Contribution

Overview

This year, our team designed, constructed, and characterized a highly integrated and controllable engineered E. coli system for the bioremediation of aromatic hydrocarbons. We developed and optimized multiple new genetic parts, including a quorum sensing (QS) circuit with a positive feedback loop, degradation gene clusters for dibenzofuran and toluene, and a degradation-tagged resistance module.To enhance the practicality and safety of the system, we also introduced a temperature-sensitive suicide switch and a physical encapsulation method. Our work provides a reliable, efficient, and safe platform for pollutant degradation and offers well-documented parts for future iGEM teams working in environmental remediation.

1. Parts Contribution

1. 1 Improvements in the Quorum Sensing System

We adopted and improved the QS system from the UCAS-China 2023 (BBa_K4619012) team by introducing a positive feedback loop through the strategic placement of the pLuxm promoter upstream of luxI. This modification significantly enhanced the system’s sensitivity and response speed under low AHL conditions, enabling density-dependent activation of downstream degradation and resistance genes with reduced metabolic burden. This optimized QS circuit serves as a reliable and efficient regulatory tool for synthetic biology applications requiring population-based control.

Fig. 1 | Quorum sensing efficiency test.
Note: QS1-Circuit without pLuxm-LuxI , QS2: Circuit with pLuxm-LuxI.

1.2 Degradation Module: New Composite Parts for Aromatic Compound Breakdown

We constructed and validated two key degradation gene clusters:

The dfd gene cluster (dfdA₁₂₃₄BC), which degrades dibenzofuran into environmentally benign salicylic acid, achieving 92.5% degradation efficiency in experimental conditions.
The xyl gene cluster (xylABCMNUW), designed for toluene degradation into less toxic benzoic acid. Although not experimentally tested due to regulatory restrictions, the cluster was fully assembled and is available for future validation.
These clusters are organized as polycistronic units and placed under the control of the QS system, ensuring high-level expression only at sufficient cell densities.

Fig. 2 | Schematic diagram of salicylic acid production and oxygen fluorene degradation efficiency in the oxygen fluorene degradation experiment

1.3 Resistance Module: Tag-Based Optimization for Functional Stability

To enhance bacterial survival under high-salinity and toxic conditions, we integrated two resistance mechanisms: the NhaA antiporter for sodium ion efflux and pH homeostasis and the AcrAB-TolC efflux pump for broad-spectrum toxin removal.

A key innovation was the systematic comparison of degradation tags (SsrA vs. LAA-LAA) appended to these proteins. We demonstrated that the LAA-LAA tag significantly improves degradation efficiency, particularly for membrane-bound proteins like NhaA, reducing unwanted protein accumulation and enhancing bacterial fitness under stress conditions. This tag optimization strategy is broadly applicable for regulating membrane protein expression in synthetic circuits.

Fig. 3 | The growth curves of strains expressing NhaA-SsrA and NhaA-LAA-LAA in high-salt LB medium

1.4 Safety Module: Reduced Leakiness in Suicide Switch

We have made improvements to the component BBa_K3392000 in the biological safety system used by the team UCAS-China in 2020. We improved the safety profile of the system by incorporating a temperature-sensitive suicide switch using a high-efficiency LAA-LAA degradation tag (replacing the traditional SsrA tag) to minimize leaky expression of the toxic Doc protein. This modification resulted in better bacterial survival under normal conditions and increased genetic stability, reducing the risk of unintended release or plasmid loss. This part represents a valuable contribution to biosafety applications in engineered organisms.

2. Platform-Level Innovation

Beyond individual parts, our project introduces a modular and scalable platform for pollutant degradation that integrates: density-dependent gene expressionvia QS regulation to balance growth and function; degradation capability targeting multiple aromatic hydrocarbons; enhanced stress resistancemechanisms for operation in real-world environments; and layered biocontainmentcombining genetic suicide switches and physical encapsulation. This system demonstrates how synthetic biology can be used to address complex environmental challenges with both efficiency and responsibility.

3. Documentation for Future Teams

We have thoroughly documented the design, construction, and testing of all new parts, including: characterization data for the improved QS circuit; degradation kinetics of the dfdcluster; growth curves under salt stress for LAA-LAA-tagged resistance proteins; and stability tests for the suicide switch. These resources are intended to assist future iGEM teams and researchers in the construction of robust and efficient biological systems for environmental applications.