We aimed to develop a comprehensive bacterial system with three core functions: sensing, therapy, and a safety suicide mechanism. First, we designed a biosensor to detect gut inflammation, initially using tetrathionate as a biomarker, but later shifting to thiosulfate for improved stability. Through iterative cycles, we optimized the sensing system by incorporating the ThsS/ThsR operon and developing Thiosulfate biosensor-B, which showed enhanced sensitivity and specificity. To make the detection visually accessible, we added the lacZ gene, allowing the bacteria to turn blue upon sensing inflammation. For the therapeutic component, we engineered bacteria to secrete human epidermal growth factor (hEGF) in response to inflammation, which significantly improved 293T cell viability in vitro. Our next step involves integrating these systems into E. coli Nissle 1917, using CRISPR-Cas9 to knock out its native β-galactosidase, thus ensuring the entire system can function safely and effectively as an oral therapy for gut inflammation, with a suicide mechanism to prevent environmental release.
The construction of the three systems—the sensing system, the therapeutic system, and the suicide system—follows engineering design principles.
Design
In the initial phase, we aimed to create a biosensor for detecting tetrathionate, a key biomarker of inflammatory bowel disease (IBD). We designed the biosensor by incorporating the TtrS/TtrR operon, which enables the detection of tetrathionate, along with the red fluorescent protein mRFP1 as a reporter. This setup would allow us to detect tetrathionate levels in real time based on fluorescence signals.
Build
We synthesized the TtrS (BBa_K5084000) and TtrR (BBa_K5084001) operon and used the PphsA promoter (BBa_K5084002) to drive the expression of mRFP1. These genetic components were then cloned into the pSB1A3 plasmid to form a recombinant construct. After assembling the plasmid, it was transformed into E. coli DH5α and cultured in LB medium with ampicillin (100 μg/mL) to ensure stable growth and expression of the tetrathionate biosensor.
Test
To evaluate the performance of the Tetrathiosulfate biosensor, E. coli strains were inoculated into LB media containing different concentrations of tetrathionate (0 µM to 1000 µM). After 8 hours of incubation, fluorescence intensity and OD600 were measured, and the normalized fluorescence ratio (Fluorescence/OD600) was calculated. Results showed a rapid increase in fluorescence at lower concentrations (0 to 50 µM), indicating high sensitivity, with a significant response between 10 µM and 50 µM. Beyond 100 µM, the signal plateaued, suggesting saturation.
Learn
Although our sensor performed well in detecting tetrathionate in vitro, literature review revealed that tetrathionate is rapidly metabolized by gut microbiota, reducing its effectiveness as an IBD biomarker in vivo. This led us to reconsider tetrathionate as the primary marker, shifting our focus to thiosulfate for more stable detection in future designs.
Design
To address the limitations of tetrathionate, we switched to thiosulfate, a more stable biomarker for gut inflammation. We designed a new biosensor named Thiosulfate biosensor-A, using the ThsS/ThsR operon to specifically detect thiosulfate. The sensor is coupled with mRFP1, which is driven by the PphsA promoter, allowing us to visually track thiosulfate levels through fluorescence.
Build
We synthesized the ThsS (BBa_K5084010) and ThsR (BBa_K5084011) operon and used the PphsA promoter to drive the expression of mRFP1. These components were cloned into the pSB1A3 plasmid, forming the thiosulfate biosensor construct. The plasmid was transformed into E. coli DH5α and cultured in LB medium containing ampicillin (100 μg/mL) to generate the engineered strain.
Test
To validate the function of Thiosulfate biosensor-A, we inoculated the strain into LB medium with varying concentrations of sodium thiosulfate (0 mM, 0.2 mM, and 1 mM) and incubated it at 37°C for 8 hours. After incubation, fluorescence intensity (excitation wavelength at 584 nm, emission wavelength at 607 nm) and OD600 values were measured, and the normalized fluorescence ratio (Fluorescence/OD600) was calculated. The results showed that in the absence of sodium thiosulfate, the sensor displayed very low fluorescence, indicating minimal background noise and a low likelihood of false positives. At 0.2 mM sodium thiosulfate, fluorescence increased significantly, indicating clear sensor activation. At 1 mM, fluorescence further increased, demonstrating a strong concentration-dependent response.
Learn
This round of development demonstrated that our sensor reliably detects thiosulfate and exhibits a strong response to increasing concentrations. However, to improve the sensor’s performance further, future work will focus on optimizing its sensitivity to lower concentrations of thiosulfate and testing it in more complex biological environments.
