iGEM 2024 — Team rdfz-china

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{% block title %}Notebook{% endblock %}
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![](https://static.igem.wiki/teams/5468/cover/{{self.title()|lower}}.png)

![](https://static.igem.wiki/teams/5468/special/6.png)
[Summary of the Experimental Plan for Alkane Monooxygenase (AlkM) and Laccase-engineered Strain Construction and Activity Analysis](#a1)

![](https://static.igem.wiki/teams/5468/special/6.png)
[Summary of the surfactant module](#a2)

![](https://static.igem.wiki/teams/5468/special/6.png)
[Summary of the Experimental Plan for IAA-Producing Engineered Strains and Plant Growth Assays](#a3)

![](https://static.igem.wiki/teams/5468/special/6.png)
[Summary of the Experimental Plan for Alkane-Inducible Promoter Construction and Testing](#a4)

![](https://static.igem.wiki/teams/5468/special/6.png)
[Here are the raw numbers for all of our results：](#a5)

![](https://static.igem.wiki/teams/5468/special/7.png)

Summary of the Experimental Plan for Alkane Monooxygenase (AlkM) and Laccase-engineered Strain Construction and Activity Analysis

This experiment involves constructing several engineered strains expressing Alkane Monooxygenase (AlkM), BsLac, and INP-BsLac proteins using pET23b plasmid. After plasmid construction and transformation into E. coli DH5α for storage and BL21 for protein expression, the activities of the expressed proteins will be tested. The activity of laccase will be measured using the ABTS assay, and the effect of pH and temperature on laccase activity will be analyzed. The experiment also includes molecular docking to evaluate the binding affinity of ligands to AlkM.

The entire procedure from June 20 can be estimated to take 30 days, covering cloning, transformation, expression, laccase activity analysis, and optimization.

Timeline and Summary

|
|  |

Date |

Experiment Plan |

Goal |

Notes |

|
|  |

June 20 |

Start cloning AlkM, BsLac, and INP-BsLac into pET23b via NdeI and XhoI |

Construct plasmids with synthesized genes (AlkM, BsLac, INP-BsLac) |

Use restriction enzymes for cloning, confirm by sequencing |

|
|  |

June 21-22 |

Sequence verification of the recombinant plasmids |

Confirm plasmid construction by sequencing |

Use Genewiz or Qingke for sequencing |

|
|  |

June 23-25 |

Transform plasmids into E. coli DH5α for storage |

Ensure plasmid stability in DH5α for long-term storage |

Perform transformation with heat shock |

|
|  |

June 26-28 |

Transform plasmids into E. coli BL21 for protein expression |

Prepare engineered strains for AlkM, BsLac, INP-BsLac protein expression |

Culture at 37°C with Ampicillin |

|
|  |

June 29 |

Inoculate BL21 strains for expression of AlkM, BsLac, and INP-BsLac |

Begin growing BL21 cultures for protein expression |

Use LB media with Amp (50 μg/mL) |

|
|  |

June 30 |

Optimize protein expression for AlkM and BsLac |

Induce protein expression with IPTG, optimize growth conditions |

Sample for Western blot to confirm expression |

|
|  |

July 1-2 |

Analyze protein expression by SDS-PAGE and Western Blot |

Confirm the expression of AlkM, BsLac, and INP-BsLac proteins |

Analyze molecular weights on SDS-PAGE |

|
|  |

July 3-5 |

Perform molecular docking analysis with AlkM |

Evaluate binding affinity of ligands to AlkM using Gnina and visualize with Maestro |

Use docking to rank binding affinities |

|
|  |

July 6-8 |

Prepare crude enzyme extracts from BL21 engineered strains |

Extract enzymes using ultrasonication and analyze enzyme concentrations |

Collect samples for further laccase activity testing |

|
|  |

July 9-10 |

Analyze laccase activity of crude extracts using ABTS assay |

Measure laccase activity in BsLac and INP-BsLac strains |

Spectrophotometric reading at 420 nm |

|
|  |

July 11-12 |

Test the effect of pH on laccase activity |

Measure laccase activity at different pH levels (2.0–6.0) using ABTS as substrate |

Use Glycine-HCl and Na2HPO4-citrate buffers |

|
|  |

July 13-14 |

Test the effect of temperature on laccase activity |

Measure laccase activity at different temperatures (20–60°C) using ABTS |

Maintain constant pH (3.2) during temperature optimization |

|
|  |

July 15-17 |

Optimize reaction conditions for maximum enzyme activity |

Determine optimal conditions for pH and temperature |

Compare BsLac and INP-BsLac activity profiles |

|
|  |

July 18-19 |

Conduct final laccase activity analysis using spectrophotometry |

Final measurements of enzyme activity at 480 nm |

Express results as enzyme activity units per dry weight |

|
|  |

July 20 |

Conclude data analysis, prepare reports, and plan follow-up experiments |

Summarize findings, create a report, and identify future directions |

Compile all experiment notes and results |

Summary of the surfactant module

The experiment begins on July 1st with the preparation of media and sterilization of equipment, followed by the transformation and screening of Bacillus subtilis and E. coli using plasmid constructs. PCR and plasmid extraction procedures are performed to confirm successful transformations, with sequencing used to verify accuracy.

