iGEM 2025 — Team ucs-a

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"title": "",
"content": '

**Team calendar 2025**

|  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Event/date | February | March | April | June | July | August | September |
| Solid medium | ✅ |  |  |  |  |  |  |
| Liquid medium | ✅ |  |  |  |  |  |  |
| Kanamycin sulfate | ✅ |  |  |  |  |  |  |
| Zygomycotina | ✅ |  |  |  |  |  |  |
| Extract pET28a-SQR/FCSD plasmid |  | ✅ |  | ✅ |  |  |  |
| Digest pET28a, gel electrophoresis，and recover DNA |  | ✅ |  |  |  |  |  |
| Prepare agarose gel, electrophorese PCR products, and recover fragments |  | ✅ |  |  |  |  |  |
| Homologous recombination with target fragments |  | ✅ |  |  |  |  |  |
| Transform *E. coli* BL21 competent cells and culture |  | ✅ |  |  |  |  |  |
| Colony PCR; culture positive colonies |  |  | ✅ |  |  |  |  |
| Extract and sequence plasmids |  |  | ✅ |  |  |  |  |
| Activate and expand cultures. |  |  | ✅ |  |  |  |  |
| Protein expression |  |  | ✅ |  |  |  |  |
| SQR ELISA |  |  |  | ✅ |  |  |  |
| Test H₂S production with L-cysteine induction. |  |  |  |  | ✅ |  |  |
| Optimize SQR:FCSD ratios for H₂S reduction |  |  |  |  |  | ✅ |  |
| Validate hardware H₂S detection |  |  |  |  |  | ✅ |  |
| Human practice | ✅ | ✅ |  | ✅ | ✅ | ✅ |  |
| Business plan |  |  |  | ✅ | ✅ | ✅ |  |
| Education | ✅ | ✅ | ✅ | ✅ | ✅ | ✅ | ✅ |

'
},{
"title": "Date: February, 2025",
"content": '

**A. Goal:**

1. Prepare solid/liquid LB medium and antibiotics
(Kana Sulfate, 50 mg/mL) configuration

2. Inoculate and culture pET28a vector

3. PCR amplification of fragments (SQR/FCSD)

**Notes:**

1. Dissolve solutions completely using a vortex mixer

2. Filter sterilization: Use BryoGold Syringe Filters (0.22 µm green filter for this experiment)

3. Laminar Flow Bench:

- UV light: 30 min (pre-use)

- White light: On during work

- Fan: Circulates HEPA-filtered sterile air.

**Result:** Color changes before/after
inoculation observed.

**B. Goal:**

1. Extract pET28a plasmid and measure concentration.

2. Digest pET28a, verify by gel electrophoresis, and
recover DNA.

3. Prepare agarose gel, electrophorese PCR products,
and recover fragments.

4. Homologous recombination with target fragments.

5. Transform *E. coli* BL21 competent cells
and culture.

**Content :**

1. **Plasmid Extraction from Overnight Culture：**

1.1. Preparation：

- Confirm RNase A added to Buffer SP1.

- Confirm ethanol added to Wash Solution.

- Check for precipitates in Buffers SP2/SP3.

1.2. Column equilibration：

- Add 500 µL Buffer S to adsorption column.

- Centrifuge at 12,000 ×g, 1 min. Discard
flow-through.

1.3. Bacterial pellet collection：

- Centrifuge 3 mL culture at 8,000 ×g, 2 min.
Discard supernatant.

1.4. Resuspend pellet in 250 µL Buffer SP1.

1.5. Add 250 µL Buffer SP2; invert 5–10x. Incubate
RT, 2–4 min.

1.6. Add 350 µL Buffer SP3; invert immediately 5–10x.

1.7. Centrifuge at 12,000 ×g, 5–10 min. Transfer
supernatant to column; centrifuge 8,000 ×g, 30 sec. Discard flow-through.

1.8. Add 500 µL Wash Solution; centrifuge 9,000 ×g,
30 sec. Discard flow-through.

1.9. Repeat Step 8.

1.10. Centrifuge empty column at 9,000 ×g, 1 min.

1.11. Elute DNA: Add 50–100 µL Elution Buffer to
column; incubate RT, 1 min. Centrifuge 9,000 ×g, 1 min. Store DNA.

2. **Agarose Gel Preparation (1%)**

- For 100 mL TAE: 1 g agarose powder.

- Microwave until clear. Add 7 µL dye (1:1000
dilution).

- Pour into tray; insert comb. Cool to solidify.

3. **Agarose Gel Electrophoresis Verification**

-Load sample onto 1% agarose gel.

-Run electrophoresis (DNA → anode).

-Image gel.

4. **Gel Extraction of DNA Fragments**

1. Excise target band; weigh gel slice.

2. Add 3× volume solubilization buffer; incubate
55°C, 10 min (invert frequently).

3. Transfer solution to column; centrifuge 12,000
rpm, 30–60 sec. Discard flow-through.

4. Add 600 µL Wash Buffer; centrifuge 12,000 rpm, 1
min. Discard flow-through.

5. Repeat Wash step.

6. Centrifuge 12,000 rpm, 2 min. Dry column (RT or
50°C).

7. Elute DNA: Add pre-warmed Elution Buffer (65°C);
incubate RT, 2 min. Centrifuge 12,000 rpm, 1 min.

8. Store DNA at –20°C.

5. **DNA Homologous Recombination**

- Mix: 4 µL ClonExpress Mix + 6 µL SQR/FCSD fragment
+ 10 µL vector.

- Incubate: 50°C, 10 min (gradient cycler); cool to
4°C.

- Nucleic acid concentration: 23.55 ng/µL
(spectrophotometer).

6. **Transformation of BL21 Competent Cells**

1. Thaw competent cells on ice (25 min).

2. Add 2–3 µL plasmid (100 ng/µL); mix gently.

3. Ice, 10 min.

4. Heat shock: 42°C, 120 sec.

5. Ice, 4 min.

6. Add 800 µL LB; shake 37°C, 200 rpm, 40 min.

7. Plate on LB + Kanamycin (100 µg/mL).

8. Incubate 37°C overnight (inverted plate).

'
}, {
"title": "Date: March, 2025",
"content": '

**A. Goal**

1. Colony PCR; culture positive colonies.

2. Extract and sequence plasmids.

3. Activate and expand cultures.

4. Induce protein expression with IPTG.

**Content：**

1. **Positive Colony Culture：**

1. Add 100 µL Kanamycin (50 mg/mL) to LB medium.
Label tubes (FCSD/SQR).

2. Inoculate at 1:20 (culture:medium).

3. Shake at 37°C, 2–3 h.

**Colony Verification：PCR or plasmid
sequencing.**

2. **Colony PCR：**

-Reaction Mix：

- Hieff PCR Master Mix: 25 µL

- Primer-F/R (10 µM): 1 µL each

- DNA Template: 1 µL

- ddH₂O: 22 µL

-Thermal Profile:Denaturation → Annealing →
Extension.

-Electrophoresis：Run gel; image bands.

3. **IPTG Induction (Lac Operon Theory)：**

- Repressor binds operator without lactose → no
transcription.

- IPTG (C₉H₁₈O₅S, MW=238.3 g/mol, stored at –20°C)
mimics allolactose → induces expression.

- Add 100 µL IPTG to 100 mL culture (1:1000).

- Shake overnight at 16°C.

'
}, {
"title": "Date: April 2025",
"content": '

**A. Goal**

1. **Isolate recombinant plasmids (pET28a-SOR/pET28a-FCSD) from *E. coli*.**

**Content**：

-Bacterial Pellet：Centrifuge 1.5–5 mL culture at
8,000 ×g, 2 min.

-Column Equilibration：Add 500 µL Buffer S; centrifuge
12,000 ×g, 1 min.

- Resuspend pellet in 250 µL Buffer SP1.

-Add 250 µL Buffer SP2; invert 5–10x. Incubate RT, 3
min.

-Add 350 µL Buffer SP3 (white filaments form); invert
5–10x.

-Centrifuge 12,000 ×g, 5–10 min. Transfer supernatant
to column; centrifuge 8,000 ×g, 30 sec.

-Repeat Step 6 .

-Add 500 µL Buffer DWI; centrifuge 9,000 ×g, 30 sec.

- Wash: Add 500 µL Wash Solution + ethanol;
centrifuge 9,000 ×g, 30 sec (×2).

- Centrifuge empty column 9,000 ×g, 1 min.

-Elute with 50 µL ddH₂O; centrifuge 9,000 ×g, 1 min.

-DNA Quantification (NanoDrop):

**- Blank: ddH₂O (A = 0.3)**

**- pET28a-sqr: 17.3 ng/µL**

**- pET28a-fcsd: 142.5 ng/µL**

2. **SDS-PAGE Gel Preparation**

Step 1: Rinse gel plates with water.

Step 2: Assemble plates (1.5 mm spacer).

Step 3: Separating Gel (per gel)

-4 mL resolving gel solution + 4 mL buffer + 80 µL
accelerator.

- Inject into chamber; overlay with ethanol.

-Polymerize 8–10 min (RT).

