This project aims to develop a multifunctional engineered bacterial strain to efficiently degrade petroleum pollutants, enhance plant growth, and ensure biosafety. The system was optimized across four modules and seven engineering cycles:

Module 1: Alkane Degradation System

⏳ Cycle 1: Engineered strain with AlkM to degrade short-chain alkanes, validated by molecular docking.

⏳ Cycle 2: BsLac strain for long-chain alkane degradation, showing promising enzyme activity.

⏳ Cycle 3: Surface-displayed INP-BsLac, enhancing degradation efficiency.

Module 2: Surfactant Secretion System

⏳ Cycle 4: Bacillus subtilis strain secreting surfactin, increasing oil emulsification and degradation.

Module 3: Plant-Microbe Collaboration System

⏳ Cycle 5: IAM pathway strain producing IAA, promoting plant root growth.

⏳ Cycle 6: IPA pathway strain with higher IAA production for enhanced plant growth.

Module 4: Biosafety System

⏳ Cycle 7: Alkane-induced suicide system, ensuring bacterial survival only in polluted environments.

Design:

To degrade short-chain alkanes, we designed an AlkM-engineered strain. AlkM, derived from Acinetobacter, is particularly efficient in catalyzing the terminal hydroxylation of alkanes, which initiates the degradation of these hydrocarbons. This enzyme system has been shown to degrade long-chain alkanes. In our study, Escherichia coliwas used as the chassis microorganism, and a recombinant plasmid containing the alkM gene was constructed under the control of the T7 promoter, with the B0015 terminator. The engineered strain promoted the degradation of long-chain alkanes by catalyzing the oxidation of inert alkanes into more reactive alcohols through the activity of AlkM.

Build:

The alkM gene was synthesized and codon-optimized for expression in E. coli. Using NdeI and XhoI restriction sites, the gene was cloned into the pET23b plasmid, constructing a recombinant plasmid (Azenta, USA). The recombinant plasmid was designed to express the alkM gene under the control of the constitutive T7 promoter. After sequence verification (Genewiz, China), the plasmid was extracted using a plasmid extraction kit (Tiangen, China). The recombinant plasmid was transformed into E. coli DH5α for plasmid storage and E. coli BL21 for protein expression. The engineered strains were cultured in LB medium containing ampicillin (50 μg/mL) at 37°C.

Test:

Due to time constraints, conducting a full experimental validation of petroleum degradation was not feasible. Instead, we used molecular docking simulations to predict the interaction between AlkM and various alkanes as a faster and more practical approach. This method allowed us to estimate the efficiency of alkane degradation by the enzyme without requiring a lengthy experimental process.

We used Gnina for molecular docking and Schrödinger’s Maestro for protein visualization to study the interaction between AlkM and alkanes ranging from C17 to C36. The docking process involved using AlkM as the receptor and various alkanes as ligands. The docking results were analyzed by calculating the equilibrium dissociation constant (KD), which indicates the binding affinity between the enzyme and its substrates. A lower KD value represents stronger binding affinity, suggesting a higher likelihood of alkane degradation by AlkM, while a higher KD indicates weaker interactions and reduced degradation potential.

Through this analysis, we observed that the binding affinity of AlkM for alkanes decreases as the chain length increases from C17 to C36. Specifically, shorter-chain alkanes like C17 and C18 showed lower KD values, indicating stronger binding and effective degradation by AlkM. In contrast, longer-chain alkanes such as C34 and C36 exhibited higher KD values, suggesting weaker interactions and reduced capability for AlkM to degrade these molecules.

Learn:

Unfortunately, due to the team's limited experience in biological experiments, we failed to properly document the experiment and lost the gel image for this gene fragment. The image shown above was taken during the gel recovery stage. Following this incident, we implemented stricter protocols to ensure the proper documentation and preservation of all experimental records.