In the second phase of the experiment, starting from July 16th, protein expression is induced in bacterial strains. The extracted proteins are analyzed using SDS-PAGE to separate them based on size. The experiment continues with Western Blot (WB), transferring the separated proteins to a membrane and detecting them with specific antibodies to ensure correct protein expression.

The experiment concludes on July 20th with the analysis of WB results and the planning of further experiments based on the collected data. The schedule ensures that all necessary steps are taken to validate the success of the plasmid construction and protein expression.

Timeline and Summary

|
|  |

Date |

Experiment Plan |

Goal |

Notes |

|
|  |

July 1 |

Prepare media and reagents, sterilize equipment |

Prepare media and sterilize to ensure a sterile environment |

UV sterilization for 30 minutes |

|
|  |

July 2 |

Prepare media for Bacillus subtilis, sterilize equipment |

Prepare a contamination-free medium for Bacillus subtilis |

Add glucose separately to avoid contamination |

|
|  |

July 3 |

Prepare antibiotic stock solutions (Amp) and LB media |

Provide media and antibiotics for transformation experiments |

Ensure complete antibiotic dissolution |

|
|  |

July 4 |

Liquid culture of Bacillus subtilis |

Start bacterial culture in preparation for transformation |

Incubate overnight at 37°C, monitor shaking speed |

|
|  |

July 5 |

Bacillus subtilis transformation and plating |

Transform plasmids and screen for positive colonies via plating |

Plates should be incubated overnight |

|
|  |

July 6 |

Analyze transformation results, PCR verification of colonies |

Verify positive colonies by PCR to confirm successful transformation |

Use positive controls to avoid false positives |

|
|  |

July 7 |

Plasmid extraction from positive colonies, measure concentration |

Extract plasmids for further experiments |

Store extracted plasmids at -20°C |

|
|  |

July 8 |

Repeat PCR for result confirmation |

Further verify gene integration in transformed strains |

Optimize temperature and primer annealing conditions |

|
|  |

July 9 |

Prepare new media, repeat transformation |

Ensure stability of transformation results by repeating experiments |

Ensure contamination-free media, appropriate antibiotic concentration |

|
|  |

July 10 |

Amplify target genes via PCR, perform gel extraction |

Amplify target gene fragments for cloning and plasmid construction |

Ensure high efficiency during gel extraction |

|
|  |

July 11 |

Plasmid construction, Golden Gate assembly |

Perform plasmid construction via Golden Gate to integrate target genes |

Control enzyme digestion conditions carefully |

|
|  |

July 12 |

Transform plasmids into E. coli, bacterial culture |

Transform and culture colonies in preparation for screening |

Ensure incubation time is appropriate |

|
|  |

July 13 |

Colony screening, PCR verification |

Verify plasmid transformation and integration via PCR |

Use high-fidelity polymerase during PCR |

|
|  |

July 14 |

Further plasmid extraction, sequencing |

Extract plasmids and send for sequencing to confirm accuracy of construction |

Careful analysis of sequencing data to avoid errors |

|
|  |

July 15 |

Analyze experimental results, document process |

Confirm plasmid construction success and prepare for subsequent experiments |

Record detailed steps, plan for the next phase |

|
|  |

July 16 |

Prepare for protein expression, culture strains |

Induce bacterial expression of target proteins, collect samples |

Control temperature and inducer concentration accurately |

|
|  |

July 17 |

Protein extraction, start SDS-PAGE |

Extract proteins and perform SDS-PAGE to separate proteins by size |

Add loading buffer and heat samples before loading |

|
|  |

July 18 |

Complete SDS-PAGE, transfer to membrane for Western Blot |

Transfer proteins from SDS-PAGE to PVDF membrane for WB preparation |

Ensure proper voltage and timing for membrane transfer |

|
|  |

July 19 |

Perform Western Blot, antibody incubation and detection |

Use primary and secondary antibodies for WB to detect protein expression |

Ensure accurate incubation time and antibody concentrations |

|
|  |

July 20 |

Analyze WB results, organize data, plan next experiments |

Analyze target protein expression, evaluate experiment success |

Record detailed analysis and ensure accurate data |

Summary of the Experimental Plan for IAA-Producing Engineered Strains and Plant Growth Assays

This experiment involves constructing IAA-producing engineered strains via two pathways (IAM and IPA) using synthetic gene sequences inserted into the pET23b plasmid. The plasmids will be transformed into E. coli BL21 for expression. The IAA production in engineered strains will be measured using the Salkowski reagent assay. The supernatants from the strains will then be tested for their effects on seed germination and root growth under normal and petroleum stress conditions.

The entire procedure is expected to take around 20 days, starting on July 20. This timeline includes the cloning of genes, transformation into E. coli, measurement of IAA production, and seed germination/root growth assays.

