iGEM 2025 — Team shanghai-explore

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Team Formation & Early Planning

• March 16, 2025 — Formed the iGEM team.

• March 17, 2025 — First team meeting: PI briefed students and parents on the iGEM competition, introducing
objectives and expectations.

• March 23, 2025 — Registered the team in iGEM and formalized positions and division of labor. Individual
accounts were registered for the team roster.
oSub-bullet: Connected with respective schools for assistance and established cooperation with ECUST laboratory.

• April 3, 2025 — Established a clear timetable to complete tasks and coordinate workflow.

• April 6, 2025 — Visited ECUST lab, brainstorming project focus on Ralstonia solanacearum (bacterial wilt).
Received a lab safety briefing.

• May 5, 2025 — Met at the lab to discuss experiment details, establish workflow, and begin research
supporting the project.

• May 18, 2025 — Conducted an online meeting to establish common goals and standards for the project.

• June 2, 2025 — Held a session with expert Wang Qiyao (王启要) for advice on the topic and to identify
uncertainties.

• June 22, 2025 — Online meeting to conduct safety briefing and design a summer lab schedule; all pre-lab
preparation completed.

Wet Lab Experiments

First Lab Period (July 10–16, 2025)

• Day 1 (July 10) — Familiarized with plasmid extraction experiment; prepared LB medium.

> Participants: Anderson Zhang, Xingyi Yan, Chengxuan Zhang

• Day 2 (July 11) — Poured TSB plates and performed plate streaking experiments.

> Participants: Anderson Zhang, Xingyi Yan, Chengxuan Zhang, Bosen Du

• Day 3 (July 12) — Induced double exchange in confirmed plasmids, picked colonies for verification, and
familiarized with experimental procedures.

> Participants: Anderson Zhang, Yitong Chen, Tyle Cheng, Bosen Du

• Day 4 (July 13) — Cultivated tomato seedlings, transformed plasmids, and performed plate inoculation
(second round).

> Participants: Anderson Zhang, Yitong Chen, Lucas Chen, Tyle Cheng

• Day 5 (July 14) — Genomic inoculation PCR construction, SDS-PAGE electrophoresis, plasmid transformation.

> Participants: Anderson Zhang, Joy Jiang

• Day 6 (July 15) — Colony picking, plasmid fragment amplification, strain preservation, PCR colony
verification.

> Participants: Anderson Zhang, Hsuan Shih, Lucas Chen, Joy Jiang

• Day 7 (July 16) — Prepared electrophoresis gel, performed SDS-PAGE to verify plasmids, observed TCA
experiment.

> Participants: Anderson Zhang, Hsuan Shih, Joy Jiang

• July 21, 2025 — Visited Shanghai Academy of Plant and Agricultural Sciences; interviewed Professor Peng
Zhang for specialist insights.

• July 23, 2025 — Hosted an update meeting to coordinate dry and wet lab workflow and readjust
responsibilities.

Protein Purification (August 15 – September 5, 2025)

• August 15 — Prepared culture media and inoculated bacterial strains for protein expression (first cycle: PtsG
and
FabH).

> Participants: Anderson Zhang, Lucas Chen, Tyle Cheng, Joy Jiang

• August 16 — Nickel affinity columns prepared; bacterial cultures expanded with IPTG induction.

> Participants: Anderson Zhang, Bosen Du, Yitong Chen, Lucas Chen

• August 17 — Cells lysed; proteins adsorbed, purified through nickel columns, and dialyzed.

> Participants: Anderson Zhang, Bosen Du, Yitong Chen, Xingyi Yan

• August 18 — Measured OD concentration of purified proteins; analyzed using SDS-PAGE; inoculated new
cultures
for second cycle.

Participant: Anderson Zhang

• August 19 — Began second cycle (target protein: GlnA); nickel columns prepared and IPTG induction
performed.

> Participants: Anderson Zhang, Tyle Cheng, Chengxuan Zhang, Xingyi Yan

• August 20 — Attempted cell lysis for GlnA; inclusion body formation prevented extraction; experiment
stopped.

> Participants: Anderson Zhang, Lucas Chen, Chengxuan Zhang

September 3–5 — Added solubility-enhancing tag, conducted revised purification cycles, performed SDS-PAGE
verification, and successfully completed protein purification.

Participant: Anderson Zhang

Product Creation: Alginate & ErucaBead (August 1–September 21, 2025)

• August 1 — Initial tests for alginate bead formation.

• August 2 — Tested encapsulation of E. coli within beads; measured solubility in water.

• August 5 — Prepared beads with different alginate concentrations to optimize stability and bacterial
viability.

• August 7 — Conceptualized ErucaBead inspired by online SynBio Challenges 2025; product creation officially
began.

• August 29 – September 28, 2025 — Began tomato experiments, selecting pots for four different treatment
groups. Conducted initial experiments and collected ongoing data to evaluate the effects of treatments over time.

> Participants: Anderson Zhang, Joy Jiang, Lucas Chen

• September 21 — Completed final ErucaBead and tested strength and durability.

> Participants: Anderson Zhang, Joy Jiang

Human Practice & Collaborations

• July 23 — Interviewed Zhang Li, founder of Euny Biotech, for insights on biotech entrepreneurship and
synthetic biology applications.

> Participants: Anderson Zhang, Bosen Du

• July 30 — Visited Shanghai Academy of Agricultural Sciences; met Professor Wen Guangyue and Professor Gao
Shigang to gather insights on market trends, practices, and regulations.

> Participants: Anderson Zhang, Bosen Du

• September 7 — Trip to the fruit and vegetable market to collect data and feedback from tomato producers,
providing insights for understanding market needs and trends.

> Participant: Chengxuan Zhang

• September 20 — Met with Team Crouton (SHSID) to discuss collaboration.

> Participants: Anderson Zhang, Joy Jiang, Yitong Chen

• September 24 — Hosted a lecture for students and STEM teachers to introduce our iGEM project.

> Participants: Anderson Zhang, Joy Jiang

Art, Design & Wiki

• May 5 — Completed team logo designs.

> Participants: Hsuan Shih, Joy Jiang

• September 9 — Finished art design of wiki home page.

> Participant: Hsuan Shih

• September 20 — Designed team gifts and pins.

> Participants: Hsuan Shih, Anderson Zhang

• September 25 — Designed team uniforms for production.

> Participants: Lucas Chen, Joy Jiang

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Overview

In our project, to prevent and control tomato bacterial wilt caused by Ralstonia
solanacearum, we have completed
work on five modules, namely the fatty acid induction system, the erucamide production sensing system, the
hydrogel encapsulation system, and the verification system for erucamide secretion by tomato seedlings infected
with Ralstonia solanacearum、verification of pressure-bearing performance for ErucaBead product beads. The fatty
acid induction system responds to the glycolytic system, initiates fatty acid synthesis, and activates the
downstream erucamide production sensing system. The hydrogel encapsulation system, on the other hand, encapsulates
the components required for the above two systems in gel particles, facilitating storage and use. The infection
experiment verified the functionality, disease resistance, and specificity of the functional Escherichia coli.The
pressure-bearing experiment is aimed at finding the optimal ratio concentration of sodium alginate to optimize the
comprehensive performance of ErucaBead products.

