This project utilizes synthetic biology to construct various engineered strains focusing on four key areas: oil degradation, surfactant secretion, plant-microbe collaboration, and biosafety. For oil degradation, alkane monooxygenase (AlkM) and surface-displayed laccase (BsLac) were employed to efficiently degrade short-chain and long-chain alkanes, respectively, with molecular docking simulations verifying the binding efficiency between the enzymes and alkanes. In the surfactant secretion module, B. subtilis was designed to secrete surfactin, enhancing the degradation of oil pollutants. In the plant collaboration module, engineered strains producing indole-3-acetic acid (IAA) promoted root growth in plants, forming a plant-microbe co-remediation system that increased plant resilience in polluted environments. For biosafety, a suicide system was introduced to ensure that engineered strains automatically self-destruct after completing their remediation task, preventing gene leakage. This research provides a comprehensive solution for oil pollution remediation.
Objective and Methods
The gene sequence encoding alkane monooxygenase (AlkM) was synthesized and codon-optimized for E. coli. This sequence was cloned into the pET23b plasmid using NdeI and XhoI restriction sites to create 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 then transformed into E. coli DH5α for plasmid preservation and E. coli BL21 for protein expression. The engineered strains were cultured in LB medium containing ampicillin (50 μg/mL) at 37°C.
Results and Conclusion
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.
Objective and Methods
According to scientific literature, the degradation of petroleum by engineered bacteria is a very slow process, often taking more than 180 days to show significant results. Due to the limited time available for our project, conducting a full experimental validation of petroleum degradation was not feasible. Therefore, we opted to use molecular docking simulations as a faster and more practical approach to predict the interaction between AlkM and various alkanes. This method allows us to estimate the enzyme’s efficiency in degrading alkanes without needing an extended experimental timeframe.
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 different 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, implying that the enzyme is more likely to bind with and degrade the alkanes, whereas a higher KD indicates weaker binding and reduced degradation potential.
Results and Conclusion
Through this analysis, we observed a trend where the binding affinity of AlkM for alkanes decreases as the chain length increases from C17 to C36. Specifically, the shorter-chain alkanes, such as C17 and C18, exhibited much lower KD values, suggesting strong binding and effective degradation by AlkM. Conversely, longer-chain alkanes, such as C34 and C36, showed higher KD values, indicating weaker interactions and a reduced capacity for AlkM to degrade these molecules efficiently.
These findings suggest that while AlkM is highly efficient at degrading medium-chain alkanes, its efficiency significantly decreases for long-chain alkanes, limiting its effectiveness in decomposing these larger hydrocarbon molecules. Molecular docking thus provides valuable insight into AlkM’s capabilities without the need for lengthy biodegradation experiments.
Objective and Methods
To construct the BsLac and INP-BsLac engineered strains, the gene encoding BsLac was first synthesized and optimized for E. coli codons. 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, with the T7 promoter controlling the expression of the downstream genes. The recombinant plasmids were synthesized and verified through sequencing (Azenta, USA; Genewiz, China).
Following successful sequencing verification, the plasmids were extracted using a plasmid extraction kit (Tiangen, China). The recombinant plasmids were then transformed into E. coli DH5α for plasmid storage and into 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.
Results and Conclusion
The gel electrophoresis results confirm the successful construction of both strains:BsLac: 1542 bp；INP-BsLac: 2079 bp.
These results validate the successful cloning and setup of both BsLac and INP-BsLac engineered strains, enabling further studies on protein expression and function.
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, which becomes blue-purple upon dilution. When oxidized by laccase, the solution changes from blue to green or blue-green, with strong absorbance at 420 nm.
Results and Conclusion
The engineered strain (BL21-pET23b-BsLac) showed significantly higher absorbance at 420 nm compared to control strains (BL21 and BL21-pET23b), confirming active BsLac expression. The higher 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, and highlighting its application potential in environmental remediation.
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.
Objective and Methods
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 biodegradation, surface-displayed enzymes are essential for effective 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 above, the experimental setup and results for the 12 samples (four groups with three replicates each) demonstrate the outcomes clearly.