Stacking Gel (per gel):

- 2 mL stacking solution + 2 mL buffer + 20 µL
accelerator.

- Inject; insert comb; polymerize 10 min.

3. **Lysis Protocol**

1. Vortex lysis buffer; add lysozyme; vortex.
Incubate on ice, 30 min.

2. Sonication (on ice)：

- 10 sec pulse / 10 sec rest (×6 cycles).

- Repeat if suspension unclear.

3. Centrifuge 10,000 ×g, 20 min, 4°C.

**4. Affinity Chromatography (His-Tag
Purification)**

- Use Ni²⁺ column for pET28a-derived proteins.

(Detailed protocols for chromatography, SDS-PAGE,
ELISA, and enzyme assays; formatted similarly to above.)

'
},{
"title": "Date: June, 2025",
"content": '

**A. Plasmid Extraction (DH5α-SQR/FCSD)**

1. Centrifuge 2.5 mL culture (10,000 rpm, 1 min).

2. Resuspend in 250 µL Buffer P1 (+ RNase A); vortex.

3. Add 250 µL Buffer P2; invert 10x.

4. Add 350 µL Buffer P3; invert 10x. Centrifuge
12,000 rpm, 10 min.

5. Transfer supernatant to column; centrifuge 12,000
rpm, 60 sec.

6. Wash with 600 µL Buffer PW1 (×2).

7. Dry column (12,000 rpm, 1 min).

8. Elute with 100 µL buffer; centrifuge 12,000 rpm, 1
min.

**B. ELISA Protocol**

1. Load 50 µL samples/standards into pre-coated
plate.

2. Add 100 µL HRP conjugate; seal. Incubate 37°C, 1
h.

3. Wash (1× buffer).

4. Add 50 µL substrate; incubate 15 min (dark).

5. Stop with 50 µL stop solution; read OD450.

6. Plot standard curve; calculate concentrations.

**C. SQR Enzyme Activity Assay**

- Principle: DUQ decomposition (A₂₇₅ decrease).

- Reagents：

- Tris-HCl buffer (50 mM)

- Glucose (20 mM)

- DUQ (50 µM)

- Glucose oxidase (1 U/mL)

- Catalase (1 U/mL)

- SQR (1 mg/mL)

- Initiate with Na₂S (100 µM); monitor A₂₇₅ every 5
sec for 5 min.

**D. MG1655 Culture**

- Inoculate 1 mL stock into 4 mL LB (25 tubes). Shake
37°C, 20 h.

'
},{
"title": "Date: July, 2025",
"content": '

**A. Test H₂S production with L-cysteine induction**

Protocol：

1. Add L-cys (0–2 mM) to MG1655 cultures.

2. Shake 37°C, 220 rpm, 6 h.

3. Quantify H₂S with lead acetate test strips.

4. Measure turbidity (OD values provided).

5. Add SQR/FCSD (0–10 mg/mL); re-assess H₂S after 1
h.

'
},{
"title": "Date: August, 2025",
"content": '

**A. Optimize SQR:FCSD ratios for H₂S reduction**

**B. Validate hardware H₂S detection**

Protocol：

- SQR:FCSD Ratios：1:1, 1:2, 1:3, 2:1, 3:1 (10 mg/mL
each).

- Add to 4 mL bacterial culture; shake 6 h.

- Test H₂S with strips/hardware.

Test Groups: H₂S (no L-cys) \H₂S + 1 mM L-cys\ H₂S +
2 mM L-cys\SQR + L-cys\FCSD + L-cys\SQR+FCSD + L-cys

**Result**

- Optimal SQR:FCSD ratio = 1:3

'
},{
"title": "Date: September, 2025",
"content": '

**A.Cell Line and Culture Conditions**

The human colorectal adenocarcinoma cell line
NCI-H716 was utilized in this study. Cells were maintained in RPMI 1640 medium supplemented with 10% fetal bovine
serum (FBS) at 37°C in a 5% CO₂ atmosphere. As a suspension cell line, NCI-H716 cells were cultured and passaged using
standard protocols for non-adherent cells. All seeding, treatment, and transfection procedures were optimized for
suspension culture.

**B.Modeling Mechanism**

A cell model of impaired glucagon-like peptide-1
(GLP-1) signaling was established by treating NCI-H716 cells with the supernatant from E. coli MG1655 cultures
pre-induced with L-cysteine. This induction triggers the bacterial production of hydrogen sulfide (H₂S), which is
present in the supernatant. Exposure to H₂S inhibits cellular GLP-1 production, leading to the downregulation of GLP-1
gene expression and a consequent disruption of its mediated metabolic regulatory pathways.

**C.Experimental Method: Model Validation by
qPCR**

**Cell Seeding:** NCI-H716 cells were
seeded in 12-well plates at a density of 5 × 10⁵ cells per well.

**Treatment:** When the cell density
reached 70-80%, the culture medium was replaced with fresh medium containing the L-cysteine-induced E. coli MG1655
supernatant (hereafter referred to as the \'conditioned supernatant\').

**Incubation:** Cells were co-cultured
with the conditioned supernatant for 4 hours. Following this incubation, the treatment medium was aspirated, and the
cells were gently washed twice with phosphate-buffered saline (PBS) to remove residual supernatant.

**Recovery:** Cells were then
replenished with fresh complete RPMI 1640 medium and further incubated for an additional 20 hours.

**RNA Extraction and Analysis:** After
the total 24-hour incubation period, total RNA was isolated from the cells. Reverse transcription quantitative
polymerase chain reaction (RT-qPCR) was performed to quantify the expression levels of GLP-1 mRNA. Subsequent
experiments were conducted only after successful validation of significant GLP-1 mRNA downregulation in the treatment
group.

**D. Experimental Groups**

**Blank Control:** Untreated NCI-H716
cells (cultured in standard complete medium).

**Treatment Group:** NCI-H716 cells
treated with the conditioned supernatant from L-cysteine-induced E. coli MG1655.

**E. Analysis of GLP-1 mRNA Expression by RT-qPCR Following Various Treatments**

**Cell Seeding and Treatment**

NCI-H716 cells were seeded in 12-well plates at a
density of 5 × 10⁵ cells per well in complete growth medium (RPMI 1640 supplemented with 10% FBS). Upon reaching
70-80% confluence, the culture medium was replaced with fresh medium containing the respective treatments (detailed in
Section 2). The cells were then co-incubated with the treatments for 4 hours. After this incubation period, the
treatment media were aspirated, and the cells were gently washed twice with phosphate-buffered saline (PBS) to
terminate the exposure. Subsequently, the cells were replenished with fresh complete medium and further incubated for
either 20 hours or 44 hours to achieve total post-treatment time points of 24 hours and 48 hours, respectively.

**Experimental Groups**

The experiment included the following treatment
groups, with the volume of bacterial supernatant added being consistent with the optimized conditions established in
Investigation 1:

**Blank Control:** Untreated NCI-H716
cells (cultured in standard complete medium).

**Negative Control:** NCI-H716 cells
treated with supernatant from the standard E. coli MG1655 culture medium (uninduced with L-cysteine).

**Positive Control:** NCI-H716 cells
treated with supernatant from L-cysteine-induced E. coli MG1655 cultures.

**Treatment Group 1 (SQR):** NCI-H716
cells treated with L-cysteine-induced E. coli MG1655 supernatant that had been pre-incubated with SQR protein
solution.

**Treatment Group 2 (FCSD)**: NCI-H716
cells treated with L-cysteine-induced E. coli MG1655 supernatant that had been pre-incubated with FCSD protein
solution.

**Treatment Group 3 (SQR+FCSD):**NCI-H716 cells treated with L-cysteine-induced E. coli MG1655 supernatant that had been pre-incubated with a
combined solution of SQR and FCSD proteins.

**F. RNA Extraction and RT-qPCR**

After the designated incubation periods (24 h total),
total RNA was isolated from all cell groups using according to the manufacturer\'s instructions. RNA concentration and
purity were determined spectrophotometrically. Complementary DNA (cDNA) was synthesized from 1 μg of total RNA using a
reverse transcription kit. Quantitative PCR (qPCR) was then performed using and SYBR Green master mix with
gene-specific primers for human GLP-1 and the housekeeping gene β-actin. The relative expression of GLP-1 mRNA was
calculated using the 2–ΔΔCt method, normalized to the housekeeping gene and relative to the blank control group.

'
}]%}
{% block title %}Notebook{% endblock %}
{% block lead %}

![](https://static.igem.wiki/teams/5765/header/notebook.webp)

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{% set table\_data = [{
"title": "Overview",
"content": '

Obesity and type II diabetes are two of the most
prevalent public health issues worldwide, with existing treatments often involved high costs and undesired side
effects. Therefore, our project seeks to develop a novel enzyme-based therapy that provides a mild and indirect
treatment through synthetic biology. Specifically, we designed and expressed two enzymes, SQR and FCSD, capable of
degrading hydrogen sulfide (H₂S) within the intestine. Presence of excess H₂S would inhibit the GLP-1 signaling
pathway, which plays a key role in insulin secretion and fat metabolism. By reducing H₂S levels, our approach aims to
restore GLP-1 expression, thereby promoting glucose and fat reduction. The recombinant enzymes were expressed in E.
coli and purified for functional test. In future applications, these enzymes may be used to produce oral capsule ,
enabling efficient and stable delivery to the patients’ intestine, where they function.