This experience highlighted that while AlkM has a high binding affinity for short-chain alkanes, its efficiency significantly decreases for longer-chain alkanes. Furthermore, the importance of rigorous documentation and careful execution during gene cloning was underscored by the challenges we faced. These lessons will guide future improvements in experimental practices and enzyme system optimization.

Design:

We designed a BsLac-engineered strain to degrade long-chain alkanes. To address the challenge of degrading polycyclic aromatic hydrocarbons (PAHs), laccase, a multi-copper oxidase, was introduced into the system. Laccase efficiently catalyzes the oxidation of phenolic and aromatic compounds, making it highly effective in breaking down PAHs.

Build:

The BsLac gene was synthesized and optimized, then cloned into the pET23b expression plasmid for expression in E. coli. To construct the BsLac-engineered strain, the gene encoding BsLac was synthesized and codon-optimized for efficient expression. The gene was inserted into the pET23b plasmid using NdeI and XhoI restriction sites. Under the control of the T7 promoter, the BsLac gene was expressed in E. coli. After successful sequencing (Azenta, USA; Genewiz, China), plasmids were extracted using a plasmid extraction kit (Tiangen, China). The recombinant plasmids were transformed into E. coli DH5α for plasmid storage and E. coli BL21 for protein expression. The engineered strains were cultured in LB medium containing ampicillin (50 μg/mL) at 37°C to ensure proper growth and plasmid maintenance.

Test 1: Analysis of Laccase Activity in Crude Enzyme Extracts from Engineered Strains

Objective and Methods:

The objective of this experiment was to measure the laccase (BsLac) activity in crude enzyme extracts from engineered E. coli strains to verify successful expression and catalytic function.

1.Culture and Extraction: The engineered strains were cultured overnight at 37°C. After centrifuging 2 mL of the culture at 8000 rpm for 10 minutes, the cell pellet was resuspended in PBS (pH 7.4) and disrupted by ultrasonication to obtain crude enzyme extracts.

2.Laccase Activity Assay: Laccase activity was measured using the ABTS oxidation method. The reaction mix included 930 μL Na₂HPO₄-citric acid buffer (50 mM, pH 3.2), 20 μL ABTS (10 mM), and 50 μL enzyme extract. Absorbance at 420 nm was recorded for 1 minute to calculate enzyme activity.

3.Reaction Principle: ABTS stock solution is deep blue, turning blue-purple upon dilution. When oxidized by laccase, the solution changes to green or blue-green, with strong absorbance at 420 nm.

Results and Conclusion:

The engineered strain (BL21-pET23b-BsLac) exhibited significantly higher absorbance at 420 nm compared to control strains (BL21 and BL21-pET23b), confirming active BsLac expression. The increased laccase activity in the engineered strain suggests efficient oxidation of ABTS, as evidenced by the color change to green/blue-green. This indicates that the BsLac strain has strong laccase activity, supporting its potential for environmental applications like oil degradation.

The BsLac-engineered strain demonstrated significantly enhanced laccase activity, confirming its ability to oxidize ABTS efficiently, highlighting its potential for use in bioremediation.

Test 2: Effects of pH and Temperature on Laccase Activity

Objective and Methods:

The aim of this experiment was to determine the optimal pH and temperature for laccase (BsLac) activity, providing insights for its application. Laccase activity was measured at 30°C across a pH range of 2.0 to 6.0, using glycine-HCl buffer for pH 2.0 and Na₂HPO₄-citric acid buffer for pH 3.0–6.0. To assess the temperature effect, activity was measured from 20°C to 60°C in Na₂HPO₄-citric acid buffer (pH 3.2), using ABTS as the substrate.

Results and Conclusion:

Laccase activity was highest at pH 3.2 and peaked at 40°C. Activity decreased significantly at lower and higher pH values, and temperatures above 40°C caused a marked decline, indicating enzyme instability. These findings highlight that BsLac is most active under moderately acidic conditions and at 40°C, guiding its potential use in biotechnological applications.

