iGEM 2024 — Team whhs-china

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CONTENTS

[Abstract：](#a1)
[Phase 1: Preparation and Learning (Day 1 - Day 5)](#a2)
[Phase 2: Gene Synthesis, Cloning, and Strain Construction (Day 6 - Day 15)](#a3)
[Phase 3: Time-Course Experiments and Optimization (Day 16 - Day 25)](#a4)
[Phase 4: Cold-Inducible and Suicide Systems Testing (Day 26 - Day 30)](#a5)
[Daily notebook](#a6)
[WHHS-China raw data](#a7)

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Abstract：

The notebooks provide a detailed timeline of a comprehensive iGEM project focused on engineering E. coli strains for glyphosate absorption and degradation. Key activities include the cloning of phnE1/E2 and phnJ genes to enhance glyphosate degradation, along with the construction of a strain expressing phnO for AMPA degradation. In addition, cold-inducible systems were designed, including a PcspA-mRFP reporter for temperature-sensitive fluorescence and a PcspA-mazF suicide system to induce cell death at low temperatures. Throughout the project, key molecular biology techniques—such as gene cloning, PCR, plasmid extraction, transformation, and ELISA assays—were used to measure strain performance, enzyme kinetics, and functionality under different conditions, culminating in the successful creation of engineered strains and detailed experimental results.

Phase 1: Preparation and Learning (Day 1 - Day 5)

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Day |

Task |

Objective |

Steps |

Expected Results |

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Day 1 |

Study Experimental Procedures and Theory |

Familiarize students with all experimental steps and principles |

- Read related literature and protocols |

- Discuss the experimental design

Students understand the purpose and methods of the experiment |

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Day 2 |

Learn Basic Molecular Biology Techniques |

Understand basic techniques such as PCR, plasmid extraction, and transformation |

- Train students in PCR, plasmid extraction, and digestion |

- Watch instructional videos

Students acquire basic experimental skills |

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Day 3 |

Prepare Equipment and Materials |

Ensure that all equipment and reagents are ready |

- Prepare LB media, antibiotics, and PCR reagents |

- Check lab equipment

Materials and equipment are ready |

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Day 4 |

Pilot Experiment |

Conduct a small-scale pilot experiment to ensure smooth workflow |

- Prepare LB media and test the quality |

- Perform a small-scale plasmid transformation

Pilot test results are normal, preparing for full experiment |

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Day 5 |

Learn Data Analysis Software |

Understand how to analyze ELISA and enzyme activity results |

- Learn how to use software like GraphPad Prism for data analysis |

Students can analyze experimental data |

Phase 2: Gene Synthesis, Cloning, and Strain Construction (Day 6 - Day 15)

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Day |

Task |

Objective |

Steps |

Expected Results |

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Day 6 |

Synthesize and Optimize phnE1/E2 Genes |

Generate codon-optimized phnE1/E2 genes for expression |

- Order the synthesized phnE1/E2 genes |

- Prepare the plasmid vector

Receive synthesized genes and plasmid |

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Day 7 |

Clone Genes into pSB1A3 Vector |

Complete cloning of phnE1/E2 genes |

- Use restriction digestion and ligation to clone the genes into pSB1A3 |

- Verify via agarose gel electrophoresis

Cloning confirmed with correct size bands |

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Day 8 |

Transform Plasmid |

Transform the plasmid into E. coli BL21 |

- Transform recombinant plasmid into E. coli BL21 |

- Plate on LB + ampicillin plates

Colonies appear on the plate, indicating successful transformation |

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Day 9 |

Colony PCR Verification |

Verify the positive clones via PCR |

- Select colonies and perform colony PCR to check for phnE1/E2 genes |

Positive PCR results confirm the presence of target genes |

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Day 10 |

Plasmid Extraction |

Extract plasmids for sequencing |

- Extract plasmids using a plasmid extraction kit |

- Measure DNA concentration

High concentration of plasmid DNA obtained |

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Day 11 |

Sequencing Verification |

Confirm the correctness of the cloned genes |

- Send the plasmid for sequencing |

- Analyze sequencing results

Sequencing confirms the correct gene sequence |

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Day 12 |

Strain Growth for Absorption Test |

Prepare the engineered strain for absorption testing |

- Inoculate positive clones into fresh LB media and grow overnight |

Active strain ready for absorption test |

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Day 13 |

Prepare Media |

Prepare LB media containing glyphosate |

- Prepare LB media containing 80 mg/L glyphosate and 50 µg/mL ampicillin |

Media is ready for upcoming experiments |

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Day 14 |

Initial Glyphosate Absorption Test |

Verify the engineered strain’s glyphosate absorption capability |

- Inoculate engineered strain into glyphosate media, take samples after 3 hours |

- Prepare samples for ELISA

Samples are ready for ELISA testing |

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Day 15 |

ELISA Testing |

Measure the glyphosate concentration |

- Use glyphosate ELISA kit to measure absorption levels |

Absorption data obtained from ELISA |

Phase 3: Time-Course Experiments and Optimization (Day 16 - Day 25)

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Day |

Task |

Objective |

Steps |

Expected Results |

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Day 16 |

Glyphosate Absorption Time-Course Test |

Measure the time dynamics of glyphosate absorption |

- Take hourly samples, centrifuge |

- Measure glyphosate concentration using ELISA

Data for the absorption time course |

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Day 17 |

Data Analysis and Optimization |

Analyze time-course results and optimize conditions |

- Analyze the absorption curve to identify the best absorption time |

- Optimize conditions such as shaking speed and temperature

Optimal experimental conditions determined |

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Day 18 |

Repeat Absorption Experiment |

Confirm absorption efficiency under optimized conditions |

- Repeat glyphosate absorption experiment under optimal conditions |

Reliable and repeatable data obtained |

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Day 19 |

Construct phnE1/E2-phnJ Strain |

Add the phnJ gene to the engineered strain |

- Clone and transform the phnJ gene into the existing strain |

New phnE1/E2-phnJ strain constructed |

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Day 20 |

Test Glyphosate Degradation Ability |

Measure degradation efficiency of the phnE1/E2-phnJ strain |

- Perform glyphosate degradation experiment |

- Measure degradation via ELISA

Glyphosate degradation efficiency data |

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Day 21 |

Construct phnO Strain |

Clone and transform the phnO gene into E. coli |

- Clone and transform phnO into E. coli |

phnO strain successfully constructed |

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Day 22 |

AMPA Degradation Test |

Test the AMPA degradation ability of the phnO strain |

- Prepare crude enzyme extract |

- Measure AMPA degradation activity

AMPA degradation data obtained |

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Day 23 |

Repeat AMPA Degradation Test |

Ensure reproducibility of results |

- Repeat the AMPA degradation experiment |

Stable degradation data confirmed |

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Day 24 |

Enzyme Activity Test |

Measure the kinetic parameters of phnO enzyme |

- Perform enzyme kinetics experiment with different AMPA concentrations |

Km and Vmax data obtained |

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Day 25 |

Data Analysis and Report Writing |

Analyze all data and start writing the report |

- Analyze data using GraphPad Prism |

- Begin writing the experimental results and conclusions

Data analyzed, and part of the report drafted |

Phase 4: Cold-Inducible and Suicide Systems Testing (Day 26 - Day 30)

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Day |

Task |

Objective |

Steps |

Expected Results |

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Day 26 |

Construct Cold-Inducible Reporter Strain |

Build a cold-inducible fluorescent reporter strain |

- Clone the cold-inducible PcspA promoter and transform into the vector |

Cold-inducible reporter strain successfully constructed |

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Day 27 |

Cold-Inducible Reporter Test |

Test reporter gene expression at different temperatures |

- Grow at 16°C, 25°C, and 37°C |

- Measure fluorescence and OD600

Temperature-dependent fluorescence data |

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Day 28 |

Construct Cold-Inducible Suicide System |

Build a cold-inducible suicide system with the mazF toxin gene |

- Clone PcspA-mazF and transform into E. coli |

Cold-inducible suicide system constructed |

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Day 29 |

Suicide System Test |

Test the suicide effect at cold temperatures |

- Grow strain at 16°C and measure OD600 changes |

Confirmation of the suicide system’s functionality |

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Day 30 |

Final Report Writing and Presentation Preparation |

Complete the experimental report and prepare for the presentation |

- Compile all experimental data |

- Finalize the report and prepare the presentation materials

Final report and presentation materials completed |

Daily notebook

WHHS-China raw data

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CONTENTS

[Abstract](#a1)
[First Cycle: phnE1/E2 Engineered Strain](#a2)
[Second Cycle: phnE1/E2-phnJ Engineered Strain](#a3)
[Third Cycle: phnO Engineered Strain](#a4)
[Fourth Cycle: Cold-Inducible Suicide System](#a5)
[Conclusion](#a6)

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Abstract

This project aimed to engineer E. coli strains capable of efficient glyphosate absorption, degradation, and biocontainment. The first cycle focused on constructing a strain expressing phnE1 and phnE2 genes to enhance glyphosate uptake. In the second cycle, the phnJ gene was added to improve degradation, although limited success led to addressing AMPA byproduct degradation in the third cycle through the introduction of phnO. Finally, to address biosafety concerns, a cold-inducible suicide system using the PcspA promoter and mazF toxin was developed to ensure controlled bacterial growth in the environment.