Timeline and Summary

|
|  |

Date |

Experiment Plan |

Goal |

Notes |

|
|  |

July 20 |

Clone iaaM, iaaH, aro8, kdc, and puuc genes into pET23b |

Construct plasmids for IAM and IPA pathways |

Use NdeI and XhoI sites for cloning, verify sequences |

|
|  |

July 21-22 |

Sequence verification and plasmid extraction |

Confirm correct insertion of genes into plasmids |

Use sequencing and plasmid miniprep |

|
|  |

July 23 |

Transform plasmids into E. coli BL21 for protein expression |

Prepare engineered strains for IAA production |

Perform heat-shock transformation, plate on LB-Amp plates |

|
|  |

July 24 |

Induce IAA production in BL21 strains |

Grow engineered strains and induce IAA production |

Incubate at 37°C, 180 rpm, wrap flasks in foil to avoid light degradation |

|
|  |

July 25 |

Measure IAA concentration using Salkowski assay |

Detect and quantify IAA in the supernatant |

Use Salkowski reagent, read absorbance at 530 nm |

|
|  |

July 26 |

Optimize IAA production conditions |

Adjust media composition, induction time, and temperature for maximum IAA production |

Perform multiple conditions for comparison |

|
|  |

July 27-28 |

Prepare seeds for germination assay |

Treat seeds with supernatant from IAM pathway-engineered strain |

Soak seeds for 12 hours, begin germination observation |

|
|  |

July 29-30 |

Record germination rate and growth |

Monitor and record the seed germination rate for treated and control groups |

Compare treated vs. control groups |

|
|  |

July 31 |

Prepare seeds for root growth assay under petroleum stress |

Soak seeds in IAM pathway strain supernatant, transfer to petroleum-stressed soil |

Treat experimental group with supernatant |

|
|  |

August 1-2 |

Record and analyze root growth |

Monitor root growth of treated and control seeds under petroleum stress |

Compare root lengths and general growth patterns |

Summary of the Experimental Plan for Alkane-Inducible Promoter Construction and Testing

This experiment involves constructing an alkane-inducible promoter system in E. coli DH5α. The genes AlkS and PAlkB are cloned into the pSB1A3 vector using XbaI and SpeI restriction sites, positioned upstream of the mRFP gene. The transcription factor AlkS is regulated by the constitutive J23100 promoter. The recombinant plasmid will then be transformed into E. coli DH5α, and the promoter will be tested by culturing the recombinant strain in M9 medium supplemented with different concentrations of n-dodecane. The fluorescence output of the mRFP gene will be measured to evaluate promoter activity.

The entire procedure is expected to take 16 days, starting from August 1st. This timeline includes cloning, transformation, testing of the alkane-inducible promoter, and data collection.

Timeline and Summary

|
|  |

Date |

Experiment Plan |

Goal |

Notes |

|
|  |

August 1 |

Clone AlkS and PAlkB into pSB1A3 |

Construct the alkane-inducible promoter in pSB1A3 |

Use XbaI and SpeI for cloning |

|
|  |

August 2-3 |

Sequence verification and plasmid extraction |

Confirm correct insertion of AlkS and PAlkB into plasmid |

Perform plasmid miniprep and sequencing |

|
|  |

August 4 |

Transform recombinant plasmid into E. coli DH5α |

Prepare the engineered strain for promoter testing |

Heat-shock transformation, plate on LB-Amp plates |

|
|  |

August 5 |

Inoculate and culture engineered strain in LB medium |

Activate the strain in LB medium overnight |

Incubate at 37°C, 200 rpm overnight |

|
|  |

August 6 |

Transfer to M9 medium with n-dodecane |

Induce the alkane-inducible promoter with different n-dodecane concentrations |

Include controls with and without alkane |

|
|  |

August 7 |

Measure OD600 and fluorescence of the cultures |

Evaluate the mRFP gene expression and promoter response |

Use microplate reader to measure fluorescence |

|
|  |

August 8 |

Calculate normalized fluorescence ratio (Fluorescence/OD600) |

Analyze the alkane promoter’s induction strength |

Compare fluorescence at different alkane concentrations |

|
|  |

August 9-10 |

Repeat induction experiments with varying alkane concentrations |

Optimize promoter induction with different n-dodecane levels |

Ensure consistency and repeatability |

|
|  |

August 11 |

Test reproducibility of fluorescence measurements |

Perform replicates to verify the stability and reproducibility of the system |

Measure triplicate values for statistical accuracy |

|
|  |

August 12-13 |

Final data collection and analysis |

Summarize the results from fluorescence measurements |

Perform statistical analysis |

|
|  |

August 14 |

Prepare final report on alkane-inducible promoter activity |

Compile all experimental data, results, and conclusions |

Generate graphs and tables for the report |

|
|  |

August 15-16 |

Review and finalize report for submission |

Final edits and review of the experimental report |

Ensure completeness and accuracy of all sections |

Here are the raw numbers for all of our results：

{% endblock %}

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{% block title %}Engineering{% endblock %}
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![](https://static.igem.wiki/teams/5468/cover/{{self.title()|lower}}.png)

![](https://static.igem.wiki/teams/5468/special/6.png)
[Abstract](#a1)

![](https://static.igem.wiki/teams/5468/special/6.png)
[Module 1: Petroleum degradation system](#a2)

![](https://static.igem.wiki/teams/5468/special/6.png)
[Module 2: Surfactant Secretion System](#a3)

![](https://static.igem.wiki/teams/5468/special/6.png)
[Module 3: Plant-Microbe Collaboration System](#a4)

![](https://static.igem.wiki/teams/5468/special/6.png)
[Module 4: Biosafety systems](#a5)

![](https://static.igem.wiki/teams/5468/special/6.png)
[Conclusion：](#a6)

![](https://static.igem.wiki/teams/5468/special/6.png)
[Reference：](#a7)

![](https://static.igem.wiki/teams/5468/special/7.png)

Abstract

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.

