Primer 1, 2 μL DNA Primer 2, and 2μL 2×Hieff PCR Master Mix
3. Add 19 μL
   of water to the mixture
4. Mix the
   things
5. Set PCR to
   following setting

|  |  |  |  |
| --- | --- | --- | --- |
| **Step** | **Temperature (°C)** | **Time** | **Cycles** |
| **Initial Denaturation** | **94** | **5 minutes** | **1** |
| **Denaturation** | **94** | **30 seconds** |  |
| **Annealing** | **50 – 60\*** | **30 seconds** | **35 Cycles** |
| **Extension** | **72** | **30-60 sec/kb\*\*** |  |
| **Final Extension** | **72** | **10 minutes** | **1** |

\* Annealing
Temperature: Try a temperature 2–5°C lower than the calculated melting
temperature (Tm) of your primers.

## **2.2 Agarose gel electrophoresis AGE****「**

Material:

* Agarose
  powder
* TAE
  Buffer
* DNA
  Samples
* DNA Ladder
* Gel Box
  & Lid
* Gel Tray
  & Combs
* Power
  Supply
* Microwave
  or Hot Plate
* UV Light
  Box
* Pipette and
  Tips

## **2.3 Preparing the Gel**

1. Weigh 0.5g
   Agarose Gel powder and 50 mL TAE Buffer
2. **Carefully**heat
   the flask in a microwave for 30-60 seconds. **It will be very hot!**
3. Swirl the flask
   gently and heat in short bursts until the liquid is completely clear with no floating
   bits.
4. Let the
   melted agarose sugar cool for 60 seconds then pour the liquid into prepared gel tray.
5. Set the gel
   for 20-30 minutes

## **2.4 Setting Up and Running the Gel**

1. Place the
   gel in the box. Wells should be at the negative side.
2. **Pour the TAE buffer into the
   box that barely covers the gel**
3. Use a pipette
   to suck up the DNA sample with the loading sample.
4. Put it into
   the wells
5. Set the gel for 20-30
   minutes
6. Put on the
   lids, and the lids should be plugged into the power supply.
7. Turn on the
   machine and let it run for 20-40 minutes

## **2.5 Visualize the DNA**

1. Turn off
   the power supply and carefully take the gel out
2. Put the gel
   into the blue UV light. Bright band is where the DNA is

## **2.6 Enzyme-digested vector**

Materials:​**​**

1. Purified
   pET-28a plasmid DNA (100-500 ng/μL)
2. BamHI
   restriction enzyme (e.g., from NEB)
3. 10x
   CutSmart Buffer (or compatible buffer)
4. Nuclease-free water
5. Thermostatic water bath or PCR
   machine

|  |  |  |
| --- | --- | --- |
| Component | Volume (μL) | Amount |
| Nuclease-free water | to 20 μL | - |
| **10xCutSmart Buffer** | ​**2.0**​ | ​**1x**​ |
| pET-28a plasmid DNA | ​**X**​ | ​**500 ng**​ |
| **BamHI** | ​**1.0**​ | **10-20 units**​ |

## **2.7 Homologous recombination**

Materials:

1. CloneExpress Mix (2×)
2. PCR
   products of NAS (wild type and mutants), MtnN, and AdeD
3. Linearized
   pET28a backbone

Procedure:

1. Measure the DNA concentration of gel-purified
fragments (NAS variants, MtnN, AdeD).

2. Prepare the recombination system in PCR tubes
for the construction of pET28a-NAS:

* 5 μL
  CloneExpress Mix
* 2.5 μL
  linearized pET28a backbone
* 2.5 μL NAS
  variant fragment (wild type or mutant)

3. Prepare additional recombination systems in PCR
tubes for the construction of pET28a-MtnN and pET28a-AdeD:

* 5 μL
  CloneExpress Mix
* 2.5 μL
  linearized pET28a backbone
* 2.5 μL MtnN
  or AdeD fragment

4. Place PCR tubes into a PCR thermal cycler and
perform ligation reaction at 50 °C for 30 minutes.

## **2.8 Transformation into competent E. coli cells**

Materials:

1. Ligated recombination
   plasmid
2. Liquid LB solution
   without antibiotics
3. LB Agar plates with
   Ampicillin and spectinomycin antibiotics

Procedures:

1. Add recombination plasmid
   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 Kanamycin antibiotics, incubate for 12-16 hours at 37˚C.

## **2.9 Colony PCR**

Material：

1. Plasmid
2. DNA Primer
   1
3. DNA Primer 2
4. 2×Hieff PCR
   Master Mix
5. Water
6. PCR
   machine

Procedure：

1. Add 2 μL
   bacteria
2. Add 2 μL
   DNA Primer 1, 2 μL DNA Primer 2, and 2μL 2×Hieff PCR Master Mix
3. Add 19 μL
   of water to the mixture
4. Mix the
   things
5. Set PCR to
   following setting

|  |  |  |  |
| --- | --- | --- | --- |
| **Step** | **Temperature (°C)** | **Time** | **Cycles** |
| **Initial Denaturation** | **94** | **5 minutes** | **1** |
| **Denaturation** | **94** | **30 seconds** |  |
| **Annealing** | **50 – 60\*** | **30 seconds** | **35 Cycles** |
| **Extension** | **72** | **30-60 sec/kb\*\*** |  |
| **Final Extension** | **72** | **10 minutes** | **1** |

\* Annealing
Temperature: Try a temperature 2–5°C lower than the calculated melting temperature (Tm)
of your primers.

## **2.10 Seed Culture**

Material:

* Positive
  Bacterial Colony
* Liquid LB
  Medium
* Antibiotic
* Sterile Culture Tubes
* Sterile
  Pipette Tips and a pipette that can measure 500-1000 µL.
* Incubator
  Shaker

Procedure:

1. Label
   Tube
2. Using a sterile
   pipette tip, add the 5 μL antibiotic to
   the tube.
3. Add 5 mL of
   sterile liquid LB medium into the tube.
4. Secure the
   lid and put it in the incubator shaker set 37°C,
   220 rpm for 12-16 hours.

## **2.11 Scale-Up Culture**

Material:

* Seed
  Culture
* Fresh
  Liquid LB Medium
* Antibiotic
* Sterile
  Erlenmeyer Flasks
* Sterile
  Pipettes or pipette tips
* ncubator
  Shaker
* Spectrophotometer

Procedure:

1. Label
   flask
2. Prepare 100
   mL LB cultural medium
3. Use a
   sterile pipette to suck 1 mL seed culture liquid into the medium
4. Put the
   flask in the incubator shaker, speed 220 rpm, temperature 37°C for 2 hours

# **3.** **Protein Expression and Purification**

## **3.1 Preliminary Experiment for Protein Expression**

3.1.1 Amplification
Culture for Correct Colony

Goal: To obtain
sufficient amounts of target NAS mutant proteins for SDS-PAGE verification.

Materials:

1. Liquid LB
   medium
2. NAS mutant
   plasmids and corresponding cofactor expression constructs (e.g., AtNAS1-Wildtype,
   AtNAS1-Truncated, AtNAS1-TRG\_to\_AAA, and cofactor-related vectors)
3. Ampicillin
   and spectinomycin antibiotics
4. Inducer:
   IPTG
5. Iso-thermic
   shaker

Procedures:

1. Inoculate
   1000 μL of bacterial culture containing NAS
   mutant plasmids and cofactor plasmids into 100 mL of liquid LB medium, and add 100μL of both
   Ampicillin and spectinomycin
   antibiotics.
2. Incubate
   the culture in an iso-thermic shaker at 37℃ for ~3 hours until the absorbance at
   OD600 reaches 0.4–0.6.
3. Add IPTG to
   the culture at a final concentration of 1 mM to induce protein expression.
4. Continue
   incubation at 37℃ for 6 hours, and collect samples for
   subsequent analysis.