'
},{
"title": "Part 1: pET28a-SQR-His",
"content": '

**Design 1**

To construct the recombinant plasmid pET28a-SQR-His,
the coding sequence of sulfide:quinone oxidoreductase (SQR) was inserted into the pET28a expression vector via
homologous recombination. The template plasmid incorporates several critical functional elements: a kanamycin
resistance gene (KanR) for antibiotic selection; LacI repressor and Lac operator sequences enabling IPTG-inducible
expression; a His-tag and T7 promoter to facilitate affinity purification and efficient transcription initiation,
respectively. The SQR gene—encoding a eukaryotic enzyme involved in hydrogen sulfide (H₂S) decomposition—was cloned
into the pET28a backbone to generate the expression construct. In this study, the recombinant SQR protein is designed
for oral delivery to the intestinal tract, where it is anticipated to degrade microbial-derived excess H₂S. This
reduction in H₂S levels is expected to restore GLP-1 signaling, a pathway critically involved in appetite suppression
and lipid metabolism.

![3](https://static.igem.wiki/teams/5765/engineering/1.webp)

**Figure 1. Plasmid map of pET28a-SQR-His**

**Build 1**

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.

**![组图1_画板 1](https://static.igem.wiki/teams/5765/engineering/2.webp)**

**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.

![SQR鉴定组图_画板 1](https://static.igem.wiki/teams/5765/engineering/3.webp)

**Figure 3. The gel electrophoresis validation of pET28a-SQR**

**Test 1**

1. **Protein Expression**

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.

![BL21菌落平板](https://static.igem.wiki/teams/5765/engineering/4.webp)

**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.

**![SQR蛋白图_画板 1](https://static.igem.wiki/teams/5765/engineering/5.webp)**

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

2. **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.

![ELISA标准曲线_画板 1](https://static.igem.wiki/teams/5765/engineering/6.webp)

**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.

![Elisa测试SQR酶含量](https://static.igem.wiki/teams/5765/engineering/7.webp)

**Figure 7. ELISA assay showing enzyme concentration**

3. **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.

![DUQ还原法测试SQR酶活性](https://static.igem.wiki/teams/5765/engineering/8.webp)

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

4. **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.

![H2S生成组图_画板 1](https://static.igem.wiki/teams/5765/engineering/9.webp)

**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.

**![SQR试纸条验证组图_画板 1](https://static.igem.wiki/teams/5765/engineering/10.webp)**

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

5. **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.

![SQR传感器验证_画板 1](https://static.igem.wiki/teams/5765/engineering/11.webp)

**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.

6. **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.

![qPCR检测建模情况](https://static.igem.wiki/teams/5765/engineering/12.webp)

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

| **24h** |  | **Ct** | **SD** | **Average** | **▲Ct** | **2-▲▲Ct** |
| --- | --- | --- | --- | --- | --- | --- |
| Control group | ACT1 | 20.303 | 0.139145008 | 20.22366667 |  |  |
| 20.305 |  |  |
| 20.063 |  |  |
| GLP-1 | 27.401 | 0.078564199 | 27.32066667 | 7.097518921 | 0.007301867 |
| 27.317 | 7.011953354 | 0.007748037 |
| 27.244 | 7.1804142 | 0.006894138 |
| Negative group | ACT1 | 19.94723991 | 0.462218603 | 20.30385177 |  |  |
| 20.82605743 |  |  |
| 20.13825798 |  |  |
| GLP-1 | 27.09345779 | 0.410917483 | 27.52850367 | 7.14621788 | 0.007059502 |
| 27.91005249 | 7.083995056 | 0.007370637 |
| 27.58200073 | 7.443742752 | 0.005743943 |
| SQR-treated group | ACT1 | 21.13188362 | 0.03791144 | 21.12831879 |  |  |
| 21.08875084 |  |  |
| 21.1643219 |  |  |
| GLP-1 | 26.63872528 | 0.042969904 | 26.59593836 | 5.50684166 | 0.021992544 |
| 26.55278778 | 5.464036942 | 0.022654839 |
| 26.59630203 | 5.431980133 | 0.023163866 |
| Positive group | ACT1 | 15.651 | 0.159104787 | 15.81333333 |  |  |
| 15.82 |  |  |
| 15.969 |  |  |
| GLP-1 | 25.87 | 0.308002164 | 26.22533333 | 10.42091846 | 0.000729442 |
| 26.416 | 10.59577942 | 0.000646179 |
| 26.39 | 10.21927261 | 0.000838866 |

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.

![细胞样本处理组_画板 1](https://static.igem.wiki/teams/5765/engineering/13.webp)

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

**Learn 1**

While our current design successfully demonstrated
the in vitro activity of SQR, there remain several perspectives which requires future exploration. For instance, the
functional impact of SQR on GLP-1 signaling pathway has not yet been validated in animal models, and there remains a
lack of understanding of the physiological impact of our approach. In addition, the enzyme’s long-term stability and
resilience under intestinal conditions still require more investigation. We believe our projects would provide some
insights for obesity research, and we hope future teams can build upon our work to bridge the gap between concept and
clinical practice.

'
},{
"title": "Part 2: pET28a-FCSD-His",
"content": '

**Design 2**

We constructed the recombinant plasmid
pET28a-FCSD-His by cloning the FCSD coding sequence into the pET28a vector using homologous recombination. The
resulting plasmid contains the following key functional elements: a kanamycin resistance gene (KanR) for antibiotic
selection, the LacI repressor and Lac operator system for IPTG-inducible expression, a His-tag sequence in conjunction
with the T7 promoter to facilitate protein purification and controlled transcription initiation, as well as the FCSD
gene integrated into the pET28a backbone as the expression cassette.

![16](https://static.igem.wiki/teams/5765/engineering/14.webp)

**Figure 14. Plasmid map of pET28a-FCSD-His**

**Build 2**

The FCSD gene sequence underwent PCR amplification.
Figure 15A displays a distinct band at the anticipated size, confirming that amplification was successful. Meanwhile,
the pET28a vector was linearized through double enzymatic digestion with XhoI and BamHI. Figure 15B exhibits a single
band, verifying successful linearization.

![组图1_画板 1](https://static.igem.wiki/teams/5765/engineering/15.webp)

**Figure 15. Gel electrophoresis of PCR-amplified FCSD gene and pET28a linear**

The recombinant plasmid pET28a-FCSD-His was
transformed into *E.coli* DH5α competent cells. The transformed culture was spread onto LB plates containing
kanamycin and incubated overnight at 37°C to obtain a single colony. Figure 16A shows a distinct single colony formed
on the plate, indicating preliminary success of the transformation experiment. Subsequently, multiple positive clones
were randomly selected and validated via colony PCR and sequencing. Figure 16B shows that the FCSD sequence matches
the expected target sequence perfectly, further confirming the correct construction of the recombinant plasmid.

![FCSD鉴定组图_画板 1](https://static.igem.wiki/teams/5765/engineering/16.webp)

**Figure 16. Map for pET28a-FCSD-His Transformation Verification**

**Test 2**

1. **Protein Expression**

For protein expression purposes, the validated
recombinant plasmid was transformed into *E.coli* BL21. As seen in Figure 17, single colonies grew on the
plate, proving the transformation worked.

![BL21菌落平板](https://static.igem.wiki/teams/5765/engineering/17.webp)

**Figure 17. pET28a-FCSD-His single colony map**

Cultures were induced with 0.5 mM IPTG at 16°C, and
cells were disrupted by ultrasonication to release the target proteins. Purification was carried out using
nickel-affinity chromatography, leveraging the His-tag. Four fractions were collected: crude lysate, flow-through,
wash, and elution. SDS-PAGE analysis revealed a clear band in the elution fraction at the anticipated molecular weight
of FCSD (~43 kDa), confirming that expression and purification were successful.

**![FCSD蛋白图_画板 1](https://static.igem.wiki/teams/5765/engineering/18.webp)**

**Figure 18. The SDS-PAGE protein gel of
samples from different purification stages**

2. **Lead Acetate Test Strips**

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.

![H2S生成组图_画板 1](https://static.igem.wiki/teams/5765/engineering/19.webp)

**Figure 19. Different L-cysteine derivatives induce varying amounts of H₂S production**

When FCSD 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 FCSD 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 FCSD concentration reached 5mg/L,
H₂S levels decreased significantly, approaching concentrations observed without L-cysteine induction. This result
strongly demonstrates that the FCSD enzyme efficiently and rapidly catalyzes H₂S degradation, highlighting its
potential application in systems requiring H₂S control.

**![FCSD试纸条检测组图_画板 1](https://static.igem.wiki/teams/5765/engineering/20.webp)**

**Figure 20. Reduction in greyscale of lead
acetate test strips with increasing FCSD concentration**

3. **Hardware Validation**

We subsequently verified the activity of FCSD using
sensor-based devices for real-time monitoring of sulfur metabolites. In untreated samples, sensor measurements for
target metabolites were high(Figure 21A). By contrast, samples treated with FCSD showed reduced sensor readings, which
confirms that the metabolites were processed effectively(Figure 21B).