First Cycle: phnE1/E2 Engineered Strain

Design

The goal of this cycle was to construct an E. coli BL21 strain that could absorb glyphosate more efficiently by expressing the phnE1 and phnE2 genes from Sinorhizobium meliloti 1021. These genes are responsible for encoding enzymes that facilitate the transport of glyphosate into the bacterial cell. By introducing both genes, we aimed to improve the uptake of glyphosate, which is a crucial first step before its degradation.

Key considerations in the design:

phnE1 and phnE2 genes were selected for their known role in glyphosate transport.

The genes were codon-optimized for efficient expression in E. coli BL21.

The pSB1A3 plasmid was chosen as the vector, which includes an ampicillin resistance gene for selection purposes.

![](https://static.igem.wiki/teams/5448/engineering/img1.png)

Figure 1. Diagram showing glyphosate transport and genetic circuit.

Build

The construction process involved several steps to ensure the successful expression of phnE1 and phnE2 in E. coli:

Gene Synthesis and Optimization: The phnE1 and phnE2 genes were synthesized with codon optimization to enhance expression in E. coli. This step is crucial to ensure that the bacterial system can effectively translate these genes into functional proteins.

Cloning into Vector: Both genes were cloned into the pSB1A3 vector. This vector was selected due to its compatibility with E. coli and its use in iGEM standards for modular cloning. The cloning involved using restriction enzyme digestion followed by ligation to insert the genes into the plasmid.

Transformation: The recombinant plasmid was transformed into E. coli BL21. The transformed cells were grown on LB agar plates containing ampicillin (50 µg/mL) to select for successful transformants. Only cells that successfully incorporated the plasmid would grow in the presence of ampicillin.

Verification of Construction: The successful integration of the phnE1 and phnE2 genes was confirmed through PCR and agarose gel electrophoresis, verifying that the genes had been correctly inserted into the plasmid.

![](https://static.igem.wiki/teams/5448/engineering/img2.png)

Figure 2: Agarose gel electrophoresis of phnE1 and phnE2 nucleic acids after PCR amplification.

Test

Once the engineered strain expressing phnE1/E2 was constructed, two key tests were performed to evaluate the strain’s ability to absorb glyphosate.

Test 1: Glyphosate Absorption Efficiency

**Objective and Methods**

The purpose of this experiment was to evaluate the glyphosate absorption capacity of the engineered E. coli strain expressing phnE1 and phnE2. The engineered strain was first cultured overnight in LB medium containing 100 µg/mL ampicillin. The next day, the culture was diluted 1:100 into LB medium supplemented with 80 mg/L glyphosate and 50 µg/mL ampicillin and incubated for 3 hours. After incubation, the cultures were centrifuged, and the supernatant was collected for glyphosate concentration analysis using an ELISA detection kit. Absorbance was measured at 450 nm to determine the glyphosate levels.

![](https://static.igem.wiki/teams/5448/engineering/img3.png)

**Results and Conclusion**

The engineered E. coli strain expressing phnE1 and phnE2 exhibited a significant reduction in glyphosate concentration in the medium after 3 hours of incubation. This indicates that the strain was able to absorb glyphosate efficiently. The results confirm the functional expression of the phnE1 and phnE2 genes, enhancing the glyphosate uptake ability of the engineered strain.

Test 2: Time-Course of Glyphosate Absorption

**Objective and Methods**

The objective of this experiment was to evaluate the glyphosate absorption capacity of the engineered E. coli strain expressing phnE1 and phnE2 over a 5-hour period. The strain was first cultured overnight and then diluted 1:100 into LB medium containing 80 mg/L glyphosate and 50 µg/mL ampicillin. The cultures were incubated at 37°C with shaking, and 1 mL samples were taken at hourly intervals for 5 hours. After centrifugation, the supernatant was collected, and glyphosate concentration was analyzed using an ELISA detection kit. Absorbance at 450 nm was measured, and the glyphosate concentration was calculated based on a standard curve.

![](https://static.igem.wiki/teams/5448/engineering/img4.png)

Figure 4: Time course of glyphosate absorption by the phnE1/E2 engineered strain over 5 hours. Glyphosate concentration was measured hourly using ELISA detection, with significant reduction seen over time.

**Results and Conclusion**

The time-course analysis showed a steady decrease in glyphosate concentration over the 5-hour period. After 1 hour, there was a substantial reduction in glyphosate levels, indicating rapid uptake by the engineered strain. Over the next four hours, the absorption continued, though at a slightly slower rate. By the 5th hour, glyphosate concentration in the medium had decreased by over 60%, confirming the strain’s sustained absorption capability.

In conclusion, the phnE1/E2 engineered strain demonstrated effective and continuous absorption of glyphosate over the 5-hour testing period, with the highest rate of uptake occurring in the first hour. This time curve highlights the strain’s potential for extended applications in glyphosate removal.

Learn

From the results of the tests, we confirmed that the engineered phnE1/E2 strain was capable of absorbing glyphosate effectively. However, this is only the first step in the overall process of glyphosate degradation. Once absorbed, the glyphosate needs to be broken down within the cell to mitigate its environmental impact.

Second Cycle: phnE1/E2-phnJ Engineered Strain

Design

Following the successful absorption of glyphosate in the first cycle, the focus of this cycle was to enhance the degradation of glyphosate after it was absorbed by the phnE1/E2 engineered strain. The degradation of glyphosate requires a series of enzymatic steps, and we hypothesized that the expression of the phnJ gene could accelerate this process.

phnJ encodes a phosphonatase enzyme responsible for catalyzing the breakdown of glyphosate into less harmful byproducts. The goal was to co-express phnJ along with phnE1/E2 to form a more efficient system capable of both absorbing and degrading glyphosate.

The J23100 promoter was selected to drive the expression of phnJ, ensuring constitutive expression of the enzyme in the engineered strain. The plasmid vector pSB1A3 was again used for consistency with previous cycles, and for maintaining ampicillin resistance as a selection marker.

![](https://static.igem.wiki/teams/5448/engineering/img5.png)

Figure 5. Diagram illustrating C-P cleavage and function of phnJ.

Build

The construction process in this cycle involved cloning the phnJ gene into the plasmid already containing the phnE1 and phnE2 genes, and ensuring that all three genes were properly expressed in E. coli BL21.

Gene Synthesis and Insertion: The phnJ gene from Enterobacter cloacae K7 was synthesized with codon optimization for expression in E. coli. It was then inserted downstream of the phnE1/E2 gene sequence in the pSB1A3 plasmid using restriction enzyme digestion and ligation.

Plasmid Construction and Transformation: The recombinant plasmid, now containing phnE1/E2 and phnJ, was transformed into E. coli BL21 via heat shock. The cells were grown on LB agar plates containing ampicillin to select for transformants carrying the plasmid.

Verification: PCR amplification was used to verify the correct insertion of phnJ. Gel electrophoresis confirmed the presence of the desired gene fragments, ensuring that the plasmid had been successfully constructed.

![](https://static.igem.wiki/teams/5448/engineering/img6.png)

Figure 6: Agarose gel electrophoresis showing the successful amplification and insertion of the phnE1, phnE2, and phnJ genes into the pSB1A3 vector.