3.1.2 Protein Crude
Extraction and SDS-PAGE

Goal: To confirm
the expression of the desired NAS mutant proteins.

Materials for
Protein Crude Extraction:

1. 500
   μL of bacterial culture from different
   induction conditions
2. Centrifuge
3. Double
   distilled water (ddH2O)
4. 6×Protein loading buffer

3.1.3 Procedures
for Protein Crude Extraction

1. Centrifuge
   the samples at 12,000 rpm for 1 minute.
2. Discard the
   supernatant and resuspend the cell pellet in 50μL of 1×Protein loading buffer diluted with
   ddH2O.
3. Heat the
   resuspended samples at 95℃ for 15 minutes using a PCR thermal
   cycler.

Materials for
SDS-PAGE:

1. 12.5%
   SDS-PAGE Color Preparation kit (Sangon)
2. Electrophoresis buffer
   (Tris-Glycine)
3. Protein
   ladder
4. 6×Protein loading buffer
5. Vertical
   electrophoresis system
6. Coomassie
   blue staining solution

Procedures for
SDS-PAGE:

1. Mix 2.2 mL
   2×separating gel solution, 2.2 mL 2× separating gel buffer, and 44 μL catalyst in a centrifuge
   tube for preparing the separating gel.
2. Pour the
   mixture slowly into the casting frame to avoid bubble formation.
3. Overlay
   with 1 mL ddH2O and allow the separating gel to solidify for ~8 minutes.
4. Remove the
   water carefully after polymerization.
5. Mix 825 μL
   2× stacking gel solution, 825 μL 2× stacking gel buffer, and 11 μL catalyst for stacking gel
   preparation.
6. Pour
   stacking gel mixture on top of the separating gel, insert comb gently to avoid air bubbles, and
   allow to solidify for ~12 minutes.
7. After
   polymerization, carefully remove the comb and wash the wells with electrophoresis buffer.
8. Load 10 μL
   of protein ladder into the first well, followed by sample loading into successive wells.
9. Place the
   gel into the vertical electrophoresis system.
10. Run
    electrophoresis at 120 V for ~90 minutes.
11. Stain the
    gel with Coomassie blue for 10 minutes, then destain repeatedly until the background becomes
    clear and the protein bands are visible.

## **3.2 His-Tag Affinity Purification of Proteins**

Protein
Purification Using a Nickel Column (Ni-NTA)

Materials:

* Bacterial
  Pellet
* Lysis
  Buffer
* Equilibration/Wash Buffer
* Elution
  Buffer
* Ni-NTA
  Resin
* Empty
  Chromatography Column
* Centrifuge
  and Tubes
* Ice
  Bucket

Procedure:

1. Grow a
   bacterial culture expressing the NAS mutant plasmid with cofactors to an OD600≈0.6, induce with
   IPTG (final concentration 1 mM), and incubate at 16℃ for 12 hours.
2. Harvest
   the cells by centrifugation at 5,000 × g for 15 minutes at 4℃.
3. Resuspend
   the pellet in lysis buffer (10 mL of buffer per 1 g of wet cell pellet).
4. Add
   lysozyme (1 mg/mL) and PMSF (1 mM) and incubate on ice for 30 minutes.
5. Disrupt
   the cells by sonication (2 seconds on, 5 seconds off, for a total of 10 minutes, incubate on
   ice).
6. Centrifuge
   the lysate at 12,000 × g for 30 minutes at 4℃ and collect the supernatant (soluble
   fraction).
7. Equilibrate the Ni-NTA resin with 5
   column volumes (CV) of lysis buffer.
8. Ensure the
   resin is completely resuspended, avoiding the introduction of bubbles.
9. Add the
   clarified supernatant to the equilibrated Ni-NTA resin.
10. Incubate
    at 4℃ with gentle shaking for 30-60 minutes to allow binding of the His-tagged NAS
    protein.
11. Wash the
    resin with 10 column volumes (CV) of wash buffer (20-40 mM imidazole) to remove nonspecifically
    bound proteins.
12. Elute the
    bound His-tagged NAS mutant protein with elution buffer (250 mM imidazole).
13. Collect
    the eluate (e.g., 1 mL aliquots) and monitor protein elution by measuring absorbance at 280
    nm.

# **4. Functional Tests Part1: Mutation Sites Selection**

## **4.1 Enzymatic Reactions Detection of AtNAS1 Mutated Proteins' Catalytic Ability**

Materials:

* SAM
  (S-adenosyl-methionine) substrate
* Purified
  mtnN enzyme
* Purified
  adaD enzyme
* AtNAS1
  protein sample (various mutants: AtNAS1-Wildtype, AtNAS1-Truncated, AtNAS1-TRG\_to\_AAA, etc.),
  quantified (mg/mL) and stored on ice
* 50 mM
  Tris-HCl buffer, pH 8.7 (fresh or filter-sterilized)
* ddH2O (purified deionized
  water)
* 1× PBS or a suitable dilution
  buffer
* Appropriate
  protein dilution buffer (if diluting the protein to 0.15 mg/mL)
* BioTek
  microplate reader
* Multichannel pipette and filter
  tips
* Ice-cold
  box, centrifuge
* Protein
  quantification reagent (Bradford/BCA)
* Sterile
  microcentrifuge tubes, vortexer, timer
* Septum film
  or cover plate

Procedure:

1. Prepare a
   reaction mixture containing 1 mg/mL mtnN, 1 mg/mL adaD, 50 mM Tris-HCl buffer (pH 8.7), and 0.15
   mg/mL purified NAS1 mutant protein.
2. Preincubate the mixture at 37℃ for 3
   minutes.
3. Initiate
   the enzymatic reaction by adding 0.125 mM SAM substrate and gently mix five times.
4. Immediately add the reaction mixture
   to a 96-well microplate.
5. Place the
   microplate in a BioTek microplate reader, set the temperature to 37℃ and the wavelength to 265
   nm, and measure in kinetic mode (continuous reading).
6. Record
   the absorbance change over time for each well, using five technical replicates for each NAS1
   mutant protein.
7. Calculate
   the initial reaction rate using the Lambert-Beer law and the molar extinction coefficient of
   adenine (ε = 6700 M⁻¹·cm⁻¹ at 265 nm).
8. Enzyme
   activity was normalized to protein concentration and expressed as nkat/mg protein.
9. Enzyme
   activity of NAS1 mutants was compared with that of wild-type AtNAS1 and truncated AtNAS1
   proteins to identify the most potent variant.

## **4.2 Molecular Docking and Prediction of AtNAS1 Mutated Proteins with Substrates**

Materials:

1. Computer workstation with internet access and
sufficient computational performance.

2. Online databases and software:

* UniProtKB
  (for protein sequence acquisition)
* SWISS-MODEL (for homology modeling and
  PDB structure prediction)
* Small
  molecule databases (e.g., PubChem, ZINC, or ChEMBL; for ligand acquisition in SDF format)
* Discovery
  Studio (DS) software (for ligand optimization and molecular modeling)
* PyMOL (for
  protein visualization and preprocessing)
* AutoDock
  Tools (ADT) (for receptor/ligand preparation, pdbqt conversion, and grid box setup)
* AutoDock
  Vina or AutoDock 4.2 (for molecular docking)

3. Standard computational chemistry
resources:

* Force
  fields and energy minimization tools integrated in DS or AutoDock.
* Visualization software for docking
  results (DS, PyMOL).

