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).
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.
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.
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.
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.
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.
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.
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).
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).
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:
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) .
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.
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).
(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).
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).
(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.
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.
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 (<30), the erucamide content was significantly increased (16-17 μg/g fresh weight), and the plant survival rate reached 75%.
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 (<30), the erucamide content was significantly increased (16-17 μg/g fresh weight), and the plant survival rate reached 75%.
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<0.05), 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 (<30) 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.
Erucamide secretion is directly associated with disease resistance: The disease index of the functional E. coli suspension group (<30) 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).
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).
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):
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):
