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

✅

✅

✅

✅

✅

✅

✅
Date: February, 2025

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：

    2. 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%)

    3. - 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

    4. -Load sample onto 1% agarose gel.

    -Run electrophoresis (DNA → anode).

    -Image gel.

  4. Gel Extraction of DNA Fragments

    5. 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

    6. - 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

    7. 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).

Date: March, 2025

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：

    2. 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：

    3. -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)：

    4. - 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.

Date: April 2025

A. Goal

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

    2. 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

    3. 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

    4. 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.)

Date: June, 2025

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.

Date: July, 2025

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.

Date: August, 2025

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

Date: September, 2025

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.