Engineering Success

Cycle 1

1. Design
Regarding the operational mode of the Degradation and Resistance modules, our initial concept involved the direct constitutive expression of heterologous pollutant-degrading gene clusters and resistance-related proteins (the NhaA and AcrAB-TolC systems) in the engineered bacteria. Inspired by the suggestion raised during human practices that engineered bacteria need to balance growth and functional execution, we introduced a quorum sensing system to act as the "accelerator" for the functional modules. The intended "brake" was to be selected between two degradation tags, SsrA and LAA-LAA.

2. Build
Based on literature findings indicating the stronger degradation capability of the LAA-LAA tag for membrane proteins, we selected NhaA as the experimental subject. We constructed variants by appending the SsrA tag and the LAA-LAA tag to the C-terminus of NhaA, respectively. The plan was to evaluate the suitability of each degradation tag by assessing the salt tolerance of the corresponding engineered bacterial strains.

3. Test
We prepared liquid LB medium with a NaCl concentration of 30 g/L and cultured the engineered strains in it. Growth curves for the NhaA-SsrA and NhaA-LAA-LAA strains were measured. The results indicated that the NhaA-LAA-LAA strain grew more slowly, suggesting a higher degradation efficiency for the LAA-LAA tag against this membrane protein.

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

4. Learn
Through the comparative experiment, we confirmed that the LAA-LAA degradation tag was more suitable as the "brake" for the functional modules. This ensures that heterologous proteins expressed at high population density can be degraded and recycled by the bacteria at low density. Inspired by this result, we further tested the application of the LAA-LAA tag in the anti-leakage component of a temperature-sensitive kill switch, another context requiring a degradation tag. This subsequent experiment again verified that the LAA-LAA tag possesses stronger degradation efficiency compared to the widely used SsrA tag, thereby improving the fitness of engineered bacteria carrying the biosafety gene circuit.

Cycle 2

1. Design
Our design for the quorum sensing system was adapted from the validated system developed by the 2023 iGEM team UCAS-China. The core components of this system include LuxI (responsible for synthesizing the AHL signal molecule), a mutant receptor protein LuxRm, and its corresponding specific promoter pLuxm. To better simulate the logic of gene expression triggered by external inducers (such as a phenol-responsive promoter or other small-molecule biosensors) in practical applications, a key adjustment was made to the induction strategy: LuxI was set as the initial element for induced expression in our experiments. Furthermore, we conducted a comparative experimental analysis of the response curves between two system variants: one containing the pLuxm-LuxI positive feedback loop and one without it.

2. Build
Using standard molecular biology techniques, the genetic elements described above were successfully assembled into plasmid vectors and transformed into E. coli chassis cells. This resulted in the construction of two stable engineered strains: one carrying the QS1 circuit (without positive feedback) and the other carrying the QS2 circuit (containing the pLuxm-LuxI positive feedback loop). The expression of the uppermost LuxI/ gene in both strains was controlled by the lac operon.

3. Test
Functional characterization experiments were performed on the constructed strains. By inducing the expression of LuxI, the dynamic response curves of the systems were monitored. The results clearly demonstrated that the QS2 system, which incorporates the pLuxm-LuxI positive feedback loop, exhibited a superior response profile compared to the basic QS1 system. This finding aligns with the observations reported by the UCAS-China team, indicating that the QS2 system shows faster activation kinetics under low AHL concentrations (simulating real application conditions).

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

4. Learn
The experimental results confirm that incorporating the pLuxm-LuxI positive feedback loop into the quorum sensing system within our project design effectively enhances its response speed and sensitivity under low signal molecule concentrations. This validates the effectiveness of this design strategy for optimizing system performance. This finding provides a key design principle for constructing more complex and robust synthetic gene circuits in the future: positive feedback architectures can be utilized to precisely modulate the dynamic behavior of systems, making them better adapted to real-world application environments.