Test: Glyphosate Degradation Efficiency

**Objective and Methods**

The goal of this experiment was to evaluate the glyphosate degradation capability of the phnE1/E2-phnJ engineered E. coli strain. The strain was first cultured overnight, then diluted 1:100 into LB medium containing 80 mg/L glyphosate and 50 µg/mL ampicillin. The cultures were incubated at 37°C with shaking for 3 hours. Samples were collected at 0 hours and 3 hours, centrifuged, and the supernatant was analyzed using an ELISA detection kit. Absorbance was measured at 450 nm to determine the glyphosate concentration, which was then calculated using a standard curve.

![](https://static.igem.wiki/teams/5448/engineering/img7.png)

Figure 7: Comparison of glyphosate concentration at 0 and 3 hours in the phnE1/E2-phnJ and phnE1/E2 engineered strains, showing no significant difference in degradation ability between the two strains.

**Results and Conclusion**

The results showed that both the phnE1/E2-phnJ and phnE1/E2 engineered strains degraded glyphosate over the 3-hour incubation period, but there was no significant difference between the two strains in terms of degradation efficiency. Despite the additional expression of the phnJ gene in the phnE1/E2-phnJ strain, glyphosate concentration in the medium decreased similarly in both strains.

Upon further analysis and discussion, it was hypothesized that the rate-limiting step in glyphosate degradation may be the transport of glyphosate into the cells, rather than the intracellular enzymatic degradation. This could explain why the extra expression of phnJ did not result in a significant improvement in degradation efficiency. Future experiments may focus on optimizing the transport mechanisms to enhance the overall degradation process.

In conclusion, although the phnE1/E2-phnJ strain demonstrated effective glyphosate degradation, the results suggest that transport, rather than enzymatic breakdown, may be the bottleneck in the degradation pathway. Further work is needed to address this limitation.

Learn:

The results from this cycle indicated that despite the successful introduction and expression of the phnJ gene, no significant improvement in glyphosate degradation was observed compared to the phnE1/E2 strain. This suggested that the degradation bottleneck might not be the enzymatic breakdown within the cell but rather the presence of AMPA, a toxic byproduct of glyphosate degradation, which remained in the medium. Therefore, the focus for the next cycle shifted towards degrading AMPA, leading to the design of the phnO engineered strain.

Third Cycle: phnO Engineered Strain

Design

After observing AMPA accumulation during glyphosate degradation in the second cycle, the third cycle aimed to degrade AMPA, a toxic byproduct of glyphosate breakdown. The strategy involved the expression of the phnO gene, which encodes an enzyme responsible for converting AMPA into a less harmful product via N-acetylation.

phnO from Salmonella enterica LT2 was selected for this task. The enzyme it encodes, AMPA N-acetyltransferase, catalyzes the acetylation of AMPA, rendering it non-toxic.

The pET23b plasmid was chosen for this cycle to provide robust expression of phnO under control of the T7 promoter. This vector also contains an ampicillin resistance gene, ensuring consistency in selection with previous cycles.

![](https://static.igem.wiki/teams/5448/engineering/img8.png)

Figure 8. Diagram showing AMPA degradation and the function of phnO.

Build

To construct the engineered strain capable of degrading AMPA, the following steps were carried out:

Gene Synthesis and Cloning: The phnO gene was codon-optimized for expression in E. coli and synthesized. It was cloned into the pET23b plasmid using EcoRI and XhoI restriction sites to ensure correct orientation and expression.

Transformation: The recombinant phnO plasmid was transformed into E. coli BL21 using heat shock transformation. Successful transformants were selected on LB agar plates containing ampicillin (50 µg/mL).

Verification: To confirm successful plasmid construction, colony PCR was conducted followed by gel electrophoresis to verify the presence of phnO in the plasmid. Sequencing was also performed to ensure the correct insertion of the gene.

![](https://static.igem.wiki/teams/5448/engineering/img9.png)

Figure 9: Agarose gel electrophoresis confirming the successful insertion of the phnO gene into the pET23b vector.

Test

With the engineered phnO strain constructed, several tests were performed to assess its ability to degrade AMPA and the efficiency of the enzyme.

Test 1: AMPA Degradation Test

**Objective and Methods**

The purpose of this experiment was to assess the ability of the phnO engineered strain to degrade aminomethylphosphonic acid (AMPA), a toxic byproduct of glyphosate breakdown. Accumulation of AMPA in the environment poses potential ecological and health risks, making its degradation crucial for effective bioremediation. The phnO gene, encoding an AMPA N-acetyltransferase, was expressed in E. coli BL21 to enable the degradation of this harmful compound.

The engineered strain was cultured overnight in LB medium containing 50 µg/mL ampicillin at 37°C. The next day, the culture was diluted 1:100 into fresh LB medium and grown until an OD600 of 0.6 was reached. Cells were collected by centrifugation, resuspended in pre-cooled Tris-HCl buffer, and lysed via sonication to obtain a crude enzyme extract. The reaction mixture, containing 1 mM AMPA, acetyl-CoA, and magnesium chloride, was incubated with the crude extract at 37°C for 3 hours. The degradation of AMPA was monitored by measuring CoA production using a DTDP-based colorimetric assay at 324 nm.

![](https://static.igem.wiki/teams/5448/engineering/img10.png)

Figure 10: Degradation of toxic AMPA by the phnO engineered strain, as indicated by CoA production. Absorbance was measured at 324 nm using the DTDP assay.

**Results and Conclusion**

The phnO engineered strain demonstrated a significant capacity to degrade AMPA, as evidenced by the increase in CoA production in the reaction mixture. The absorbance at 324 nm confirmed that the phnO enzyme successfully catalyzed the acetylation of AMPA, leading to its degradation. The reduction of this toxic byproduct is a critical step in mitigating the environmental and health hazards associated with glyphosate use.

In conclusion, the phnO strain efficiently degraded the toxic compound AMPA, highlighting the strain’s potential for comprehensive glyphosate degradation. By breaking down AMPA, this engineered strain provides a valuable tool for reducing the harmful effects of glyphosate byproducts in bioremediation efforts.

Test 2: Time-Course of AMPA Degradation

**Objective and Methods**

The objective of this experiment was to evaluate the time-dependent degradation of aminomethylphosphonic acid (AMPA) by the phnO engineered strain. AMPA, a toxic byproduct of glyphosate degradation, poses environmental and health risks, making its efficient degradation critical. The engineered strain expressing the phnO gene was tested for its ability to degrade AMPA over a 5-hour period.

The strain was cultured overnight in LB medium containing 50 µg/mL ampicillin. The following day, the culture was diluted 1:100 into fresh LB medium and incubated at 37°C until an OD600 of 0.6 was reached. After centrifugation, the cells were resuspended in pre-cooled Tris-HCl buffer and lysed by sonication to prepare a crude enzyme extract.

The reaction mixture containing 1 mM AMPA, acetyl-CoA, and magnesium chloride was incubated with the crude enzyme extract at 37°C. Samples were taken at hourly intervals for 5 hours, and CoA production was measured using the DTDP assay by recording the absorbance at 324 nm. The production of CoA indicated the degradation of AMPA by the phnO enzyme over time.

![](https://static.igem.wiki/teams/5448/engineering/img11.png)

Figure 11: Time-dependent degradation of AMPA by the phnO engineered strain, measured by CoA production at hourly intervals over 5 hours. Absorbance was recorded at 324 nm using the DTDP assay.

**Results and Conclusion**

The time-course analysis revealed a steady increase in CoA production over the 5-hour period, indicating continuous degradation of AMPA by the phnO enzyme. The rate of degradation was highest during the first two hours, after which it slowed slightly but remained consistent throughout the experiment. By the 5th hour, a significant reduction in AMPA levels was observed, confirming the enzyme’s sustained activity.

In conclusion, the phnO engineered strain efficiently degraded AMPA over time, with the most rapid degradation occurring in the initial stages. The ability of the strain to maintain enzyme activity over several hours highlights its potential for use in bioremediation processes aimed at mitigating the harmful effects of glyphosate byproducts, such as AMPA.

Test 3: Kinetic Analysis of phnO Enzyme

**Objective and Methods**

The objective of this experiment was to determine the kinetic parameters of the phnO enzyme expressed in E. coli BL21. The enzyme catalyzes the degradation of aminomethylphosphonic acid (AMPA), a toxic byproduct of glyphosate degradation. Understanding the enzyme’s kinetics is crucial for optimizing its application in bioremediation.

