WHHS-China has contributed the biobrick BBa_K3332067, encoding the phnE1 and phnE2 genes from Sinorhizobium meliloti 1021, which enhance the uptake of phosphonate compounds like glyphosate into E. coli cells. This part plays a crucial role in improving the efficiency of glyphosate absorption, making it a key element in bioremediation projects targeting glyphosate-contaminated environments.

The functional expression of these genes has been validated, demonstrating efficient glyphosate absorption by engineered E. coli, which significantly reduces glyphosate concentration in the environment. This contribution serves as a foundational tool for future iGEM teams aiming to tackle herbicide pollution through microbial bioremediation.

WHHS-China’s project goes beyond the transport of glyphosate. They have developed an integrated system that includes:

Multi-pathway Glyphosate Degradation: In addition to the BBa_K3332067 part, the team incorporated genes for the C-P cleavage pathway, which degrades glyphosate into non-toxic compounds like phosphate and glycine. This pathway avoids the generation of AMPA, a toxic byproduct of the C-N cleavage pathway.

AMPA Degradation Module: The team introduced the phnO gene to degrade AMPA, the toxic byproduct of glyphosate breakdown. This ensures that the entire degradation process is environmentally safe and minimizes residual toxicity.

This multi-module design showcases a well-rounded approach to bioremediation, and future iGEM teams can adopt this strategy to develop more efficient and safer microbial systems.

WHHS-China’s experimental process provides valuable lessons for future teams:

Optimizing Transport and Degradation: While their engineered strain successfully absorbed glyphosate, the team identified that glyphosate degradation may be limited by its transport into cells rather than the intracellular breakdown. This insight suggests that optimizing the transport system (such as improving the expression or regulation of phnE1/phnE2) could further enhance the degradation efficiency.

Time-Course Analysis: The team performed time-course experiments to measure glyphosate absorption, observing rapid uptake within the first hour, followed by a slower but consistent absorption over five hours. This data provides a practical guideline for other teams on how to structure similar experiments and measure success over time.

These experimental insights and data will be helpful for teams working on similar projects, allowing them to optimize their experiments and avoid potential bottlenecks.

WHHS-China also made a significant contribution in the area of biosafety by designing a cold-inducible suicide system. This system utilizes the cold-sensitive PcspA promoter to control the expression of the mazF toxin gene, which induces cell death under low-temperature conditions. This biocontainment system ensures that the engineered bacteria self-destruct once they finish degrading glyphosate, preventing the release of genetically modified organisms into the environment.

This safety mechanism is vital for future teams who are concerned about environmental containment and regulatory compliance in field applications.

The team engaged in meaningful collaborations with other iGEM teams, exchanging knowledge and sharing experiences related to bioremediation and synthetic biology tools. This collaboration fostered innovation and problem-solving on a broader scale.

In terms of human practices, WHHS-China conducted outreach activities, interacting with farmers, local experts, and environmental organizations. They focused on understanding the real-world implications of glyphosate use, particularly in tea plantations, and tailored their project to provide practical solutions. These activities helped ensure their project was not only scientifically sound but also aligned with community needs.

WHHS-China actively promoted their project and synthetic biology through social media platforms, raising public awareness about glyphosate pollution and the potential of bioremediation technologies. Their social media strategy serves as a valuable example for future teams looking to enhance public engagement and outreach efforts. Effective communication of science to non-expert audiences is crucial for building public support and understanding of synthetic biology.

WHHS-China’s project has wide-ranging applications in agricultural and environmental management, particularly for areas contaminated with glyphosate, such as farmlands and tea plantations. By applying their engineered bacteria, these environments can be detoxified efficiently, preventing the accumulation of harmful residues that affect crops and water sources.

Their work paves the way for future iGEM teams to expand the scope of microbial bioremediation, and further optimize these systems for diverse environmental challenges. The integration of biocontainment measures ensures that these applications can be implemented safely in the field.

WHHS-China has contributed the biobrick BBa_K3332067, encoding the phnE1 and phnE2 genes, which are derived from Sinorhizobium meliloti 1021. These genes enable the transport of phosphonate compounds, particularly glyphosate, into E. coli cells for further degradation. The development of this part allows engineered bacteria to absorb glyphosate more efficiently, a significant step toward biodegradation of this widely used herbicide. This contribution is invaluable for future iGEM teams working on bioremediation projects targeting herbicide pollution or similar environmental contaminants.

The part BBa_K3332067 has been experimentally validated to enhance the absorption of glyphosate, contributing to effective bioremediation strategies, especially when coupled with downstream degradation pathways such as C-P cleavage.

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.

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.

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.

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.

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.

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.

This part, BBa_K3332067, serves as a foundational tool for projects aiming to bioremediate environments contaminated with phosphonate herbicides like glyphosate. It can be integrated into more complex systems involving degradation enzymes like PhnJ (for C-P bond cleavage) or PhnO (for AMPA detoxification).

The part also holds potential for agricultural applications where glyphosate contamination is a concern, enabling microbial treatment solutions to mitigate residue levels in soil and water.

While the engineered strain showed impressive glyphosate absorption, experiments revealed that improving the transport of glyphosate might further enhance degradation efficiency. Future teams working on similar projects could focus on optimizing the transport mechanisms or combining phnE1/E2 with more effective degradation enzymes to increase the overall bioremediation potential.

WHHS-China's work provides a crucial piece for glyphosate bioremediation. Future iGEM teams are encouraged to build upon this by integrating additional safety features, such as suicide switches or cold-inducible biocontainment systems, to prevent the engineered microbes from escaping into natural ecosystems.

In conclusion, BBa_K3332067 is a well-characterized and validated part that enables efficient glyphosate absorption, making it a valuable resource for future teams aiming to tackle environmental pollution through synthetic biology.

WHHS-China’s contribution extends beyond the development of BBa_K3332067. They have provided an integrated approach to solving the issue of glyphosate pollution through their innovative multi-module system, combining glyphosate absorption, efficient degradation, and safety features. Their experimental insights, troubleshooting methods, and safety systems offer a robust framework for future iGEM teams to build upon.

By addressing both the technical challenges of glyphosate bioremediation and the safety concerns related to environmental release, WHHS-China has laid a solid foundation for future innovations in synthetic biology. Their project not only demonstrates the power of engineering microbes for environmental cleanup but also highlights the importance of interdisciplinary approaches, including biosafety, collaboration, and public engagement.

Future teams can take inspiration from WHHS-China’s comprehensive strategy, especially in:

Developing integrated bioremediation systems that combine transport, degradation, and detoxification of pollutants.

Incorporating biosafety mechanisms like cold-inducible suicide systems to meet regulatory and ethical standards for field deployment.

Enhancing collaboration between teams and engaging with local communities to ensure that synthetic biology projects address real-world needs.

WHHS-China’s work represents a valuable contribution to the iGEM community and beyond, offering both practical tools (such as BBa_K3332067) and strategic insights that can guide future efforts in bioremediation and environmental management.