In our project, we followed the DBTL (Design-Build-Test-Learn) principle, tested the
functions of these four
systems during the project cycle, and made improvements and iterations based on the test results, which finally
enabled them to achieve our expected effects.

![](https://static.igem.wiki/teams/5889/engineering/image2.webp)

Figure 1 Flow Chart of Erucamide Production

Cycle 1: Fatty Acid Induction System

Design

After pathogen infection, tomatoes activate their glycolytic pathway to resist the invasion of external
pathogens. Since fatty acid production requires the PtsG gene to rapidly accumulate acetyl-CoA through glycolysis
and initiate the expression of FabH, thereby starting fatty acid synthesis, we performed expression and protein
purification of these two genes to activate our fatty acid induction system. We chose the most common bacterial
strain, Escherichia coli, for fermentation production. We used pET-28a(+) as the vector plasmid for these two
genes (Figure 2), and Escherichia coli BL21 (DE3) as the candidate strain for protein expression to obtain our
target proteins.

![](https://static.igem.wiki/teams/5889/engineering/image3.webp)
![](https://static.igem.wiki/teams/5889/engineering/image4.webp)

Figure 2 Plasmid Maps of fabH and PtsG

Build

To verify that our FabH and PtsG genes were successfully inserted into the pET-28a(+) plasmid and that this
plasmid was successfully introduced into Escherichia coli BL21 (DE3), we used specific primers for PCR
amplification of the plasmids extracted from Escherichia coli (Figure 3).

![](https://static.igem.wiki/teams/5889/engineering/4.webp)

Figure 3. Plasmids after agarose gel electrophoresis. Left panel: PtsG. Middle panel: FabH. Right
panel: Size of
bands corresponding to the Marker. Plasmids were extracted from Escherichia coli.

Test

We conducted protein expression experiments using Escherichia coli BL21 (DE3) that had been successfully
transformed with pET-28a(+) carrying either FabH or PtsG. The proteins were purified, and the Coomassie Brilliant
Blue method was used to perform electrophoretic analysis of protein bands in the solutions obtained during protein
purification (Figure 4). As shown in the figure, Elution 3 is the final collected protein solution.

![](https://static.igem.wiki/teams/5889/engineering/3.webp)

Figure 4. Protein size distribution map after protein electrophoresis. Left panel: PtsG. Middle
panel: FabH. Right panel: Size map of bands corresponding to the Marker. Proteins were extracted from Escherichia
coli.

Learn

Before column chromatography, the original sample showed numerous and dense impurity bands, indicating a high
content of impurity proteins. Both PtsG and FabH proteins were obscured by these impurity proteins, making it
difficult to identify them clearly. After column chromatography, the impurity bands in both gels were
significantly reduced, demonstrating that the column chromatography step can initially remove some impurity
proteins for both proteins. During the Washing 1 and Washing 2 stages, the impurity bands continued to change:
non-specifically bound impurity proteins were gradually eluted, and their abundance showed a decreasing trend.

In the specific elution (Elution 1, Elution 2, Elution 3) step, the PtsG protein band (around 53 kD on the
left) and the FabH protein band (around 35 kD on the right) both exhibited a consistent pattern: "the band in
Elution 1 was the clearest, the band in Elution 2 faded, and the band in Elution 3 faded further". This reflects
that the PtsG and FabH proteins were gradually eluted under specific conditions (such as imidazole, pH variation,
etc.), and both were mainly enriched in Elution 1. The contents of the two proteins in the subsequent elution
fractions decreased sequentially.

Overall, the purification effect of the two proteins was favorable: Elution 1 was the main collection
fraction for both PtsG and FabH proteins, with the highest purity and concentration; Elution 2 and Elution 3 could
recover part of the corresponding proteins, but their contents decreased gradually.

Cycle 2: Erucamide Production Sensing System

Design

The synthesis of erucamide relies on an amidation reaction: erucic acid + glutamine → erucamide + glutamic
acid. GlnA provides the amide group donor (glutamine) required for the reaction in erucamide production, serving
as the direct nitrogen source for erucamide synthesis, and maintains the intracellular glutamine pool to support
the activity of fatty acid amide hydrolases FAA1/FAA2. We chose the most common bacterial strain, Escherichia
coli, for fermentation production. We used pET-28a(+) as the vector plasmid for this gene (Figure 5), and
Escherichia coli BL21 (DE3) as the candidate strain for protein expression to obtain our target protein.

![](https://static.igem.wiki/teams/5889/engineering/5.webp)

Build

To verify that our GlnA gene was successfully inserted into the pET-28a(+) plasmid and that this plasmid was
successfully introduced into Escherichia coli BL21 (DE3), we used specific primers for PCR amplification of the
plasmids extracted from Escherichia coli (Figure 6: Left).

Test

We conducted protein expression experiments using Escherichia coli BL21 (DE3) that had been successfully
transformed with pET-28a(+) carrying GlnA. The protein was purified, and the Coomassie Brilliant Blue method was
used to perform electrophoretic analysis of protein bands in the solutions obtained during protein purification
(Figure 6: Right).

Learn

Analysis of protein purification in each lane from left to right is as follows: The original sample before
loading onto the column showed multiple impurity bands, indicating a high content of impurity proteins—this made
the target protein GlnA (51kD) indistinct. The flow-through fraction (corresponding to "flow rate" mentioned)
still exhibited numerous bands, which means some unbound proteins (including impurity proteins and the target
protein) were washed out. During the high-salt washing step for impurity removal, a high-salt buffer was used to
elute non-specifically bound proteins; the presence of many bands in this lane indicated that impurity proteins
were washed off. Washing 1 and Washing 2 continued to elute impurity proteins, leading to gradually fading bands
and a slight reduction in the number of impurity proteins. For Elution 1, Elution 2, and Elution 3, specific
elution conditions (such as imidazole, pH variation, etc.) were applied to elute the target protein GlnA. A
distinct single band appeared around 51kD, with the target protein concentration being the highest in Elution 1
and gradually decreasing in Elution 2 and 3—this confirms that the target protein was successfully eluted and
enriched.

Overall, the protein purification achieved a good effect: the elution fractions showed a clear single band
at 51kD with very few impurity proteins. Among these fractions, Elution 1 was the main collection fraction, with
the highest purity and concentration. Although Elution 2 and 3 could recover part of the protein, their purity
might be slightly lower.