Module 1: Petroleum degradation system

Cycle 1: Alkane Monooxygenase (AlkM) Engineered Strain

**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.

![](https://static.igem.wiki/teams/5468/{{self.title()|lower}}/img1.png)

Figure 1. Gel recovery stage for the AlkM gene fragment.

**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.

![](https://static.igem.wiki/teams/5468/{{self.title()|lower}}/img2.png)

Figure 2. Molecular docking between AlkM and alkanes (C17-C36).

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.

Cycle 2: Laccase (BsLac) Engineered Strain

**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.

![](https://static.igem.wiki/teams/5468/{{self.title()|lower}}/img3.png)

Figure 3. Schematic of laccase degrading petroleum.

**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.

![](https://static.igem.wiki/teams/5468/{{self.title()|lower}}/img4.png)

Figure 4.Gel electrophoresis of the BsLac gene constructs.

**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.

![](https://static.igem.wiki/teams/5468/{{self.title()|lower}}/img5.png)

Figure 5. Laccase activity assay for the BL21-pET23b-BsLac strain.

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.

![](https://static.igem.wiki/teams/5468/{{self.title()|lower}}/img6.png)

Figure 6. Effects of pH and temperature on laccase activity.

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.

Cycle 3: Surface-Displayed Laccase (INP-BsLac) Engineered Strain

**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.

![](https://static.igem.wiki/teams/5468/{{self.title()|lower}}/img7.png)

Figure 7. Genetic circuit of laccase degradation.

**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.

![](https://static.igem.wiki/teams/5468/{{self.title()|lower}}/img8.png)

Figure 8. Gel electrophoresis of INP-BsLac gene constructs.

**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.

![](https://static.igem.wiki/teams/5468/{{self.title()|lower}}/img9.png)

Figure 9. Laccase activity of engineered strains with surface-displayed enzymes.

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.

Module 2: Surfactant Secretion System

Cycle 4: B. subtilis Secretion of Surfactin

**Design****Design**

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.

![](https://static.igem.wiki/teams/5468/{{self.title()|lower}}/img10.png)

Figure 10. Genetic circuits for enhancing surfactin secretion in B. subtilis through the co-expression of secA, ftsE, and ftsY under the T7 promoter.

**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.

![](https://static.igem.wiki/teams/5468/{{self.title()|lower}}/img11.png)

Figure 11. Gel electrophoresis of B. subtilis strain expressing secA, ftsY, and ftsE genes.

**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.

Module 3: Plant-Microbe Collaboration System

Cycle 5: Auxin (IAA) Production via the IAM Pathway

**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).

![](https://static.igem.wiki/teams/5468/{{self.title()|lower}}/img12.png)

Figure 12. Schematic of the IAM pathway.

**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.

![](https://static.igem.wiki/teams/5468/{{self.title()|lower}}/img13.png)

Figure 13. Gel electrophoresis of the iaaM and iaaH gene constructs producing IAA.

**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.

![](https://static.igem.wiki/teams/5468/{{self.title()|lower}}/img14.png)

Figure 14. Standard curve of IAA using the Salkowski reagent.

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.

![](https://static.igem.wiki/teams/5468/{{self.title()|lower}}/img15.png)

Figure 15. IAA production by engineered strains via the IAM pathway.

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.

![](https://static.igem.wiki/teams/5468/{{self.title()|lower}}/img16.png)

Figure 16. Time-course of IAA production by IAM pathway strain.

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.

![](https://static.igem.wiki/teams/5468/{{self.title()|lower}}/img17.png)

Figure 17. Effects of light on IAA production.

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.

![](https://static.igem.wiki/teams/5468/{{self.title()|lower}}/img18.png)

Figure 18. Seed germination results with IAM pathway strain supernatant.

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.

![](https://static.igem.wiki/teams/5468/{{self.title()|lower}}/img19.png)

Figure 19. Root growth under oil stress with IAM pathway strain supernatant.

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.

Cycle 6: Auxin (IAA) Production via the IPA Pathway

**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).

![](https://static.igem.wiki/teams/5468/{{self.title()|lower}}/img20.png)

Figure 20. Differences between IAM and IPA pathways

**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.

![](https://static.igem.wiki/teams/5468/{{self.title()|lower}}/img21.png)

Figure 21. Gel electrophoresis of aro8, kdc, and puc genes in the IPA pathway.

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.

![](https://static.igem.wiki/teams/5468/{{self.title()|lower}}/img22.png)

Figure 22. Comparison of IAA production between the IAM and IPA pathways.

**Learn**ing:

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.

Module 4: Biosafety systems

Cycle 7: Biosafety System

**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.

![](https://static.igem.wiki/teams/5468/{{self.title()|lower}}/img23.png)

Figure 23. Suicide system diagram

**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.

![](https://static.igem.wiki/teams/5468/{{self.title()|lower}}/img24.png)

Figure 24. Gel Electrophoresis of Suicide System Components (PalkB, mazF, mazE)

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.

![](https://static.igem.wiki/teams/5468/{{self.title()|lower}}/img25.png)

Figure 25. Alkane Promoter Test Using mRFP Fluorescence

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.

![](https://static.igem.wiki/teams/5468/{{self.title()|lower}}/img26.png)

Figure 26. Suicide system test

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.

Conclusion：

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.