Procedure:

1. Protein sequence acquisition and
modeling

* Search the
  target gene in UniProtKB and download its protein sequence in FASTA format.
* Upload the
  sequence to SWISS-MODEL to perform homology modeling.
* Select the
  model with the highest sequence identity and model quality.
* Download
  and save the resulting protein structure as PDB format for later use.

2. Ligand acquisition and optimization

* Search the
  target small molecule in a chemical database (e.g., PubChem or ZINC).
* Download
  the ligand in SDF format.
* Open the
  ligand in DS software, perform energy minimization to optimize its geometry, and save the
  optimized structure in PDB format.

3. Protein preprocessing

* Load the
  protein PDB file into PyMOL.
* Remove
  water molecules and any non-relevant ligands or ions.
* Save the
  cleaned protein structure as PDB format.

4. Receptor preparation

* Import the
  protein into AutoDock Tools (ADT).
* Add polar
  hydrogens and assign Gasteiger charges.
* Save the
  processed protein as pdbqt format, defined as the receptor.

5. Ligand preparation

* Import the
  optimized ligand into ADT.
* Add
  hydrogens, assign charges, and define rotatable bonds.
* Save the
  ligand structure as pdbqt format, defined as the ligand.

6. Grid box setup

* Since the
  binding pocket is unknown, define the grid box to cover the entire protein structure.
* Save the
  grid parameter file for docking.

7. Docking simulation

* Perform
  docking using AutoDock.
* Run at
  least 50 independent docking simulations to ensure reliability and reproducibility of
  results.

8. Result analysis

* Analyze
  docking outputs and compare binding energies.
* Select the
  conformation with the lowest binding energy and the highest clustering frequency as the optimal
  binding pose.
* Visualize
  docking results using DS or PyMOL, displaying the 3D binding conformation, protein surface
  interaction, and binding pocket environment.

## **4.3 Molecular Dynamics Fitting Analysis of Catalytic Efficiency in AtNAS1 Mutated Proteins**

Materials:

1. Software and computational resources

* GROMACS
  2025 molecular dynamics simulation package
* ACPYPE or
  AmberTools (for ligand topology generation with GAFF2 force field)
* VMD,
  PyMOL, or Chimera (for visualization of trajectories and results)
* g\_mmpbsa
  or GMXPBSA tool (for MM-PBSA calculations)
* High-performance computing (HPC)
  cluster or workstation with sufficient CPU/GPU resources

2. Force fields and parameters

* AMBER99SB-ILDN force field (for
  protein)
* GAFF2
  force field (for ligand parameterization)
* TIP3P
  water model (for solvation)

3. Input files

* Protein
  structure file (PDB format, pre-processed and minimized)
* Ligand
  structure file (optimized and converted into topology using GAFF2)

Procedure:

1. System setup

* Import the
  protein structure into GROMACS.
* Generate
  topology for the protein using the AMBER99SB-ILDN force field.
* Generate
  ligand topology and parameters using GAFF2 (via ACPYPE/AmberTools).
* Merge the
  protein and ligand topology files, checking for atom type compatibility and resolving
  conflicts.

2. Define simulation box

* Place the
  protein–ligand complex in a cubic box.
* Ensure the
  minimum distance from the solute surface to the box boundary is 1.2 nm.

3. Solvation and ion addition

* Solvate
  the system with TIP3P water model.
* Neutralize
  the system and adjust ionic concentration to 0.15 M NaCl, mimicking physiological
  conditions.

4. Energy minimization

* Perform
  steepest descent energy minimization to remove steric clashes.
* Ensure the
  maximum force on the system is below the threshold (e.g., <1000 kJ/mol/nm).

5. Equilibration

* Conduct
  NVT equilibration (constant number of particles, volume, and temperature) for 1,000,000 steps
  (~2 ns) at 310 K.
* Conduct
  NPT equilibration (constant number of particles, pressure, and temperature) for 1,000,000 steps
  (~2 ns) at 310 K and 1 bar.
* Use
  coupling constants of 0.1 ps for temperature and pressure.

6. Production molecular dynamics
simulation

* Perform
  NPT production MD for 100 ns.
* Simulation
  parameters:
* Time step:
  2 fs
* Number of
  steps: 50,000,000
* Temperature: 310 K (using Nose-Hoover
  thermostat)
* Pressure:
  1 bar (using Parrinello-Rahman barostat)
* Periodic
  boundary conditions (PBC) applied in all directions
* Save
  trajectory data every 1000 steps for later analysis.
* 7. Trajectory analysis
* Calculate
  RMSD (root mean square deviation) to evaluate structural stability.
* Calculate
  RMSF (root mean square fluctuation) to assess residue-level flexibility.
* Measure
  radius of gyration (Rg) to monitor molecular compactness.
* Compute
  solvent accessible surface area (SASA) to evaluate solvent exposure.
* Monitor
  the number of hydrogen bonds between protein and ligand over time to assess interaction
  stability.

8. Free energy calculations

* Construct
  free energy landscapes (2D and 3D FELs) based on principal components or RMSD/Rg
  coordinates.
* Perform
  MM-PBSA analysis using g\_mmpbsa/GMXPBSA:
* Compute
  the average binding free energy between protein and ligand.
* Perform
  per-residue energy decomposition to identify key residues contributing to binding
  affinity.

9. Result visualization and reporting

* Use
  PyMOL/VMD for trajectory visualization.
* Present
  energy profiles, RMSD/RMSF plots, Rg, SASA, hydrogen bond occupancy, and FEL graphs.
* Summarize
  MM-PBSA results in tables and highlight critical binding residues.

# **5. Functional Tests Part2: Plant Model Validation**

## **5.1 Construction of** **N. benthamiana** **Transient Expression Plasmids**

The method of this
step is similar to 2.7, except that the vector and gene are different.

## **5.2 Transient** **N. benthamiana** **Expression Assay**

### **5.2.1 Detection the Effect of AtNAS1 Mutant Proteins on Subcellular Localization Using Confocal Microscopy**

Materials:

* Expression
  vector
* Control
  vector
* Competent
  cells of the Agrobacterium tumefaciens strain (GV3101) for transient expression.
* LB medium
  and antibiotics for Agrobacterium culture.
* Infiltration buffer: 10 mM MgCl2, 10
  mM MES pH 5.6, 100 µM acetosyringone.
* 4-6 leaf
  Nicotiana benthamiana (or tobacco) plants for Agrobacterium-mediated transient
  expression.
* Syringe
  (without needle) for leaf infiltration.
* Sterile
  microcentrifuge tubes, pipettes, and tips.
* Glass
  slides, coverslips, vacuum grease or gaskets, and mounting medium.
* Fluorescent organelle stain.
* Confocal
  laser scanning microscope.
* Image
  analysis software (ImageJ).