![FCSD传感器验证_画板 1](https://static.igem.wiki/teams/5765/engineering/21.webp)

**Figure 21. Sensor display when FCSD is added**

4. **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.

![qPCR检测建模情况](https://static.igem.wiki/teams/5765/engineering/22.webp)

**Figure 22. 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 FCSD protein
(FCSD-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 FCSD administration**

| **24h** |  | **Ct** | **SD** | **Average** | **▲Ct** | **2-▲▲Ct** |
| --- | --- | --- | --- | --- | --- | --- |
| Control group | ACT1 | 20.303 | 0.139145008 | 20.22366667 |  |  |
| 20.305 |  |  |
| 20.063 |  |  |
| GLP-1 | 27.401 | 0.078564199 | 27.32066667 | 7.097518921 | 0.007301867 |
| 27.317 | 7.011953354 | 0.007748037 |
| 27.244 | 7.1804142 | 0.006894138 |
| Negative group | ACT1 | 19.94723991 | 0.462218603 | 20.30385177 |  |  |
| 20.82605743 |  |  |
| 20.13825798 |  |  |
| GLP-1 | 27.09345779 | 0.410917483 | 27.52850367 | 7.14621788 | 0.007059502 |
| 27.91005249 | 7.083995056 | 0.007370637 |
| 27.58200073 | 7.443742752 | 0.005743943 |
| FCSD-treated group | ACT1 | 21.8233223 | 0.093600657 | 21.92073758 |  |  |
| 21.92890167 |  |  |
| 22.00998878 |  |  |
| GLP-1 | 27.58271027 | 0.260798516 | 27.70004082 | 5.75938797 | 0.01846084 |
| 27.51851654 | 5.589614868 | 0.020766259 |
| 27.99889565 | 5.98890686 | 0.015745607 |
| Positive group | ACT1 | 15.651 | 0.159104787 | 15.81333333 |  |  |
| 15.82 |  |  |
| 15.969 |  |  |
| GLP-1 | 25.87 | 0.308002164 | 26.22533333 | 10.42091846 | 0.000729442 |
| 26.416 | 10.59577942 | 0.000646179 |
| 26.39 | 10.21927261 | 0.000838866 |

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

![细胞样本处理组_画板 1](https://static.igem.wiki/teams/5765/engineering/23.webp)

**Figure 23. The map of GLP-1 gene expression level changes under different treatments**

**Learn 2**

Our current design shows FCSD works in lab tests, but
there’s more to explore. For example, we haven’t checked how FCSD affects the GLP-1 signaling pathway in animal
models, and we don’t know enough about its effects in living organisms. Also, we need to study how stable the enzyme
is long-term in the gut. We think this project can help with obesity research, and we hope future teams will build on
this work to turn the idea into real clinical use.

'
}]%}
{% block title %}Engineering{% endblock %}
{% block lead %}

![](https://static.igem.wiki/teams/5765/header/engineering.webp)

{% endblock %}

{% extends "layout.html" %}
{% set table\_data = [{
"title": "Disposition of LB Culture Base",
"content": "

**Disposition of LB culture base
(1L)**

",
"children": [{
"title": "A. Liquid LB Culture",
"content": "

**Materials:**

1. Trypotone
2. Yeast extract
3. Sodium chloride

**Procedures:**

1. Add 10g Trypotone + 10g Sodium chloride + 5g Yeast culture
2. Add 1L of ddH2O

"
}, {
"title": "B. Solid LB Culture",
"content": "

**Materials:**

1. Trypotone
2. Yeast extract
3. Sodium chloride
4. Agar

**Procedures:**

1. Add 10g Trypotone + 10g Sodium chloride + 5g Yeast culture + 15g Agar
2. Add 1L of ddH2O

"
}, {
"title": "C. Kana 50mg/ml",
"content": "

**Materials:**

1. kana
2. ddH2O
3. BryoGold Syring Filters

**Procedures:**

1. add 50mg Kana(stored in -4°C) + 1ml ddH2O
2. filter and remove the bacteria by BryoGold Syring Filters(using 0.22 μm)
3. finally being stored at -20°C if we do not use that immediately.

"
}]
}, {
"title": "Plasmid Construction",
"content": "

**Plasmid construction**

",
"children": [{
"title": "A. PCR Amplified Fragments",
"content": "

**Procedures:**

1. Add 2\*Taq PCR Mix 25μl, SQR\\FCSD template 2μl, primer-R 2μl, and primer-F 2μl to centrifuge tube. Add
   ddH2O to the centrifuge tube until reaching 50μl.
2. Centrifuge the mixture for a few seconds to remove the bubbles.
3. Insert mixture into a PCR thermal cycler.
4. Perform the cycle, Step2-4 repeat 30 cycles.

|  |  |  |
| --- | --- | --- |
| 98˚C | 5 minutes | Step 1 |
| 98˚C | 30 seconds | Step 2 |
| 55˚C | 30 seconds | Step 3 |
| 72˚C | 90 seconds | Step 4 |
| 72˚C | 10 minutes | Step 5 |

"
}, {
"title": "B. Inoculated and Cultured pET28a Vector",
"content": "

**Materials：**

1. LB medium
2. Tube
3. pET28a vector

**Procedures：**

1. The sample was mixed in LB solvent.
2. A 1:1000 inoculation was performed into fresh LB medium.
3. The bacteria were cultivated in 4 mL of medium

"
}, {
"title": "C. Gel Extraction",
"content": "

**Materials:**clean scalpel\\ 1.5 mL
Eppendorf tube\\pipettes and sterilized pipette heads\\ hot water bath\\buffer B2\\purification column and
collection tube\\centrifuge\\wash solution containing pure ethanol\\ddH2O.

**Procedure:**

1. Cut the slice of gel containing SQR and FCSD, cutting off as much unneeded gel as possible, and place it in an
   1.5 mL Eppendorf tube.
2. Add 500uL of buffer B2 and put the tube into a 50°C hot water bath until gel has completely melted.
3. Transfer the solution containing melted gel into a purification column that's in a collection tube and perform
   centrifugation at 8000 g for 30 seconds.
4. Empty the collection tube, put the purification column back in, and add 500uL of wash solution containing pure
   ethanol. Perform centrifugation at 9000 g for 30 seconds, and empty collection tube again.
5. Repeat (4).
6. Perform centrifugation one more time at 9000 g for 2 minute, then open the cap of the tube and let it sit for
   one minute to allow the ethanol to evaporate.
7. Transfer purification column to a 1.5 mL EP tube. Add 20uL of ddH2O at the center of the purification
   column.
8. Cap the lid and let the tube sit for 1 minute. Perform centrifugation at 9000 g for 1 minute.
9. Discard the purification column. Store DNA at 4°C.

"
}, {
"title": "D. Homologous Recombination",
"content": "

**Materials:**

1. ClonExpress Mix
2. SQR/FCSD gene fragment
3. pET28a linear vector

**Procedures:**

1. Add 4 μL of ClonExpress Mix + 6 μL of the SQR/FCSD gene fragment + 10 μL of the gene vector
2. Place it in a gradient thermal cycler and incubate at 50°C for 10 minutes.
3. Cool it to 4°C and place it on ice

"
}, {
"title": "E. Transform Competent E. coli BL21/DH5α Cells and Culture",
"content": "

**Procedures:**

1. Thaw competent cells on ice.
2. Add 10 μL recombinant product to 100 μL competent cells, mix gently by flicking the tube (do not vortex), and
   incubate on ice for 30 minutes.
3. Heat-shock at 42 ℃ water bath for 45 seconds.
4. Place immediately on ice for 1 minute.
5. Add 890 μL LB medium (without antibiotics) and incubate at 37 ℃ for 60 minutes.
6. Spread 100 μL bacterial culture via the spread plate method.
7. Incubate at 37 ℃ for 12–16 hours.

"
}, {
"title": "F. Colony PCR",
"content": "

**Materials:**

1. 2\*Hieff PCR Master Mix 25 μL
2. Primer-F(10uM) & Primer-R(10uM) 1μL + DNA Template 1μL+ ddH2O 22μL

**Procedures:**

|  |  |  |
| --- | --- | --- |
| 98˚C | 5 minutes | Step 1 |
| 98˚C | 30 seconds | Step 2 |
| 55˚C | 30 seconds | Step 3 |
| 72˚C | 90 seconds | Step 4 |
| 72˚C | 10 minutes | Step 5 |

"
}, {
"title": "G. Extract Plasmids",
"content": "

**Materials:**

1. cultured bacterial liquid
2. reagent: buffer P1, buffer P2, buffer P3, buffer PW1, buffer PW2, ddH2O
3. absorption column

**Extracting Procedures:**

1. Preparation work：Check whether RNase A has been added to Buffer P1. Check whether anhydrous ethanol has been
   added to the buffer PW2.
2. Take 1-5 ml of overnight cultured of bacterial solution, add it to the centrifuge tube, 10,000 rpm centrifuge
   for 1 min. Discard the culture medium.
3. Add 250 μL Buffer P1 and mix well with a vortex meter.
4. Add 250 μL Buffer P2, gently mix it up and down 8-10 times to make the bacteria fully cracked.
5. Add 350 μL Buffer P3, and immediately gently reverse 8-10 times to completely neutralize the buffer P2 of the
   solution.
6. Place the FastPure DNA Mini Columns adsorption column in the Collection Tube 2ml collection tube. Carefully
   transfer step 4 supernata with pipette to the adsorption column, 12,000rpm centrifuge for 1 min. Pour out the
   waste liquid in the collection tube and put the adsorption column back into the collection tube.
7. Add 600 μL Buffer PW2. 12,000rpm centrifuge for 1 min. Pour out the waste liquid in the collection tube and put
   the adsorption column back into the collection tube.
8. Repeat step 7.
9. Put the adsorption column back into the collection tube. 12,000 rpm centrifuge for 1 min drying adsorption
   column is to completely remove the residual rinse solution in the adsorption column.
10. Place the adsorption column in a new sterilized 1.5ml centrifuge tube. Add Elution Buffer to the center of the
    membrane of the column adsorption column. 2 min ar room temperature. 12,000rpm centrifuge for 1min to elute DNA.