Design
To enhance the sensitivity and accuracy of our thiosulfate biosensor, we optimized Thiosulfate biosensor-A and developed an improved version, Thiosulfate biosensor-B (BBa_K5084013). The primary optimization strategy involved modifying the promoter of ThsR and adjusting the ribosome binding site (RBS) sequences for both ThsS and ThsR. These changes were designed to balance the expression of the sensing system, improving both the response speed and sensitivity to thiosulfate (Figure 7).
Build
During the optimization process, we replaced the promoter of ThsR and adjusted the RBS sequences of both ThsS and ThsR to enhance their translation efficiency and sensing capabilities. This fine-tuning of the regulatory elements allowed Thiosulfate biosensor-B to detect changes in thiosulfate concentrations more efficiently and sensitively than Thiosulfate biosensor-A (Figure 8).
Test
To evaluate the performance of Thiosulfate biosensor-B, we conducted multiple rounds of screening and functional testing under conditions identical to those used for Thiosulfate biosensor-A. The engineered bacterial strains were inoculated into LB medium containing different concentrations of sodium thiosulfate (0 mM and 1 mM) and incubated at 37°C for 8 hours. After incubation, we measured the fluorescence intensity (excitation at 584 nm, emission at 607 nm) and OD600 using a microplate reader. The normalized fluorescence ratio (Fluorescence/OD600) was calculated to assess the biosensor’s sensitivity. The results demonstrated that both Thiosulfate biosensor-A and B exhibited low and comparable fluorescence signals in the absence of thiosulfate, indicating minimal background noise. However, Thiosulfate biosensor-B showed a significantly higher response to 1 mM sodium thiosulfate, with markedly increased fluorescence intensity compared to Thiosulfate biosensor-A. This indicates that the modifications to the promoter and RBS sequences successfully enhanced the sensitivity of Thiosulfate biosensor-B (Figure 9).
To evaluate the dose-dependence of Thiosulfate biosensor-B, we measured its fluorescence response under different concentrations of sodium thiosulfate. The engineered strain was inoculated into LB medium containing 0, 0.125, 0.25, 0.5, and 1 mM of sodium thiosulfate and incubated at 37°C for 8 hours. After incubation, fluorescence intensity was measured using a microplate reader, and the values were normalized to OD600 (Fluorescence/OD600) to account for variations in cell growth. The results showed a clear increase in the normalized fluorescence ratio as the concentration of sodium thiosulfate increased. At 0 mM, the fluorescence signal was very low, indicating that the sensor was inactive. At 0.125 mM, the fluorescence ratio rose sharply, demonstrating the sensor’s responsiveness to low concentrations of thiosulfate. As the concentration increased to 0.25 mM, the fluorescence signal continued to strengthen, nearing saturation at 0.5 mM. Beyond 1 mM, the response plateaued, indicating that the sensor’s response had stabilized at higher concentrations. These results demonstrate that Thiosulfate biosensor-B exhibits clear dose-dependence, with saturation occurring around 0.5 mM and stabilization at 1 mM, suggesting optimal performance at approximately 0.5 mM.
To evaluate the specificity of Thiosulfate biosensor-B, we tested its response to various sulfate-based compounds, including sodium sulfate, sodium sulfite, sodium tetrathionate, and sodium thiosulfate, all at 1 mM concentration. After 8 hours of incubation at 37°C, fluorescence values were measured and normalized to OD600. The results showed very low responses to sulfate and sulfite, indicating that the biosensor was not activated by these compounds. There was a moderate response to tetrathionate, suggesting some cross-reactivity, but given its role as an inflammatory biomarker, this level is acceptable. The strongest response was observed with thiosulfate, confirming that the biosensor is highly specific to thiosulfate and minimally affected by structurally similar compounds. These findings demonstrate the high specificity and reliability of the biosensor for detecting thiosulfate.
Learn
Through optimization, we successfully developed Thiosulfate biosensor-B and conducted dose-dependence and specificity experiments. The results demonstrated that the sensor’s sensitivity and specificity for detecting thiosulfate met our expectations and performed well. Moving forward, we plan to further improve the sensor by making the detection results visually accessible, enhancing its practical application.
Design
In this phase, our goal was to visually confirm the detection of gut inflammation. We designed a system where, upon detecting inflammation, the patient's stool would turn blue. This was achieved by incorporating the lacZ gene downstream of our sensory system, as lacZ encodes β-galactosidase, an enzyme capable of breaking down X-gal to produce a blue color. This design allows for a clear, visual indicator of inflammation.
Build
We constructed the lacZ system by synthesizing the thiosulfate operon (including ThsS and ThsR) and coupling it with the lacZ gene under the control of the PphsA promoter. The gene fragments were cloned into the pSB1A3 plasmid and successfully validated through sequencing. The plasmid was then transformed into E. coli DH5α, creating the engineered strain capable of producing β-galactosidase upon sensing thiosulfate.