Procedure:

1. Construct
   Preparation
2. Agrobacterium Transformation and
   Cultivation
3. Transform
   the construct into Agrobacterium (GV3101) and select transformants on appropriate antibiotic
   plates.
4. Pick a
   single colony and culture in LB medium containing antibiotics at 28°C with shaking
   overnight.
5. The next
   day, subculture the cell into fresh LB medium and grow to an OD600 of approximately
   0.6–1.0.
6. Prepare
   the Infiltration Suspension
7. Resuspend
   the pellet in infiltration buffer and adjust the final OD600 of each construct to approximately
   0.3–0.5.
8. If not
   already added, add acetosyringone to a final concentration of 100 µM.
9. Incubate
   the suspension at room temperature for 2–3 hours to activate the Agrobacterium virulence genes.
10. Using a
    needleless syringe, infiltrate the abaxial surface of the selected Nicotiana benthamiana leaf
    with the Agrobacterium suspension.
11. Mark the
    infiltrated area and maintain the plant under normal growth conditions (22-25°C, 8 hours of
    darkness).
12. Allow
    expression for 48-72 hours.
13. Sample
    preparation for imaging
14. At the
    selected time point, excise a small leaf disc from the infiltrated area.
15. Place the
    leaf disc, abaxially facing down, on a glass slide in a drop of water or mounting medium.
16. Use
    spacers or vacuum aspiration to avoid squeezing the tissue and cover with a coverslip.
17. Confocal
    imaging
18. Image
    processing and analysis

### **5.2.2 Detection the Effect of AtNAS1 Mutant Proteins on the Expression of Iron Cycling Related Genes Using Real-time Quantitative PCR** **In Vivo**

Materials:

1. Plants
   expressing wild-type and mutant AtNAS1 or leaf samples transiently expressed via Agrobacterium,
   and corresponding controls (empty vector/wild-type).
2. Disposable
   scissors and forceps, quick-freezing equipment (liquid nitrogen or dry ice box), and an
   RNase-free reagent environment.
3. RNA
   extraction reagents/kits
4. Reverse
   transcription reagents/kits: Reagents or kits used to reverse transcribe total RNA into
   cDNA.
5. qPCR
   primers: Specific primers for target iron cycle-related genes and internal reference
   genes.
6. Real-time
   fluorescence quantitative PCR reagents
7. RNase-free
   pipette tips, RNase-free centrifuge tubes, 96/384-well qPCR reaction plates, and optical cover
   film.
8. Real-time
   qPCR instrument
9. Analysis
   software for Ct value processing, ΔΔCt calculation, graphing, and statistical testing.

Procedure:

1. Collect
   similar tissues from each treatment group at the designated time points.
2. Rapidly
   process and immediately freeze to preserve RNA integrity.
3. Extract
   total RNA from frozen tissues and perform DNA removal.
4. Reverse
   transcribe the quality-controlled RNA into cDNA using an appropriate reverse transcription
   reagent.
5. qPCR
   primer design.
6. qPCR
   reaction preparation.
7. qPCR run
   and raw data collection.
8. Data
   processing and relative quantification.
9. Statistical analysis and result
   interpretation.

## **5.3 Construction of** **B. subtilis** **Expression Plasmids**

The method of this
step is similar to 2.7, except that the vector and gene are different.

## **5.4 Biofortification Experiment of Soil-Based** **N. benthamiana** **Cultivation Using** **B. subtilis** **Stably Expressing AtNAS1 Mutated Proteins**

### **5.4.1 Effect of** **B. subtilis** **Expressing AtNAS1 Mutated Proteins on** **N. benthamiana** **Growth and Biomass**

Materials:

1. N.
   benthamiana seedlings for soil cultivation.
2. Bacillus
   subtilis strains stably expressing AtNAS1 variants (wild-type and mutant) and a control
   strain.
3. Sterile or
   specialized potting soil suitable for N. benthamiana.
4. Growth
   chamber with controlled light, temperature, and humidity.
5. Drying
   oven for handling and measuring fresh and dry biomass.

Procedure:

1. Grow
   Nicotiana benthamiana until it reaches a growth stage suitable for microbial treatment.
2. Apply a
   strain of Bacillus subtilis to the plant rhizosphere, maintaining a control treatment.
3. Regularly
   observe plant growth, developmental changes, and health.
4. Collect
   plant growth data, including fresh and dry biomass.

### **5.4.2 Effect of** **B. subtilis** **Expressing AtNAS1 mutated Proteins on Iron and Zinc Trace Element Contents in Tobacco Leaves Using ICP-MS**

Materials:

1. Nicotiana
   benthamiana leaves harvested from soil
2. Bacillus
   subtilis strains stably expressing AtNAS1 variants and a control strain
3. Leaf
   harvesting tools, a desiccator or freeze dryer for tissue dehydration, and a mortar and pestle
   for grinding dried tissue
4. Acids and
   buffers suitable for trace element digestion, compatible with ICP-MS instrumentation
5. ICP-MS
   instrument
6. Digestion
   tubes
7. Volumetric
   flasks
8. Sterile
   tweezers
9. Sample
   labeling material

Procedure:

1. Define
   treatment groups (e.g., uninoculated, parental B. subtilis, B. subtilis expressing wild-type
   AtNAS1, and each AtNAS1 mutant) and ensure sufficient biological replicates for statistical
   comparisons.
2. Leaf
   Sampling: Collect comparable leaves from each plant to avoid contamination; label and record
   sample metadata.
3. Tissue
   Drying and Homogenization: Dry leaves using standard methods and then grind into a fine, uniform
   powder for consistent analysis.
4. Sample
   Digestion: Digest the powdered tissue with acid or other suitable reagents to release trace
   elements into solution.
5. ICP-MS
   Measurement: Analyze the digested samples using ICP-MS to determine iron and zinc
   concentrations.

{% endcall %} {% endblock %}

{% extends "layout.html" %} {% block title %}Results{% endblock %} {% block lead
%}Present the results of your project, along with a detailed analysis and
discussion of their significance. Also outline future plans and reflections on
the impact of your project.{% endblock %} {% block page\_content %} {% from
"/components/article-content.html" import articleContent %} {% call
articleContent("RESULTS", [
{"content": "Overview","anchor": "#title\_1"},
{"content": "Molecular Docking and Prediction of AtNAS1 Mutated Proteins with Substrates","anchor": "#title\_2"},
{"content": "Construction of Plasmids","anchor": "#title\_3", "children": [
{"content": "Construction of E. coli Expression Plasmids","anchor": "#level2\_1"}
]},
{"content": "Protein Expression and Purification","anchor": "#title\_4", "children": [
{"content": "Construction of E. coli Expression Strains","anchor": "#level2\_2"},
{"content": "Preliminary Experiment for Protein Expression","anchor": "#level2\_3"},
{"content": "His-Tag Affinity Purification of Proteins","anchor": "#level2\_4"}
]},
{"content": "Functional Tests Part1: Mutation Sites Selection","anchor": "#title\_5", "children": [
{"content": "Enzymatic Reactions Detection of AtNAS1 Mutated Proteins' Catalytic Ability","anchor": "#level2\_5"},
{"content": "Molecular Dynamics Fitting Analysis of Catalytic Efficiency in AtNAS1 Mutated Proteins","anchor":
"#level2\_5\_2"}
]},
{"content": "Functional Tests Part2: Plant Model Validation","anchor": "#title\_6", "children": [
{"content": "Construction of N. benthamiana Transient Expression Plasmids","anchor": "#level2\_6"},
{"content": "Transient N. benthamiana Expression Assay","anchor": "#level2\_7", "children": [
{"content": "Detection the Effect of AtNAS1 Mutant Proteins on Subcellular Localization Using Confocal
Microscopy","anchor": "#level3\_1"},
{"content": "Detection the Effect of AtNAS1 Mutant Proteins on the Expression of Iron Cycling Related Genes Using
Real-time Quantitative PCR In Vivo","anchor": "#level3\_2"}
]},
{"content": "Construction of B. subtilis Expression Plasmids","anchor": "#level2\_8"},
{"content": "Biofortification Experiment of Soil-Based N. benthamiana Cultivation","anchor": "#level2\_9",
"children": [
{"content": "Effect of B. subtilis Expressing AtNAS1 Mutated Proteins on N. benthamiana Growth and
Biomass","anchor": "#level3\_3"},
{"content": "Effect of B. subtilis Expressing AtNAS1 mutated Proteins on Iron and Zinc Trace Element Contents in
Tobacco Leaves Using ICP-MS","anchor": "#level3\_4"}
]}
]},
{"content": "Future plans","anchor": "#title\_7"},
{"content": "References","anchor": "#title\_8"}
]) %}