"
}, {
"title": "H. Double Digestion with XhoI and BamHI",
"content": "

**Materials:**

1. XhoI and BamHI restrictive enzyme
2. 10\*loading buffer
3. pET28a vector
4. ddH2O

**Procedures:**

1. Add 1μL of each of the XhoI and BamHI restrictive enzymes + 5μL of 10\*loading buffer + 3.04 μL of the plasmid
   vector + 40μL of ddH2O
2. Transfer the mixed sample into a PCR amplifier. set the PCR amplifier to maintain a constant temperature of 37°C
   for 20 minutes.

"
}]
},{
"title": "Protein Expression",
"content": "

**Protein expression**

",
"children": [{
"title": "A. Strain Activation and Expansion of Culture",
"content": "

**Materials:**

1. Kana (50mg/ml)
2. LB medium

**Procedures:**

1. Add 100μL Kana to the LB medium
2. The ratio of bacterial liquid and LB medium is one to twenty
3. Repeat the procedure with another enzyme and put them into the shaker at 37°C for 2 to 3 hours.
4. Add 100μL IPTG to the 100ml expanded cultivation of bacteria (1:1000)
5. culture it in the shaker overnight before further steps at 16°C

"
}, {
"title": "B. Extraction of Crude Protein",
"content": "

**Procedures:**

1. Clean the sonicator to ensure it is free of contaminants.
2. Place the sample on ice to maintain a low temperature throughout the process.
3. Add protease to the sample and perform sonication under the following conditions: 3 seconds of sonication
   followed by 3 seconds of rest, repeated for 10 min.

"
}, {
"title": "C. Purification of His-tagged Protein",
"content": "

**Materials:**

1. non-denatured lysis solution
2. lyase
3. 50% BeyoGold His-tag Purification Resin
4. washing buffer
5. elution buffer

**Procedures:**

1. Simple preparation, lysate is mixed with nickel ion afginity resin.
2. Washing buffer removes weakly bound impurities.
3. Elution buffer elutes the target protein.
4. Purigication process: Crude protein-resin mixture → gravity flow fitrition column → Collect flow-through →
   Washing → Elution
5. Simple preservation, collect the flow-through liqiud and elution fractions and temporarily store them in ice.

"
}, {
"title": "D. Preparation of SDS-PAGE Gel",
"content": "

**Materials:**

1. gel casting plate (long + short)
2. gel casting module
3. upper/lower gel solution
4. dyed stacking gel buffer
5. polymerization accelerator

**Procedures:**

1. **Cleaning Casting Plates**

Rinse the vertical gel casting plates thoroughly
with tap water.

2. **Assembling the Gel Casting Module**

- Position the long plate (1.5 mm thickness,
selected as the thickest option) with the short plate with its groove facing the user (long plate at the back,
short
plate at the front).

- Insert it into the red spacer, press to ensure
flat alignment.

3. **Gel Preparation**

**Lower (Separating) Gel:**

1. Prepare the resolving gel for protein separation
   based on target molecular weights.

- For two gels (SQR and FCSD):

- Each gel: 4 mL resolving gel solution + 4 mL
resolving gel buffer + 80 μL polymerization accelerator.

2. Mix thoroughly and inject the solution evenly
   into the casting chamber using a 1 mL pipette.
3. Add ethanol to:

- Flatten the gel surface via gravitational
pressure.

- Form a boundary to prevent desiccation of the
resolving gel.

4. Allow polymerization for 8-10 min at room
   temperature.
5. Invert the casting module in a sink to drain
   ethanol.

**Upper (Stacking) Gel:**

1. Prepare the stacking gel for protein
   concentration:

- For each gel: 20 μL polymerization accelerator
+
2 mL stacking gel buffer + 2 mL stacking gel solution.

- Avoid bubble formation during mixing.

2. Inject the stacking gel solution into the
   chamber with a pipette.
3. Insert a vertically aligned comb and allow
   polymerization for 10 min.

"
}]
},{
"title": "Functional Testing",
"content": "",
"children": [{
"title": "A. Elisa Kit",
"content": "

**Materials:**

1. 96-well ELISA microplate
2. 5 standard wells: known SQR concentrations
3. 4 experimental wells: crude protein, flow-through, wash buffer, and elution buffer
4. HRP
5. wash solution
6. stop solution

**Procedures:**

1. **Sample Preparation and Loading**

* Prepare a 96-well ELISA microplate
* Pre-warm the plate to room temperature for 20 minutes before use. The plate is pre-coated with SQR antibodies.
* Add 50 µL of each sample to designated wells: 5 standard wells: Known SQR concentrations;4 experimental wells:
  Crude protein, flow-through, wash buffer, and elution buffer.
* Add 100 µl HRP to each well.
* Seal the plate with a microplate sealer.
* Incubate at 37°C for 1 hour.

2. **Washing and Detection**

* remove the liquid by tapping the plate upside-down on absorbent paper.
* Fill each well with 1× wash solution (diluted from 20× stock: 1 mL stock + 19 mL ddH₂O).
* Repeat this wash 5 times total.
* Add 50 µL HRP substrate to each well.
* Incubate for 15 minutes in the dark
* Stop the reaction by adding 50 µL stop solution.
* Measure OD450

3. **Data Analysis**

* Plot the standard curve:X-axis: SQR concentration of standards;Y-axis: OD450 values.
* Obtain the curve equation
* Calculate sample concentrations by Substituting experimental OD values into the equation.

"
}, {
"title": "B. Spectrophotometric Detection Based on DUQ Reduction",
"content": "

**Materials:**

1. Tris-HCl buffer solution
2. Glucose stock solution
3. DUQ
4. Ethyl alcohol
5. Glucose oxidiase
6. Hydrogen peroxidase
7. SQR protein solution

**Procedures:**

1. **Preparation**

* Prepare 20mL of Tris-HCl buffer solution:Take 1mL of stock solution and 19 mL of ddH2O, shake well.
* Prepare glucose stock solution: weigh 72.064mg of glucose and dissolve in 20mL ddH2O.
* DUQ:weigh 1.612mg of DUQ and dissolve by absolute ethyl alcohol to 10mL.
* Glucose oxidise: dilute enzyme precursor solution by 10000 times using ddH2O.
* Hydrogen peroxidase: dissolve 1 µL catalse in 10mL ddH2O.
* The concentration of SQR protein solution should be 1mg/mL.

2. **reaction system**

* Add 130mL of 50m mol/L Tris-HCl + 20mL of 20 m mol/L glucose + 20mL of 50 µ mol/L DUQ + 10mL of 1 unit/mL
  glucose oxidase + 10mL of 1 unit/mL catalase + 10mL of SQR protein solution
* Add 10 µl 100 µM Na2S and measure absorbance of 275nm

3. **Establish a control group**

* Negative control: no SQR protein, other consistency
* Blank control: no Na2S, other consistency
* Enzyme inactivation control: use PCR amplifier to inactivate SQR protein solution, other consistency
* Parallel test: repeat each sample 3 times

"
}, {
"title": "C. Treatment of MG1655 with SQR/FCSD Proteins",
"content": "

**Procedures:**

1. Add 1ml of MG1655 stock solution into 4ml solution for 25 tect tubes
2. Place in a 37°C shake for 20h
3. Using hydrogen sulfide induces *E.coli*  MG1655 by the addition of L-cysteine.
4. Stock solution diluted to achieve concentrations 0mM, 0.5mM, 1mM, 1.5mM and 2mM
5. Corresponding add 0μm, 20μm, 40μm, 60μm, 80μm of L-cys
6. Incubated in a shaker at 37℃ and 220 rpm for six hours

"
}, {
"title": "D. Detection of Hydrogen Sulfide Concentration Changes Using Test Strips",
"content": "

**Materials:**

1. L-cysteine
2. Lead acetate test paper
3. Microplate reader

**Procedures:**

The study investigated the production of hydrogen
sulfide following the addition of L-cys at concentrations ranging from 0 to 2 mM.