Learn:

The successful expression of BsLac and the achievement of strong activity tests indicate that the enzyme can be effectively utilized for environmental bioremediation. However, challenges remain in enhancing the efficiency of long-chain alkane degradation, as these compounds are more difficult to penetrate through the cell membrane.

Design

The design involved fusing the BsLac gene with the ice nucleation protein (INP) to enhance surface display and improve the degradation of long-chain alkanes. Laccase, known for oxidizing aromatic hydrocarbons, can be more effective when displayed on the bacterial surface, allowing for direct interaction with hydrophobic compounds like petroleum oils.

Build:

The INP-BsLac fusion gene was synthesized, cloned into the pET23b plasmid, and expressed in E. coli. To construct the INP-BsLac-engineered strain, the BsLac gene was first synthesized and codon-optimized. For the INP-BsLac fusion strain, a truncated ice nucleation protein (INP) sequence was synthesized and placed upstream of the BsLac gene to create the fusion protein sequence. Both constructs were cloned into the pET23b plasmid using NdeI and XhoI restriction sites. The T7 promoter controlled the expression of the downstream genes. After successful sequencing verification (Azenta, USA; Genewiz, China), the plasmids were extracted using a plasmid extraction kit (Tiangen, China). The recombinant plasmids were transformed into E. coli DH5α for plasmid storage and E. coli BL21 for protein expression. The engineered strains were cultured in LB medium containing ampicillin (50 μg/mL) at 37°C to ensure proper growth and plasmid maintenance.

Test:

Laccase activity of surface-displayed enzymes

The aim of this experiment was to validate the laccase activity of engineered E. coli strains expressing laccase (BsLac) on the cell surface using cell surface display technology. Previously, only crude enzyme extracts from intracellular expression were tested. However, for real-world applications, such as bioremediation, surface-displayed enzymes are essential for efficient function in live bacterial systems. In this experiment, ABTS was used as a substrate to measure the activity of live engineered strains. The reaction mixture consisted of 1 mL of 5 mM ABTS, 0.5 mL of cell suspension, and 1.5 mL of 0.1 M acetate buffer (pH 5.0). The samples were incubated at 30°C in the dark, and laccase activity was measured at 420 nm using spectrophotometry. Enzyme activity (U) was defined as the amount of enzyme required to oxidize 1 μmol of ABTS per minute, calculated per gram of bacterial dry weight.

Results and Conclusion:

The results showed that the engineered strain BL21-pET23b-BsLac with surface-displayed laccase had significantly higher laccase activity compared to the control strain (BL21) and the strain expressing laccase intracellularly (BL21-pET23b). Additionally, the fusion strain (BL21-pET23b-INP-BsLac), which further enhances enzyme display on the cell surface, exhibited the highest activity, suggesting an improvement in the efficiency of laccase activity when displayed on the bacterial surface. These findings confirm that surface-displayed BsLac in live engineered strains can be a powerful tool for applications such as bioremediation. As shown in the image, the experimental setup and results for the 12 samples (four groups with three replicates each) clearly demonstrate the outcomes.

Learn:

Surface display significantly enhanced the degradation efficiency of laccase, making it more effective at degrading petroleum components. However, since E. coli’s interaction with hydrophobic oil residues is limited, the degradation speed could be slow. To address this issue, we integrated biosurfactants produced by Bacillus subtilis to emulsify oil residues into a water-soluble state. This increased the interaction between the engineered strain and the oil, thereby significantly enhancing the degradation speed.

DesignDesign

We designed a B. subtilis strain to express and secrete surfactin, a biosurfactant that enhances the dispersion and degradation of oil pollutants. Efficient secretion of surfactin is critical for its activity in breaking down oil residues. Our focus was on enhancing the Sec-SRP pathway, which is responsible for translocating proteins across membranes. Specifically, we integrated the secA, ftsY, and ftsE genes, which work together to promote the secretion of surface proteins:

⏳ SecA is a key component of the Sec pathway, driving protein translocation across membranes.