# Overview

Iron (Fe) and zinc (Zn) are essential trace
elements for human health[1]. However, iron and zinc deficiency is widespread worldwide, affecting
approximately 2 billion people, particularly infants, young children[2], and pregnant women[3]. The risk
of iron and zinc deficiency is particularly high in sub-Saharan Africa, where the diet relies heavily on
maize, which provides at least 30% of total caloric intake[4]. However, the iron and zinc concentrations
in maize endosperm are extremely low, and long-term maize-based diets can easily lead to iron and zinc
deficiency symptoms. While iron and zinc supplements can improve human iron and zinc nutrition, these
measures have limited success in developing countries due to economic constraints and low consumer
acceptance[5]. Biofortification through genetically modified crops, by contrast, shows greater
potential.

In recent years, genetic engineering of areas
related to iron absorption and metabolism has successfully increased the iron content of edible crop
parts[6]. For example, the coordinated expression of AtNAS1 (nicotinamide synthase 1), PvFERRITIN
(ferritin), and AfPHYTASE (phytase) increased iron concentration in rice endosperm[7]. These studies
demonstrate the broad potential of genetic engineering for crop iron fortification. Nicotinamide
synthase is a key enzyme in the synthesis of nicotinamide (NA) in plants[8]. Nicotinamide is an
important metal chelator that can form stable complexes with divalent metal ions such as iron (Fe) and
zinc (Zn). The NAS gene directly influences the absorption, transport, and distribution of iron and zinc
in plants by regulating nicotinamide synthesis, thus playing a central role in plant iron and zinc
nutrition[9].

However, some literature reports confirmed the
existence of an autoinhibitory signal at the C-terminus of the AtNAS1 protein through in vitro
truncation experiments, thereby affecting enzyme activity[8]. This may be the reason why the effect of
increasing iron and zinc in transgenic rice overexpressing AtNAS1 is not obvious[8]. Therefore, based on
the above research background, this project intends to use the key amino acid sites at the C-terminus of
the AtNAS1 protein predicted in published literature to affect enzyme activity, and mutate the key sites
of the full-length protein. After purifying the AtNAS1 mutant protein through prokaryotic expression in
Escherichia coli, the enzyme activity mechanics analysis was performed. Compared with the wild-type
AtNAS1 protein, and using the truncated AtNAS1 protein reported in the literature as a positive control
for increased enzyme activity, the point mutation combination with stronger activity of the AtNAS1
full-length protein was screened for Bacillus subtilis expression vector construction, and the iron and
zinc content in tobacco leaves and roots was measured after tobacco soil culture. In addition, the
mutant AtNAS1 protein was verified by tobacco transient expression experiments, and the transcription
level of the known genes regulating iron and zinc absorption and metabolism was detected by qPCR to
confirm the mutant AtNAS1 Whether the mutation affects the intracellular distribution and localization
characteristics of AtNAS1 can be determined by fusing it with fluorescent protein and observing its
subcellular localization under a confocal microscope. This study uses tobacco as a model organism and
provides new ideas and theoretical support for solving the "hidden hunger" problem caused by the lack of
trace elements such as iron and zinc in crops.

Results of our
work

Outline

1. Molecular Docking and Prediction of AtNAS1 Mutated Proteins with Substrates

2. Construction of **E. coli** Expression Plasmids

pET28a-AtNAS1\_Wildtype

pET28a-AtNAS1\_Truncated

pET28a-AtNAS1\_T287A

pET28a-AtNAS1\_R288A

pET28a-AtNAS1\_G289A

pET28a-AtNAS1\_TRGtoAAA

pET28a-MtnN

pET28a-AdeD

3. Protein Expression and Purification

3.1 Construction of **E. coli** Expression Strains

3.2 Preliminary Experiment for Protein Expression

3.3 His-Tag Affinity Purification of Proteins

4. Functional Tests Part1: Mutation Sites Selection

4.1 Enzymatic Reactions Detection of AtNAS1 Mutated Proteins' Catalytic Ability

4.2 Molecular Dynamics Fitting Analysis of Catalytic Efficiency in AtNAS1 Mutated
Proteins

5. Functional Tests Part2: Plant Model Validation

5.1 Construction of **N. benthamiana** Transient Expression Plasmids

pCambia1305-AtNAS1wt-eGFP-6myc

pCambia1305-AtNAS1truncated-eGFP-6myc

pCambia1305-AtNAS1\_TRG\_to\_AAA-eGFP-6myc

5.2 Transient **N. benthamiana** Expression Assay

5.3.1 Detection the Effect of AtNAS1 Mutant Proteins on Subcellular Localization
Using Confocal Microscopy

5.3.2 Detection the Effect of AtNAS1 Mutant Proteins on the Expression of Iron
Cycling Related Genes Using Real-time Quantitative PCR In Vivo

5.3 Construction of **B. subtilis** Expression Plasmids

pHT43-AtNAS1wt

pHT43-AtNAS1truncated

pHT43-AtNAS1\_TRG\_to\_AAA

5.4 Biofortification Experiment of Soil-Based **N. benthamiana**
Cultivation Using **B. subtilis** Stably Expressing AtNAS1 Mutated Proteins

5.4.1 Effect of **B. subtilis** Expressing AtNAS1 Mutated Proteins on
**N. benthamiana** Growth and Biomass

5.4.2 Effect of **B. subtilis** Expressing AtNAS1 mutated Proteins on
Iron and Zinc Trace Element Contents in Tobacco Leaves Using ICP-MS

# 1. Molecular Docking and Prediction of AtNAS1 Mutated Proteins with Substrates

Based on key amino acid sites
predicted in the literature, we constructed AtNAS1 mutants and conducted molecular docking analysis
using its natural substrate S-adenosylmethionine (SAM) as a ligand. By comparing the differences in the
binding patterns of wild-type and mutant AtNAS1 proteins with SAM, we can assess at the molecular level
whether the mutations may improve substrate binding affinity and enzyme activity.

First, we searched for the
protein sequence based on the gene name on the Uniprotkb website. The searched sequence was imported
into the SWISS-MODLE website, where the optimal structure was found and saved in PDB format. The small
molecule was then searched in the Small Molecule Database and downloaded in sdf format for subsequent
operations. The first step was energy optimization, minimizing the energy of the selected ligand to
reduce the energy of the docked system and achieve a stable state. The ligand was then optimized using
DS software, and the optimized structure was saved in PDB format for future use.

The second step was protein
preprocessing. The protein file was imported into PYMOL software, where redundant ligands and water
molecules were deleted and saved in PDB format for future use. The protein was hydrogenated and charged,
and then configured as a receptor and saved as a pdbqt file for future use. Finally, the small molecule
was hydrogenated and charged, configured as a ligand, and torsion angles and bonds were checked and
saved in pdbqt format for future use.