1. Hydrogen sulfide was induced in Escherichia coli MG1655 by the addition of L-cysteine (L-cys).
2. A stock solution of 100 mM was prepared and subsequently diluted to achieve concentrations of 0 mM, 0.5 mM, 1
   mM, 1.5 mM, and 2 mM, corresponding to the addition of 0 μL, 20 μL, 40 μL, 60 μL, and 80 μL of L-cys,
   respectively.
3. The five test tubes were then incubated in a shaker at 37°C and 220 rpm for six hours.
4. The hydrogen sulfide production was quantified using lead acetate test paper. Following a subsequent 10 minutes
   incubation at 37°C, photographs were taken.
5. Turbidity measurements were performed using a microplate reader, with 300 μL aliquots from each tube yielding
   the following optical density (OD) values corresponding to the 0 mM, 0.5 mM, 1 mM, 1.5 mM, and 2 mM
   concentrations, respectively.
6. SQR and FCSD were then added to the bacterial cultures at concentrations of 0, 1, 2, 5, and 10 mg/mL, followed
   by a one-hour incubation in the shaker.
7. The hydrogen sulfide concentration was subsequently re-evaluated using lead acetate test paper.

"
}, {
"title": "E. Observe the Effects in Different Concentration Ratios to the Bacterial Culture",
"content": "

**Materials:**

1. SQR
2. FCSD
3. Shaking incubator
4. Bacterial solution
5. Test strips

**Procedures:**

1. Test the proportions of SQR and FCSD when mixed with different concentrations; Each concentration 10mg/mL; SQR +
   FCSD ratio: 1:1\\1:2\\1:3\\2:1\\3:1.
2. Continue the culture in the shaking incubator for another six hours.
3. 4 mL of bacterial solution was prepared, and each was added to the bacterial solution(add in a solution).
4. The bacterial solution was cultured in a shaker for two hours.
5. All the concentration ratios were measured using test strips.

"
}]
},{
"title": "Hardware Testing",
"content": "",
"children": [{
"title": "A. Test Whether Hardware Can Detect Changes in Hydrogen Sulfide",
"content": "

**Test Groups:**

1. H2S without L-cys
2. H2S with 1 mM (40 ml) L-cys
3. H2S with 2 mM (80 ml) L-cys

These three tubes are used to test whether the
equipment can accurately measure hydrogen sulfide.

* SQR + L-cys 60ml
* FCSD + L-cys 60ml
* S&F + L-cys 60ml

1. These three tubes are used to test whether our protease can break down hydrogen sulfide.
2. Wait for the culture medium and then conduct the hardware test.
3. Attach the cultured hydrogen sulfide to the instrument for real-time monitoring.

"
}]
},{
"title": "In Vitro Cell Model Establishment",
"content": "",
"children": [{
"title": "A. Cell Line and Culture Conditions",
"content": "

The human colorectal adenocarcinoma cell line NCI-H716 was utilized in this study. Cells were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) at 37°C in a 5% CO₂ atmosphere. As a suspension cell line, NCI-H716 cells were cultured and passaged using standard protocols for non-adherent cells. All seeding, treatment, and transfection procedures were optimized for suspension culture.

"
}, {
"title": "B. Modeling Mechanism",
"content": "

A cell model of impaired glucagon-like peptide-1 (GLP-1) signaling was established by treating NCI-H716 cells with the supernatant from E. coli MG1655 cultures pre-induced with L-cysteine. This induction triggers the bacterial production of hydrogen sulfide (H₂S), which is present in the supernatant. Exposure to H₂S inhibits cellular GLP-1 production, leading to the downregulation of GLP-1 gene expression and a consequent disruption of its mediated metabolic regulatory pathways.

"
}, {
"title": "C. Experimental Method: Model Validation by qPCR",
"content": "

* **Cell Seeding:** NCI-H716 cells were seeded in 12-well plates at a density of 5 × 10⁵ cells per well.
* **Treatment:** When the cell density reached 70-80%, the culture medium was replaced with fresh medium containing the L-cysteine-induced E. coli MG1655 supernatant (hereafter referred to as the 'conditioned supernatant').
* **Incubation:** Cells were co-cultured with the conditioned supernatant for 4 hours. Following this incubation, the treatment medium was aspirated, and the cells were gently washed twice with phosphate-buffered saline (PBS) to remove residual supernatant.
* **Recovery:** Cells were then replenished with fresh complete RPMI 1640 medium and further incubated for an additional 20 hours.
* **RNA Extraction and Analysis:** After the total 24-hour incubation period, total RNA was isolated from the cells. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) was performed to quantify the expression levels of GLP-1 mRNA. Subsequent experiments were conducted only after successful validation of significant GLP-1 mRNA downregulation in the treatment group.
* **Note:** The volume of bacterial supernatant added (e.g., 10%, 20% v/v) and the duration of incubation may be optimized based on initial experimental outcomes to achieve consistent downregulation.

"
}, {
"title": "D. Experimental Groups",
"content": "

* **Blank Control:** Untreated NCI-H716 cells (cultured in standard complete medium).
* **Treatment Group:** NCI-H716 cells treated with the conditioned supernatant from L-cysteine-induced E. coli MG1655.

"
}]
},{
"title": "Analysis of GLP-1 mRNA Expression by RT-qPCR Following Various Treatments",
"content": '',
"children": [{
"title": "1. Cell Seeding and Treatment",
"content": "

NCI-H716 cells were seeded in 12-well plates at a density of 5 × 10⁵ cells per well in complete growth medium (RPMI 1640 supplemented with 10% FBS). Upon reaching 70-80% confluence, the culture medium was replaced with fresh medium containing the respective treatments (detailed in Section 2). The cells were then co-incubated with the treatments for 4 hours. After this incubation period, the treatment media were aspirated, and the cells were gently washed twice with phosphate-buffered saline (PBS) to terminate the exposure. Subsequently, the cells were replenished with fresh complete medium and further incubated for either 20 hours or 44 hours to achieve total post-treatment time points of 24 hours and 48 hours, respectively.

"
}, {
"title": "2. Experimental Groups",
"content": "

The experiment included the following treatment groups, with the volume of bacterial supernatant added being consistent with the optimized conditions established in Investigation 1:

**Blank Control:** Untreated NCI-H716 cells (cultured in standard complete medium).

**Negative Control:** NCI-H716 cells treated with supernatant from the standard E. coli MG1655 culture medium (uninduced with L-cysteine).

**Positive Control:** NCI-H716 cells treated with supernatant from L-cysteine-induced E. coli MG1655 cultures.

**Treatment Group 1 (SQR):** NCI-H716 cells treated with L-cysteine-induced E. coli MG1655 supernatant that had been pre-incubated with SQR protein solution.

**Treatment Group 2 (FCSD):** NCI-H716 cells treated with L-cysteine-induced E. coli MG1655 supernatant that had been pre-incubated with FCSD protein solution.

**Treatment Group 3 (SQR+FCSD):** NCI-H716 cells treated with L-cysteine-induced E. coli MG1655 supernatant that had been pre-incubated with a combined solution of SQR and FCSD proteins.

"
}, {
"title": "3. RNA Extraction and RT-qPCR",
"content": "

After the designated incubation periods (24 h or 48 h total), total RNA was isolated from all cell groups using according to the manufacturer's instructions. RNA concentration and purity were determined spectrophotometrically. Complementary DNA (cDNA) was synthesized from 1 μg of total RNA using a reverse transcription kit. Quantitative PCR (qPCR) was then performed using and SYBR Green master mix with gene-specific primers for human GLP-1 and the housekeeping gene β-actin. The relative expression of GLP-1 mRNA was calculated using the 2–ΔΔCt method, normalized to the housekeeping gene and relative to the blank control group.

"
}]
}]%}
{% block title %}Experiments{% endblock %}
{% block lead %}

![](https://static.igem.wiki/teams/5765/header/experiments.webp)

{% endblock %}

{% extends "layout.html" %}
{% set table\_data = [{
"title": "Overview",
"content": "

Our goal of this research project is obesity
management. The reason for obesity is that excess amount of hydrogen sulfide may lead 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.

![流程图](https://static.igem.wiki/teams/5765/results/1.webp)

**Figure 1. Roadmap of Experimental Techniques**

"
}, {
"title": "Construction of Plasmid",
"content": '

1. **The PCR amplification of pET28a-SQR and pET28a-FCSD**

As shown in the Figure 2, the target genes for SQR
(1350 bp) and FCSD (1293 bp) were successfully amplified. The amplified bands align precisely with the expected
molecular sizes when compared to the DNA ladder, confirming the specificity and efficiency of the amplification
process. These results demonstrate the successful preparation of DNA templates for subsequent cloning experiments.

![](https://static.igem.wiki/teams/5765/results/2.webp)

**Figure 2. Target gene PCR amplification diagram**

2. **Double digestion of pET28a**

Following digestion with the restriction enzymes
XhoI
and BamHI and subsequent PCR amplification at 37 °C for 10 minutes, agarose gel electrophoresis was performed.
Comparison with the DNA ladder on the left reveals two distinct bands for the undigested pET28a plasmid, which
correspond to its supercoiled and open circular conformations. In contrast, the digested pET28a sample appears as a
single band, confirming complete linearization of the plasmid by the restriction enzymes. This result indicates
successful enzymatic cleavage and conversion of the supercoiled and open circular forms into a uniform linear DNA
fragment.