⏳ FtsY serves as the signal recognition particle (SRP) receptor, playing a critical role in targeting proteins to the membrane for translocation.

⏳ FtsE, together with ftsX, forms an ATP-binding cassette (ABC) transporter complex that provides the energy required for the transport of proteins such as surface-active proteins.

These three genes work together to increase the efficiency of protein secretion in B. subtilis, ensuring that the surfactant is effectively exported out of the cell into the environment, where it emulsifies oil.

Build:

We synthesized the secA, ftsY, and ftsE genes and constructed an engineered B. subtilis strain capable of secreting the surfactant. To construct the strain, we first synthesized the gene sequences encoding these proteins, followed by codon optimization for expression in B. subtilis. The genes were amplified via PCR and cloned into plasmids using restriction enzymes such as NdeI and XhoI. The ftsE and ftsY genes were combined into one construct, while secA was inserted into a separate plasmid. After assembling the recombinant plasmids, colony PCR and gel electrophoresis were used to verify successful gene insertion. Once confirmed, plasmid digestion and ligation were performed, followed by transformation into B. subtilis. The transformed strains were cultured on antibiotic selection plates containing ampicillin, and plasmid extraction confirmed successful gene integration into the bacterial genome.

Test:

Protein Extraction and Validation: Challenges Faced

To validate protein expression in the engineered strain, we performed ultrasonic cell disruption, protein extraction, SDS-PAGE, and Western blot analysis. Unfortunately, due to limitations in our experimental techniques, we were unable to obtain conclusive results.

Process involved:

1.Ultrasonication for 1 hour to extract proteins from bacterial cells

2.Protein extraction using a commercial kit.

3.SDS-PAGE electrophoresis of the samples, followed by Coomassie staining for one gel and Western blot transfer for another.

4.Western Blot: The membrane was incubated with primary and secondary antibodies before developing the blot.

Despite completing these steps, we were unable to obtain clear results, highlighting areas where our experimental technique and documentation need improvement.

Learn:

While surfactin significantly improved oil degradation efficiency, according to the literature, the bacterial degradation of petroleum remains slow. Therefore, we plan to adopt a plant-microbe symbiosis remediation approach, utilizing the symbiotic relationship between plants and microbes to remediate hydrocarbon-contaminated soil. Plants improve the soil environment, providing habitat and nutrients for microbes, while the microbes secrete amino acids, sugars, and other compounds that support plant growth. This reduces the stress and toxicity caused by hydrocarbons on plants and enhances overall soil restoration.

Design

We engineered a bacterial strain to produce indole-3-acetic acid (IAA) through the indole-3-acetamide (IAM) pathway to promote root growth in plants. The IAM pathway is a conventional method for E. coli to synthesize IAA. Tryptophan is first converted to IAM by tryptophan-2-monooxygenase (encoded by the iaaM gene). IAM is then hydrolyzed to IAA by IAM hydrolase (encoded by the iaaH gene).

Build:

The iaaM and iaaH genes were synthesized and cloned into the pET23b plasmid for expression in E. coli. The purpose of this experiment was to construct an engineered strain capable of producing IAA via the IAM pathway. The biosynthesis starts with tryptophan, which is converted into IAM by tryptophan-2-monooxygenase (encoded by iaaM). IAM is then hydrolyzed into IAA by IAM hydrolase (encoded by iaaH).

The synthesized iaaM and iaaH genes were arranged in a polycistronic structure with ribosome binding sites (RBS B0034) between the genes, and they were cloned into the pET23b vector using NdeI and XhoI restriction sites (Azenta, USA). After sequencing validation, the recombinant plasmids were transformed into E. coli BL21 for expression.

Test:

Quantifying IAA Production Using the Salkowski Reagent and Testing its Effects on Seed Germination and Root Growth.