The engineered strain was cultured overnight in LB medium containing 50 µg/mL ampicillin at 37°C. The next day, the culture was diluted 1:100 into fresh LB medium and grown until an OD600 of 0.6. Cells were harvested by centrifugation, resuspended in pre-cooled Tris-HCl buffer, and lysed via sonication to prepare a crude enzyme extract.

To determine the enzyme kinetics, reaction mixtures were prepared with varying concentrations of AMPA and incubated with the crude enzyme extract at 37°C for 3 hours. CoA production was measured using the DTDP assay, and absorbance was recorded at 324 nm. The Michaelis constant (Km) and maximum reaction velocity (Vmax) were calculated using nonlinear regression analysis of the data, plotted as a Michaelis-Menten curve.

![](https://static.igem.wiki/teams/5448/engineering/img12.png)

Figure 12: Michaelis-Menten kinetics curve for the phnO enzyme, showing the relationship between AMPA concentration and reaction velocity (CoA production).

**Results and Conclusion**

The Michaelis-Menten kinetic analysis revealed that the phnO enzyme followed saturation kinetics with increasing concentrations of AMPA. The calculated Km value was 0.5958 mM, indicating a moderate affinity for AMPA. The Vmax was 0.1828 µmol/min/mg protein, reflecting the maximum reaction velocity achieved when the enzyme was saturated with AMPA.

These kinetic parameters demonstrate that the phnO enzyme is effective at catalyzing AMPA degradation at moderate substrate concentrations. The relatively low Km value suggests that the enzyme is well-suited for bioremediation purposes where the environmental concentration of AMPA is likely to be low to moderate.

In conclusion, the kinetic analysis of the phnO enzyme confirmed its ability to efficiently degrade the toxic byproduct AMPA. The specific values of Km and Vmax provide valuable insights for optimizing its application in environmental detoxification strategies.

Learn:

The results from this cycle demonstrated that the engineered phnO strain was able to effectively degrade AMPA, reducing the accumulation of this toxic byproduct. However, while the strain successfully handled AMPA degradation, there were concerns regarding the potential uncontrolled spread of the engineered bacteria in the environment, raising biosafety issues. This realization led to the development of a biocontainment system for the next cycle, focused on creating a cold-inducible suicide system to prevent the bacteria from proliferating outside of controlled conditions.

Fourth Cycle: Cold-Inducible Suicide System

Design

Following the success in degrading AMPA in the third cycle, the next focus was on biosafety. To prevent the potential uncontrolled spread of the engineered bacteria in natural environments, a cold-inducible suicide system was designed. This system would trigger cell death when the bacteria are exposed to lower temperatures, ensuring containment outside of laboratory conditions.

The PcspA promoter, which is cold-inducible, was selected to control the expression of the mazF gene. mazF encodes a toxin that cleaves cellular mRNA, leading to cell death. The idea was that at temperatures below 16°C, PcspA would activate mazF expression, inducing the suicide mechanism.

The plasmid pSB1A3 was chosen to maintain consistency with previous cycles, with ampicillin resistance used for selection.

![](https://static.igem.wiki/teams/5448/engineering/img13.png)

Figure 13. Diagram of the cold-inducible suicide system.

Build

The construction of the cold-inducible suicide system involved cloning the cold-sensitive regulatory elements and ensuring functional expression of the mazF toxin.

Gene Cloning: The mazF gene was codon-optimized for expression in E. coli. It was cloned downstream of the PcspA promoter in the pSB1A3 vector, ensuring that its expression would only occur under cold temperatures.

Transformation: The recombinant plasmid was transformed into E. coli BL21 via heat shock, and transformants were selected on LB agar plates containing ampicillin.

Verification: Colony PCR was performed to verify the correct insertion of the PcspA-mazF construct, followed by sequencing to ensure the accurate assembly of the plasmid.

![](https://static.igem.wiki/teams/5448/engineering/img14.png)

Figure 14: Schematic representation of the cold-inducible reporter system. The PcspA promoter regulates mRFP expression in response to low-temperature conditions.

![](https://static.igem.wiki/teams/5448/engineering/img15.png)

Figure 15: Agarose gel electrophoresis showing the PCR amplification of the PcspA promoter and mazF gene, confirming successful cloning.

Test

With the cold-inducible suicide system in place, several tests were carried out to verify the functionality of the PcspA-mazF system in response to different temperatures.

Test 1: Cold-Inducible Reporter System (mRFP)

**Objective and Methods**

The objective of this experiment was to test the cold-inducible reporter strain constructed with the PcspA promoter driving the expression of mRFP (monomeric red fluorescent protein). The goal was to evaluate the induction of mRFP expression at different temperatures and determine the temperature sensitivity of the cold-inducible system.

The overnight culture of the engineered strain was diluted 1:100 into fresh LB medium containing 50 µg/mL ampicillin. The cultures were then incubated at different temperatures (16°Cand 37°C) with shaking at 180 rpm for 12 hours. After incubation, 200 µL samples were taken from each culture, and fluorescence intensity (excitation at 584 nm and emission at 607 nm) was measured using a microplate reader. Optical density at 600 nm (OD600) was also measured to normalize the fluorescence values. The normalized fluorescence intensity (Fluorescence/OD600) was calculated to compare mRFP expression levels across different temperature conditions.

![](https://static.igem.wiki/teams/5448/engineering/img16.png)

Figure 16: Normalized fluorescence intensity (Fluorescence/OD600) of the cold-inducible reporter strain at various temperatures (16°C, 37°C). Fluorescence was measured at excitation 584 nm and emission 607 nm.

**Results and Conclusion**

The results showed a significant increase in mRFP expression at lower temperatures, with the highest fluorescence observed at 16°C. As the temperature increased, the fluorescence intensity decreased, with minimal expression at 37°C. This indicates that the PcspA promoter is highly sensitive to cold temperatures, and mRFP expression is strongly induced under cold-inducible conditions.

In conclusion, the cold-inducible reporter strain successfully demonstrated temperature-dependent expression of mRFP, with optimal induction occurring at 16°C. This strain can be effectively used to study cold-inducible gene expression systems and could be applied to temperature-controlled biotechnological processes.

Test 2: Cold-Induced Cell Death

**Objective and Methods**

The purpose of this experiment was to test the functionality of the cold-inducible suicide system in E. coli, which utilizes the PcspA promoter to control the expression of the mazF toxin gene. The mazF gene induces cell death by cleaving mRNA, and this system is designed to activate at lower temperatures, offering a temperature-sensitive biocontainment strategy.

The engineered strain was cultured overnight in LB medium containing 50 µg/mL ampicillin at 37°C. The following day, 100 µL of the culture was inoculated into 5 mL of fresh LB medium containing ampicillin and incubated at either 16°C or 37°C with shaking at 180 rpm for 12 hours. After incubation, 200 µL samples were taken from each condition, and cell growth was measured by determining the optical density at 600 nm (OD600). Cell viability at low temperatures was compared with that of control strains and normal growth conditions at 37°C.

![](https://static.igem.wiki/teams/5448/engineering/img17.png)

Figure 17: Comparison of cell growth (OD600) of the cold-inducible suicide strain at 16°C and 37°C after 12 hours of incubation, demonstrating activation of the suicide system at low temperatures.

**Results and Conclusion**

The results showed that at 37°C, the engineered strain grew normally, with no signs of mazF-induced cell death. However, when incubated at 16°C, the growth of the engineered strain was significantly inhibited, with a drastic reduction in OD600, indicating the activation of the suicide system. The control strain without the mazF gene showed normal growth at both 16°C and 37°C, confirming that the observed growth inhibition was due to the cold-inducible expression of mazF.

In conclusion, the cold-inducible suicide system effectively triggered cell death at 16°C, validating the functionality of the PcspA-regulated mazF system. This system provides a potential safety mechanism for biocontainment, ensuring that the engineered bacteria can be selectively eliminated under specific environmental conditions.

Learn

The cold-inducible suicide system proved effective in triggering cell death at low temperatures, providing a reliable biocontainment mechanism. This system ensures that the engineered bacteria can be safely controlled in the environment, as the suicide gene is activated when the bacteria leave the controlled, warmer laboratory environment. With the cold-inducible PcspA promoter successfully regulating gene expression, the system is a valuable tool for biosafety, reducing risks associated with the release of genetically modified organisms into the wild.

