Hydrogen sulfide (H₂S) is a gaseous signaling molecule that promotes GLP-1 secretion and modulates GLP-1 degradation. Appropriate levels of H₂S contribute to enhancing GLP-1 function, whereas excessive H₂S may exert counterproductive effects. Elevated H₂S concentrations can lead to increased oxidative stress and cellular damage, which may subsequently impair the function of enteroendocrine L-cells, thereby reducing GLP-1 secretion. Consequently, regulating intestinal H₂S levels has emerged as a pivotal target for innovative fat management strategies. Sulfide quinone oxidoreductase (SQR) and flavocytochrome c sulfide dehydrogenase (FCSD) are two sulfide dehydrogenases that can decompose hydrogen sulfide gas, thereby achieving the goal of regulating GLP-1 secretion and addressing obesity.

In this study, we contributed 2 new parts to iGEM, including 1 basic parts and 2 composite parts. Our goal of this research project is obesity management. The reason for obesity is that excess amount of hydrogen sulfide may leas to increased oxidative stress and cell damage, thus reducing the secretion of GLP-1. So we aim to decompose hydrogen sulfide by two enzymes FCSD + SQR. During this experiment, we first transformed competent Escherichia coli BL21 and DH5α cells, then used IPTG to induce protein expression and finally examined the concentration of hydrogen sulfide. The design of this project reflects the synthetic biotechnology: high-yield expression of key enzymes (FCSD + SQR) in hydrogen sulfide metabolism using E.coli BL21 and DH5α as a prokaryotic host. The relevant parts are listed in Table 1.

Table 1. Part contributions

Construction Design:

In order to construct the recombinant plasmid pET28a-SQR-His, we used homologous recombination to insert the SQR gene, which codes for an eukaryotic enzyme that breaks down H₂S into the vector pET28a. SQR catalyzes the degradation of H₂S and thereby reduces H₂S levels. In our project, an orally delivered SQR capsule would act in the intestinal environment to degrade excess H₂S produced by microbes. This reduction would restore GLP-1 activity, which can promote fat metabolism and suppress appetite.

This plasmid includes several key units to ensure successful and efficient expression and purification. The backbone includes a kanamycin resistance gene (KanR) for antibiotic selection, as well as LacI and the Lac operator for IPTG-induced expression, and a His-tag attached to a T7 promoter to enable protein purification and initiation of transcription.

By integrating the SQR gene into this plasmid, we created a system capable of producing functional SQR enzyme in E.coli. Once the protein is expressed and purified, this enzyme would degrade excess H₂S in the gut, therefore lowering H₂S levels and reactivating the GLP-1 signaling pathway, which is a key regulator of appetite and fat metabolism. This offers a potential therapy for obesity and type II diabetes.

Figure 1. The plasmid map of pET28a-SQR-His

Experimental Approach:

We successfully amplified the SQR gene sequence using PCR technology. The amplified product was verified by agarose gel electrophoresis, as shown in Figure 2A. A clear, specific band was observed at the expected size position, with no non-specific amplification products or primer dimers, confirming successful amplification of the SQR gene. Simultaneously, to construct the recombinant vector, the pET28a empty vector was double-digested with XhoI and BhoI restriction enzymes for linearization. The digestion products were analyzed by agarose gel electrophoresis (Figure 2B). The results showed only a single, clear band, indicating complete linearization of the vector with no uncut or partially digested products remaining, confirming successful linearization. Building upon this, the purified SQR gene fragment was ligated with the linearized pET28a vector via homologous recombination, successfully inserting the SQR sequence into the vector backbone. This laid the foundation for subsequent recombinant plasmid construction and protein expression studies.