Reference：

[1]Sui, Xin, Xuemei Wang, Yuhuan Li, and Hongbing Ji. 2021. "Remediation of Petroleum-Contaminated Soils with Microbial and Microbial Combined Methods: Advances, Mechanisms, and Challenges" Sustainability 13, no. 16: 9267. https://doi.org/10.3390/su13169267

[2]https://openknowledge.fao.org/server/api/core/bitstreams/fe5df8d6-6b19-4def-bdc6-62886d824574/content/src/html/chapter-04-2.html

[3]Wei Yang, 2020,The harm and present situation analysis of soil oil pollution, 38(04)China Resources Comprehensive Utilization

[4]Zhao Zhirui, Li Shuyuan, Zuo Tianyuan, Hu Ziru, Li Shuoyang, Liu Hui, Hu Qing, He Yuhuan, Li Fanghong. Fu Characteristics of remediation of petroleum contaminated soil by microbial communities [J/OL]. Environmental Science https://doi.org/10.13227/j.hjkx.202401164

[5]Xie Yun, efficient petroleum hydrocarbon degrading bacteria and crude oil degrading genes Research on the construction of engineering bacteria, https://www.cnki.net/20140610

[6]Lim MW, Lau EV, Poh PE. A comprehensive guide of remediation technologies for oil contaminated soil - Present works and future directions. Mar Pollut Bull. 2016 Aug 15;109(1):14-45. doi: 10.1016/j.marpolbul.2016.04.023. Epub 2016 Jun 4. PMID: 27267117.

[7]Dhaka A, Chattopadhyay P. A review on physical remediation techniques for treatment of marine oil spills. J Environ Manage. 2021 Jun 15;288:112428. doi: 10.1016/j.jenvman.2021.112428. Epub 2021 Apr 7. PMID: 33831635.

[8]https://www.noaa.gov/explainers/oil-spills-major-marine-ecosystem-threat

[9]Yuanfei Lv, Jianfeng Bao, Liandong Zhu,A comprehensive review of recent and perspective technologies and challenges for the remediation of oil-contaminated sites

[10]Amber J. Pete, Bhuvnesh Bharti, and Michael G. Benton ACS ES&T Engineering 2021 1 (6), 928-946 DOI: 10.1021/acsestengg.0c00217

[11]https://www.differencebetween.com/difference-between-biodegradation-and-vs-bioremediation/

[12]https://www.biotechniques.com/drug-discovery-development/evolutionary-mapping-of-e-coli-offers-novel-drug-target/

[13]https://mybyome.com/the-mighty-bacillus-subtilis-a-promising-probiotic-for-gut-health-and-overall-wellness/

[14]Ratajczak A, Geißdörfer W, Hillen W.1998.Alkane Hydroxylase from Acinetobactersp. Strain ADP1 Is Encoded by alkM and Belongs to a New Family of Bacterial Integral-Membrane Hydrocarbon Hydroxylases. Appl Environ Microbiol64:.https://doi.org/10.1128/AEM.64.4.1175-1179.1998

[15]Diefenbach T, Sumetzberger-Hasinger M, Braunschmid V, Konegger H, Heipieper HJ, Guebitz GM, Lackner M, Ribitsch D, Loibner AP. Laccase-mediated degradation of petroleum hydrocarbons in historically contaminated soil. Chemosphere. 2024 Jan;348:140733. doi: 10.1016/j.chemosphere.2023.140733. Epub 2023 Nov 15. PMID: 37977536.

[16]Li R, Zhou T, Khan A, Ling Z, Sharma M, Feng P, Ali G, Saif I, Wang H, Li X, Liu P. Feed-additive of bioengineering strain with surface-displayed laccase degrades sulfadiazine in broiler manure and maintains intestinal flora structure. J Hazard Mater. 2021 Mar 15;406:124440. doi: 10.1016/j.jhazmat.2020.124440. Epub 2020 Nov 1. PMID: 33302188.

[17]Xia L, Hou Z, Zhu F, Wen J. Enhancing surfactin production in Bacillus subtilis: Insights from proteomic analysis of nitrate-induced overproduction and strategies for combinatorial metabolic engineering. Bioresour Technol. 2024 Apr;397:130499. doi: 10.1016/j.biortech.2024.130499. Epub 2024 Feb 27. PMID: 38417461.

[18]Wang A, Fu W, Feng Y, Liu Z, Song D. Synergetic effects of microbial-phytoremediation reshape microbial communities and improve degradation of petroleum contaminants. J Hazard Mater. 2022 May 5;429:128396. doi: 10.1016/j.jhazmat.2022.128396. Epub 2022 Feb 1. PMID: 35236043.