Conclusion

The project successfully developed engineered E. coli strains that absorbed and degraded glyphosate and AMPA efficiently while implementing a cold-inducible suicide system for environmental biosafety. While the introduction of the phnJ gene did not enhance degradation, the addition of phnO effectively degraded AMPA. The cold-inducible suicide system provided an essential biocontainment mechanism, making the engineered bacteria safer for environmental applications in glyphosate bioremediation.

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CONTENTS

[Construction of phnE1/E2 Glyphosate-Engineering Strain](#a1)
[Verification of Glyphosate Absorption by phnE1/E2 Engineered Strain](#a2)
[Testing Time Course of Glyphosate Absorption in phnE1/E2 Engineered Strain](#a3)
[Testing Glyphosate Degradation by phnE1/E2-phnJ Engineered Strain](#a4)
[Construction of phnO Engineered Strain](#a5)
[Testing AMPA Degradation by phnO Engineered Strain](#a6)
[Time-Course Testing of AMPA Degradation by phnO Engineered Strain](#a7)
[Kinetic Analysis of phnO Enzyme Activity](#a8)
[Construction of Cold-Inducible Reporter Strain](#a9)
[Testing of Cold-Inducible Reporter Strain](#a10)
[Construction of Cold-Inducible Suicide System](#a11)
[Testing of Cold-Inducible Suicide System](#a12)
[Statistical Analysis](#a13)
[Related kit instructions](#a14)

---

Construction of phnE1/E2 Glyphosate-Engineering Strain

The phnE1 and phnE2 genes from Sinorhizobium meliloti 1021 were synthesized, and codon-optimized for expression in E. coli. These genes were cloned into the pSB1A3 vector as polycistronic units to form a recombinant plasmid. The plasmid was then transformed into E. coli BL21 to create the engineered strain. The strain was grown in LB medium containing 50 µg/mL ampicillin at 37°C.

Verification of Glyphosate Absorption by phnE1/E2 Engineered Strain

The engineered strain was cultured overnight in LB medium containing 100 µg/mL ampicillin at 37°C with shaking at 180 rpm. The next day, the culture was diluted 1:100 into LB medium containing 80 mg/L glyphosate (Sigma-Aldrich) and 50 µg/mL ampicillin, then incubated under the same conditions for 3 hours. 1 mL samples were taken, centrifuged at 12,000 g for 10 minutes at 4°C, and the supernatant was collected. Glyphosate concentration was measured using a Glyphosate ELISA kit (JI-ICHI), following the manufacturer’s protocol. Samples and standards were added to ELISA wells, incubated, and washed. Enzyme conjugates were added, followed by another incubation and washing step. Substrates were added, and after color development, a stop solution was added. Absorbance was measured at 450 nm, and the glyphosate concentration was calculated based on a standard curve.

Testing Time Course of Glyphosate Absorption in phnE1/E2 Engineered Strain

After overnight activation, the engineered strain was diluted 1:100 into 50 mL LB medium containing 80 mg/L glyphosate and 50 µg/mL ampicillin and incubated at 37°C with shaking at 180 rpm. 1 mL samples were taken hourly for 5 hours, centrifuged at 10,000 g for 10 minutes at 4°C, and the supernatant was collected. Glyphosate concentration was measured as described previously, with absorbance at 450 nm.

Construction of phnE1/E2-phnJ Engineered Strain

Building on the phnE1/E2 recombinant plasmid, the J23100-B0034-phnJ sequence from Enterobacter cloacae K7 was synthesized and cloned downstream of the phnE1 and phnE2 genes. The recombinant plasmid was then transformed into E. coli BL21 to form the engineered strain, which was cultured in LB medium containing 50 µg/mL ampicillin at 37°C.

Testing Glyphosate Degradation by phnE1/E2-phnJ Engineered Strain

After overnight activation, the engineered strain was diluted 1:100 into 50 mL LB medium containing 80 mg/L glyphosate and 50 µg/mL ampicillin, then incubated at 37°C with shaking at 180 rpm for 3 hours. Samples were taken at 0 and 3 hours, centrifuged at 10,000 g for 10 minutes at 4°C, and the supernatant was collected. Glyphosate concentration was measured using the ELISA method at 450 nm. The degradation efficiency of different strains was compared.

Construction of phnO Engineered Strain

The phnO gene from Salmonella enterica LT2 was synthesized and codon-optimized for expression in E. coli. It was cloned into the pET23b vector (using EcoRI and XhoI sites) to form a recombinant plasmid. The plasmid was then transformed into E. coli BL21, and the engineered strain was cultured in LB medium containing 50 µg/mL ampicillin at 37°C.

Testing AMPA Degradation by phnO Engineered Strain

The phnO-overexpressing strain was inoculated into LB medium containing 50 µg/mL ampicillin and incubated overnight at 37°C with shaking at 200 rpm. The next day, the culture was diluted 1:100 into fresh LB medium and incubated for 2-3 hours until OD600 reached 0.6. Cells were harvested by centrifugation at 10,000 g for 5 minutes and resuspended in pre-cooled 25 mM Tris-HCl buffer (with 0.3 M NaCl, 10 mM imidazole, and 1 mM EDTA, pH 8.0). Cells were lysed by sonication (1s on, 3s off, 250 W, 10 minutes), and the supernatant was collected by centrifugation at 13,000 g for 20 minutes to prepare a crude enzyme extract.

To test phnO activity, 100 µL of crude enzyme extract was added to a 6 mL reaction system containing 1 mM aminomethylphosphonate (AMPA), 3 mM acetyl-CoA, 3 mM magnesium chloride, and 25 mM Tris-HCl buffer. The reaction was incubated at 37°C for 3 hours. phnO acts as an AMPA N-acetyltransferase, catalyzing the reaction between AMPA and acetyl-CoA to produce CoA. A 0.5 mM 44'-dithiodipyridine (DTDP) solution in PBS was freshly prepared, and 1 mL of the reaction mixture was mixed with 100 µL DTDP solution in a 96-well plate and incubated for 30 minutes at room temperature. CoA reacts with DTDP to form thiopyridone, and absorbance at 324 nm was measured to quantify CoA production and assess AMPA degradation.

Time-Course Testing of AMPA Degradation by phnO Engineered Strain

After overnight activation, the engineered strain was diluted into 50 mL LB medium containing ampicillin and incubated at 37°C with shaking at 180 rpm until OD600 reached 0.6. Cells were harvested, resuspended in Tris-HCl buffer, and lysed by sonication. The crude enzyme extract was prepared as previously described. 100 µL of crude extract was added to a 6 mL AMPA reaction system and incubated at 37°C for 3 hours. CoA production was measured using the DTDP method to assess AMPA degradation.

Kinetic Analysis of phnO Enzyme Activity

The engineered strain was cultured as described above, and crude enzyme extract was prepared. The enzyme kinetics were measured by adding 100 µL of crude extract to a 6 mL reaction system containing varying concentrations of AMPA. After a 3-hour incubation at 37°C, CoA production was measured using the DTDP method. One enzyme activity unit was defined as the amount of enzyme that catalyzes the degradation of 1 mM AMPA per minute per mg of protein. The kinetic parameters Km and Vmax were calculated using GraphPad Prism 8.0 software based on the Michaelis-Menten curve.

Construction of Cold-Inducible Reporter Strain

The cold-inducible PcspA promoter (BBa\_K3289001) was synthesized, and the mRFP gene was placed downstream of PcspA. The mRFP gene was codon-optimized for E. coli, with removal of EcoRI, XbaI, SpeI, and PstI restriction sites to comply with RFC#10 standards (Genewiz, USA). The PcspA-mRFP construct was cloned into the pSB1A3 vector (between XbaI and SpeI sites), and the recombinant plasmid was transformed into E. coli BL21. Positive clones were selected on LB plates containing 50 µg/mL ampicillin at 37°C and verified by sequencing. The engineered strain was cultured in LB medium with ampicillin, and optical density at 600 nm (OD600) was measured to monitor growth.

Testing of Cold-Inducible Reporter Strain

The cold-inducible reporter strain was activated overnight and diluted 1:100 into fresh LB medium. The cultures were incubated at different temperatures (16°C, 25°C, and 37°C) with shaking at 180 rpm for 12 hours. 200 µL samples were taken, and fluorescence intensity (excitation at 584 nm, emission at 607 nm) was measured using a microplate reader. OD600 was also measured, and the normalized fluorescence intensity (Fluorescence/OD600) was calculated.