Objective and Methods
To construct the engineered B. subtilis strains expressing SecA, FtsY, and FtsE, the process started by synthesizing the gene sequences for these proteins, followed by codon optimization for expression in B. subtilis . The genes were amplified using PCR and cloned into plasmids through a multi-step process involving restriction enzymes such as NdeI and XhoI. For the construction, the ftsE and ftsY genes were combined into one construct, while individual plasmids for each gene were also created. Once the recombinant plasmids were assembled, verification through colony PCR and gel electrophoresis was performed to ensure successful gene insertion. After confirmation, plasmid digestion and ligation were carried out for transformation into B. subtilis . The transformed strains were cultured on antibiotic selection plates containing Ampicillin, and plasmid extraction was conducted to confirm successful integration of the genes into the bacterial genome. This entire process ensured the correct construction of the SecA, FtsY, and FtsE engineered strains in B. subtilis for further experimental validation.
Results and Conclusion
This image represents the gel electrophoresis results following the DNA recovery process. The lanes show the presence of DNA bands at varying molecular weights, corresponding to different gene fragments that were recovered. The brighter bands indicate successful DNA recovery, confirming that the expected fragments were isolated. The bands are well-defined, suggesting good quality recovery, which is crucial for further cloning or experimental processes.
Each lane corresponds to a different sample, and the molecular weight markers on the left help identify the size of the recovered fragments, ensuring that the correct gene segments were successfully retrieved for subsequent experimental steps.
In an effort to verify protein expression in the engineered strains, we performed ultrasonication, protein extraction, SDS-PAGE, and Western blot (WB) analysis. Unfortunately, due to limitations in our experimental technique, we were unable to obtain successful results.
The process involved:
Ultrasonication for 1 hour to extract proteins from bacterial cells.
Protein Extraction using a commercial extraction kit.
SDS-PAGE electrophoresis of the samples, followed by Coomassie staining for one gel and Western blot transfer for the other.
Western Blot: The membrane was incubated with primary and secondary antibodies, followed by exposure.
Despite completing these steps, we were unable to achieve clear results, highlighting areas for improvement in our experimental technique and documentation.
Objective and Methods
In this experiment, the goal was to construct an engineered strain that produces indole-3-acetic acid (IAA) via the IAM pathway. The biosynthesis begins with tryptophan, which is first converted to indole-3-acetamide (IAM) by tryptophan-2-monooxygenase, encoded by the iaaM gene. In the second step, IAM is hydrolyzed to IAA by IAM hydrolase, encoded by the iaaH gene.
The synthesized iaaM and iaaH genes, arranged in a polycistronic structure with ribosome binding sites (RBS B0034) between the genes, were cloned into the pET23b vector (Azenta, USA) using NdeI and XhoI restriction sites. After sequencing verification, the recombinant plasmids were transformed into E. coli BL21 for expression.
Results and Conclusion
The gel electrophoresis results shown above demonstrate successful amplification of the iaaM and iaaH genes, with bands at approximately 1635 bp and 1341 bp, respectively, confirming the correct size of the inserted genes. This indicates that the genes were successfully cloned into the vector, laying the foundation for the production of IAA via the IAM pathway.
Objective and Methods
To construct the IAA test standard curve, various concentrations of IAA were dissolved in PBS (pH 7.4). For each sample, 1 mL of IAA solution was mixed with 1 mL of Salkowski reagent. The Salkowski reagent contains 0.5% FeCl₃ in 35% perchloric acid. The mixtures were incubated in the dark for 30 minutes.
Reaction Principle:
In a strongly acidic environment created by sulfuric acid, IAA is oxidized to form an indole derivative with a conjugated double bond. This derivative reacts with iron ions (Fe³⁺) to form a colored complex, turning the solution pink or purple. The intensity of the color correlates with the concentration of IAA, which can be measured by detecting the absorption value at 530 nm.
