Results

Overview

Following the completion of the project design, we categorized the conceptual wastewater treatment platform into functional modules: the Degradation Module, the Resistance Module, and the Safety Module. A quorum sensing system was incorporated to couple the expression of relevant proteins in the Degradation and Resistance Modules with the density of the engineered bacteria. Consequently, the experimental section involved separate evaluations of the degradation efficiency of the degradation gene cluster, the extent of enhanced resistance in engineered bacteria conferred by heterologously expressed resistance proteins, the escape rate of the toxin-antitoxin system, and the construction of the quorum sensing system. Throughout this process, data were collected and analyzed according to the engineering cycle (refer to the Engineering page) to refine our experiments.

Degradation Module

As introduced in the design section, we selected the dfd gene cluster (dfdA₁₂₃₄BC), which degrades dibenzofuran, and the xyl gene cluster (xylABCMNUW), which degrades toluene, for expression in the engineered bacteria. Through literature review, we identified and constructed both clusters. Due to the difficulty in procuring toluene, the substrate for the xyl cluster, we focused on testing the degradation capability of the dfd cluster against dibenzofuran.

The dfdA₁₂₃₄BC genes were constructed in a polycistronic format downstream of a lac operon promoter. To mitigate potential reduction in expression levels caused by excessive distance from the promoter, the gene cluster was split and constructed onto two separate vectors. When the engineered E. coli cultures reached the logarithmic growth phase (OD₆₀₀ = 0.4-0.6), expression was induced with 1 mM IPTG. After 8 hours of induction, 0.2 mM dibenzofuran was added to the culture medium. The concentration of salicylic acid in the medium was measured at 24 hours and 48 hours post-addition using a plant salicylic acid ELISA kit. After 48 hours, the measured salicylic acid concentration was 2.56 × 10⁴ ng/mL (equivalent to 0.185 mM), indicating a degradation efficiency of 92.5%.

Fig.1 Schematic diagram of salicylic acid production and oxygen fluorene degradation efficiency in the oxygen fluorene degradation experiment

Resistance Module

Considering potential application scenarios like wastewater treatment plants, where engineered bacteria might encounter high salinity or high levels of toxic substances, we heterologously expressed the NhaA sodium-proton antiporter and the drug efflux pump AcrAB-TolC to enhance the resistance of E. coli.

1.NhaA
NhaA is the first discovered sodium/proton antiporter in microorganisms, actively transporting sodium ions to maintain osmotic balance and pH homeostasis across the cell membrane. To assess its ability to confer resistance to hyperosmotic stress, engineered bacteria induced to express heterologous NhaA were inoculated into liquid LB media containing salt concentrations of 10 g/L and 30 g/L, with a negative control group included. Growth curve analysis indicated that strains expressing heterologous NhaA exhibited faster initial growth in high-salt LB medium. Since E. coli endogenously expresses NhaA, the control group eventually reached a comparable cell density after a sufficiently long incubation period even under high-salt conditions.

Fig. 2 The growth curves of engineered bacteria and wild-type bacteria in different salt concentration culture media

Although introducing the NhaA gene can improve the environmental adaptability of the engineered bacteria to some extent, excessive expression of heterologous genes can burden cellular resources and potentially reduce fitness. Therefore, the expression of this resistance protein was placed under the control of the quorum sensing system, aiming to couple its expression with bacterial cell density. This strategy allows the bacteria to prioritize growth and reproduction at low densities and activate heterologous gene expression at high densities. Correspondingly, proteins expressed at high density should be degradable and recyclable by the bacteria at low density. To achieve this, degradation tags were appended to the C-terminus of the heterologous proteins. We tested the commonly used SsrA tag and the LAA-LAA tag, which is reported to be more effective for membrane proteins like NhaA, as part of our engineering cycle. Similarly, we measured the growth curves of strains expressing NhaA-SsrA and NhaA-LAA-LAA in high-salt LB medium. The results showed that the NhaA-LAA-LAA strain grew more slowly, suggesting that the LAA-LAA tag confers higher degradation efficiency for this membrane protein.

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

2.AcrAB-TolC
The AcrAB-TolC complex is a key system in E. coli responsible for the active efflux of a wide range of toxic substances. This tripartite pump recognizes and extrudes compounds with diverse chemical structures, effectively mitigating their cellular damage and thereby enhancing bacterial survival in adverse environments. Literature review informed us that many complex aromatic pollutants exhibit significant physiological toxicity to E. coli. These substances (e.g., polycyclic aromatic hydrocarbons, substituted phenols) can inhibit bacterial growth, disrupt cell membrane integrity, and interfere with normal metabolic activities. We designed experiments to test whether introducing heterologous acrAB-tolC genes could enhance the stress resistance of E. coli. However, as some toxic aromatic pollutants were unavailable for purchase, we alternatively tested the growth curves of engineered bacteria expressing this gene cluster under triple the normal concentration of kanamycin (the standard working concentration is 50 µg/mL), including a negative control group. The results indicated that heterologous expression of AcrAB-TolC provided only a modest improvement in bacterial adaptability to high-concentration kanamycin. In subsequent experiments, we hope to identify a more suitable testing environment that better simulates aromatic pollutant stress.

Fig. 4 The growth curves of engineered bacteria and wild-type bacteria in different salt concentration culture media

Safety Module

For engineered bacteria intended for specific environments, incorporating a suicide switch triggered by environmental cues is a common and effective containment strategy. Among various environmental factors, temperature serves as a suitable trigger. Initially, we planned to utilize a cold-induced suicide system coupled with the Doc toxin protein, based on the design from the 2020 UCAS-China team. However, inspired by the degradation tags used in the Resistance Module, we replaced the SsrA tag in the anti-leakage part of this system with the more efficient and smaller LAA-LAA tag and conducted experiments. The results demonstrated that at 37°C, after switching the degradation tag from SsrA to LAA-LAA, the engineered bacteria exhibited better survival. Leaky expression of the toxin protein can lead to increased mortality under normal conditions, reducing fitness and increasing the risk of plasmid loss; therefore, a more efficient degradation tag can enhance the stability of the suicide system.

Fig. 5 The different behaviors of the cold-inducible suicide system equipped with LAA-LAA tags (the L part) and SsrA tags (the S part) at 37℃.
The picture shows the gradient dilution of the bacterial solution and its subsequent cultivation on a solid culture medium.

Quorum Sensing System

The quorum sensing system we initially intended to apply was derived from the 2023 UCAS-China team's design, comprising LuxI, a mutant LuxR (LuxRm), and its corresponding promoter pLuxm. Without the positive feedback loop formed by pLuxm drivin luxI expression, this system could still function and exhibited a certain threshold effect. However, unlike the experiments conducted by the UCAS-China team, we induced the expression of LuxI at the uppermost stream of the genetic circuit in our experiments, rather than LuxRm. This was done to better simulate the behavior of the quorum sensing system when initiated by a phenol-inducible promoter or other small molecule biosensors in practical application scenarios. Subsequently, we tested a quorum sensing system incorporating the pLuxm - luxI positive feedback loop. The positive feedback between LuxI and pLuxm makes the system more likely to exhibit a distinct threshold inflection point. The experimental results showed that the response curve of the system with the positive feedback loop was superior to that without it. This aligns with the findings of the UCAS-China team, indicating that at lower AHL concentrations (which we anticipate in real-world applications), QS2 (with the positive feedback loop) responds faster than QS1 (without the feedback loop).

Fig.6 Quorum sensing efficiency test. Note: QS1-Circuit without pLuxm-LuxI , QS2: Circuit with pLuxm-LuxI.