Construction of Cold-Inducible Suicide System

The mazF toxin gene was cloned downstream of the PcspA promoter. The mazF gene was codon-optimized for E. coli and had EcoRI, XbaI, SpeI, and PstI restriction sites removed to comply with RFC#10 standards. The PcspA-mazF construct was cloned into the pSB1A3 vector and transformed into E. coli BL21.

Testing of Cold-Inducible Suicide System

The cold-inducible suicide strain and control strain were cultured overnight. 100 µL of the culture was inoculated into 5 mL LB medium containing 50 µg/mL ampicillin and incubated at 16°C and 180 rpm for 12 hours. 200 µL samples were taken to measure OD600 to assess cell growth.

Statistical Analysis

All data were analyzed using GraphPad Prism software. Results are presented as mean ± standard deviation (SD). One-way ANOVA followed by Tukey’s post-hoc test was used for multi-group comparisons, while a two-tailed Student’s t-test was used for two-group comparisons. A p-value of <0.05 was considered statistically significant.

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CONTENTS

[Abstract](#a1)
[phnE1/E2 glyphosate engineering strain:](#a2)
[Verification of Glyphosate Absorption Ability in phnE1/E2 Engineered Strain:](#a3)
[Testing the Time Curve of Glyphosate Absorption in the phnE1/E2 Engineered Strain](#a4)
[Construction of the phnE1/E2-phnJ Engineered Strain:](#a5)
[Testing Glyphosate Degradation by the phnE1/E2-phnJ Engineered Strain](#a6)
[Construction of the phnO Engineered Strain:](#a7)
[Testing the Degradation of AMPA by the phnO Engineered Strain:](#a8)
[Testing the Time Course of AMPA Degradation by the phnO Engineered Strain:](#a9)
[Kinetic Analysis of the phnO Enzyme Activity](#a10)
[Construction of the Cold-Inducible Reporter Strain:](#a11)
[Testing the Cold-Inducible Reporter Strain:](#a12)
[Construction of the Cold-Inducible Suicide System:](#a13)
[Testing the Cold-Inducible Suicide System:](#a14)
[Statistical Analysis:](#a15)
[Summary](#a16)
[References:](#a17)

---

Abstract

This study aimed to construct and evaluate several genetically engineered Escherichia colistrains for glyphosate degradation and cold-inducible biocontainment systems. The engineered strains expressing **phnE1/E2, phnE1/E2-phnJ, and **phnO** genes were successfully constructed, and their abilities to absorb and degrade glyphosate and its byproduct AMPA were tested. Additionally, a cold-inducible reporter strain and a cold-inducible suicide system using the **PcspA** promoter and **mazF** gene were developed and validated. Statistical analysis confirmed that the experimental results were significant, providing a robust foundation for further bioremediation and biocontainment applications.**

phnE1/E2 glyphosate engineering strain:

Objective and Methods

The aim was to construct an engineered E. coli BL21 strain by synthesizing the codon-optimized **phnE1** and **phnE2** genes from Sinorhizobium meliloti 1021. These genes were co-expressed in the pSB1A3 vector. The recombinant plasmid was transformed into E. coli BL21 and cultured in LB medium containing 50 µg/mL ampicillin at 37°C to select for the engineered strain.

![](https://static.igem.wiki/teams/5448/results/img1.png)

Figure 1: Agarose gel electrophoresis of phnE1 and phnE2 nucleic acids after PCR amplification.

Results and Conclusion

The recombinant plasmid containing the **phnE1** and **phnE2** genes was successfully constructed and transformed into E. coli BL21. The engineered strain grew in LB medium supplemented with ampicillin, confirming successful plasmid incorporation and selection. This strain is now ready for further testing related to glyphosate absorption.

Verification of Glyphosate Absorption Ability in phnE1/E2 Engineered Strain:

Objective and Methods

The purpose of this experiment was to evaluate the glyphosate absorption capacity of the engineered E. coli strain expressing **phnE1** and **phnE2.** The engineered strain was first cultured overnight in LB medium containing 100 µg/mL ampicillin. The next day, the culture was diluted 1:100 into LB medium supplemented with 80 mg/L glyphosate and 50 µg/mL ampicillin and incubated for 3 hours. After incubation, the cultures were centrifuged, and the supernatant was collected for glyphosate concentration analysis using an ELISA detection kit. Absorbance was measured at 450 nm to determine the glyphosate levels.

![](https://static.igem.wiki/teams/5448/results/img2.png)

Results and Conclusion

The engineered E. coli strain expressing **phnE1** and **phnE2** exhibited a significant reduction in glyphosate concentration in the medium after 3 hours of incubation. This indicates that the strain was able to absorb glyphosate efficiently. The results confirm the functional expression of the **phnE1** and **phnE2** genes, enhancing the glyphosate uptake ability of the engineered strain.

Testing the Time Curve of Glyphosate Absorption in the phnE1/E2 Engineered Strain

Objective and Methods

The objective of this experiment was to evaluate the glyphosate absorption capacity of the engineered E. coli strain expressing **phnE1** and **phnE2** over a 5-hour period. The strain was first cultured overnight and then diluted 1:100 into LB medium containing 80 mg/L glyphosate and 50 µg/mL ampicillin. The cultures were incubated at 37°C with shaking, and 1 mL samples were taken at hourly intervals for 5 hours. After centrifugation, the supernatant was collected, and glyphosate concentration was analyzed using an ELISA detection kit. Absorbance at 450 nm was measured, and the glyphosate concentration was calculated based on a standard curve.

![](https://static.igem.wiki/teams/5448/results/img3.png)

Figure 3: Time course of glyphosate absorption by the phnE1/E2 engineered strain over 5 hours. Glyphosate concentration was measured hourly using ELISA detection, with significant reduction seen over time.

Results and Conclusion

The time-course analysis showed a steady decrease in glyphosate concentration over the 5-hour period. After 1 hour, there was a substantial reduction in glyphosate levels, indicating rapid uptake by the engineered strain. Over the next four hours, the absorption continued, though at a slightly slower rate. By the 5th hour, glyphosate concentration in the medium had decreased by over 60%, confirming the strain’s sustained absorption capability.

In conclusion, the **phnE1/E2** engineered strain demonstrated effective and continuous absorption of glyphosate over the 5-hour testing period, with the highest rate of uptake occurring in the first hour. This time curve highlights the strain’s potential for extended applications in glyphosate removal.

Construction of the phnE1/E2-phnJ Engineered Strain:

Objective and Methods

The aim of this experiment was to construct an engineered E. coli BL21 strain expressing **phnE1,** **phnE2,** and **phnJ** genes. The genes **phnE1** and **phnE2** (from Sinorhizobium meliloti 1021) were already inserted into a recombinant plasmid. In this step, the **phnJ** gene from Enterobacter cloacae K7 was synthesized and cloned downstream of the **phnE1/E2** sequence in the pSB1A3 vector, under the control of the J23100 promoter. The recombinant plasmid, containing all three genes, was then transformed into E. coli BL21. The engineered strain was selected and cultured in LB medium containing 50 µg/mL ampicillin at 37°C.

![](https://static.igem.wiki/teams/5448/results/img4.png)

Figure 4: Agarose gel electrophoresis showing the successful amplification and insertion of the phnE1, phnE2, and phnJ genes into the pSB1A3 vector.

Results and Conclusion

The recombinant plasmid containing the **phnE1**, **phnE2,** and **phnJ** genes was successfully constructed and transformed into E. coli BL21. The engineered strain grew in LB medium supplemented with ampicillin, indicating the successful incorporation of the plasmid. The agarose gel electrophoresis confirmed the correct insertion of the three target genes, validating the construction of the desired plasmid.

In conclusion, the **phnE1/E2-phnJ** engineered strain was successfully constructed, laying the foundation for subsequent experiments to evaluate its glyphosate degradation capability.

Testing Glyphosate Degradation by the phnE1/E2-phnJ Engineered Strain

Objective and Methods

The goal of this experiment was to evaluate the glyphosate degradation capability of the **phnE1/E2-phnJ** engineered E. coli strain. The strain was first cultured overnight, then diluted 1:100 into LB medium containing 80 mg/L glyphosate and 50 µg/mL ampicillin. The cultures were incubated at 37°C with shaking for 3 hours. Samples were collected at 0 hours and 3 hours, centrifuged, and the supernatant was analyzed using an ELISA detection kit. Absorbance was measured at 450 nm to determine the glyphosate concentration, which was then calculated using a standard curve.