Cycle 3: Hydrogel Encapsulation System

Design

To address the issue of protein instability and standardize pesticide dosage, we aimed to package our product
in a specific form to prevent misuse. Drawing inspiration from the design of laundry detergent pods, which consist
of individual beads enclosed in PVA films, we conducted research and found that PVA films possess excellent water
solubility, biocompatibility, degradability, high mechanical properties, as well as high strength, ease of
processing, low production cost, and strong practicality. Therefore, we decided to use PVA films to encapsulate
our three required proteins.

In the design of ErucaBead, we mixed the three protein solutions and then packaged them in water-soluble PVA
films. The resulting composite has strong stability and can quickly dissolve in water for direct application to
bacterial strains.

![](https://static.igem.wiki/teams/5889/engineering/6.webp)

Build

We dropped an aqueous solution of purified proteins at a certain concentration onto a sodium alginate solution,
then mixed the solution and dropped it into calcium chloride. Finally, we packaged these mixed liquids into PVA
films of a certain size and sealed them using a heat sealer to produce our ErucaBead (Figure 7).

Test

To verify the activity maintenance effect of functional protein solutions carrying PtsG, FabH, and GlnA genes
in a glycerol system, we performed verification through agarose gel electrophoresis. The experimental lanes from
left to right are as follows:

![](https://static.igem.wiki/teams/5889/engineering/7.webp)

1. Detectability of Proteins and Effectiveness of Expression/Purification

The left-side lanes correspond to different proteins (PtsG, FadH, GlnA) and protein combinations,
respectively. All lanes labeled with target proteins (e.g., lanes for PtsG alone, FadH alone, and GlnA alone)
showed specific bands (marked by red boxes). This indicates that after expression or purification, these proteins
could be effectively detected via SDS-PAGE, confirming the success of protein preparation.

2. Effect of 50% Glycerol on Protein Electrophoretic Behavior

When comparing the lanes of "protein alone" and "protein + 50% glycerol" (e.g., PtsG vs. PtsG + 50%
glycerol; FadH vs. FadH + 50% glycerol; GlnA vs. GlnA + 50% glycerol):

- There was no significant change in \*\*band position (molecular weight mobility)\*\*. Under SDS conditions,
proteins are denatured, and their mobility is mainly determined by molecular weight—glycerol did not alter the
molecular weight characteristics of the proteins.

- There was no significant difference in \*\*band intensity (protein content/solubility)\*\*. This suggests that
50% glycerol did not cause protein degradation or massive aggregation, and had minimal impact on protein
solubility and electrophoretic detectability.

3. Stability of the Multi-Protein Mixed System

By observing the multi-protein combination lanes (e.g., GlnA + PtsG + FadH + GlnA + 50% glycerol, PtsG +
FadH + 50% glycerol):

The specific bands of each target protein were still clearly present, with no obvious band disappearance,
new band formation, or sudden decrease in band intensity. This indicates that in the presence of 50% glycerol, no
significant degradation or aggregation-related interactions occurred between these proteins, and the mixed system
exhibited good stability.

Learn

1. The three proteins (PtsG, FadH, and GlnA) exhibit good intrinsic stability and can be effectively detected via
SDS-PAGE after expression/purification.

2. 50% glycerol can serve as a stabilizer: it neither significantly alters the electrophoretic mobility
(molecular weight characteristics) of these proteins nor causes protein degradation or aggregation. It is capable
of maintaining protein solubility and stability during sample processing/storage without interfering with
electrophoretic analysis.

3. In the multi-protein mixed system, each protein remains stable in the presence of 50% glycerol, and no
significant degradation or aggregation caused by protein-protein interactions has occurred.

4. Our ErucaBead can stably encapsulate the functional proteins (PtsG+FabH+GlnA) and release them stably
upon dissolution in water. Subsequently, it can enable the continuous secretion of functional proteins via an
Escherichia coli vector to synthesize and release erucamide, which exerts a favorable effect on tomatoes infected
with Ralstonia solanacearum. Therefore, we can conclude that ErucaBead, as our final product, can maintain the
activity of its active ingredients during storage, demonstrate favorable efficacy in application, and bring more
benefits and convenience to farmers.

Cycle 4: Verification of Erucamide Secretion in Tomato Seedlings Infected with Ralstonia
solanacearum

Design

The core objective of this phase is to "verify whether functional Escherichia coli (harboring expression
products of functional genes related to erucamide secretion) can enhance tomato resistance to Ralstonia
solanacearum by regulating the plant’s own erucamide secretion". The independent variables were defined as the
application of functional E. coli suspension (including "application or not" and "differences from negative
control E. coli") and infection treatment with Ralstonia solanacearum; the dependent variables included the
disease index of tomato plants, the in vivo erucamide secretion content, and plant survival rate. Meanwhile,
irrelevant variables were strictly controlled.

Tomato seedlings at the 4-5 leaf stage with uniform growth status were selected. A standard strain of
Ralstonia solanacearum was cultured and diluted uniformly to a bacterial suspension of specific concentration. All
plants were placed in the same greenhouse environment with a temperature of 25-28°C, humidity of 60%-70%, and a
photoperiod of 16h light/8h dark.

On this basis, following the classic framework of "blank control - positive control - negative control -
experimental group", 4 treatment groups were set up (8 seedlings per group, with 3 replicates) (Table 1).

Table 1: Experimental Grouping Table

![](https://static.igem.wiki/teams/5889/engineering/8.webp)

The blank control group was only irrigated with sterile water without Ralstonia solanacearum inoculation, aiming
to determine the basal secretion level of erucamide.

The positive control group was only irrigated with Ralstonia solanacearum suspension without E. coli BL21
(DE3) application, so as to clarify the disease pattern under natural infection conditions.

The negative control group was first irrigated with control E. coli BL21 (DE3) (without functional gene
expression products), and then irrigated with Ralstonia solanacearum suspension after 24 hours, which was designed
to rule out interference from non-functional components.

The experimental group was first irrigated with E. coli BL21 (DE3) containing expression products of
relevant functional genes, and then irrigated with Ralstonia solanacearum suspension after 24 hours, in order to
verify the core hypothesis.

In addition, a multi-time-point detection scheme was designed: the disease index was counted daily starting
from the 3rd day after infection; leaf samples were collected on the 21st day after infection to detect erucamide
content, and plant survival rate was also counted on the 21st day after infection. This design enabled dynamic
tracking of the correlation between erucamide secretion and disease progression.

Build

This phase focuses on standardized preparation of experimental materials, and establishment of the experimental
environment.

**Standardized Preparation of Experimental Materials**

Plasmids expressing three newly synthesized functional genes were transformed into Escherichia coli BL21
strain, and the bacterial solution was cultured on an expanded scale.

For the preparation of Ralstonia solanacearum suspension: first, the standard strain Ralstonia solanacearum
GMI1000 was inoculated onto NA medium and cultured at 30°C for 24 hours to pick single colonies; then, the
colonies were transferred to NB liquid medium and cultured in a shaker at 30°C and 180 rpm for 16 hours. The OD600
value was measured with a spectrophotometer and adjusted to 0.5 (approximately 10^8 CFU/mL).

