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

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

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.

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.

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

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.

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

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.

Materials:

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

Procedures:

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.

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.

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

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.

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.

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.

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.

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

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

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.

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.

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.

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.

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.

• 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.

• 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.

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.

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.

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.