Test 1: Construction of an IAA Standard Curve

Objective and Methods:

Different concentrations of IAA were dissolved in PBS (pH 7.4) to construct a standard curve for IAA detection. For each sample, 1 mL of IAA solution was mixed with 1 mL of Salkowski reagent, which contains 0.5% ferric chloride in 35% perchloric acid. The mixture was incubated in the dark for 30 minutes.

Principle of the Reaction:

In the highly acidic environment created by perchloric acid, IAA is oxidized into indole derivatives with conjugated double bonds. These derivatives react with iron ions (Fe3+) to form a colored complex, turning the solution pink or purple. The color intensity is proportional to the IAA concentration, which can be measured by detecting absorbance at 530 nm.

Results and Conclusion:

The absorbance at 530 nm was measured for different IAA concentrations, yielding the standard curve above. The linear regression equation was determined as Y = 0.005299 x + 0.05417 with an R² value of 0.98, indicating a strong linear relationship between IAA concentration and absorbance. This standard curve allows for accurate quantification of IAA in experimental samples.

Test 2: Study on IAA Production by Engineered Strain via IAM Pathway

Objective and Methods:

To detect whether the engineered strain BL21-pET23b-iaaM-iaaH could produce high levels of IAA, the following experiment was performed. The bacteria were first grown overnight in LB medium at 37°C with shaking at 180 rpm. The next day, 1 mL of bacterial culture was transferred to 50 mL of fresh LB medium and incubated under the same conditions. To prevent IAA photodegradation, the flask was wrapped in foil. After 12 hours of incubation, 1.5 mL of culture was centrifuged at 10,000 rpm for 1 minute. The supernatant (1 mL) was collected and mixed with 1 mL of Salkowski reagent. The mixture was incubated in the dark for 30 minutes, and absorbance was measured at 530 nm to determine IAA concentration using the standard curve.

Results and Conclusion:

As shown in the figure, the results indicated that the engineered strain BL21-pET23b-iaaM-iaaH produced the highest IAA concentration after 12 hours at 37°C, significantly higher than the control strains BL21 and BL21-pET23b. This confirms that the iaaM and iaaH genes in the engineered strain successfully promoted IAA production, making BL21-pET23b-iaaM-iaaH the most efficient choice for auxin production.

Test 3: Time-Course Analysis of IAA Production by Engineered Strain

Objective and Methods:

To analyze the time-dependent production of IAA by the engineered strain BL21-pET23b-iaaM-iaaH, bacteria were grown overnight in LB medium at 37°C with shaking at 180 rpm. The next day, 1 mL of culture was transferred to 50 mL of fresh medium and incubated under the same conditions. Samples were taken at 0, 4, 6, 8, 12, and 20 hours (1 mL each). After centrifugation, the supernatant was collected, and IAA concentration was measured using the Salkowski method.

Results and Conclusion:

The time-course curve shows that IAA production increased rapidly, reaching a maximum at 8 hours and peaking at 15 hours before stabilizing. Based on these results, a cultivation time of 15 hours was selected as the optimal condition for future experiments involving seed treatment.

Test 4: Effects of Light on IAA Production by IAM Pathway Engineered Strain

Objective and Methods:

To determine whether IAA undergoes photodegradation, the engineered strain BL21-pET23b-iaaM-iaaH was grown overnight in LB medium at 37°C with shaking at 180 rpm. The bacterial culture was then divided into two groups: one wrapped in foil (dark condition), and the other exposed to light. Both groups were incubated for 12 hours under the same conditions. After incubation, 1 mL of culture was collected, centrifuged, and IAA concentration in the supernatant was analyzed using the Salkowski method.

Results and Conclusion:

The results, as shown in the figure, indicate that IAA production was nearly zero in the light-exposed group, while the dark-incubated group produced normal levels of IAA. This demonstrates that IAA is highly sensitive to light and degrades upon exposure. Therefore, cultures producing IAA should be grown in the dark to prevent auxin degradation.