Commercially available tomato seedlings were selected as experimental plants. They were cultivated in a
sterile seedling substrate until reaching the 4-5 leaf stage; seedlings with uniform plant height and leaf count
were then selected and transplanted into uniform culture pots (10 cm in diameter), followed by acclimation to the
greenhouse environment for 3 days.

**Liquid chromatography (LC) detection system was established**

 C18 chromatographic column (250 mm × 4.6 mm, 5 μm) was used, with methanol-water (85:15, v/v) as the mobile
phase. The flow rate was set to 1.0 mL/min, the detection wavelength to 220 nm, and the column temperature to
30°C. A standard curve of erucamide with a concentration gradient of 0.1-100.0 μg/mL was plotted to ensure the
accuracy of subsequent content detection. As shown in Figure 9, we first prepared a standard curve using standard
samples, where the horizontal axis represents the sample peak area and the vertical axis represents the sample
concentration (µg/g). It can be observed that our standard curve is expressed as y = 3E-07x - 13.72, with R² =
0.999, indicating a high confidence level of the standard curve.

![](https://static.igem.wiki/teams/5889/engineering/9.webp)

Operations in this phase were strictly performed in accordance with the design plan:

(1) During grouping treatment（Table2), 10 mL of the corresponding E. coli BL21 (DE3) solution was irrigated
to the root of each tomato seedling in the negative control group and experimental group; the blank control group
and Ralstonia solanacearum control group were irrigated with the same volume of sterile water(Table 3).

(2) After 24 hours, 10 mL of standardized Ralstonia solanacearum suspension was irrigated to each seedling
in the positive control group, negative control group, and experimental group; the blank control group was
continuously irrigated with the same volume of sterile water. Meanwhile, all plants were numbered and their
initial growth status was recorded（Figure 10).

![](https://static.igem.wiki/teams/5889/engineering/10.webp)

(3) During the dynamic monitoring phase, the disease status of plants was observed at 9:00 a.m. daily in
accordance with the 0-4 grade standard. The disease grade of each plant was recorded, the disease index was
calculated, and a dynamic disease progression curve was plotted. Meanwhile, the leaf wilting rate, stem softening
status, and plant death time were recorded synchronously.

For sample detection: on the 7th, 14th, and 21st days post-infection, 3 plants were randomly selected from
each group. 0.5 g of tissue was collected from each of 3-4 functional leaves per plant. After freezing and
grinding with liquid nitrogen, 5 mL of chromatographically pure methanol was added for ultrasonic extraction (300
W, 25°C) for 30 minutes. The mixture was centrifuged at 12,000 rpm and 4°C for 10 minutes to collect the
supernatant. The extraction process was repeated twice, and the supernatants from the three extractions were
combined and concentrated to 1 mL. Finally, the concentrated solution was filtered through a 0.22 μm filter
membrane to obtain the sample to be tested.

Test

We performed liquid chromatography (LC) analysis on the samples to be tested (Figure 11). The highest peak
appearing at approximately 37 minutes was identified as the target sample peak for calculation. The erucamide
content of the four groups was calculated using the standard curve, and the results are shown in Table 4. These
results are consistent with our predicted content, indicating that our functional bacteria exhibit a therapeutic
effect against Ralstonia solanacearum.​​

![](https://static.igem.wiki/teams/5889/engineering/11.webp)

The blank control group had no Ralstonia solanacearum infection, so its disease index remained 0 throughout the
experiment. The erucamide content was maintained at a basal level of 14-15 μg/g fresh weight, and the plant
survival rate was 100%. The positive control group and negative control group had no effective disease resistance
intervention: their disease indices both exceeded 90, erucamide content decreased to 3.3-4.5 μg/g fresh weight,
and plant survival rate was less than 15%. There were no significant differences in all indicators between these
two groups. The experimental group had a disease index significantly lower than the previous two groups (&lt30),
erucamide content significantly increased (16-17 μg/g fresh weight), and the plant survival rate reached
75%（Figure 12/Table 5）.

![](https://static.igem.wiki/teams/5889/engineering/12.webp)

Learn

Functional E. coli suspension significantly promotes erucamide secretion in tomatoes: After infection, the
erucamide content in the functional E. coli group was significantly higher than that in the Ralstonia solanacearum
control group and the negative control E. coli group (P
&lt0.05), confirming that the E. coli suspension activates the erucamide synthesis pathway in tomatoes.​
Erucamide secretion is directly related to disease resistance: The disease index of the functional E. coli group (
&lt30) was only 1/3 to 1/2 of that of the negative and positive control groups, and the plant survival rate (75%)
was 7-8 times higher than that of the control groups. Moreover, in stages with higher erucamide content, the
disease index was lower, reflecting a negative correlation of "erucamide content ↑ → disease severity ↓" and
verifying the core hypothesis of "enhancing disease resistance by regulating erucamide secretion" .​
The
effect of functional E. coli suspension is specific: There were no significant differences in all indicators
between the negative control E. coli group and the Ralstonia solanacearum control group, which rules out the
possibility that "non-functional components of the E. coli suspension interfere with the experimental results" and
indicates that the disease resistance effect comes from the functional gene expression products in the protein
solution.

Cycle 5: Verification of Pressure Resistance of ErucaBead Product Beads

Design (Scheme Design): Clarify Objectives and Technical Framework

Design Objectives Focusing on the core needs of the ErucaBead product, address two key issues:• Verify the
pressure-bearing capacity of individual beads to ensure that the product (70g per bag, containing 15 spherical
beads with a diameter of 13mm, sealed in PVA film, and filled with calcium alginate beads + glycerin) does not
rupture during transportation and storage;• Explore the impact of sodium alginate concentration on the pressure
resistance of beads, and determine the optimal ratio by combining the degradation rate of calcium alginate in
the
natural soil environment.

Core Design Elements• Sample specification design: Fix the bead shape (spherical, 13mm in diameter), the
number per bag (15 beads), and the packaging method (PVA film sealing), and only change the sodium alginate
concentration (variables: 0.5%, 1%, 1.5%, 2%) to eliminate interference from irrelevant variables;• Testing
device
design: For the requirement of "individual bead pressure bearing", design a device with a "load-bearing platform
+
uniform pressure application module" to ensure uniform pressure loading (avoiding data deviation caused by
instantaneous impact), and record the maximum pressure value when the bead ruptures;• Analysis logic design:
Plan
to establish a comprehensive model through "actual pressure resistance data + degradation rate characteristics
(based on the hypothetical law of calcium alginate materials)", and balance the relationship between the two to
derive the optimal concentration.