[19]https://parts.igem.org/Part:BBa\_K4013003

[20]Yamaguchi, Yoshihiro & Inouye, Masayori. (2011). Yamaguchi, Y. & Inouye, M. Regulation of growth and death in Escherichia coli by toxin-antitoxin systems. Nat. Rev. Microbiol. 9, 779-790. Nature reviews. Microbiology. 9. 779-90. 10.1038/nrmicro2651.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block page\_content %}

![](https://static.igem.wiki/teams/5468/cover/{{self.title()|lower}}.png)

![](https://static.igem.wiki/teams/5468/special/6.png)
[Construction of Alkane Monooxygenase (AlkM) Engineered Strain](#a1)

![](https://static.igem.wiki/teams/5468/special/6.png)
[Molecular Docking](#a2)

![](https://static.igem.wiki/teams/5468/special/6.png)
[Construction of BsLac-engineered strain](#a3)

![](https://static.igem.wiki/teams/5468/special/6.png)
[Construction of INP-BsLac-engineered strain](#a4)

![](https://static.igem.wiki/teams/5468/special/6.png)
[Laccase activity analysis of crude enzyme extracts from engineered strains](#a5)

![](https://static.igem.wiki/teams/5468/special/6.png)
[Effect of pH and temperature on laccase activity](#a6)

![](https://static.igem.wiki/teams/5468/special/6.png)
[Analysis of laccase activity in engineered strains](#a7)

![](https://static.igem.wiki/teams/5468/special/6.png)
[Construction of indole-3-acetic acid (IAA)-producing engineered strain via IAM pathway](#a8)

![](https://static.igem.wiki/teams/5468/special/6.png)
[Construction of indole-3-acetic acid (IAA)-producing engineered strain via IPA pathway](#a9)

![](https://static.igem.wiki/teams/5468/special/6.png)
[Measurement of indole-3-acetic acid (IAA) production in engineered strains](#a10)

![](https://static.igem.wiki/teams/5468/special/6.png)
[Effect of Engineered Strain Supernatant on Seed Germination](#a11)

![](https://static.igem.wiki/teams/5468/special/6.png)
[Effect of Engineered Strain Supernatant on Root Growth of Water Spinach and Soybean Seeds Under Petroleum Stress](#a12)

![](https://static.igem.wiki/teams/5468/special/6.png)
[Construction of Alkane-Inducible Promoter](#a13)

![](https://static.igem.wiki/teams/5468/special/6.png)
[Testing of Alkane Promoter](#a14)

![](https://static.igem.wiki/teams/5468/special/6.png)
[Statistical Analysis](#a15)

![](https://static.igem.wiki/teams/5468/special/6.png)
[Reference：](#a16)

![](https://static.igem.wiki/teams/5468/special/7.png)

Construction of Alkane Monooxygenase (AlkM) Engineered Strain

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.

Molecular Docking

Tools used: Gnina [https://github.com/gnina/gnina](https://github.com/gnina/gnina)

Protein visualization tool: Schrödinger Maestro

Command: `gnina -r \*.pdb -l \*.sdf --autobox\_ligand \*.sdf -o \*.sdf.gz`

Affinity: Binding affinity refers to the strength of the interaction between a single biomolecule (e.g., protein or DNA) and its ligand/binding partner (e.g., drug or inhibitor). Binding affinity is typically measured and reported using the equilibrium dissociation constant (KD), which evaluates the strength of bimolecular interactions and ranks their intensity. The smaller the KD value, the greater the binding affinity of the ligand for its target. Conversely, a larger KD value indicates weaker attraction and binding between the target molecule and the ligand.

Construction of BsLac-engineered strain

The coding gene sequence of BsLac was synthesized and codon-optimized for E. coli. The sequence was cloned into the pET23b plasmid using NdeI and XhoI restriction sites, resulting in a recombinant plasmid (Azenta, USA). The downstream coding gene is constitutively expressed under the T7 promoter. After sequence verification (Qingke, China), the recombinant plasmid was extracted using a plasmid extraction kit (Tiangen, China). The plasmid was then transformed into E. coli DH5α for storage and BL21 for expression. The engineered strain was cultured in LB medium containing 50 μg/mL ampicillin (Amp) at 37°C.

Construction of INP-BsLac-engineered strain

The BsLac coding gene and a truncated ice nucleation protein (INP) sequence were synthesized and positioned upstream of BsLac. The fusion protein INP-BsLac coding sequence was cloned into the pET23b plasmid via NdeI and XhoI restriction sites, resulting in a recombinant plasmid (Genewiz, USA). Similarly, recombinant engineered strains based on BL21 were obtained.

Laccase activity analysis of crude enzyme extracts from engineered strains

The wild-type BL21, control strain (containing only the pET23b empty plasmid), and BsLac-overexpressing strains were inoculated at a 1:100 ratio into fresh LB medium containing 50 μg/mL ampicillin. After overnight incubation at 37°C, 2 mL of bacterial culture was collected. The cultures were centrifuged at 8000 rpm for 10 minutes, and the bacterial pellet was resuspended in PBS (pH 7.4). Ultrasonic disruption was performed using a Biosafer1000 ultrasonic homogenizer (Saifei) under ice bath conditions (75 W, 1s on, 3s off, 20 min) to obtain crude enzyme extracts. Laccase activity was measured using the ABTS method. ABTS was incubated with BsLac enzyme solution in a water bath at 30°C, and absorbance changes at 420 nm were measured using a microplate reader (Multiskan GO, Thermo Fisher Scientific, USA). The reaction system consisted of 930 μL Na2HPO4-citrate buffer (50 mM, pH 3.2), 20 μL ABTS (10 mM), and 50 μL enzyme solution. One enzyme activity unit (U) was defined as the amount of enzyme required to catalyze the oxidation of 1 μmol of ABTS per minute per mL of solution.

Effect of pH and temperature on laccase activity

To determine the optimal temperature, laccase activity was measured with ABTS as a substrate in 50 mM Na2HPO4-citrate buffer (pH 3.2) at different temperatures (20-60°C). The effect of pH on laccase activity was assessed at 30°C across a pH range of 2.0 to 6.0. Glycine-HCl buffer was used for pH 2.0, and Na2HPO4-citrate buffer was used for pH 3.0–6.0.