Since we do not know the binding
pocket area, we set the grid to the entire protein area for ligand binding and run this operation. After
the grid setting is completed, we perform 50 molecular Autodock dockings. The official recognition of
more than 50 docking times indicates that the docking results are relatively accurate. After the docking
is completed, the conformation with the highest repeatability and the best binding energy is selected as
the output result conformation, and DS or PYMOL is used to display the visualization, three-dimensional
structure and surface model.

|  |
| --- |
| ![](https://static.igem.wiki/teams/5769/image/result-1up.webp) |
| ![](https://static.igem.wiki/teams/5769/image/result-1down.webp) |
| Fig. 1 Two-dimensional, three-dimensional, and surface model images of the wild-type and mutant AtNAS1 proteins docked with SAM. |

Two-dimensional and
three-dimensional results show that wild-type AtNAS1 primarily forms multiple hydrogen bonds with SAM
through residues such as GLU77, SER133, and LEU138, resulting in a relatively stable binding. The
binding site of the truncated AtNAS1 shifts to residues such as GLN196 and ASP198, resulting in a more
exposed binding pocket and reduced stability. Among the point mutants, T287A interacts with ASN285 and
VAL284, but the binding is weak; T288A and G289A maintain moderate levels of interaction; and TRG→AAA
forms multiple hydrogen bonds around residues such as GLY277, GLY278, and GLY286, resulting in a deeper
and tighter binding pocket (Fig. 1).

The binding energy results were
consistent with the structural observations: the wild-type binding energy was -6.0 kcal/mol, while the
truncated forms decreased to -5.3 kcal/mol. T287A exhibited the weakest binding energy (-4.5 kcal/mol),
while T288A and G289A exhibited -5.7 kcal/mol, and TRG→AAA exhibited the best binding energy (-6.5
kcal/mol). Overall, this suggests that the TRG→AAA mutation enhances SAM binding stability and may
improve AtNAS1 enzymatic activity (Fig. 2).

|  |
| --- |
| ![](https://static.igem.wiki/teams/5769/image/result-2.webp) |
| Fig. 2 Binding energies between wild-type and mutant AtNAS1 proteins and SAM ligands. |

# 2.Construction of Plasmids

## 2.1 Construction of **E. coli** Expression Plasmids

We constructed the
pET28a-AtNAS1\_Wildtype and pET28a-AtNAS1\_Truncated plasmids using Infusion recombination. Initially, we
amplified the AtNAS1\_Wildtype and AtNAS1\_Truncated fragments via high-fidelity PCR, resulting in
fragment lengths of 963 bp and 861 bp, respectively. The right panel of Fig. 3 displays the correct
bands corresponding to the target sizes of the two genes. Subsequently, we used the restriction
endonuclease BamHI to linearize the pET28a vector backbone, as shown in the left panel of Fig. 3. After
performing agarose gel electrophoresis and recovering the bands, we proceeded with the Infusion assemble
kit to obtain the recombinant plasmids.

|  |
| --- |
| ![](https://static.igem.wiki/teams/5769/image/result-3.webp) |
| Fig. 3 Agarose gel electrophoresis of target genes and linearized pET28a vector |

After the Infusion reaction
assay, we transformed the recombinant plasmids into the cloned strain E. coli DH5α. Fig. 4A
shows the single colonies on the plate. We then selected six colonies from each plate. Next, we used
vector universal primers, T7-Forward and T7-Terminal, to detect the insertion of target genes using
colony PCR. As shown in Fig. 4B, the AtNAS1\_Wildtype and pET28a-AtNAS1\_Truncated gene fragments were
successfully inserted into the pET28a backbone.

|  |
| --- |
| ![](https://static.igem.wiki/teams/5769/image/result-4.webp) |
| Fig. 4 Verification of the transformation results for positive colonies of pET28a-AtNAS1\_Wildtype and pET28a-AtNAS1\_Truncated plasmids. A: Single colonies on the plate; B: Amplification results from colony PCR. |

Finally, we used the universal
primer T7-Forward to verify the positive colonies through Sanger sequencing. Fig. 5 displays the correct
sequences of the recombinant plasmids. We selected the correct colonies for further IPTG
induction.

|  |
| --- |
| ![](https://static.igem.wiki/teams/5769/image/result-5.webp) |
| Fig. 5 Sanger sequencing results of pET28a-AtNAS1\_Wildtype and pET28a-AtNAS1\_Truncated positive colony. |

To further construct single-base
and multi-base mutations, we linearized the pET28A-AtNAS1-Wildtype plasmid using **inverse
PCR** to incorporate specific mutation sites. Subsequently, we constructed the mutated
plasmids AtNAS1-T287A, AtNAS1-R288A, AtNAS1-G289A, and AtNAS1-TRGtoAAA via Infusion assay. Following the
same construction process as for pET28A-AtNAS1-Wildtype, we also used colony PCR and Sanger sequencing
to verify the positive colonies of the mutated plasmids, as shown in Fig. 6.

|  |
| --- |
| ![](https://static.igem.wiki/teams/5769/image/result-6.webp) |
| Fig. 6 The construction process of pET28A-AtNAS1 mutated plasmids. A. Agarose gel electrophoresis of mutated genes; B. Single colonies on the plate; C: Amplification results from colony PCR; D: Sanger sequencing results. |

Since the enzymatic activity
assay of nicotinamide synthase requires the substrate SAM and two coenzymes, MtnN and AdeD, we cloned
the MtnN and AdeD gene fragments from the E. coli XL-1 Blue strain. Using the same construction
strategy as for the pET28A-AtNAS1 plasmid, we constructed the expression plasmids pET28a-MtnN and
pET28a-AdeD. The construction process is shown in Fig. 7.

|  |
| --- |
| ![](https://static.igem.wiki/teams/5769/image/result-7.webp) |
| Fig. 7 The construction process of pET28A-MtnN and pET28A-AdeD plasmids. A. Agarose gel electrophoresis of MtnN and AdeD genes; B. Single colonies on the plate; C: Amplification results from colony PCR; D: Sanger sequencing results. |

# 3. Protein Expression and Purification

## 3.1 Construction of **E. coli** Expression Strains

We transformed the correct
plasmids into the E. coli expression strain BL21 and selected single colonies for
culture in preparation for the next step of prokaryotic expression. The plates are shown in Fig.
8.

|  |
| --- |
| ![](https://static.igem.wiki/teams/5769/image/result-8.webp) |
| Fig. 8 Single colonies of recombinant plasmids in E. coli BL21 strain. |

## 3.2 Preliminary Experiment for Protein Expression「

Based on the lac operator system
in the pET28a vector backbone, we used IPTG for protein expression. Initially, we applied a final
concentration of 1 mM IPTG for standard protein expression. The expected molecular weights of AtNAS1
(including the mutated proteins), AtNAS1-Truncated, MtnN, and AdeD are 35.5 kDa, 31.7 kDa, 24.4 kDa, and
63.7 kDa, respectively. As shown in Fig. 9, we can clearly observe the protein bands corresponding to
the target genes.

|  |
| --- |
| ![](https://static.igem.wiki/teams/5769/image/result-9.webp) |
| Fig. 9 SDS-PAGE analysis of preliminary experiment for target proteins expression. |

## 3.3 His-Tag Affinity Purification of Proteins

We utilized the principle of
His-tag affinity with Ni2+. This allowed us to bind the crude protein extracted from the
E. coli expression cells to Ni-NTA beads. After washing away impurities with a low
concentration of imidazole, we eluted the protein with a high concentration of imidazole. As shown in
Fig. 10, we obtained a high-purity protein with a high expression level. Subsequently, we used dialysis
to remove the high concentration of imidazole from the buffer and aliquoted the protein at a
concentration of 1 mg/mL for storage, to be used in subsequent functional assays.