Figure 2. The gel electrophoresis validation of SQR and pET28a linear

Based on the above, the purified SQR gene fragment was ligated with the linearized pET28a vector via homologous recombination, successfully inserting the SQR sequence precisely into the vector backbone to construct a recombinant plasmid. This recombinant plasmid was designated pET28a-SQR-His and transformed into E.coli DH5α competent cells. The transformed bacterial suspension was spread onto LB plates containing the corresponding antibiotic. As shown in Figure 3A, uniformly distributed, well-formed single-colony colonies were observed on the plates, preliminarily indicating successful transformation. Subsequently, several single-colony colonies were randomly selected for further validation via colony PCR and sequencing. Colony PCR results showed distinct amplification bands (Figure 3B), preliminarily confirming that the recombinant plasmid had successfully transferred into the host bacteria and possessed amplification capability. Final sequencing results (Figure 3C) confirmed that the SQR gene sequence was correct and the insertion read-through was accurate, indicating the successful construction of the recombinant plasmid pET28a-SQR-His.

Figure 3. Map for pET28a-SQR-His Transformation Verification

For protein expression, the validated recombinant plasmid was transformed into E.coli BL21(DE3) competent cells. The transformed culture was spread onto LB solid plates containing kanamycin and incubated overnight at 37°C. The results are shown in Figure 4, where uniformly distributed single colonies are visible on the plates, indicating successful transformation.

Figure 4. pET28a-SQR-His Colony Map

Subsequently, we selected a single monoclonal colony and inoculated it into LB liquid medium. After incubating at 37°C with shaking until the logarithmic growth phase (OD600 ≈ 0.6–0.8), we added IPTG to a final concentration of 0.5 mM. Expression was then induced at 16°C for 16 hours to promote soluble protein formation. After induction, bacterial cells were harvested by centrifugation. Cells were lysed using an ultrasonic cell disruptor, and the supernatant was used. Using the histidine tag (His-tag) at the C-terminus of the SQR protein, we purified the protein via nickel affinity chromatography. Four key fractions were collected: crude lysate, flow-through, wash fraction, and elution fraction. Analysis of each fraction by SDS-PAGE gel electrophoresis yielded the results shown in Figure 5. A single, dense protein band appeared at the expected molecular weight (approximately 50 kDa) in the elution fraction, while this band was faint or absent in the crude lysate, flow-through, and wash fractions. Indicating that the target protein SQR-His successfully bound specifically to the nickel column and was efficiently eluted. This confirms the successful expression of the recombinant protein in the E.coli system and the effective purification achieved through the nickel affinity chromatography step. The collected material was used to obtain the final product.

Figure 5. The SDS-PAGE protein gel of samples from different purification stages

Function Test

1. ELISA assay

In this experiment, we employed the enzyme-linked immunosorbent assay (ELISA) to quantitatively analyze the content of sulfquinone reductase (SQR) in different eluate fractions. The experiment began by pre-coating SQR antibodies onto microplate wells. Standard samples, test samples, and HRP-labeled detection antibodies were sequentially added. After incubation and thorough washing, the TMB substrate was introduced for color development. Under peroxidase catalysis, TMB initially turned blue and then converted to yellow upon acid termination. The color intensity correlated positively with SQR concentration in the samples. Absorbance (OD value) was measured at 450 nm, and SQR concentrations in each sample were calculated using the standard curve.



Figure 6. Assaying the Standard Curve for ELISA

Results showed significantly higher SQR enzyme concentrations in the eluate fraction compared to other fractions, indicating this fraction primarily enriched the target protein. A good linear relationship was observed between standard concentrations and OD450 values, with a high standard curve fit, confirming reliable detection results.



Figure7. ELISA assay showing enzyme concentration

2. DUQ assay

This study employed a spectrophotometric method based on DUQ reduction to evaluate SQR enzyme activity. The principle relies on SQR catalyzing the oxidation of sulfides and transferring electrons to the ubiquinone analog DUQ, resulting in DUQ reduction and a characteristic decrease in absorbance at 275 nm. By monitoring this absorbance change in real time, the enzymatic reaction rate can be quantitatively reflected.

Results showed that after adding SQR enzyme solution, the absorbance at 275 nm decreased significantly over time, indicating successful catalytic reduction of DUQ by the enzyme and confirming its high catalytic activity. Moreover, the established spectrophotometric method effectively and specifically detects SQR enzyme activity. The results clearly demonstrate the enzyme's high catalytic efficiency under the specified conditions.