Results and Conclusion:
Using spectrophotometry, the absorbance at 530 nm was measured for different concentrations of IAA, resulting in the standard curve shown above. The linear regression equation derived from the data is Y = 0.005299X + 0.05417, with an R² value of 0.98, indicating a strong linear relationship between IAA concentration and absorbanc. This standard curve enables accurate quantification of IAA in experimental samples.
Objective and Methods
To test whether the engineered strain BL21-pET23b- iaaM - iaaH can produce significant amounts of indole-3-acetic acid (IAA), the following experiment was conducted. 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 light-induced degradation of IAA, the flasks were covered with aluminum foil. After 12 hours of incubation, 1.5 mL of bacterial 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 the absorbance at 530 nm was measured to determine IAA concentration based on the standard curve.
Results and Conclusion:
The results, as shown in the accompanying figure, demonstrate that the engineered strain BL21-pET23b- iaaM - iaaH produced the highest concentration of IAA 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 enabled the production of IAA. Therefore, the BL21-pET23b- iaaM - iaaH strain is the most effective choice for auxin production.
This confirms the strain's capability to synthesize IAA through the IAM pathway, making it a strong candidate for applications requiring auxin production.
Objective and Methods
To analyze the time-dependent production of IAA by the engineered strain BL21-pET23b- iaaM - iaaH , the bacteria were grown overnight in LB medium at 37°C and 180 rpm. The following day, 1 mL of bacterial culture was transferred to 50 mL of fresh medium and incubated under the same conditions. Samples (1 mL) were taken at 0, 4, 6, 8, 12, and 20 hours. After centrifugation, the supernatant was collected, and the IAA concentration was determined using the Salkowski method.
Results and Conclusion:
From the time-course curve, it can be observed that IAA production increased rapidly after the start of cultivation. The yield rose significantly within the first 8 hours and stabilized between 12 and 15 hours. Based on these results, a cultivation period of 15 hours is recommended as the optimal duration for future seed treatment experiments.
Objective and Methods
To determine whether IAA is degraded by light, the engineered strain BL21-pET23b- iaaM - iaaH was grown overnight in LB medium at 37°C and 180 rpm. The bacterial culture was then divided into two groups: one covered with aluminum foil (dark condition), and the other exposed to light (light condition). Both groups were incubated for 12 hours under the same conditions. After incubation, 1 mL of culture was collected, centrifuged, and the supernatant was analyzed for IAA concentration using the Salkowski method.
Results and Conclusion:
The results, shown in the accompanying figure, demonstrate that IAA production in the light-exposed group was nearly zero, while the dark-incubated group produced normal levels of IAA. This indicates that IAA is highly sensitive to light and is degraded when exposed. Therefore, cultivation of IAA-producing strains should be conducted in the dark to prevent auxin degradation.
Objective and Methods
The indole-3-pyruvic acid (IPA) pathway involves three key enzymes that convert L-tryptophan into indole-3-acetic acid (IAA) in a stepwise manner. First, tryptophan aminotransferase (aro8), derived from yeast, converts L-tryptophan into indole-3-pyruvic acid. This is followed by indole-3-pyruvate decarboxylase (kdc), also from yeast, which transforms indole-3-pyruvic acid into 2-(1H-indol-3-yl) acetaldehyde. Finally, acetaldehyde dehydrogenase (puuc), from E. coli, converts acetaldehyde into indole-3-acetic acid (IAA).
The genes encoding aro8, kdc, and puuc were synthesized and cloned into the pET23b vector using the NdeI and XhoI restriction sites. The genes were arranged in a polycistronic structure with ribosome binding sites (RBS B0034) between them. After successful sequencing verification, the plasmid was transformed into E. coli BL21 for expression.
Results and Conclusion
The gel electrophoresis results show successful amplification of aro8 (1525 bp), kdc (1930 bp), and puuc (1448 bp), confirming the correct size of each gene, indicating successful cloning into the vector. This lays the foundation for IAA production through the IPA pathway.