![](https://static.igem.wiki/teams/5889/engineering/13.webp)

Build (Sample and Device Construction): Implement the Design Scheme

Bead Sample PreparationMass-produce samples with different concentrations according to the design
specifications, following these steps:• Prepare 4 groups of sodium alginate solutions (with concentrations of
0.5%, 1%, 1.5%, and 2% respectively). Each group of solutions reacts with a calcium salt solution (such as calcium
chloride) in a fixed proportion to form spherical calcium alginate gel beads (13mm in diameter, with size
consistency controlled by molds);• Take 15 beads from each group, mix them with a fixed amount of glycerin, and
seal them with PVA film into standard sample bags with a total weight of 70g (for later use). Meanwhile, reserve
individual bead samples for pressure resistance testing;

Construction of Pressure Testing Device• Basic structure: Use a 3D-printed load-bearing platform (area ≥ 5cm
× 5cm to ensure stable placement of beads), and add weights one by one on top;• Data collection: Observe and
manually record (range 0-1000g, accuracy ±1g), collect pressure changes in real-time until the bead ruptures (the
rupture point is determined when the pressure drops suddenly).

![](https://static.igem.wiki/teams/5889/engineering/14.webp)

Test (Acquire Key Data)

Testing Process (Individual Bead Pressure Test)• Sample placement: Place a single bead (randomly selected from
the corresponding concentration sample group; each group is tested 3 times, and the average value is taken to
reduce accidental errors) in the center of the load-bearing platform;• Pressure loading: Gradually add weights;•
Data recording: When the pressure value drops suddenly (bead ruptures), stop loading and record the pressure value
at this moment as the "individual bead pressure-bearing value";

Summary of Test ResultsAfter repeated tests, the pressure-bearing data of the 4 concentration groups are as
follows (average values):

![](https://static.igem.wiki/teams/5889/engineering/15.webp)

Meanwhile, based on the material properties of calcium alginate, the degradation rate law is supplemented (for
subsequent analysis): the higher the concentration, the denser the calcium alginate gel network, and the slower
the microbial decomposition in the natural soil environment. It is assumed that the complete degradation time (90%
mass loss) is: 12 days for 0.5% concentration, 18 days for 1% concentration, 25 days for 1.5% concentration, and
35 days for 2% concentration.

Learn (Data and Model Analysis): Derive the Optimal Ratio

1. Comprehensive Data Analysis (Establish Mathematical Model)• Model construction: Take "pressure resistance
(positive indicator, higher is better)" and "degradation rate (reverse indicator, shorter time is more
environmentally friendly)" as the core, calculate the comprehensive score through standardization (eliminate
dimension differences) and weight allocation (0.5 weight for both pressure resistance and degradation in a
balanced scenario);• Standardization and scoring:◦ Pressure resistance standardization: Map 400g (0.5%) - 580g
(2%) to 0-1 points (higher value indicates better pressure resistance);◦ Degradation standardization: Map 12 days
(0.5%) - 35 days (2%) to 0-1 points (higher value indicates faster degradation);◦ Comprehensive score = 0.5 ×
pressure resistance standardized score + 0.5 × degradation standardized score;

2. Conclusions and Insights• Derivation of optimal ratio: Through calculation, 1% concentration sodium
alginate has the highest comprehensive score (pressure resistance of 470g, better than 0.5%; degradation time of
18 days, better than 1.5% and 2%), which is a balanced solution of "pressure resistance meeting usage requirements
+ relatively environmentally friendly degradation rate" and is determined as the optimal product ratio;• Key
insights:◦ Pressure resistance law: Sodium alginate concentration is positively correlated with pressure-bearing
value (for every 0.5% increase in concentration, the pressure-bearing value increases by an average of about
45-90g). However, the degradation time is significantly prolonged (35 days) at 2% concentration, resulting in
reduced environmental friendliness;◦ Optimization directions: In the future, it is necessary to actually measure
the degradation rate of calcium alginate in natural soil (to verify the hypothetical data) and incorporate the
"cost variable" (higher concentration sodium alginate has higher raw material costs) to further improve the model.

References

[1] Xu, B. B., Wang, R. J., Guo, Y., et al. Analysis and evaluation of nutritional quality of different cherry
tomato varieties [J]. Food and Nutrition in China, 2025, 31(07): 62-67. (In Chinese)

[2] Zhang, H. L., Sun, J., Pang, Z. Q. Study on quality and storage-transportation resistance of tomatoes
[J]. Jiangsu Agricultural Sciences, 2012(8): 269-270. (In Chinese)

[3] Wang, C., Ye, H. X., Ye, F. H., et al. Comparison and evaluation of cherry tomato varieties under
protected cultivation [J]. Journal of Zhejiang Agricultural Sciences, 2015, 56(8): 1214-1216. (In Chinese)

[4] Alshatwi, A. A., Obaaid, M. A. A., Sedairy, S. A. A., et al. Tomato powder is more protective than
lycopene supplement against lipid peroxidation in rats [J]. Nutrition Research, 2010, 30(1): 66-73.

[5] Xie, L., Ying, Y. Discrimination of transgenic tomatoes based on visible near-infrared spectra [J].
Analytica Chimica Acta, 2007, 584(2): 379-384.

[6] Med, A. I. Healthy living is the best revenge: findings from the European Prospective Investigation Into
Cancer and Nutrition-Potsdam study [J]. Archives of Internal Medicine, 2009, 169(15): 1355-1362.

[7] Qin, L. J., Li, J. W., Li, H. M., et al. Comparative study on growth and quality characteristics of
different new cherry tomato varieties [J]. Journal of Chifeng University (Natural Science Edition), 2018, 34(11):
16-18. (In Chinese)

[8] Butelli, E., Titta, L., Giorgio, M., et al. Enrichment of tomato fruit with health-promoting
anthocyanins by expression of select transcription factors [J]. Nature Biotechnology, 2008, 26(11): 1301-1308.

[9] Zhao, J., Ping, A. M., Hou, L. P., et al. Comparison of quality traits among different cherry tomato
varieties [J]. Shaanxi Journal of Agricultural Sciences, 2017, 63(2): 19-22, 26. (In Chinese)

[10] Li, Y. D., Lu, Z. Q., Bai, X. L., et al. Effects of different planting densities and fruit cluster
numbers on yield and quality of early spring greenhouse tomatoes [J]. Northern Horticulture, 2018(11): 38-42. (In
Chinese)

[11] Mi, G. Q., Cheng, Z. F., Zhao, X. B., et al. Investigation on fruit traits of main tomato varieties in
protected cultivation in North China [J]. Journal of Henan Agricultural Sciences, 2013, 42(9): 91-94. (In Chinese)

[12] Li, H., Deng, Z., Liu, R., et al. Characterization of phytochemicals and antioxidant activities of a
purple tomato (Solanum lycopersicum L.) [J]. Journal of Agricultural and Food Chemistry, 2014, 62(25): 6024-6034.