Analysis of laccase activity in engineered strains

Laccase activity was measured using ABTS as the substrate at a wavelength of 480 nm. The reaction mixture was prepared as follows: 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 3 mL mixture was reacted at 30°C in the dark. Enzyme activity was measured at 480 nm by spectrophotometry, and enzyme activity units were calculated per gram of bacterial dry weight.

Construction of indole-3-acetic acid (IAA)-producing engineered strain via IAM pathway

The synthetic iaaM and iaaH genes (polycistronic structure with RBS B0034 between genes) were inserted into the pET23b vector (Azenta, USA) at the NdeI and XhoI cloning sites. After sequence verification, the plasmid was transformed into E. coli BL21.

Construction of indole-3-acetic acid (IAA)-producing engineered strain via IPA pathway

The IPA pathway requires three enzymes to convert L-tryptophan into IAA in three steps. Tryptophan transaminase (aro8) converts tryptophan into indole-3-pyruvate, which is then converted into indole-3-acetaldehyde by indole-3-pyruvate decarboxylase (kdc). Indole-3-acetaldehyde is finally converted to indole-3-acetic acid by aldehyde dehydrogenase (puuc). The aro8 and kdc genes are derived from yeast, while puuc is from E. coli. The synthetic aro8, kdc, and puuc genes (polycistronic structure, RBS B0034 between genes) were inserted into the pET23b vector (Azenta, USA) at NdeI and XhoI cloning sites. After sequence verification, the plasmid was transformed into E. coli BL21.

Measurement of indole-3-acetic acid (IAA) production in engineered strains

The engineered and control strains were activated overnight in LB medium at 37°C with shaking at 180 rpm. The following day, 1 mL of culture was transferred to 50 mL of fresh medium and incubated at 37°C, 180 rpm. The flasks were wrapped in aluminum foil to prevent IAA degradation by light. After centrifuging 1.5 mL of bacterial culture (10000 rpm, 1 min), 1 mL of the supernatant was collected. For IAA detection, 1 mL of the supernatant was mixed with 1 mL of Salkowski reagent (15 mL 0.5 M FeCl₃, 300 mL concentrated sulfuric acid, 500 mL distilled water). The mixture was incubated in the dark for 30 minutes, turning pink, indicating the presence of IAA. The absorbance was measured at 530 nm using spectrophotometry, with absorbance proportional to IAA concentration. The IAA content in the bacterial cultures was quantified using a standard curve prepared with known concentrations of IAA.

Effect of Engineered Strain Supernatant on Seed Germination

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.

Effect of Engineered Strain Supernatant on Root Growth of Water Spinach and Soybean Seeds Under Petroleum Stress

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.

Construction of Alkane-Inducible Promoter

The sequences encoding AlkS and PAlkB were obtained through gene synthesis and cloned into the pSB1A3 vector (using XbaI and SpeI restriction sites), positioned upstream of the mRFP gene coding sequence. The expression of the transcription factor is regulated by the constitutive J23100 promoter. The recombinant plasmid was transformed into E. coli DH5α.

Testing of Alkane Promoter

The alkane biosensor strain was inoculated into LB medium at a ratio of 1:100 and cultured overnight at 37°C. The following day, it was transferred to fresh M9 medium (containing 50 μg/mL ampicillin: Na2HPO4, 3.0 g/L; KH2PO4, 0.5 g/L; NaCl, 1.0 g/L; NH4Cl, 1.0 g/L; MgSO4, 5.0 mM; CaCl2, 0.1 mM; supplemented with 10 g/L glucose) at a 1:50 ratio. Meanwhile, n-dodecane was dissolved in anhydrous ethanol containing 1% Tween 80 (200 mg/L) and mixed thoroughly using a magnetic stirrer. Subsequently, solutions of n-dodecane at different concentrations were added to the culture medium. Induction was conducted at 37°C for 20 hours. A 1 mL aliquot of the culture was taken, and OD600 values and fluorescence were measured using a microplate reader (excitation wavelength 584 nm, emission wavelength 607 nm). The normalized fluorescence ratio (Fluorescence/OD600) was calculated.

Statistical Analysis

Data were analyzed and graphed using GraphPad Prism software. Results are presented as mean ± standard deviation (SD). For comparisons of multiple groups, one-way ANOVA followed by Tukey’s post-hoc test was used for difference analysis. For comparisons of two groups, a two-tailed Student’s t-test was performed. A p-value of less than 0.05 was considered statistically significant.

Reference：

(1) Gang, S.; Sharma, S.; Saraf, M.; Buck, M.; Schumacher, J. Analysis of Indole-3-acetic Acid (IAA) Production in Klebsiellaby LC-MS/MS and the Salkowski Method. Bio Protoc 2019, 9 (9), e3230. DOI: 10.21769/BioProtoc.3230 From NLM.