Objective and Methods
To compare the efficiency of the IAM and IPA pathways in producing IAA, we cultivated engineered bacteria from both pathways, as well as a control strain, under identical conditions: 37°C in the dark for 12 hours. After incubation, the supernatant was collected, and the IAA concentration was measured using the Salkowski method.
Results and Conclusion:
As shown in the image above, the engineered strain utilizing the IPA pathway produced approximately double the amount of IAA compared to the IAM pathway strain. This indicates that the IPA pathway is significantly more efficient in IAA production, making it a superior option for increasing auxin levels to promote plant growth.
Objective and Methods
To evaluate the influence of the supernatant from engineered strains on seed germination, four types of seeds were selected: water spinach, soybeans, wheat, and black wheat. For each type, 200 seeds were divided into an experimental group and a control group, with 100 seeds in each. The experimental group was treated with the supernatant from IAM pathway-engineered strains, while the control group was treated with water.
The engineered strain was cultured overnight in LB medium at 37°C and 180 rpm. The following day, 1 mL of the bacterial culture was transferred to 50 mL of fresh medium, wrapped in aluminum foil to avoid light degradation. After 12 hours of incubation, the culture was centrifuged to collect the supernatant. Seeds in the experimental group were soaked in this supernatant for 12 hours, while control seeds were soaked in water. After soaking, the seeds were transferred to germination trays with water. Seed germination was observed after two days.
Results and Conclusion:
Soybeans: Control group germination rate 68%, experimental group 91%.
Wheat: Control group 59%, experimental group 83%.
Water spinach: Control group 53%, experimental group 78%.
Black wheat: Control group 64%, experimental group 87%.
The images above compare the control and experimental groups, demonstrating a notable improvement in seed germination rates when treated with the supernatant from IAM pathway-engineered strains.
Objective and Methods
To assess the impact of engineered strain supernatant on root growth under petroleum stress, healthy seeds of water spinach and soybeans were selected. Six seeds of each type were randomly divided into two groups: the experimental group (3 seeds) and the control group (3 seeds). The experimental group was treated with the supernatant from an IAM pathway-engineered strain.
The engineered strain was cultured overnight in LB medium, and the next day, 1 mL of bacterial culture was transferred to 50 mL of fresh LB medium wrapped in aluminum foil. After 12 hours of incubation at 37°C and 180 rpm, the culture was centrifuged, and the supernatant was collected. Seeds in the experimental group were soaked in the supernatant for 12 hours, while the control group seeds were soaked in water. After soaking, the seeds were transferred to soil containing trace amounts of petroleum and root growth was observed.
Results and Conclusion:
The results showed that the root growth in the experimental group was significantly better than in the control group. This indicates that under petroleum stress, seeds treated with the supernatant from the IAA pathway-engineered strain exhibited stronger root growth. As a plant hormone, IAA promotes root development and enhances the plant's ability to adapt to adverse environmental conditions. Therefore, this treatment could potentially improve plant growth in polluted soils, facilitating root establishment and development in petroleum-contaminated environments.
This suggests that IAA-producing engineered strains not only promote early root growth but may also enhance plant survival and growth under stress conditions, offering potential applications in environmental remediation.
Objective and Methods
To construct the suicide system, sequences encoding PalkB, mazF, and mazE were synthesized and cloned into the pSB1A3 vector using XbaI and SpeI restriction sites. In this system, mazF, which encodes a toxin, is constitutively expressed, while the antitoxin gene mazE is placed downstream of the PalkB promoter. The PalkB promoter is induced by alkanes, meaning that the presence of alkanes will trigger the expression of mazE, neutralizing the toxic effect of mazF.
Results and Conclusion
The gel electrophoresis results confirm the successful amplification of the following gene sequences:
PalkB: 2966 bp
mazF: 336 bp
mazE: 252 bp
These results indicate successful cloning of the key components of the suicide system into the pSB1A3 vector. This system ensures that in the absence of alkanes, mazF will kill the host cells, preventing their survival, while in the presence of alkanes, mazE will be expressed, allowing the cells to remain viable. Such a system can be used as a biocontainment strategy in environmental applications where it is important to control the survival of engineered microorganisms.
