The project schedule is organized in monthly units, where each month is standardized to a duration of 30 days for consistency. Within this framework, the timeline is divided into smaller increments, with each individual grid cell representing a span of three consecutive days. This breakdown allows the timeline to be viewed at a fine enough scale for tracking progress.

Record of Main Experimental Content

Construction of recombination plasmids

pET-28a(+)-AsCDA/Cts2p/FjChiB

Ⅰ. Obtain target gene fragments by PCR

Goal: PCR technology is used to amplify the target gene fragment to provide DNA fragments for subsequent infusion recombination.

Materials:

1) 2x PrimeStarMix
2) Gene template
3) Double distilled water (ddH2O)
4) Primer-F
5) Primer-R
6) PCR tube
7) PCR thermal cycler

PrimeStarMix includes buffer and dNTPs for PCR. The extention rate for PrimeStarMix (2x) is 1000bp/10 s.

Procedures:

1) Aseptically prepare the PCR reaction system. The recommended total reaction volume is 50 µL: add 25 µL of 2×PrimeStar Mix, 1 µL of template DNA, 1 µL of Primer-F, and 1 µL of Primer-R. Add ddH2O to the remaining volume to 50 µL. After preparing the reaction solution, briefly centrifuge to remove any bubbles.

2) Place the reaction tube in a PCR instrument and set the reaction program:

3) Initial denaturation: 95℃ for 3 minutes to fully unwind the double-stranded structure of the template DNA;

4) Cycling: Perform 35 cycles, each cycle consisting of:

5) Denaturation at 95℃ for 30 seconds

6) Annealing at 55℃ for 30 seconds (the specific temperature can be adjusted based on primer design);

7) Extension at 72℃ for 2 minutes (the PrimeStar Mix has an extension rate of approximately 1000 bp/10 seconds; the time can be adjusted based on the target fragment size);

8) End extension: 72℃ for 1 minute to ensure complete extension of all fragments.

9) After the reaction, if not used immediately, the product can be stored at 4℃ for a short period of time.

10) To obtain purified DNA fragments, separate the PCR products by electrophoresis on a 1%–1.2% agarose gel. Cut out the target band and use a DNA recovery kit to extract the fragment for subsequent infusion recombination.

Protein expression and purification

3.1 Induction of protein expression for 6xHis-AsCDA/Cts2p/FjChiB

Ⅰ. Amplification culture for correct colony

Goal: The pET-28a(+)-AsCDA/Cts2p/FjChiB recombinant protein was efficiently expressed in Escherichia coli BL21, and bacterial samples were collected at different times and temperatures for SDS-PAGE analysis of protein expression.

Materials:

• LB liquid medium
• E. coli BL21 transformed with pET-28a(+)-AsCDA/Cts2p/FjChiB
• Kanamycin solution
• Isopropyl β-D-thiogalactopyranoside (IPTG) inducer
• Iso-thermic shaker
• Spectrophotometer (for OD600 measurement)
• 1.5 mL centrifuge tubes and sterile pipette tips

Procedures:

1) Inoculation and Preculture

2) Add 100 µL of the recombinant bacterial culture to 100 mL of LB liquid medium. Also add an appropriate amount of kanamycin (e.g., 100 µg/mL) to ensure selective pressure.

3) Incubate the culture flask at 37℃ on a constant-temperature shaker, shaking vigorously to ensure aeration.

4) Growth Monitoring

5) Measure the OD600 value of the culture every 30 minutes until the culture reaches the logarithmic growth phase (OD600 ≈ 0.4–0.6), which usually takes approximately 2–3 hours.

6) At this stage, the bacteria are actively growing and suitable for IPTG induction.

7) Protein Induction

8) Add IPTG to the culture to a final concentration of 1 mM and gently mix to initiate recombinant protein expression.

9) Parallel cultures at different temperatures (e.g., 25℃ and 37℃) can be set up to compare expression efficiency and solubility.

4. Sampling and Monitoring

10) At 0.5 h, 1 h, 3 h, and 6 h after induction, collect equal volumes of bacterial culture samples (e.g., 1–2 mL) and place them in centrifuge tubes.

11) Samples can be frozen immediately or added to lysis buffer for subsequent SDS-PAGE analysis of protein expression.

Notes:

• IPTG solution should be prepared fresh or stored frozen in aliquots to avoid repeated freeze-thaw cycles.
• The induction temperature affects protein solubility and folding. Low temperatures (25℃) generally favor soluble protein expression.
• When sampling, perform the procedure as quickly as possible to prevent continued bacterial growth or protein degradation.

Ⅱ: Protein crude extraction and SDS-PAGE

Goal: Crude protein was extracted from the induced expression E. coli BL21 culture medium, and the expression and size of the target protein were analyzed by

SDS-PAGE.

Materials for protein crude extraction:

• Induced bacterial suspension (approximately 500 µL per induction condition)
• Centrifuge
• Double-distilled water (ddH2O)
• Sangon 12.5% SDS-PAGE Preparation Kit
• Electrophoresis Buffer (Tris-Glycine)
• Protein Molecular Weight Standard
• 6×Protein Loading Buffer
• Vertical Electrophoresis System
• Coomassie Brilliant Blue Staining Solution
• Destaining Solution

Procedures for protein crude extraction and SDS-PAGE:

1. Crude Protein Extraction

• Transfer 500 µL of bacterial suspension from each induction condition to a centrifuge tube and centrifuge at 12,000 rpm for 1 minute.
• Discard the supernatant and resuspend the bacterial pellet in 50 µL of 1×protein loading buffer (6×loading buffer diluted with ddH2O).
• Heat the resuspension in a PCR thermocycler at 95℃ for 15 minutes to lyse the cells and denature the proteins.
• After completion, cool briefly or proceed immediately to SDS-PAGE analysis.