Test
To determine the β-galactosidase activity, we used the ONPG method. The engineered bacteria were cultured in LB medium and treated with varying concentrations of thiosulfate. After incubation, we measured β-galactosidase activity by assessing the hydrolysis of ONPG into ortho-nitrophenol (ONP), which can be quantified at OD400. Also, 1 mL sample of the bacterial culture was collected into a centrifuge tube, and an appropriate amount of X-gal was added. The culture was incubated at 37°C, and color changes were observed to determine β-galactosidase activity, which indicates the degradation of X-gal. The results showed a direct correlation between thiosulfate concentration and enzyme activity, confirming that the sensor activates β-galactosidase production in response to thiosulfate. Through the addition of X-gal, we successfully observed the blue color reaction produced by β-galactosidase.
Learn
With the successful construction of the sensory system, we confirmed that the engineered bacteria can detect gut inflammation and produce β-galactosidase in response, which degrades X-gal, leading to the production of a blue color. Moving forward, we aim to integrate this system with in-situ therapeutic mechanisms, allowing the engineered bacteria not only to detect inflammation but also to treat it at the site of infection.
Design
In this cycle, we aimed to develop a therapeutic system that could be activated upon detecting gut inflammation. We constructed two recombinant plasmids: pSB-PE and pSB-PEA. The pSB-PE plasmid contains the thiosulfate sensing system (ThsS/R) and the Hly-EGF expression module (encoding human epidermal growth factor, hEGF). In contrast, pSB-PEA includes the complete α-hemolysin secretion system (hlyB BBa_K5084015 and hlyD BBa_K5084016 genes) to promote hEGF secretion. These constructs were used to test the secretion efficiency of hEGF in engineered E. coli strains under simulated colitis conditions.
Build
The plasmids were successfully constructed and transformed into both E. coli DH5α and E. coli Nissle 1917 (EcN). The pSB-PE strain served as a control, lacking the secretion system, while the pSB-PEA strain included the α-hemolysin system to enhance hEGF secretion. Both strains were tested for hEGF production and secretion efficiency.
Test
To assess the secretion efficiency of hEGF in different cellular environments, E. coli Nissle 1917 (EcN) expressing hEGF was cultured in LB medium with varying concentrations of sodium thiosulfate (0 mM and 1 mM). The extracellular and intracellular hEGF levels were measured using ELISA after cell lysis and separation of supernatant. Results showed that at 1 mM thiosulfate, the EcN/pSB-PEA strain, which incorporates the α-hemolysin secretion system, significantly increased extracellular hEGF levels (19.92 ± 1.67 μg/L) compared to EcN/pSB-PE (2.37 ± 0.44 μg/L). Simultaneously, intracellular hEGF was reduced in the EcN/pSB-PEA strain (8.16 ± 1.19 μg/L) compared to EcN/pSB-PE (17.15 ± 1.18 μg/L), indicating that the α-hemolysin secretion system enhances secretion efficiency by promoting hEGF release into the extracellular environment.
To evaluate the impact of hEGF secretion on 293T cell viability, E. coli Nissle 1917 (EcN) strains expressing hEGF (pSB-PE and pSB-PEA) were cultured in LB medium with 1 mM sodium thiosulfate, followed by collection of extracellular supernatant and bacterial content samples. These were applied to 293T cells cultured to 70% confluency, and after 8 hours of incubation, cell viability was measured using a CCK8 assay. Results showed that in the supernatant group, the EcN/pSB-PEA strain (with the α-hemolysin secretion system) had the highest A450 value (2.87 ± 0.41), indicating improved 293T cell viability due to enhanced extracellular hEGF secretion. In the bacterial content group, the EcN/pSB-PE strain had a higher A450 value (4.75 ± 0.49), suggesting more intracellular hEGF accumulation. Overall, the α-hemolysin system in the EcN/pSB-PEA strain significantly boosted hEGF secretion, enhancing extracellular activity and cell viability.
Learn
The successful construction and testing of our therapeutic system showed promising results in cell-based experiments. The engineered EcN/pSB-PEA strain significantly increased the extracellular secretion of hEGF, leading to enhanced cell viability. Moving forward, our next step is to develop a safety suicide system to prevent the accidental release of engineered strains and their genetic material into the environment.
Future Work
Currently, our sensory system is developed in E. coli DH5α, while the therapeutic system is tested in EcN. However, EcN naturally produces β-galactosidase, which can break down X-gal, rendering our color-based sensory system ineffective. To address this, we plan to use CRISPR-Cas9 technology to knock out the β-galactosidase gene in EcN, allowing us to integrate both the sensory and therapeutic systems into this strain. Since EcN is non-toxic and suitable for oral administration, this will make the system safer and more effective for real-world applications.