[13] Rao, A. V., & Rao, L. G. Carotenoids and human health [J]. Pharmacological Research, 2007, 55(3):
207-216.

[14] Story, E. N., Kopec, R. E., Schwartz, S. J., & Harris, G. K. An update on the health effects of tomato
lycopene [J]. Annual Review of Food Science and Technology, 2010, 1: 189-210.

[15] Beckles, D. M. Factors affecting the postharvest soluble solids and sugar content of tomato (Solanum
lycopersicum L.) fruit [J]. Postharvest Biology and Technology, 2012, 63(1): 129-140.

[16] Mansfield, J., Genin, S., Magori, S., et al. Top 10 plant pathogenic bacteria in molecular plant
pathology [J]. Molecular Plant Pathology, 2012, 13(6): 614-629.

[17] Hayward, A. C. Biology and epidemiology of bacterial wilt caused by Pseudomonas solanacearum [J].
Annual Review of Phytopathology, 1991, 29(1): 65-87.

[18] Genin, S., & Denny, T. P. Pathogenomics of the Ralstonia solanacearum species complex [J]. Annual
Review of Phytopathology, 2012, 50: 67-89.

[19] Wang, T. L., Wang, H., Feng, J. Research progress on the pathogenic mechanism of Ralstonia solanacearum
[J]. Acta Microbiologica Sinica, 2020, 60(10): 2059-2073. (In Chinese)

[20] Vasse, J., Frey, P., & Trigalet, A. Microscopic studies of intercellular infection and protoxylem
invasion of tomato roots by Pseudomonas solanacearum [J]. Molecular Plant-Microbe Interactions, 1995, 8(2):
241-251.

[21] Tans-Kersten, J., Huang, H., & Allen, C. Ralstonia solanacearum needs motility for invasive virulence
on tomato [J]. Journal of Bacteriology, 2001, 183(12): 3597-3605.

[22] Goddard, J. M., & Hotchkiss, J. H. Polymer surface modification for the attachment of bioactive
compounds [J]. Progress in Polymer Science, 2007, 32(7): 698-725.

[23] Kenawy, E. R., Worley, S. D., & Broughton, R. The chemistry and applications of antimicrobial polymers:
a state-of-the-art review [J]. Biomacromolecules, 2007, 8(5): 1359-1384.

[24] Wang, L., Chen, G. X., Li, B. Synthesis of long-chain fatty acid amide derivatives and study on their
antimicrobial properties [J]. New Chemical Materials, 2020, 48(08): 255-259. (In Chinese)

[25] Munoz-Bonilla, A., & Fernández-García, M. Polymeric materials with antimicrobial activity [J]. Progress
in Polymer Science, 2012, 37(2): 281-339.

[26] Berg, J. M., Tymoczko, J. L., Gatto, G. J., & Stryer, L. Biochemistry (8th ed.) [M]. New York: W. H.
Freeman, 2015. (Chapter 16: Glycolysis and Gluconeogenesis)

[27] Nelson, D. L., & Cox, M. M. Lehninger Principles of Biochemistry (7th ed.) [M]. New York: W. H.
Freeman, 2017. (Chapter 14: Glycolysis, Gluconeogenesis, and the Pentose Phosphate Pathway)

[28] Vander Heiden, M. G., Cantley, L. C., & Thompson, C. B. Understanding the Warburg effect: the metabolic
requirements of cell proliferation [J]. Science, 2009, 324(5930): 1029-1033.

[29] Wang, J. Y., Zhu, S. G., Xu, C. F. Biochemistry (3rd ed.) [M]. Beijing: Higher Education Press, 2002.
(In Chinese)

{% 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 %}

## What Should this Page Contain?

---

Provide comprehensive, step-by-step protocols for all experiments conducted. Include a list of materials, reagents, and equipment used. Specify concentrations, volumes, incubation times, temperatures, and other critical parameters. Document any modifications or optimizations made to standard protocols.

Explain the purpose of each experiment and its relevance to your project goals. Describe the experimental design, including controls and replicates. Provide a clear rationale for the chosen methods and approaches.

Document any troubleshooting steps taken and optimizations made during the experimental process. Share any lessons learned that could benefit future teams.

## Inspirations

---

* [2024 Heidelberg](https://2024.igem.wiki/heidelberg/experiments)
* [2024 Marburg](https://2024.igem.wiki/marburg/experiments)
* [2024 EPFL](https://2024.igem.wiki/epfl/experiments)
* [2023 Estonia-TUIT](https://2023.igem.wiki/estonia-tuit/experiments)

{% endblock %}

{% extends "layout.html" %}
{% block lead %}Describe how and why you chose your iGEM project.{% endblock %}
{% block page\_content %}

Overview

In our project, to prevent and control tomato bacterial wilt caused by Ralstonia solanacearum, we completed
work on four modules, namely the fatty acid induction system, the erucamide production sensing system, the
hydrogel encapsulation system, and the verification experiment for erucamide secretion in plant infection. The
fatty acid induction system responds to the glycolytic system, initiates fatty acid synthesis, and activates the
downstream erucamide production sensing system. The hydrogel encapsulation system, on the other hand, encapsulates
the components required by the above two systems in gel particles, facilitating storage and application. The
infection experiment verified the functionality, disease resistance, and specificity of the functional Escherichia
coli (Figure 1).

![](https://static.igem.wiki/teams/5889/results/21.webp)

1. Fatty Acid Induction System

After pathogen infection, tomato will activate its glycolytic pathway to resist the invasion of external
pathogens. Since the generation of fatty acids requires the PtsG gene to rapidly accumulate acetyl - CoA through
glycolysis and initiate the expression of FabH, thus starting fatty acid synthesis, we carried out the expression
and protein purification of these two genes, so as to start our fatty acid induction system. We selected the most
common strain Escherichia coli for fermentation production. We used pET - 28a(+) as the vector plasmid for these
two genes, and E. coli BL21(DE3) as the candidate strain for protein expression to obtain our target proteins.

To verify that our FabH and PtsG genes were successfully inserted into the pET - 28a(+) plasmid, and that
this plasmid was successfully introduced into E. coli BL21(DE3), we used specific primers to amplify the plasmids
extracted from E. coli by PCR (Figure 2). The experimental results showed that we had successfully synthesized
these two plasmids.

![](https://static.igem.wiki/teams/5889/results/22.webp)

Before column chromatography, the original sample contained a large number of dense impurity protein bands, and
the target proteins PtsG and FabH were obscured, making them difficult to identify clearly.

After column chromatography, the impurity protein bands in the gel were significantly reduced, indicating
that this step could initially remove some impurity proteins for both proteins. During the Washing 1 and Washing 2
stages, the non-specifically bound impurity proteins were gradually eluted, and their abundance showed a
decreasing trend.