|  |
| --- |
| ![](https://static.igem.wiki/teams/5769/image/result-10.webp) |
| Fig. 10 SDS-PAGE analysis of large-scale His-tag protein purification of target proteins. Total: Total Protein; FT: Flow Through. |

# 4. Functional Tests Part1: Mutation Sites Selection

## 4.1 Enzymatic Reactions Detection of AtNAS1 Mutated Proteins' Catalytic Ability

We assessed the enzymatic activity of
nicotinamide synthase (NAS1) mutants in Functional Tests Part 1. The enzymatic activities of the
wild-type and various mutants (Truncated, T287A, R288A, G289A, TRG\_to\_AAA) of NAS1 were evaluated by
measuring the initial reaction rates (ΔA/Δt) and calculating the enzyme activities (nkat/mg) based on
the parameters of the reaction system (Fig. 11B). Compared to the slope and correlation coefficient of
AtNAS1-Wildtype, the enzyme activities of the different mutants varied significantly, with some mutants,
such as AtNAS1-T287A and AtNAS1-R288A, exhibiting markedly reduced activities. In contrast,
AtNAS1-Truncated and AtNAS1-TRG\_to\_AAA demonstrated steep slopes and high enzyme catalytic efficiency
(Fig. 11A). The AtNAS1-TRG\_to\_AAA mutant protein, unlike AtNAS1-Truncated, retains the complete
C-terminal region of the NAS1 protein, thereby avoiding potential functional redundancies associated
with the truncated form. Notably, the TRG\_to\_AAA mutant also exhibited a relatively higher level of
nicotinamide synthesis activity compared to the truncated protein (Fig. 11B). Therefore, the TRG\_to\_AAA
mutant may possess greater potential for application than the truncated variant.

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| ![](https://static.igem.wiki/teams/5769/image/result-11.webp) |
| Fig. 11 Kinetic and Thermodynamic Analysis of Engineered Variants of AtNAS1 protein. A. Enzyme Kinetics of AtNAS1 mutated protein; B. Enzyme Activity Analysis. |

## 4.2 Molecular Dynamics Fitting Analysis of Catalytic Efficiency in AtNAS1 Mutated Proteins

After testing the enzymatic
assays, we further investigated the binding affinities of the AtNAS1-Wildtype and AtNAS1 mutant forms
with the substrate S-adenosyl methionine (SAM) using molecular dynamics simulations. As shown in Figure
12, the average binding free energy (dG) between AtNAS1-Wildtype and SAM was -36.83 kcal/mol, while the
dG values for AtNAS1-Truncated is -13.63 kcal/mol and the dG values for AtNAS1-TRG\_to\_AAA is -17.06
kcal/mol. These results indicate that the ligand (SAM) can spontaneously bind to the protein, and the
resulting protein-ligand complexes are dynamically stable, suggesting they may possess biological
activity. This provides a theoretical energy-based foundation for further studying the biological
functions of these ligand-protein complexes. The favorable binding free energies imply that the
AtNAS1-Wildtype and AtNAS1-TRG\_to\_AAA can effectively interact with the SAM substrate, which is a
critical step in the enzymatic reactions catalyzed by this protein.

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| ![](https://static.igem.wiki/teams/5769/image/result-12.webp) |
| Fig. 12 Molecular dynamics simulation of the binding sffinities between AtNAS1-Wildtype and mutant forms with the substrate S-Adenosyl Methionine (SAM) |

# 5. Functional Tests Part2: Plant Model Validation

## 5.1 Construction of **N. benthamiana** Transient Expression Plasmids

Our previous results showed that the
AtNAS1-Truncated protein and the AtNAS1 triple-mutant AtNAS1-TRG\_to\_AAA protein have comparable enzyme
activity levels. Therefore, in the next step, we would like to validate our hypothesis using a plant
model system, like **N. benthamiana** . First, we constructed the gene sequences of
AtNAS1-Wildtype, AtNAS1-Truncated, and AtNAS1-TRG\_to\_AAA into the dual expression vector pCambia1305,
which has a GFP fluorescent protein tag. This will allow us to detect the subcellular localization of
the mutants using confocal microscope. As showed in Fig. 11A, we first amplified the gene fragments
containing the homologous arms of the pCambia1305 backbone, where the AtNAS1-Wildtype served as the
control. We then linearized the pCambia1305 vector using the restriction enzyme BamHI (Fig. 13B). After
that, we performed an In-Fusion assembly reaction to construct the recombinant vector (Fig. 13C).
Finally, we screened for positive clones using colony PCR (Fig. 13D).

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| ![](https://static.igem.wiki/teams/5769/image/result-13.webp) |
| Fig. 13 The construction process of pCambia1305-GFP-AtNAS1-WT, pCambia1305-GFP-AtNAS1-Truncated, and pCambia1305-GFP-AtNAS1-TRG\_to\_AAA plasmids. A. Agarose gel electrophoresis of genes; B. Linearized pCambia1305 backbone; C. Single colonies on the plate; D: Amplification results from colony PCR. |

## 5.2 Transient **N. benthamiana** Expression Assay

### 5.3.1 Detection the Effect of AtNAS1 Mutant Proteins on Subcellular Localization Using Confocal Microscopy

Given that the subcellular localization of a
protein can affect its function, we utilized transient expression in **N.
benthamiana** along with microscopy to observe whether the localization patterns
of the AtNAS1 mutant proteins differ from the AtNAS1-Wildtype protein. As shown in Fig. 14A, the process
of transient expression in tobacco involves Agrobacterium strain in acetosyringone buffer, selecting
healthy tobacco leaves, and infiltrating the bacterial suspension into the leaf abaxial surface using a
1 mL syringe. After two days of transient expression, the leaves were analyzed. We first transformed the
AtNAS1-Wildtype, AtNAS1-Truncated, and AtNAS1-TRG\_to\_AAA positive plasmids into the Agrobacterium GV3101
strain and grew them on dual-antibiotic plates containing kanamycin and rifampicin, picking single
colonies for further culturing (Fig. 14B). We initially used a handheld fluorescence instrument to
detect the protein expression levels, which revealed that the AtNAS1-Truncated and AtNAS1-TRG\_to\_AAA
proteins exhibited higher expression efficiency compared to the AtNAS1-Wildtype protein (Fig. 14C).
Further analysis using confocal microscopy showed that the AtNAS1-Truncated and AtNAS1-TRG\_to\_AAA
proteins had stronger fluorescence intensity and were more strongly localized to the endoplasmic
reticulum, providing a potential explanation for their higher enzymatic activity (Fig. 14D).