![](https://static.igem.wiki/teams/5448/results/img5.png)

Figure 5: Comparison of glyphosate concentration at 0 and 3 hours in the phnE1/E2-phnJ and phnE1/E2 engineered strains, showing no significant difference in degradation ability between the two strains.

Results and Conclusion

The results showed that both the **phnE1/E2-phnJ** and **phnE1/E2** engineered strains degraded glyphosate over the 3-hour incubation period, but there was no significant difference between the two strains in terms of degradation efficiency. Despite the additional expression of the **phnJ** gene in the **phnE1/E2-phnJ** strain, glyphosate concentration in the medium decreased similarly in both strains.

Upon further analysis and discussion, it was hypothesized that the rate-limiting step in glyphosate degradation may be the transport of glyphosate into the cells, rather than the intracellular enzymatic degradation. This could explain why the extra expression of **phnJ** did not result in a significant improvement in degradation efficiency. Future experiments may focus on optimizing the transport mechanisms to enhance the overall degradation process.

In conclusion, although the **phnE1/E2-phnJ** strain demonstrated effective glyphosate degradation, the results suggest that transport, rather than enzymatic breakdown, may be the bottleneck in the degradation pathway. Further work is needed to address this limitation.

Construction of the phnO Engineered Strain:

Objective and Methods

The objective of this experiment was to construct an engineered E. coli BL21 strain expressing the **phnO** gene, which encodes an enzyme involved in the degradation of aminomethylphosphonic acid (AMPA), a major byproduct of glyphosate breakdown. The **phnO** gene from Salmonella enterica LT2 was codon-optimized for expression in E. coli and cloned into the pET23b vector using the EcoRI and XhoI restriction sites. The recombinant plasmid was transformed into E. coli BL21, and the engineered strain was selected by growing in LB medium containing 50 µg/mL ampicillin at 37°C.

![](https://static.igem.wiki/teams/5448/results/img6.png)

Figure 6: Agarose gel electrophoresis confirming the successful insertion of the phnO gene into the pET23b vector.

Results and Conclusion

The recombinant plasmid containing the **phnO** gene was successfully constructed and transformed into E. coli BL21. The engineered strain grew robustly in LB medium containing ampicillin, indicating successful plasmid incorporation and expression of the gene. Gel electrophoresis confirmed the correct insertion of **phnO** into the vector.

In conclusion, the **phnO** engineered strain was successfully constructed, providing a basis for further studies on its role in AMPA degradation, which will be critical for advancing the glyphosate degradation pathway.

Testing the Degradation of AMPA by the phnO Engineered Strain:

Objective and Methods

The purpose of this experiment was to assess the ability of the **phnO** engineered strain to degrade aminomethylphosphonic acid (**AMPA**), a toxic byproduct of glyphosate breakdown. Accumulation of AMPA in the environment poses potential ecological and health risks, making its degradation crucial for effective bioremediation. The **phnO** gene, encoding an AMPA N-acetyltransferase, was expressed in E. coli BL21 to enable the degradation of this harmful compound.

The engineered strain was cultured overnight in LB medium containing 50 µg/mL ampicillin at 37°C. The next day, the culture was diluted 1:100 into fresh LB medium and grown until an OD600 of 0.6 was reached. Cells were collected by centrifugation, resuspended in pre-cooled Tris-HCl buffer, and lysed via sonication to obtain a crude enzyme extract. The reaction mixture, containing 1 mM AMPA, acetyl-CoA, and magnesium chloride, was incubated with the crude extract at 37°C for 3 hours. The degradation of AMPA was monitored by measuring CoA production using a DTDP-based colorimetric assay at 324 nm.

![](https://static.igem.wiki/teams/5448/results/img7.png)

Figure 7: Degradation of toxic AMPA by the phnO engineered strain, as indicated by CoA production. Absorbance was measured at 324 nm using the DTDP assay.

Results and Conclusion

The phnO engineered strain demonstrated a significant capacity to degrade AMPA, as evidenced by the increase in CoA production in the reaction mixture. The absorbance at 324 nm confirmed that the **phnO** enzyme successfully catalyzed the acetylation of AMPA, leading to its degradation. The reduction of this toxic byproduct is a critical step in mitigating the environmental and health hazards associated with glyphosate use.

In conclusion, the **phnO** strain efficiently degraded the toxic compound AMPA, highlighting the strain’s potential for comprehensive glyphosate degradation. By breaking down AMPA, this engineered strain provides a valuable tool for reducing the harmful effects of glyphosate byproducts in bioremediation efforts.

Testing the Time Course of AMPA Degradation by the phnO Engineered Strain:

Objective and Methods

The objective of this experiment was to evaluate the time-dependent degradation of aminomethylphosphonic acid (AMPA) by the **phnO** engineered strain. **AMPA**, a toxic byproduct of glyphosate degradation, poses environmental and health risks, making its efficient degradation critical. The engineered strain expressing the **phnO** gene was tested for its ability to degrade AMPA over a 5-hour period.

The strain was cultured overnight in LB medium containing 50 µg/mL ampicillin. The following day, the culture was diluted 1:100 into fresh LB medium and incubated at 37°C until an OD600 of 0.6 was reached. After centrifugation, the cells were resuspended in pre-cooled Tris-HCl buffer and lysed by sonication to prepare a crude enzyme extract.

The reaction mixture containing 1 mM AMPA, acetyl-CoA, and magnesium chloride was incubated with the crude enzyme extract at 37°C. Samples were taken at hourly intervals for 5 hours, and CoA production was measured using the DTDP assay by recording the absorbance at 324 nm. The production of CoA indicated the degradation of AMPA by the phnO enzyme over time.

![](https://static.igem.wiki/teams/5448/results/img8.png)

Figure 8: Time-dependent degradation of AMPA by the phnO engineered strain, measured by CoA production at hourly intervals over 5 hours. Absorbance was recorded at 324 nm using the DTDP assay.

Results and Conclusion

The time-course analysis revealed a steady increase in CoA production over the 5-hour period, indicating continuous degradation of AMPA by the **phnO** enzyme. The rate of degradation was highest during the first two hours, after which it slowed slightly but remained consistent throughout the experiment. By the 5th hour, a significant reduction in AMPA levels was observed, confirming the enzyme’s sustained activity.

In conclusion, the **phnO** engineered strain efficiently degraded AMPA over time, with the most rapid degradation occurring in the initial stages. The ability of the strain to maintain enzyme activity over several hours highlights its potential for use in bioremediation processes aimed at mitigating the harmful effects of glyphosate byproducts, such as AMPA.

Kinetic Analysis of the phnO Enzyme Activity

Objective and Methods

The objective of this experiment was to determine the kinetic parameters of the phnO enzyme expressed in E. coli BL21. The enzyme catalyzes the degradation of aminomethylphosphonic acid (AMPA), a toxic byproduct of glyphosate degradation. Understanding the enzyme’s kinetics is crucial for optimizing its application in bioremediation.

The engineered strain was cultured overnight in LB medium containing 50 µg/mL ampicillin at 37°C. The next day, the culture was diluted 1:100 into fresh LB medium and grown until an OD600 of 0.6. Cells were harvested by centrifugation, resuspended in pre-cooled Tris-HCl buffer, and lysed via sonication to prepare a crude enzyme extract.

To determine the enzyme kinetics, reaction mixtures were prepared with varying concentrations of AMPA and incubated with the crude enzyme extract at 37°C for 3 hours. CoA production was measured using the DTDP assay, and absorbance was recorded at 324 nm. The Michaelis constant (Km) and maximum reaction velocity (Vmax) were calculated using nonlinear regression analysis of the data, plotted as a Michaelis-Menten curve.

![](https://static.igem.wiki/teams/5448/results/img9.png)

Figure 9: Michaelis-Menten kinetics curve for the phnO enzyme, showing the relationship between AMPA concentration and reaction velocity (CoA production).

Results and Conclusion

The Michaelis-Menten kinetic analysis revealed that the **phnO e**nzyme followed saturation kinetics with increasing concentrations of AMPA. The calculated Km value was **0.5958 mM,** indicating a moderate affinity for AMPA. The Vmax was **0.1828 µmol/min/mg protein**, reflecting the maximum reaction velocity achieved when the enzyme was saturated with AMPA.