Test 5: Effects of Engineered Strain Supernatant on Seed Germination

Objective and Methods:

To evaluate the effects of the engineered strain’s supernatant on seed germination, four types of seeds were selected: water spinach, soybeans, wheat, and black wheat. 200 seeds of each type were divided into experimental (treated with IAM pathway engineered strain supernatant) and control groups (treated with water), with 100 seeds in each group.

The engineered strain was grown overnight at 37°C with shaking at 180 rpm. The next day, 1 mL of bacterial culture was transferred to 50 mL of fresh medium and wrapped in foil to prevent light degradation. After 12 hours of incubation, the supernatant was collected by centrifugation. Experimental seeds were soaked in the supernatant for 12 hours, and control seeds were soaked in water for 12 hours. After soaking, seeds were transferred to germination trays with water. Seed germination was observed two days later.

Results and Conclusion:

Soybeans: 68% germination in the control group, 91% in the experimental group.

Wheat: 59% in control, 83% in the experimental group.

Water spinach: 53% in control, 78% in the experimental group.

Black wheat: 64% in control, 87% in the experimental group.

The images compare the control and experimental groups, showing a significant improvement in seed germination when treated with supernatant from the IAM pathway engineered strain.

Test 6: Effects of Engineered Strain Supernatant on Root Growth Under Oil Stress

Objective and Methods:

To evaluate the effect of the engineered strain supernatant on root growth under oil stress, healthy water spinach and soybean seeds were selected. Six seeds of each type were randomly divided into experimental (treated with IAM pathway supernatant) and control groups (treated with water). The engineered strain was grown overnight in LB medium at 37°C with shaking at 180 rpm. The next day, 1 mL of bacterial culture was transferred to 50 mL of fresh LB medium wrapped in foil. After 12 hours of incubation, the supernatant was collected by centrifugation. Experimental seeds were soaked in the supernatant for 12 hours, while control seeds were soaked in water for the same period. After soaking, seeds were transferred to soil containing trace amounts of oil, and root growth was observed.

Results and Conclusion:

The results indicated that root growth in the experimental group was significantly better than in the control group, suggesting that IAA produced by the engineered strain enhanced root growth under oil stress. As a plant hormone, IAA promotes root development and improves plant resilience to adverse environmental conditions. This treatment shows potential in enhancing plant growth in contaminated soils, promoting root establishment and development in oil-polluted environments.

Learn:

The IAM pathway successfully produced IAA and effectively promoted seed germination and root growth, especially in contaminated environments, enhancing plant resilience. However, more efficient production methods would be beneficial.

Design

The engineered strain is designed to produce IAA via the IPA pathway, a promising method for enhancing auxin production and promoting plant root growth. The IPA pathway involves three key enzymes:

1.Aro8 (Tryptophan transaminase): Converts tryptophan into indole-3-pyruvic acid (IPA).

2.Kdc (Indole-3-pyruvic acid decarboxylase): Converts IPA to indole-3-acetaldehyde (IAAld).

3.Aldehyde dehydrogenase (PucC): Converts IAAld into indole-3-acetic acid (IAA).

Build:

The aro8, kdc, and puuc genes were synthesized, cloned into the pET23b plasmid, and expressed as a polycistronic structure in E. coli. The IPA pathway involves stepwise conversion of L-tryptophan to IAA, facilitated by Aro8, Kdc, and PucC enzymes. After verifying the gene sequences, the plasmid constructs were transformed into E. coli BL21 for protein expression.

Gel electrophoresis results confirmed successful amplification of aro8 (1525 bp), kdc (1930 bp), and puuc (1448 bp), indicating the correct gene sizes and proper cloning into the plasmid.

Test:

The IAA yield from the IPA pathway was measured using the Salkowski reagent method and compared with the IAA yield from the IAM pathway. Both pathways were evaluated under the same conditions (12 hours, 37°C, in darkness). The results highlighted significant differences in the efficiency of IAA production.