![双酶切载体.png](https://static.igem.wiki/teams/5765/results/3.webp)

**Figure 3. Double digestion with XhoI and BamHI**

3. **Recombinant plasmid construction by homologous recombination**

The purified PCR product and linearized pET28a
backbone were then assembled using homologous recombination. The resulting recombinant plasmid was transformed into
*E. coli* DH5α competent cells. Single colonies were observed on the selection plate, indicating successful
transformation. According to the sequencing results, the SQR and FCSD gene was correctly inserted into the pET28a
vector without mutations, confirming the successful construction of the pET28a -SQR-His and pET28a -FCSD-His
plasmid.

![鉴定组图SQR+fcsd_画板 1](https://static.igem.wiki/teams/5765/results/4.webp)

**Figure 4. Recombinant Plasmid Identification Map**

'
},{
"title": "Protein Expression and Purification",
"content": '

1. **Transform the recombinant plasmid into
   *E.coli* BL21(DE3)**

For protein expression, the validated recombinant
plasmid was transformed into *E. coli* BL21(DE3) competent cells. The transformed culture was then
plated onto LB agar plates supplemented with kanamycin and incubated overnight at 37 °C. As shown in Figure 5A,
well-distributed single colonies were observed on the plates, demonstrating successful transformation.Subsequently,
several single colonies were randomly selected for preliminary identification of the recombinant plasmid via colony
PCR. Agarose gel electrophoresis results (Figure 5B) showed that the amplified DNA band size matched the expected
target fragment, confirming that the constructed recombinant plasmid was successfully transformed into
*E.coli*
BL21(DE3).

![BL21鉴定组图_画板 1](https://static.igem.wiki/teams/5765/results/5.webp)

**Figure 5. Monoclonal Colony Plate Diagram**

2. **Protein Expression and
   Purification**

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 and FCSD 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 6. A single, dense protein band appeared at the expected
molecular weight 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 and FCSD-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.

![SDS-PAGE蛋白胶图](https://static.igem.wiki/teams/5765/results/0.webp)

**Figure 6. SDS-PAGE for Protein Expression Analysis**

'
},{
"title": "Functional Testing",
"content": '

1. **ELISA Test For SQR Enzyme**

The assay principle of the ELISA kit relies on the
specific binding between the antigen and antibody, followed by the use of an enzyme-labeled antibody or antigen to
catalyze a chromogenic reaction of the substrate. The presence and concentration of the target analyte (SQR) are
determined based on the intensity of the color developed.

In the microplate layout, the left column was
designated for the standard curve. The right column wells were loaded with samples including Crude Enzyme,
Flow-through fluid, Washing buffer, and Elution Solution. After incubation with the corresponding ELISA reagents,
varying degrees of color development were observed in the wells, reflecting the amount of target protein present in
each sample.

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.

![ELISA标准曲线_画板 1](https://static.igem.wiki/teams/5765/results/7.webp)

**Figure 7. Assaying the Standard Curve for ELISA**

The wells corresponding to the Crude Enzyme,
Flow-through fluid, and Washing buffer exhibited extremely faint color (almost colorless), indicating that the
content
of the SQR enzyme in these samples was very low and insufficient to effectively catalyze the substrate to produce a
significant amount of yellow product. In contrast, the well containing the Elution Solution displayed a distinct
yellow color, demonstrating a high concentration of SQR enzyme. This sample efficiently catalyzed the chromogenic
reaction, yielding a markedly stronger yellow coloration compared to the other three sample types.

Following the visual observation of color
development
in the microplate wells, the plate was transferred to a microplate reader for quantitative analysis. Absorbance was
measured at 275 nm, and enzyme activity was calculated in units of U/L based on the obtained values:

**Elution Solution:** The enzyme
activity peaked at approximately 300 U/L, significantly exceeding that of all other samples. This result indicates
that the SQR enzyme retained high catalytic activity in this fraction, efficiently generating product per unit time
and corresponding to a strong absorbance signal.

**Crude Enzyme, Flow-through, and Washing
Buffer:** The measured enzyme activity in these samples was nearly 0 U/L, demonstrating that the SQR enzyme
exhibited minimal catalytic activity and was unable to effectively facilitate substrate conversion. These
quantitative
results are consistent with the observed lack of visible color development in the corresponding wells.

![Elisa测试SQR酶含量](https://static.igem.wiki/teams/5765/results/8.webp)

**Figure 8. ELISA assay showing enzyme concentration**

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.

Based on the ELISA results, it can be concluded
that
a higher concentration of the target enzyme leads to increased production of yellow product and correspondingly
deeper
color development. Throughout the experimental procedure, the SQR enzyme was effectively enriched and maintained its
biological activity following the elution step. The ELISA assay confirmed both the high content and strong activity
of
the SQR enzyme in the elution solution through two complementary approaches: qualitative color visualization and
quantitative activity measurement. These results provide critical support for subsequent functional studies and
practical applications of this enzyme.

2. **SQR Activity Assay by DUQ Reduction**

The assay principle is based on the catalytic
activity of the SQR enzyme, which oxidizes sulfide while simultaneously reducing DUQ. The reaction is monitored by
measuring the change in absorbance at 275 nm (ΔOD₂₇₅) using a microplate reader. Enzyme activity is calculated
according to the formula incorporating the molar extinction coefficient of DUQ. The experiment included multiple
control groups-negative control, blank control, and enzyme-inactivated control-with three technical replicates
performed for each sample.

As shown in the results, the enzyme activity in the
experimental group exhibited a continuous increase over time. In contrast, the blank control, negative control, and
enzyme-inactivated groups all showed fluctuations near baseline without a significant upward trend. These findings
demonstrate that the SQR enzyme effectively catalyzes the reaction, and that the assay system robustly detects its
activity. The control results effectively rule out non-specific interference, confirming the reliability of the
experimental outcomes.

![DUQ还原法测试SQR酶活性](https://static.igem.wiki/teams/5765/results/9.webp)

**Figure 9. spectrophotometric determination of SQR enzyme activity based on DUQ reduction**

3. **Lead acetate test strip**
   1. **Construction of *E.coli* MG1655 for H₂S production with L-cys**

   To verify the ability of the enzymes (SQR and
   FCSD)
   to decompose hydrogen sulfide, lead acetate test strips were employed. The principle of this method is that
   sulfur-containing compounds (such as H₂S) generated in the enzyme-catalyzed reaction react with lead acetate,
   forming
   a black precipitate of lead sulfide (PbS). The intensity of the color change on the test strip reflects the
   level of
   enzyme activity.

   As shown in Figure 10A, samples containing 1.5
   mM and
   2.0 mM L-cysteine exhibited a noticeably darker color compared to those with lower concentrations, indicating
   more
   successful construction of *E.coli* MG1655 for enhanced hydrogen sulfide production. Figure 10B presents
   a
   grayscale value analysis of the test strips for semi-quantitative evaluation, demonstrating a clear correlation
   between higher L-cysteine concentration and increased production, consistent with a dose-dependent response.

   ![H2S生成组图_画板 1](https://static.igem.wiki/teams/5765/results/10.webp)

   **Figure 10. Different L-cysteine derivatives induce varying amounts of H₂S production**

   Figure 11 presents a bar chart of OD₆₀₀ values,
   demonstrating that the absorbance reaches a relatively high level at 1.5 mM L-Cys. Correspondingly, the lead
   acetate
   test strip also exhibited the most pronounced color change at this concentration. These results indicate that
   within
   the concentration range of 0–2 mM, the amount of hydrogen sulfide produced increases with L-Cys concentration
   and
   peaks at 1.5 mM. This suggests that L-Cys influences hydrogen sulfide generation in the system, and an
   appropriate
   concentration (1.5 mM under these experimental conditions) can enhance H₂S production, thereby subsequently
   affecting
   enzyme-mediated decomposition processes.

   The optimal concentration of L-cysteine for
   hydrogen
   sulfide (H₂S) production was determined to be 1.5 mM. At this concentration, a significant increase in H₂S
   generation
   was observed, indicating efficient utilization of the supplemented precursor by the enzymatic system. Notably,
   this
   enhancement in H₂S yield was achieved without exerting inhibitory effects on bacterial growth, as confirmed by
   OD₆₀₀
   measurements, which showed robust microbial density comparable to controls. These results suggest that 1.5 mM
   L-cysteine effectively supports the metabolic demand for sulfide synthesis while maintaining cellular viability,
   establishing this concentration as suitable for subsequent applications requiring maximal H₂S production under
   non-toxic conditions.

   ![L-Cys OD600图](https://static.igem.wiki/teams/5765/results/11.webp)

   **Figure 11. OD600 absorbance value of different concentration of H₂S**

   2. **Hydrogen sulfide decomposition ability validation**

   As the concentrations of SQR and FCSD increased
   (from
   0 to 10 mg/mL), the color change of the lead acetate test strips became progressively more pronounced. This
   observation indicates that both enzymes are capable of promoting the decomposition of hydrogen sulfide.
   Furthermore,
   the results suggest a positive correlation between enzyme concentration and the extent of H₂S decomposition
   within
   this concentration range.