Figure 8. spectrophotometric determination of SQR enzyme activity based on DUQ reduction

3. Lead Acetate Test Strip

To assess SQR activity visually, we used lead acetate test strips, which turn black when exposed to H₂S. For this purpose, Escherichia coli MG1655 was cultured in media containing different concentrations of L-cysteine (0 mM, 0.5 mM, 1 mM, 1.5 mM, 2 mM) to induce H₂S production. The generation of H₂S was verified by the blackening of lead acetate paper strips. Simultaneously, we further analyzed H₂S yield using ImageJ. Results demonstrated that H₂S production progressively increased with rising L-cysteine concentrations.



Figure 9. Different L-cysteine derivatives induce varying amounts of H₂S production

When SQR was introduced into the testing system, a pronounced concentration-dependent response was observed: the color intensity of the test strip gradually weakened as the amount of added SQR increased. This visual change was further confirmed by grayscale analysis—grayscale values exhibited a corresponding decrease with rising enzyme concentration, verifying the quantitative reduction of H₂S content in the sample.

Notably, when the SQR concentration reached 10 mg/L, H₂S levels decreased significantly, approaching concentrations observed without L-cysteine induction. This result strongly demonstrates that the SQR enzyme efficiently and rapidly catalyzes H₂S degradation, highlighting its potential application in systems requiring H₂S control.



Figure 10. Reduction in greyscale of lead acetate test strips with increasing SQR concentration

4. Hardware Validation

We further employed sensor-based detection equipment to quantitatively validate the enzymatic activity of SQR in real time, enabling real-time quantitative monitoring of hydrogen sulfide (H₂S) concentration. In samples without enzyme treatment, sensor readings consistently remained at elevated levels (Figure 11A). Conversely, in the SQR-treated experimental group, sensor signals exhibited a significant decline (Figure 11B). This result clearly demonstrates that SQR efficiently catalyzes H₂S decomposition, confirming its strong hydrogen sulfide degradation capability.



Figure 11. H₂S concentration with corresponding sensor display

The results above demonstrate that pET28a-SQR-His is a novel functional construct capable of efficiently expressing active sulfquinone reductase (SQR). This construct exhibits excellent biological functionality and practicality, enabling efficient H₂S degradation. It provides a key technological and theoretical basis for subsequent indirect regulation of the GLP-1 signaling pathway.

5. GLP-1 Pathway Detection

To further investigate the effects of H₂S on intestinal endocrine cell function, we established the following in vitro model: the supernatant from L-cysteine-induced E.coli MG1655 was added to the NCI-H716 cell culture system. This supernatant, rich in H₂S produced by bacterial metabolism, significantly inhibited the synthesis and secretion of glucagon-like peptide-1 (GLP-1) by NCI-H716 cells. Results indicate that H₂S intervention disrupts GLP-1-mediated metabolic regulatory pathways, leading to downregulation of GLP-1 gene expression. This process mimics the potential regulatory role of microbial metabolites in the intestinal microenvironment on host cell endocrine function.

Figure 12. Map of GLP-1 Gene Expression Level Changes

NCI-H716 cells were seeded at a density of 5 × 10⁵ cells per well in a 12-well plate. After the cells adhered and reached 70–80% confluence, they were treated with different drugs: One group received supernatant from the L-cysteine-induced Escherichia coli MG1655 strain (Negative group), another group received supernatant from the L-cysteine-induced strain pretreated with SQR protein (SQR-treated group), with corresponding control groups established. After co-incubation for 4 hours, the supernatant was aspirated, cells were washed with PBS to remove drug residues, and fresh medium was added for continued culture for 24 hours, respectively. Following culture, total cellular RNA was extracted, and the expression level of glucagon-like peptide-1 (GLP-1) mRNA was detected using RT-qPCR.

Table 2 qPCR detection of GLP-1 mRNA expression levels in cells following SQR administration

Results demonstrated that GLP-1 mRNA expression was significantly upregulated in the SQR-treated group compared to the Negative group (p < 0.05). This indicates that SQR protein effectively mitigates H₂S-induced suppression of GLP-1 gene expression by degrading H₂S in bacterial supernatant, thereby restoring GLP-1 transcriptional levels.

Figure 13. The map of GLP-1 gene expression level changes under different treatments