iGEM 2025 — Team cfls

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# Notebook

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# 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.

![](https://static.igem.wiki/teams/5804/engineering/e1.webp)

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).

![](https://static.igem.wiki/teams/5804/engineering/e2.webp)

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.

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# Experiments

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# 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%.

![](https://static.igem.wiki/teams/5804/result/r1.webp)

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.

![](https://static.igem.wiki/teams/5804/result/r2.webp)

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.

![](https://static.igem.wiki/teams/5804/result/r3.webp)

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.

![](https://static.igem.wiki/teams/5804/result/r4.webp)

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.

![](https://static.igem.wiki/teams/5804/result/r5.webp)

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).

![](https://static.igem.wiki/teams/5804/result/r6.webp)

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

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