2. SDS-PAGE Gel Preparation

• Combine 2.2 mL of 2×separating gel solution, 2.2 mL of 2×separating gel buffer, and 44 µL of catalyst in a centrifuge tube. Mix gently.
• Slowly pour the separating gel into the gel mold, avoiding the formation of bubbles.
• Add 1 mL of ddH2O to the top of the separating gel and let it stand for approximately 8 minutes to allow the gel to fully polymerize.
• After the gel solidifies, discard the top layer of water.
• Combine 825 µL of 2×stacking gel solution, 825 µL of 2×stacking gel buffer, and 11 µL of catalyst. Mix gently.
• Pour the stacking gel into the mold until full and slowly insert the comb to avoid creating bubbles.
• Let it stand for approximately 12 minutes to solidify, then carefully remove the comb and rinse the wells with running buffer.

3. Sample Loading and Electrophoresis

• Add 10 µL of protein molecular weight standard to the first well, followed by an appropriate amount of protein sample to each well.
• Place the gel in a vertical electrophoresis tank and add running buffer.
• Set the electrophoresis condition to 120 V and run for approximately 90 minutes until the protein bands are clearly separated.

4. Staining and Destaining

• After electrophoresis, stain the gel in Coomassie Brilliant Blue solution for approximately 10 minutes.
• Rinse the gel repeatedly in destaining solution until the background becomes transparent and protein bands are clearly visible.

Notes:

• Be gentle when preparing the separating and stacking gels to avoid creating bubbles, which can affect protein band resolution.
• Load a moderate amount of sample to avoid excessive sample loading, which can cause band smearing.
• Adjust the staining and destaining times based on band appearance to achieve optimal contrast.

Optimal free enzyme activity reaction conditions

Goal:

Determine the optimal reaction conditions for three chitin-related enzymes, AsCDA, Cts2p, and FjChiB, including optimal pH, temperature, pH stability, temperature stability, and the effects of metal ions on enzyme activity, to provide experimental basis for subsequent enzymatic applications and optimization.

Materials:

• Purified AsCDA, Cts2p, and FjChiB enzyme samples (0.5 mg)
• Colloidal chitin solution (1% w/v)
• Buffer system:
• 0.1 M glycine-HCl (pH 2.0–3.0)
• 0.1 M citric acid-Na2HPO4 (pH 3.0–8.0)
• 0.1 M Tris-HCl (pH 8.0–9.0)
• 0.1 M glycine-NaOH (pH 9.0–11.0)
• 50 mM Na2PO4-citric acid buffer (pH 4.0–6.0)
• 50 mM Na2PO4 buffer (pH 6.0–8.0)
• 50 mM Tris-HCl buffer (pH 8.0–9.0)
• 50 mM Glycine-NaOH buffer (pH 9.0–11.0)
• Metal ion solution (final concentration 10 mM): Mg²⁺, Co²⁺, Fe³⁺, Ni²⁺, Cu²⁺, Na⁺, K⁺, Mn²⁺, Zn²⁺
• Spectrophotometer (OD540, for DNS assay of reducing sugars)
• Centrifuge tube (1.5 mL)
• Water bath

Procedures:

a. Determination of Optimal pH

1. Centrifuge 1% colloidal chitin, discard the supernatant, and resuspend in an equal volume of buffer covering the pH range of 2.0–11.0 (glycine-HCl, citric acid-Na2HPO4, Tris-HCl, glycine-NaOH).
2. Add each enzyme to the colloidal chitin system in different pH buffers and incubate at 37℃ for 1 hour.
3. After the reaction, determine the reducing sugar yield using the DNS method. The highest enzyme activity was set as 100%. A relative enzyme activity versus pH curve was plotted to determine the optimal pH.

b. Optimal Temperature Determination

1. Dissolve the enzyme in the optimal pH buffer.
2. Set a temperature gradient (20, 30, 40, 50, 60, 70, and 80℃) and add colloidal chitin for a 1-hour reaction.
3. Determine reducing sugar production by DNS. Plot a curve of relative enzyme activity versus temperature to determine the optimal reaction temperature.

c. Effect of Metal Ions on Enzyme Activity

1. At the optimal pH, add a single metal ion (final concentration of 10 mM, Mg²⁺, Co²⁺, Fe³⁺, Ni²⁺, Cu²⁺, Na⁺, K⁺, Mn²⁺, or Zn²⁺) to the enzyme solution.
2. Incubate at 37℃ for 1 hour, measure reducing sugar production, calculate relative enzyme activity, and analyze the promoting or inhibiting effect of each metal ion on enzyme activity.

Notes

• When conducting pH or temperature gradient experiments, perform at least three replicates per group.
• When determining reducing sugars using the DNS method, ensure that the sample temperature is consistent to prevent non-enzymatic factors from influencing the results.
• For metal ion experiments, it is recommended to include a blank control (without metal ions) to correct for background.
• The optimal pH and temperature for each enzyme are determined based on the highest relative enzyme activity and are then used for optimization in subsequent experiments.