These kinetic parameters demonstrate that the phnO enzyme is effective at catalyzing AMPA degradation at moderate substrate concentrations. The relatively low Km value suggests that the enzyme is well-suited for bioremediation purposes where the environmental concentration of AMPA is likely to be low to moderate.

In conclusion, the kinetic analysis of the phnO enzyme confirmed its ability to efficiently degrade the toxic byproduct AMPA. The specific values of Km and Vmax provide valuable insights for optimizing its application in environmental detoxification strategies.

Construction of the Cold-Inducible Reporter Strain:

Objective and Methods

The objective of this experiment was to construct a cold-inducible reporter strain using a cold-inducible promoter to regulate the expression of the mRFP (monomeric red fluorescent protein) gene. This strain allows for the detection of gene expression under low-temperature conditions.

The cold-inducible promoter **PcspA**was synthesized and cloned upstream of the mRFP gene in the pSB1A3 vector. The mRFP gene was codon-optimized for expression in E. coli, and restriction sites (EcoRI, XbaI, SpeI, PstI) were removed to comply with RFC#10 standards. The recombinant plasmid was transformed into E. coli BL21 cells. Positive clones were selected on LB agar plates containing 50 µg/mL ampicillin at 37°C and confirmed by sequencing.

The engineered strain was cultured in LB medium containing ampicillin to evaluate the induction of mRFP expression under different temperatures.

![](https://static.igem.wiki/teams/5448/results/img10.png)

Figure 10: Schematic representation of the cold-inducible reporter system. The PcspA promoter regulates mRFP expression in response to low-temperature conditions.

Results and Conclusion

The recombinant plasmid containing the cold-inducible **PcspA** promoter and the mRFP gene was successfully constructed and transformed into E. coli BL21. Sequencing confirmed the correct insertion of the promoter and reporter gene into the pSB1A3 vector. The engineered strain exhibited normal growth in LB medium containing ampicillin, demonstrating successful plasmid incorporation and expression capability.

In conclusion, the construction of the cold-inducible reporter strain was successful, providing a functional system for studying temperature-dependent gene expression. This strain will be used in subsequent experiments to monitor mRFP expression under varying cold conditions.

Testing the Cold-Inducible Reporter Strain:

Objective and Methods

The objective of this experiment was to test the cold-inducible reporter strain constructed with the PcspA promoter driving the expression of mRFP (monomeric red fluorescent protein). The goal was to evaluate the induction of mRFP expression at different temperatures and determine the temperature sensitivity of the cold-inducible system.

The overnight culture of the engineered strain was diluted 1:100 into fresh LB medium containing 50 µg/mL ampicillin. The cultures were then incubated at different temperatures (16°Cand 37°C) with shaking at 180 rpm for 12 hours. After incubation, 200 µL samples were taken from each culture, and fluorescence intensity (excitation at 584 nm and emission at 607 nm) was measured using a microplate reader. Optical density at 600 nm (OD600) was also measured to normalize the fluorescence values. The normalized fluorescence intensity (**Fluorescence/OD600**) was calculated to compare mRFP expression levels across different temperature conditions.

![](https://static.igem.wiki/teams/5448/results/img11.png)

Figure 11: Normalized fluorescence intensity (Fluorescence/OD600) of the cold-inducible reporter strain at various temperatures (16°C, 37°C). Fluorescence was measured at excitation 584 nm and emission 607 nm.

Results and Conclusion

The results showed a significant increase in mRFP expression at lower temperatures, with the highest fluorescence observed at 16°C. As the temperature increased, the fluorescence intensity decreased, with minimal expression at 37°C. This indicates that the PcspA promoter is highly sensitive to cold temperatures, and mRFP expression is strongly induced under cold-inducible conditions.

In conclusion, the cold-inducible reporter strain successfully demonstrated temperature-dependent expression of mRFP, with optimal induction occurring at 16°C. This strain can be effectively used to study cold-inducible gene expression systems and could be applied to temperature-controlled biotechnological processes.

Construction of the Cold-Inducible Suicide System:

Objective and Methods

The aim of this experiment was to construct a cold-inducible suicide system in E. coli using the cold-inducible PcspA promoter to control the expression of the mazF gene, which encodes a toxin that inhibits cellular growth by cleaving mRNA. This system is designed to activate cell death under low-temperature conditions, providing a potential biocontainment strategy.

The mazF gene, codon-optimized for E. coli, was cloned downstream of the PcspA promoter in the pSB1A3 vector. The **PcspA-mazF** construct was transformed into E. coli BL21 cells, and positive clones were selected on LB agar plates containing 50 µg/mL ampicillin at 37°C. The presence of the PcspA-mazF system was confirmed by sequencing.

The engineered strain was cultured under standard growth conditions (37°C), and subsequent experiments were planned to assess the activation of the suicide system at lower temperatures.

![](https://static.igem.wiki/teams/5448/results/img12.png)

Figure 12: Agarose gel electrophoresis showing the PCR amplification of the PcspA promoter and mazF gene, confirming successful cloning.

Results and Conclusion

The recombinant plasmid containing the PcspA promoter and mazF toxin gene was successfully constructed and transformed into E. coli BL21. Sequencing confirmed the correct insertion of the mazF gene under the control of the cold-inducible promoter. The engineered strain grew normally at 37°C in LB medium containing ampicillin, confirming successful plasmid incorporation and maintenance of growth under non-inducing conditions.

In conclusion, the construction of the cold-inducible suicide system was successful, providing a functional system that can potentially trigger cell death in response to cold temperatures. This strain will be used in subsequent experiments to test the effectiveness of the suicide system at inducing cell death under low-temperature conditions.

Testing the Cold-Inducible Suicide System:

Objective and Methods

The purpose of this experiment was to test the functionality of the cold-inducible suicide system in E. coli, which utilizes the PcspA promoter to control the expression of the mazF toxin gene. The mazF gene induces cell death by cleaving mRNA, and this system is designed to activate at lower temperatures, offering a temperature-sensitive biocontainment strategy.

The engineered strain was cultured overnight in LB medium containing 50 µg/mL ampicillin at 37°C. The following day, 100 µL of the culture was inoculated into 5 mL of fresh LB medium containing ampicillin and incubated at either 16°C or 37°C with shaking at 180 rpm for 12 hours. After incubation, 200 µL samples were taken from each condition, and cell growth was measured by determining the optical density at 600 nm (OD600). Cell viability at low temperatures was compared with that of control strains and normal growth conditions at 37°C.

![](https://static.igem.wiki/teams/5448/results/img13.png)

Figure 13: Comparison of cell growth (OD600) of the cold-inducible suicide strain at 16°C and 37°C after 12 hours of incubation, demonstrating activation of the suicide system at low temperatures.

Results and Conclusion

The results showed that at 37°C, the engineered strain grew normally, with no signs of mazF-induced cell death. However, when incubated at 16°C, the growth of the engineered strain was significantly inhibited, with a drastic reduction in OD600, indicating the activation of the suicide system. The control strain without the mazF gene showed normal growth at both 16°C and 37°C, confirming that the observed growth inhibition was due to the cold-inducible expression of mazF.

In conclusion, the cold-inducible suicide system effectively triggered cell death at 16°C, validating the functionality of the PcspA-regulated mazF system. This system provides a potential safety mechanism for biocontainment, ensuring that the engineered bacteria can be selectively eliminated under specific environmental conditions.

Statistical Analysis:

All statistical analyses were performed using **GraphPad Prism** software. Data are presented as mean ± standard deviation (SD), with experiments conducted in triplicate to ensure reliability. For multiple group comparisons, one-way ANOVA followed by Tukey’s post-hoc test was applied. Comparisons between two groups were analyzed using a two-tailed Student’s t-test. A p-value of < 0.05 was considered statistically significant. These analyses verified that the observed differences, such as in growth inhibition and temperature-dependent gene expression, were statistically significant and not due to random variation, confirming the validity of the experimental results.

Summary

In conclusion, the successful construction of the **phnE1/E2**, **phnE1/E2-phnJ,** and **phnO** engineered strains demonstrated effective glyphosate and AMPA degradation, confirming the functional roles of these genes. The cold-inducible reporter and suicide systems were also validated, with the **PcspA** promoter effectively regulating gene expression and inducing cell death at low temperatures. These results highlight the potential of the engineered strains for environmental bioremediation and biosafety applications, offering innovative solutions for glyphosate degradation and biocontainment.

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