   ![SQR+FCSD试纸条_画板 1](https://static.igem.wiki/teams/5765/results/12.webp)

   **Figure 12. Reduction in greyscale of lead acetate test strips with increasing SQR and FCSD
   concentration**

   3. **Effect of different proportion of mixed
      enzymes on inhibiting hydrogen sulfide**

   When SQR and FCSD were mixed in different
   proportions
   (1:1, 1:2, 1:3, 2:1, 3:1), distinct color changes were observed on the lead acetate test strips. These results
   indicate that the ratio of the two enzymes influences the efficiency of hydrogen sulfide decomposition, and an
   appropriately optimized ratio may enhance H₂S degradation capability. Notably, the 1:3 mixture (SQR:FCSD)
   exhibited
   the most pronounced color change, suggesting that this ratio yields the highest decomposition activity. These
   findings
   imply a potential synergistic or regulatory interaction between SQR and FCSD during hydrogen sulfide
   decomposition.

   ![复配比试纸条_画板 1](https://static.igem.wiki/teams/5765/results/13.webp)

   **Figure 13. Effect of different proportion of mixed enzymes on inhibiting hydrogen sulfide**
4. **Hardware Result**

The experiment covered the variation ranges of
humidity (63 - 75) and temperature (35.0 - 38.0). However, regardless of how the temperature and humidity
fluctuated,
the sensor will block out some interfering data. The sensor\'s displayed value is positively correlated with the
hydrogen sulfide concentration (the higher the concentration, the larger the value), and within the experimental
temperature and humidity range, this rule is stable, verifying the reliability of the sensor in detecting the H₂S
concentration gradient.

![不同浓度H2S_画板 1](https://static.igem.wiki/teams/5765/results/14.webp)

**Figure 14. Sensor response positively correlates with H₂S concentration**

Compared to the control groups with "no enzyme" and
those with "enzyme added," the introduction of either SQR or FCSD resulted in a significant decrease in the hydrogen
sulfide sensor readings. This reduction clearly indicates that both enzymes are functionally active in decomposing
hydrogen sulfide present in the system, thereby effectively lowering the ambient concentration of H₂S.

![传感器验证组合图_画板 1](https://static.igem.wiki/teams/5765/results/15.webp)

**Figure 15. Sensor display when SQR or FCSD is added**

The SQR enzyme exhibited a more pronounced effect,
reducing the sensor reading by approximately 129 units (from 1714 to 1585), demonstrating a stronger capacity for
hydrogen sulfide decomposition. In contrast, the FCSD enzyme showed a relatively weaker effect, decreasing the
sensor
value by only about 58 units (from 1714 to 1656), indicating a lower efficiency in H₂S degradation compared to SQR.

'
},{
"title": "GLP-1 Pathway Detection",
"content": '

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.

![qPCR检测建模情况](https://static.igem.wiki/teams/5765/results/16.webp)

**Figure 16. 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 1 qPCR detection of GLP-1 mRNA
expression levels in cells following SQR administration**

| **24h** |  | **Ct** | **SD** | **Average** | **▲Ct** | **2-▲▲Ct** |
| --- | --- | --- | --- | --- | --- | --- |
| Control group | ACT1 | 20.303 | 0.139145008 | 20.22366667 |  |  |
| 20.305 |  |  |
| 20.063 |  |  |
| GLP-1 | 27.401 | 0.078564199 | 27.32066667 | 7.097518921 | 0.007301867 |
| 27.317 | 7.011953354 | 0.007748037 |
| 27.244 | 7.1804142 | 0.006894138 |
| Negative group | ACT1 | 19.94723991 | 0.462218603 | 20.30385177 |  |  |
| 20.82605743 |  |  |
| 20.13825798 |  |  |
| GLP-1 | 27.09345779 | 0.410917483 | 27.52850367 | 7.14621788 | 0.007059502 |
| 27.91005249 | 7.083995056 | 0.007370637 |
| 27.58200073 | 7.443742752 | 0.005743943 |
| SQR-treated group | ACT1 | 21.13188362 | 0.03791144 | 21.12831879 |  |  |
| 21.08875084 |  |  |
| 21.1643219 |  |  |
| GLP-1 | 26.63872528 | 0.042969904 | 26.59593836 | 5.50684166 | 0.021992544 |
| 26.55278778 | 5.464036942 | 0.022654839 |
| 26.59630203 | 5.431980133 | 0.023163866 |
| FCSD-treated group | ACT1 | 21.8233223 | 0.093600657 | 21.92073758 |  |  |
| 21.92890167 |  |  |
| 22.00998878 |  |  |
| GLP-1 | 27.58271027 | 0.260798516 | 27.70004082 | 5.75938797 | 0.01846084 |
| 27.51851654 | 5.589614868 | 0.020766259 |
| 27.99889565 | 5.98890686 | 0.015745607 |
| SQR+FCSD-treated group | ACT1 | 22.19654846 | 0.402187651 | 22.6494058 |  |  |
| 22.7866993 |  |  |
| 22.96496964 |  |  |
| GLP-1 | 26.14676476 | 0.415753946 | 26.58778636 | 3.950216293 | 0.064694358 |
| 26.64405251 | 3.85735321 | 0.068995534 |
| 26.97254181 | 4.007572174 | 0.06217282 |
| Positive group | ACT1 | 15.651 | 0.159104787 | 15.81333333 |  |  |
| 15.82 |  |  |
| 15.969 |  |  |
| GLP-1 | 25.87 | 0.308002164 | 26.22533333 | 10.42091846 | 0.000729442 |
| 26.416 | 10.59577942 | 0.000646179 |
| 26.39 | 10.21927261 | 0.000838866 |

Quantitative real-time PCR analysis revealed that
GLP-1 mRNA expression was significantly upregulated in both the SQR-treated group and the FCSD-treated group compared
to the Negative control group (p < 0.05). This indicates that both SQR and FCSD proteins effectively alleviate
H₂S-mediated suppression of GLP-1 gene expression by degrading extracellular H₂S present in the bacterial supernatant,
leading to the recovery of GLP-1 transcription levels.

Furthermore, the combined administration of SQR and
FCSD resulted in a more pronounced upregulation of GLP-1 expression than either enzyme alone, with the efficacy
following the order: SQR + FCSD > SQR > FCSD. This suggests a synergistic effect between the two enzymes in
enhancing GLP-1 expression, likely due to more efficient and sustained removal of inhibitory H₂S.

![细胞样本处理组_画板 1](https://static.igem.wiki/teams/5765/results/17.webp)

**Figure 17. The map of GLP-1 gene expression level changes under different treatments**

'
},{
"title": "Discussion",
"content": '

The primary objective of this study was to
investigate the potential of using synthetically produced enzymes-sulfide:quinone oxidoreductase (SQR) and FCSD-to
degrade hydrogen sulfide (H₂S) as a therapeutic strategy for obesity-related metabolic dysregulation. Our findings
demonstrate that both enzymes effectively decompose H₂S in a concentration-dependent manner, with SQR exhibiting
significantly higher activity than FCSD. Notably, a mixed ratio of SQR to FCSD at 1:3 resulted in the most pronounced
degradation effect, suggesting synergistic interaction between the two enzymes. Real-time monitoring using a
specialized H₂S sensor confirmed the notable decrease in H₂S concentration following enzymatic treatment, reinforcing
the reliability and quantitative accuracy of our detection system.

Excessive H₂S has been implicated in promoting
oxidative stress and cellular damage, thereby suppressing GLP-1 secretion-a key regulator of satiety and lipid
metabolism. In this study, we achieved high-yield expression and purification of both SQR and FCSD in *E.coli*BL21(DE3), and validated their functional efficacy *in vitro*. The observation that 1.5 mM L-cysteine
optimally supported H₂S production without inhibiting bacterial growth highlights a suitable concentration range for
future *in vivo* applications or synthetic consortia design. Furthermore, the enhanced degradation efficiency
observed with the SQR-FCSD mixture suggests the potential for engineering multi-enzyme complexes for improved H₂S
scavenging.

Despite promising *in vitro* results, several
challenges must be addressed before clinical translation. The stability, immunogenicity, and retention of enzymatic
activity within the gastrointestinal environment remain to be thoroughly evaluated. Additionally, potential
interference from other sulfur compounds or volatile metabolites in real biological samples warrants further
investigation. Future work should focus on *in vivo* validation using animal models of obesity, as well as the
development of efficient enzyme delivery systems to enhance localized H₂S degradation and functional persistence.

In conclusion, this study establishes a robust
pipeline for the molecular cloning, expression, purification, and functional characterization of H₂S-degrading
enzymes. We provide compelling evidence that SQR and FCSD act synergistically to reduce H₂S levels, supporting their
potential application as enzyme-based therapeutics for metabolic disorders associated with H₂S accumulation.

'
}]%}
{% block title %}Results{% endblock %}
{% block lead %}

![](https://static.igem.wiki/teams/5765/header/results.webp)

{% endblock %}