In the specific elution (Elution 1, Elution 2, Elution 3) step, PtsG (around 53 kD on the left) and FabH
(around 35 kD on the right) showed a consistent pattern: the band in Elution 1 was the clearest, the band in
Elution 2 faded, and the band in Elution 3 faded further. This indicated that both proteins were gradually eluted
under specific conditions (such as imidazole, pH variation, etc.) and were mainly enriched in Elution 1, with the
contents of the two proteins in the subsequent elution fractions decreasing sequentially.

In conclusion, the purification effect of the two proteins was good: Elution 1 was the main collection
fraction for both PtsG and FabH, with the highest purity and concentration; Elution 2 and Elution 3 could recover
part of the target proteins, but their contents decreased gradually.

2. Erucamide Production Sensing System

The synthesis of erucamide relies on an amidation reaction: Erucic acid + Glutamine → Erucamide + Glutamic
acid. GlnA provides the amide group donor (glutamine) required for the reaction in erucamide production, serving
as the direct nitrogen source for erucamide synthesis. Additionally, it maintains the intracellular glutamine pool
and supports the activity of fatty acid amide hydrolases FAA1/FAA2.

We selected the most common strain, Escherichia coli, for fermentation production. We used pET-28a(+) as the
vector plasmid for these two genes (GlnA and FAA1/FAA2) and E. coli BL21(DE3) as the candidate strain for protein
expression to obtain our target proteins.

To verify that our GlnA gene was successfully inserted into the pET-28a(+) plasmid and that this plasmid was
successfully introduced into E. coli BL21(DE3), we used specific primers to amplify the plasmids extracted from E.
coli via PCR (Figure 4). The experimental results showed that we had successfully constructed these two plasmids.

![](https://static.igem.wiki/teams/5889/results/23.webp)

Figure 4 Plasmids after Agarose Gel Electrophoresis;Left panel: Plasmid containing GlnA gene;Middle panel: Band
size reference map corresponding to the marker;Right panel: Protein size distribution map of GlnA after protein
electrophoresis
The plasmids were extracted from Escherichia coli.

Analysis of protein purification in each lane from left to right is as follows: The original
sample before
loading onto the column showed multiple impurity bands, indicating a high content of impurity proteins—this made
the target protein GlnA (51kD) indistinct. The flow-through fraction (corresponding to "flow rate" mentioned)
still exhibited numerous bands, which means some unbound proteins (including impurity proteins and the target
protein) were washed out. During the high-salt washing step for impurity removal, a high-salt buffer was used to
elute non-specifically bound proteins; the presence of many bands in this lane indicated that impurity proteins
were washed off. Washing 1 and Washing 2 continued to elute impurity proteins, leading to gradually fading bands
and a slight reduction in the number of impurity proteins. For Elution 1, Elution 2, and Elution 3, specific
elution conditions (such as imidazole, pH variation, etc.) were applied to elute the target protein GlnA. A
distinct single band appeared around 51kD, with the target protein concentration being the highest in Elution 1
and gradually decreasing in Elution 2 and 3—this confirms that the target protein was successfully eluted and
enriched.

Overall, the protein purification achieved a good effect: the elution fractions showed a clear single band
at 51kD with very few impurity proteins. Among these fractions, Elution 1 was the main collection fraction, with
the highest purity and concentration. Although Elution 2 and 3 could recover part of the protein, their purity
might be slightly lower.

3. Hydrogel Encapsulation System

To address the issue of protein instability and standardize pesticide dosage, we aimed to package our product
in a specific form to prevent misuse. Drawing inspiration from the design of laundry detergent pods— which consist
of individual beads enclosed in PVA (polyvinyl alcohol) films—we conducted research and found that PVA films
exhibit excellent water solubility, biocompatibility, and degradability, along with high mechanical strength, ease
of processing, low production costs, and strong practicality. Therefore, we decided to use PVA films to
encapsulate the three target proteins we required.

In the design of ErucaBead, we mixed the three protein solutions and then encapsulated the mixture in water
- soluble PVA films. The resulting composite exhibits high stability, can dissolve quickly in water, and can be
directly applied to the strains.

We dropped an aqueous solution of purified proteins at a specific concentration onto a sodium alginate
solution, then dripped the mixture into calcium chloride. Finally, we packaged this mixed liquid into PVA films of
a specific size and sealed the films using a heat sealer to produce our ErucaBead (Figure 5).

![](https://static.igem.wiki/teams/5889/results/24.webp)

To verify the activity maintenance effect of the functional protein solution carrying the PtsG, FabH, and GlnA
genes in the glycerol system, we conducted verification via agarose gel electrophoresis. The experimental lanes
from left to right are as follows:

![](https://static.igem.wiki/teams/5889/results/25.webp)

1. Detectability of Proteins and Effectiveness of Expression/Purification

The left-side lanes correspond to different proteins (PtsG, FadH, GlnA) and protein combinations,
respectively. All lanes labeled with target proteins (e.g., lanes for PtsG alone, FadH alone, and GlnA alone)
showed specific bands (marked by red boxes). This indicates that after expression or purification, these proteins
could be effectively detected via SDS-PAGE, confirming the success of protein preparation.

2. Effect of 50% Glycerol on Protein Electrophoretic Behavior

When comparing the lanes of "protein alone" and "protein + 50% glycerol" (e.g., PtsG vs. PtsG + 50%
glycerol; FadH vs. FadH + 50% glycerol; GlnA vs. GlnA + 50% glycerol):

- There was no significant change in band position (molecular weight mobility). Under SDS conditions,
proteins are denatured, and their mobility is mainly determined by molecular weight—glycerol did not alter the
molecular weight characteristics of the proteins.

- There was no significant difference in band intensity (protein content/solubility). This suggests that 50%
glycerol did not cause protein degradation or massive aggregation, and had minimal impact on protein solubility
and electrophoretic detectability.

3. Stability of the Multi-Protein Mixed System

By observing the multi-protein combination lanes (e.g., GlnA + PtsG + FadH + GlnA + 50% glycerol, PtsG +
FadH + 50% glycerol):

The specific bands of each target protein were still clearly present, with no obvious band disappearance,
new band formation, or sudden decrease in band intensity. This indicates that in the presence of 50% glycerol, no
significant degradation or aggregation-related interactions occurred between these proteins, and the mixed system
exhibited good stability（Figure 6) .

Summary

The three proteins (PtsG, FadH, and GlnA) exhibit good inherent stability and can be effectively detected by
SDS-PAGE after expression and purification.

50% glycerol can serve as a stabilizer: it neither significantly alters the electrophoretic mobility
(molecular weight characteristics) of these proteins nor causes protein degradation or aggregation. It maintains
the solubility and stability of the proteins during sample processing and storage, without interfering with
electrophoretic analysis.

In the multi-protein mixture system in the presence of 50% glycerol, each protein remains stable, and no
significant degradation or aggregation caused by protein-protein interactions occurs.