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| ![](https://static.igem.wiki/teams/5769/image/result-14.webp) |
| Fig. 14 Subcellular localization of AtNAS1-Wildtype, AtNAS1-Truncated, and AtNAS1-TRG\_to\_AAA proteins. A. Process of transient transfection in N. benthamiana; B. Single colonies of recombinant plasmids in Agrobacterium GV3101 strain. The plate contains Kanamycin and Rifampicin dual-antibiotics; C. Detection of GFP protein expression in N. benthamiana leaves using a handheld fluorescence instrument; D. The subcellular localization patterns of AtNAS1-Wildtype, AtNAS1-Truncated, and AtNAS1-TRG\_to\_AAA in N. benthamiana leaves using confocal microscope. |

### 5.3.2 Detection the Effect of AtNAS1 Mutant Proteins on the Expression of Iron Cycling Related Genes Using Real-time Quantitative PCR In Vivo

To further investigate whether mutations in the
AtNAS1 gene could affect the expression of genes related to the absorption and activation of iron and
zinc micronutrients, we validated this hypothesis using a **N. benthamiana** -based
culture transient system. First, we used the previously constructed Agrobacterium strains carrying the
AtNAS1-Wildtype, AtNAS1-Truncated, and AtNAS1-TRG\_to\_AAA to infiltrate tobacco leaves. After 3 days of
culture, we extracted RNA from the leaves, reverse transcribed it into cDNA, and used quantitative
real-time PCR to detect the expression levels of the target genes. The expression levels were normalized
using the tobacco NtActin1 gene as an internal reference. As shown in Fig. 15A, compared to the
non-transformed tobacco leaves, the tobacco leaves expressing the AtNAS1-Wildtype, AtNAS1-Truncated, and
AtNAS1-TRG\_to\_AAA plasmids significantly enhanced AtNAS1 gene expression, indicating that the transient
expression of AtNAS1 in tobacco was successful. Further analysis revealed that the transient
overexpression of both the wild-type and mutant AtNAS1 significantly increased the expression of the
iron and zinc absorption and transport genes NtIRT1 and NtMTP8 (Fig. 15B-C), while the expression of the
iron activation-related gene NtFRO2 remained unchanged (Fig. 15D). Notably, the AtNAS1-Truncated, and
AtNAS1-TRG\_to\_AAA exhibited even higher expression levels of the iron and zinc absorption and transport
genes NtIRT1 and NtMTP8, suggesting their greater potential for iron biofortification (Fig.
15B-C).

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| ![](https://static.igem.wiki/teams/5769/image/result-15.webp) |
| Fig. 15 Overexpression of wild-type and mutant AtNAS1 affects the expression of genes related to iron and zinc homeostasis. A: Relative expression levels of the AtNAS1 gene in tobacco leaves; B: Relative expression levels of the iron transporter gene NtIRT1 in tobacco leaves; C: Relative expression levels of the zinc transporter gene NtMTP8; D: Relative expression levels of the iron activation gene NtFRO2 in tobacco leaves. |

## 5.3 Construction of **B. subtilis** Expression Plasmids

To further evaluate the application potential
of AtNAS1 mutants in plants, we also used **N. benthamiana** as a model system and
used **B. subtilis** harboring expression plasmids as a bioinoculant for soil-grown
tobacco. Using the same construction strategy as for the pET28A-AtNAS1 plasmid, we constructed the
B. subtilis expression plasmids pHT43-AtNAS1-Wildtype, pHT43-AtNAS1-Truncated, and
pHT43-AtNAS1-TRG\_to\_AAA. The construction process is shown in Fig. 16.

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| ![](https://static.igem.wiki/teams/5769/image/result-16.webp) |
| Fig. 16 The construction process of pHT43-AtNAS1-WT, pHT43-AtNAS1-Truncated, and p pHT43-AtNAS1-TRG\_to\_AAA plasmids. A. Agarose gel electrophoresis of genes; B. Linearized p pHT43 backbone; C. Single colonies of DH5alpha on the plate; D: Single colonies of B. subtilis on the plate. |

## 5.4 Biofortification Experiment of Soil-Based **N. benthamiana** Cultivation Using **B. subtilis** Stably Expressing AtNAS1 Mutated Proteins

### 5.4.1 Effect of **B. subtilis** Expressing AtNAS1 Mutated Proteins on **N. benthamiana** Growth and Biomass

The previous research results have shown that
compared to the AtNAS1-Wildtype, both the AtNAS1-Truncated and the AtNAS1-TRG\_to\_AAA mutants have higher
enzyme activity and a high efficiency to activate genes related to iron cycling. Since the
AtNAS1-Truncated removes the C-terminal domain of the protein, it may lead to the partial loss of other
biological functions, so we are more focused on the application of the AtNAS1-TRG\_to\_AAA mutant. In our
tobacco pot culture experiment, we used **B. subtilis** as a probiotic. When the
tobacco plants grew at the 4-leaf stage, we applied 10 mL of B. subtilis at different
concentrations to the root system for root colonization. After two weeks of growth, we measured the
plant fresh biomass and then dried and ground the shoot to determine the iron and zinc micronutrient
contents using ICP-MS. As shown in Figure 17, we found that after applying B. subtilis, the
fresh biomass of the tobacco plants actually showed a decreasing trend, and the leaves exhibited partial
curling, which may be related to the applied concentration and still requires further
optimization.

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| ![](https://static.igem.wiki/teams/5769/image/result-17.webp) |
| Fig. 17 Phenotypic analysis of AtNAS1 transient expressing tobacco plants. A: Images showing the growth of pHT43-AtNAS1-Wildtype and pHT43-AtNAS1-TRG\_to\_AAA transient expressing tobacco plants under different B. subtilis optical densities (PBS Control, OD=0.5, OD=1.0); B: The fresh weight of tobacco plants. |

### 5.4.2 Effect of **B. subtilis** Expressing AtNAS1 mutated Proteins on Iron and Zinc Trace Element Contents in Tobacco Leaves Using ICP-MS

After harvesting the tobacco samples with
**B. subtilis** root application, as shown in Figure 18A, we dried the shoot of the
tobacco plants at 60°C and ground them into powder. We then performed microwave digestion of the samples
using nitric acid and hydrogen peroxide, and used ICP-MS to measure the micronutrient contents. The raw
data from the instrument was converted to mg/kg units using standard calibration curves. As shown in
Figure 18B, compared to the PBS control, the tobacco plants inoculated with B. subtilis
carrying the pHT43-AtNAS1-Wildtype and pHT43-AtNAS1-TRG\_to\_AAA plasmids showed increased iron (Fe) and
zinc (Zn) micronutrient contents in the shoot at different B. subtilis concentrations. However,
at the OD=0.5 concentration, there was no significant difference in either Fe or Zn content between the
AtNAS1-Wildtype and AtNAS1-TRG\_to\_AAA treatments. At the OD=1 concentration, the tobacco plants
inoculated with the B. subtilis carrying the pHT43-AtNAS1-TRG\_to\_AAA plasmid had significantly
higher Fe content in the shoot compared to the pHT43-AtNAS1-Wildtype, while the Zn content did not
differ. This suggests the potential application of the B. subtilis strain carrying the
pHT43-AtNAS1-TRG\_to\_AAA plasmid for iron biofortification. Overall, our experiment has achieved
preliminary success, but further optimization is still needed to fully understand the effects of the
different B. subtilis treatments on plant biomass and micronutrient accumulation.

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| ![](https://static.igem.wiki/teams/5769/image/result-18.webp) |
| Fig. 18 A: Sample processing process, B: Fe element determination results, C: Zn element determination results |

# Future plans

Our preliminary results demonstrate that
compared to the AtNAS1-Wildtype, the AtNAS1-Truncated and AtNAS1-TRG\_to\_AAA mutant exhibit higher
enzymatic reaction rates. Importantly, the AtNAS1-TRG\_to\_AAA, which retains the complete C-terminal
amino acid sequence of AtNAS1, has greater potential for practical applications. We further validated
our hypotheses in pot experiments, but the application dosage of B. subtilis may not have
reached the optimal conditions. We only detected higher iron content in the shoot of tobacco plants
treated with the AtNAS1-TRG\_to\_AAA mutant strain compared to the AtNAS1-Wildtype, when the application
concentration was at OD=1. This indicates the need for further optimization of the experimental
conditions to achieve better results. In the future, we can explore the application of the homologous
genes of AtNAS1 in other crop plants, building on the insights gained from this study. The favorable
enzymatic properties of the AtNAS1 mutant forms, combined with the potential for improved micronutrient
accumulation, provide a promising foundation for developing biofortification strategies using these
genetic resources.

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