Objective and Methods
To test the functionality of the alkane-inducible promoter, sequences encoding AlkS and PalkB were synthesized and cloned into the pSB1A3 vector using XbaI and SpeI restriction sites. These were placed upstream of the mRFP fluorescent protein gene. The expression of the transcription factor AlkS was controlled by the constitutive J23100 promoter. The recombinant plasmid was transformed into E. coli DH5α cells.
The alkane biosensor strains were inoculated into LB medium at a 1:100 ratio and cultured overnight at 37°C. The next day, the culture was diluted 1:50 into fresh M9 medium supplemented with 50 μg/mL ampicillin and 10 g/L glucose. Solutions of n-dodecane dissolved in 1% Tween80 ethanol were prepared at a concentration of 200 mg/L and stirred thoroughly. Different concentrations of n-dodecane solution were added to the culture medium and induced at 37°C for 20 hours. After incubation, 1 mL of the culture was collected, and OD600 and fluorescence values were measured (excitation wavelength: 584 nm, emission wavelength: 607 nm) using a plate reader. The fluorescence was normalized by calculating the fluorescence/OD600 ratio.
Results and Conclusion:
As shown in the figure above, the fluorescence/OD600 ratio increased with higher concentrations of n-dodecane, indicating that the alkane sensor was successfully activated. This result confirms that the PalkB promoter effectively induces expression in the presence of n-dodecane, demonstrating its potential as a biosensor for detecting alkane levels.
Objective and Methods
The objective of this experiment was to test the functionality of the alkane-inducible suicide system in E. coli BL21 engineered strains. The engineered system included the toxin gene mazF under the control of the PBAD promoter and the antitoxin gene mazE downstream of the PalkB promoter, which is induced by alkanes. After cloning and verification of the recombinant plasmid, it was transformed into E. coli BL21. To evaluate the effectiveness of the system, the engineered strains were first grown overnight in LB medium at 37°C. The next day, cultures were diluted into M9 medium supplemented with ampicillin, glucose, 0.02% arabinose, and varying concentrations of n-dodecane (alkane). OD600 values were measured to monitor bacterial growth and the functionality of the suicide system under different conditions.
Results and Conclusion
The results, as shown in the image, demonstrate that the engineered strain BL21-PBAD-MazF showed inhibited growth in the presence of 0.02% arabinose due to the expression of the MazF toxin. However, in the presence of n-dodecane, the PalkB-induced expression of MazE (the antitoxin) partially neutralized the toxic effects of MazF, allowing for moderate bacterial growth. The highest growth recovery was observed when both n-dodecane and 0.02% arabinose were present, indicating that the suicide system can be effectively controlled by the presence of alkanes. The control strain (BL21 without the suicide system) grew normally, showing the highest OD600 values. This experiment confirms the functionality and controllability of the engineered suicide system, with potential applications in biocontainment.
This project achieved significant progress in the four core areas of oil pollution remediation. First, in the oil degradation module, engineered strains expressing alkane monooxygenase (AlkM) and surface-displayed laccase (BsLac) efficiently degraded short-chain and long-chain alkanes, respectively. Molecular docking simulations confirmed that AlkM exhibited high binding affinity for short-chain alkanes, while BsLac enhanced the degradation of long-chain alkanes, with both enzymes working synergistically to improve overall oil degradation efficiency. Second, in the surfactant secretion module, surfactin secretion by B. subtilis significantly improved oil dispersion and biodegradation efficiency. Third, in the plant-microbe co-remediation module, IAA-producing engineered strains promoted root growth, and experimental results demonstrated that the system effectively increased plant survival and growth rates in polluted soils. Finally, in the biosafety module, an alkane-inducible suicide system was constructed, ensuring that the engineered strains self-destruct after completing their remediation task, preventing gene leakage.
Through the synergy of these four modules, this study not only demonstrated the great potential for oil pollution remediation but also ensured the safety and controllability of the engineered strains in real-world applications, offering a robust and secure technology for environmental remediation.