Design
The goal of constructing a rhamnose-inducible reporter strain was to develop a controllable gene expression system, using L-rhamnose as the inducer. The design began with selecting the rhamnose-inducible promoter (PRha) and ribosome binding site (RBS) B0034. These genetic elements were synthesized and cloned into the pSB1A3 plasmid using the XbaI and SpeI restriction sites. The coding sequence of the red fluorescent protein (mRFP) was inserted downstream of the promoter, serving as the reporter. This gene circuit, as depicted in Figure 15, allows the measurement of fluorescence as an output signal in response to L-rhamnose.
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Build
After successful cloning, the recombinant plasmid was transformed into E. coli BL21 cells using the calcium chloride heat-shock method, ensuring that the bacterial strain would express mRFP in the presence of L-rhamnose.
Test
During the test phase, we evaluated the response of the E. coli BL21 strain with the rhamnose-inducible system to different concentrations of L-rhamnose. First, a 1:100 dilution of the strain was inoculated into LB medium and cultured overnight at 37°C. The next day, the overnight culture was further diluted 1:50 into fresh M9 medium containing 50 μg/mL ampicillin and 0.4% glucose. Simultaneously, L-rhamnose at concentrations ranging from 0.1% to 1% was added to induce the system. After 5 hours of incubation at 37°C, 1 mL of the culture was sampled, and the OD600 was measured. Fluorescence was measured using a microplate reader (excitation at 584 nm, emission at 607 nm). The normalized fluorescence intensity (Fluorescence/OD600) was calculated to assess the reporter strain’s activity. The results showed that the strain exhibited very low fluorescence in the absence of L-rhamnose. When 0.1% L-rhamnose was added, the fluorescence signal increased significantly, indicating successful system activation. As the concentration of L-rhamnose increased to 0.5% and 1%, the fluorescence intensity further increased, demonstrating a concentration-dependent response (Figure 21).
Learn
When different concentrations of L-rhamnose were added, the fluorescence signal increased proportionally, demonstrating a clear dose-dependent response. At 0.1% L-rhamnose, the system was successfully activated, and at higher concentrations (0.5% and 1%), the fluorescence intensity further increased. This result demonstrates that the system is a reliable tool for controlling gene expression based on L-rhamnose concentration, making it suitable for future applications.
Design
To create a controllable suicide system, a two-component circuit was designed. The antitoxin gene (mazE) was placed under the control of the rhamnose-inducible promoter (PRha) and RBS B0034, while the toxin gene (mazF) was regulated by the arabinose-inducible promoter (pBAD). This design allows precise control over the balance between toxin and antitoxin expression depending on the presence of rhamnose and arabinose, as depicted in Figure 22. The system’s modular structure enables selective bacterial cell death based on specific environmental conditions.
Build
The synthesized mazE and mazF genes were cloned into the pSB1A3 plasmid, and the construction was verified through sequencing and gel electrophoresis (Figures 17B and 17C). The recombinant plasmid (Rha-MazTox) was then transformed into E. coli BL21 cells, preparing the strain for functional testing.
Test
To validate the functionality of the suicide system, the engineered E. coli BL21 strain with the rhamnose-inducible suicide system (BL21/PBAD-mazE-PRha) was inoculated into LB medium and cultured overnight at 37°C. The next day, the overnight culture was diluted 1:50 into fresh M9 medium containing 50 μg/mL ampicillin, 1% rhamnose, 0.2% arabinose, and 0.4% glucose. The OD600 was continuously monitored using a microplate reader to evaluate the impact of the suicide system on bacterial growth. The results showed that both the BL21 and BL21/PBAD-mazE-PRha strains (with 1% L-rhamnose) grew normally during the 8-hour period, reaching an OD600 value of approximately 0.7, indicating that these strains were unaffected by the suicide system. However, the BL21/PBAD-mazE-PRha strain(without 1% L-rhamnose) exhibited a significant decline in growth after 3 hours, indicating that the suicide system had been activated, leading to cell death (Figure 24).
Learn
The results demonstrated that the rhamnose-inducible suicide system effectively controlled cell death. In the presence of L-rhamnose, the mazE antitoxin gene was expressed, allowing the strain to grow normally. However, in the absence of L-rhamnose, the mazF toxin gene was activated, causing a significant reduction in cell growth and eventually leading to cell death. This result confirms that the suicide system can be finely regulated by the presence of L-rhamnose, providing a potential biosafety strategy to prevent the engineered strain from uncontrolled proliferation or environmental release.
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