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![](https://static.igem.wiki/teams/5468/cover/{{self.title()|lower}}.png)

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[Abstract](#a1)

![](https://static.igem.wiki/teams/5468/special/6.png)
[Petroleum degradation system](#a2)

![](https://static.igem.wiki/teams/5468/special/6.png)
[The Surfactin system](#a3)

![](https://static.igem.wiki/teams/5468/special/6.png)
[Plant-Microbe Collaboration for Co-degradation](#a4)

![](https://static.igem.wiki/teams/5468/special/6.png)
[Biosafety system](#a5)

![](https://static.igem.wiki/teams/5468/special/6.png)
[Conclusion](#a6)

![](https://static.igem.wiki/teams/5468/special/7.png)

Abstract

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.

Petroleum degradation system

Alkane monooxygenase (AlkM)

Construction of Alkane Monooxygenase (AlkM) Engineered Strain

**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.

![](https://static.igem.wiki/teams/5468/{{self.title()|lower}}/img1.png)

Figure 1. Gel Recovery Stage for AlkM Gene Fragment

**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.

Molecular Docking

**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.

![](https://static.igem.wiki/teams/5468/{{self.title()|lower}}/img2.png)

Figure 2. Molecular Docking of AlkM with Alkanes (C17-C36)

**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.

Laccase

Construction of BsLac and INP-BsLac Engineered Strains

**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.

![](https://static.igem.wiki/teams/5468/{{self.title()|lower}}/img3.png)

Figure 3. Gel Electrophoresis of BsLac and INP-BsLac Gene Constructs

**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.

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, 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.

![](https://static.igem.wiki/teams/5468/{{self.title()|lower}}/img4.png)

Figure 4. Laccase Activity Assay for BL21-pET23b-BsLac Strain

**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.

pH and Temperature Effects 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.

![](https://static.igem.wiki/teams/5468/{{self.title()|lower}}/img5.png)

Figure 5. pH and Temperature Effects on Laccase Activity

**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.

Laccase Activity of the Engineered Strain

**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.

![](https://static.igem.wiki/teams/5468/{{self.title()|lower}}/img6.png)

Figure 6. Surface Display Laccase Activity of Engineered Strains

**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.

The Surfactin system

Construction and Validation of  B. subtilis  Strains for Surfactin Secretion and Protein Expression

**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.

![](https://static.igem.wiki/teams/5468/{{self.title()|lower}}/img7.png)

Figure 7. Gel Electrophoresis of Bacillus subtilis Strains Expressing SecA, FtsY, and FtsE Genes

**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.

Protein Extraction and Validation: Challenges Faced

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.

Plant-Microbe Collaboration for Co-degradation

Construction of IAA-Producing Engineered Strains via the IAM Pathway

**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.

![](https://static.igem.wiki/teams/5468/{{self.title()|lower}}/img8.png)

Figure 8. Gel Electrophoresis of IaaM and IaaH Gene Constructs for IAA Production

**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.

Construction of the IAA Test Standard Curve

**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.

![](https://static.igem.wiki/teams/5468/{{self.title()|lower}}/img9.png)

Figure 9. IAA Standard Curve Using Salkowski Reagent

**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.

Production of IAA by Engineered Strains via the IAM Pathway

**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.

![](https://static.igem.wiki/teams/5468/{{self.title()|lower}}/img10.png)

Figure 10. IAA Production by Engineered Strains via IAM Pathway

**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.

Time-Course Analysis of IAA Production by Engineered Strains via the IAM Pathway

**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.

![](https://static.igem.wiki/teams/5468/{{self.title()|lower}}/img11.png)

Figure 11. Time-Course of IAA Production by IAM Pathway Strains

**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.

Effect of Light on IAA Production in IAM Pathway Engineered Strains

**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.

![](https://static.igem.wiki/teams/5468/{{self.title()|lower}}/img12.png)

Figure 12. Effect of Light on IAA Production

**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.

Construction of IAA-Producing Engineered Strains via the IPA Pathway

**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.

![](https://static.igem.wiki/teams/5468/{{self.title()|lower}}/img13.png)

Figure 13. Gel Electrophoresis of aro8, kdc, and puuc Genes for IPA Pathway

**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.

Comparison of IPA and IAM Pathway Engineered Strains

**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.

![](https://static.igem.wiki/teams/5468/{{self.title()|lower}}/img14.png)

Figure 14. Comparison of IAA Production Between IAM and IPA Pathways

**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.

Effect of Engineered Strain Supernatant on Seed Germination

**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.

![](https://static.igem.wiki/teams/5468/{{self.title()|lower}}/img15.png)

Figure 15. Seed Germination Results with IAM Pathway Strain Supernatant

**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.

Effect of Engineered Strain Supernatant on Root Growth of Water Spinach and Soybean Seeds Under Petroleum Stress

**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.

![](https://static.igem.wiki/teams/5468/{{self.title()|lower}}/img16.png)

Figure 16. Root Growth Under Petroleum Stress with Supernatant from IAM Pathway Strain

**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.

Biosafety system

Construction of the Suicide System

**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.

![](https://static.igem.wiki/teams/5468/{{self.title()|lower}}/img17.png)

Figure 17. Gel Electrophoresis of Suicide System Components (PalkB, mazF, mazE)

**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.

Alkane Promoter Test

**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.

![](https://static.igem.wiki/teams/5468/{{self.title()|lower}}/img18.png)

Figure 18. Alkane Promoter Test Using mRFP Fluorescence

**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.

Suicide system test

**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.

![](https://static.igem.wiki/teams/5468/{{self.title()|lower}}/img19.png)

Figure 19. Suicide system test

**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.

Conclusion

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.

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