Learning:

The IPA pathway produced nearly twice as much IAA as the IAM pathway, as shown in the experimental data. This demonstrates that the IPA pathway is a more efficient and effective method for producing IAA. These findings suggest that the IPA pathway could be a superior choice for applications aiming to enhance plant growth, though biosafety issues will need to be addressed before scaling the solution.

Design

Our engineered suicide system is designed to ensure biosafety by preventing engineered bacteria from surviving outside of oil-polluted environments. The system incorporates the alkane-responsive promoter (PalkB) and the toxin-antitoxin system (mazF/mazE).

⏳ The PalkB promoter is specifically activated by the presence of alkanes, which are common hydrocarbons found in oil. In the presence of alkanes, the PalkB promoter drives the expression of the antitoxin mazE, which neutralizes the toxic effects of MazF. This allows the engineered bacteria to survive and function in oil-polluted environments rich in alkanes.

⏳ In the absence of alkanes, the PalkB promoter becomes inactive, halting the expression of mazE. Without MazE, MazF, a toxin that degrades mRNA and arrests growth, remains active, leading to cell death. This ensures that the engineered bacteria cannot survive in clean, non-polluted environments, thereby safeguarding biosafety.

Build:

The PalkB, mazF, and mazE genes were synthesized and cloned into the pSB1A3 plasmid using XbaI and SpeI restriction sites. The toxin mazF is constitutively expressed, while the antitoxin mazE is controlled by the alkane-inducible PalkB promoter.

Caption for Gel Electrophoresis Image:

Gel electrophoresis confirms the successful amplification of the key gene sequences for the biosafety system:

⏳ PalkB: 2966 bp

⏳ mazF: 336 bp

⏳ mazE: 252 bp

These results confirm the successful cloning of the essential components of the suicide system into the pSB1A3 plasmid.

Test 1: Alkane Sensor Response

Objective and Method: To validate the function of the alkane-inducible promoter, we constructed a reporter system using AlkS and PalkB to drive the expression of the mRFP (red fluorescent protein). The recombinant plasmid was transformed into E. coli DH5α, and the bacteria were incubated in media containing varying concentrations of n-dodecane. Fluorescence intensity was measured at 584 nm (excitation) and 607 nm (emission) to evaluate promoter activity.

Result: The graph shows that increasing concentrations of n-dodecane resulted in higher fluorescence levels, confirming the successful activation of the PalkB promoter in response to alkanes.

Test 2: Suicide System Functionality

Objective and Method: To assess the functionality of the engineered suicide system, we used E. coli BL21 transformed with a plasmid containing mazF under the control of the PBAD promoter and mazE downstream of the PalkB promoter. The engineered strain was incubated in media containing arabinose and varying concentrations of n-dodecane to test the system’s effectiveness.

Result: As shown in the graph, BL21-PBAD-MazF strains displayed growth inhibition under arabinose induction due to MazF toxin expression. However, in the presence of n-dodecane, the PalkB-induced MazE partially neutralized MazF’s toxic effects, allowing limited growth. The combination of arabinose and n-dodecane resulted in optimal control of the suicide system. This demonstrates the controlled function of the biosafety mechanism.

Conclusion:

The suicide system was successfully triggered by the presence of alkanes, confirming the system's efficacy in regulating bacterial survival, ensuring that the engineered bacteria can only thrive in oil-polluted environments, thus ensuring biosafety for environmental applications.

Learn:

The suicide system demonstrated reliable activation in the presence of alkanes, successfully controlling bacterial survival, and validating its potential for environmental safety applications.

Through the engineering optimization of four modules and seven cycles, we successfully developed a multifunctional engineered bacterial system capable of efficiently degrading petroleum alkanes, secreting surfactants, promoting plant root growth, and implementing biosafety controls. Each module has laid a solid foundation for future environmental remediation applications, particularly in real-world oil pollution scenarios. Moving forward, we aim to further optimize system performance, focusing on improving biosafety and strain stability to ensure efficient and safe applications in real environmental conditions.

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