Our ErucaBead can stably encapsulate the functional proteins (PtsG + FabH + GlnA) and then release them
stably after dissolving in water. Subsequently, the Escherichia coli vector can be used to continuously secrete
the functional proteins for the synthesis and release of erucamide, which exerts a good effect on tomatoes
infected with Ralstonia solanacearum. Therefore, we can conclude that ErucaBead, as our final product, can
maintain the activity of active ingredients during storage, demonstrate good efficacy in application, and bring
more benefits and convenience to farmers.

4. Plant Infection Experiment

First, we generated a standard curve using standard samples, where the x-axis represents the sample peak area
and the y-axis represents the sample concentration (µg/g). The resulting standard curve is expressed by the
equation y = 3E-07x - 13.72 with a coefficient of determination (R²) of 0.999, indicating high confidence in the
standard curve （Figure 7).

![](https://static.igem.wiki/teams/5889/results/26.webp)

(1) Grouping and Treatment

For the negative control group and experimental group: 10 mL of the corresponding Escherichia coli BL21(DE3)
suspension was irrigated onto the root of each tomato plant.

For the blank control group and Ralstonia solanacearum control group: An equal volume (10 mL) of sterile
water was irrigated instead.

(2) Inoculation with Ralstonia solanacearum (24 Hours Later)

For the positive control group, negative control group, and experimental group: 10 mL of standardized
Ralstonia solanacearum suspension was irrigated onto each plant.

For the blank control group: 10 mL of sterile water was continued to be irrigated.

Meanwhile, all plants were numbered, and their initial growth status was recorded(Figure 8).

![](https://static.igem.wiki/teams/5889/results/27.webp)

(3)Dynamic Monitoring and Sample Detection

Dynamic Monitoring

Every morning at 9:00, the disease status of plants was observed using a 0-4 grade scale. The disease grade
of each plant was recorded, the disease index was calculated, and a disease progression curve was plotted.

Simultaneously, the rate of leaf wilting, the degree of stem softening, and the plant death time were
recorded.

Sample Detection

On the 21st day after infection, 3 plants were randomly selected from each group. From each plant, 3-4
functional leaves (0.5 g each) were collected.

The leaf samples were frozen and ground in liquid nitrogen, then mixed with 5 mL of chromatographically pure
methanol for ultrasonic extraction (300 W, 25 °C) for 30 minutes.The mixture was centrifuged at 12,000 rpm at 4 °C
for 10 minutes, and the supernatant was collected. This extraction process was repeated twice, and the
supernatants were combined and concentrated to 1 mL.The concentrated solution was filtered through a 0.22 μm
filter membrane to obtain the sample for detection.

We performed liquid chromatography (LC) analysis on the samples. The largest peak appearing at approximately
37 minutes was identified as the target sample peak. Using this peak and the standard curve, we calculated the
erucamide content of the four groups, as shown in Table 4. The results were consistent with our predictions,
confirming that our functional bacteria exhibit a therapeutic effect against Ralstonia solanacearum.

![](https://static.igem.wiki/teams/5889/results/28.webp)

In the blank control group, there was no Ralstonia solanacearum infection. The disease index remained 0, the
erucamide content was maintained at a basal level of 14-15 μg/g fresh weight, and the plant survival rate was
100%.

In the positive control group and negative control group, there was no effective disease-resistant
intervention. The disease index of both groups exceeded 90, the erucamide content decreased to 3.3-4.5 μg/g fresh
weight, the plant survival rate was less than 15%, and there were no significant differences in all indicators
between the two groups.

In the experimental group, the disease index was significantly lower than that of the previous two groups
(&lt30), the erucamide content was significantly increased (16-17 μg/g fresh weight), and the plant survival rate
reached 75%.

![](https://static.igem.wiki/teams/5889/results/29.webp)
![](https://static.igem.wiki/teams/5889/results/30.webp)

The suspension of functional Escherichia coli can significantly promote erucamide secretion in tomatoes: After
infection, the erucamide content in the functional E. coli suspension group was significantly higher than that in
the Ralstonia solanacearum control group and the negative control (non-functional E. coli suspension) group
(P&lt0.05), which confirms that the E. coli suspension exerts an activating effect on the erucamide synthesis
pathway in tomatoes.

Erucamide secretion is directly associated with disease resistance: The disease index of the functional E.
coli suspension group (&lt30) was only 1/3 to 1/2 of that of the negative and positive control groups, and the
plant survival rate (75%) was 7-8 times higher than that of the control groups. Moreover, during the stages when
erucamide content was higher, the disease index was lower, reflecting a negative correlation of "↑ Erucamide
content → ↓ Disease severity" and verifying the core hypothesis of "enhancing disease resistance by regulating
erucamide secretion".

The effect of the functional E. coli suspension is specific: There were no significant differences in all
indicators between the negative control (non-functional E. coli suspension) group and the Ralstonia solanacearum
control group. This rules out the possibility that "non-functional components in the E. coli suspension interfere
with the experimental results" and indicates that the disease-resistant effect originates from the functional gene
expression products in the bacterial suspension.

5. Verification of Pressure Resistance of ErucaBead Product Beads

**Sample and Device Construction**

Bead Sample PreparationMass-produce samples with different concentrations according to the design
specifications, following these steps:• Prepare 4 groups of sodium alginate solutions (with concentrations of
0.5%, 1%, 1.5%, and 2% respectively). Each group of solutions reacts with a calcium salt solution (such as calcium
chloride) in a fixed proportion to form spherical calcium alginate gel beads (13mm in diameter, with size
consistency controlled by molds);• Take 15 beads from each group, mix them with a fixed amount of glycerin, and
seal them with PVA film into standard sample bags with a total weight of 70g (for later use). Meanwhile, reserve
individual bead samples for pressure resistance testing;

Construction of Pressure Testing Device• Basic structure: Use a 3D-printed load-bearing platform (area ≥ 5cm
× 5cm to ensure stable placement of beads), and add weights one by one on top;• Data collection: Observe and
manually record (range 0-1000g, accuracy ±1g), collect pressure changes in real-time until the bead ruptures (the
rupture point is determined when the pressure drops suddenly).

![](https://static.igem.wiki/teams/5889/results/31.webp)

**Acquire Key Data**

Testing Process (Individual Bead Pressure Test)• Sample placement: Place a single bead (randomly selected
from the corresponding concentration sample group; each group is tested 3 times, and the average value is taken to
reduce accidental errors) in the center of the load-bearing platform;• Pressure loading: Gradually add weights;•
Data recording: When the pressure value drops suddenly (bead ruptures), stop loading and record the pressure value
at this moment as the "individual bead pressure-bearing value";

Summary of Test ResultsAfter repeated tests, the pressure-bearing data of the 4 concentration groups are as
follows (average values):

![](https://static.igem.wiki/teams/5889/results/32.webp)

{% endblock %}