1. Set up 5 assay tubes and 1 control tube:

   Tube Number	Sample Name	Volume to Add
   1	Wild-type AChE (positive control)	100 μL
   2	Mutant 1	100 μL
   3	Mutant 2	100 μL
   4	Mutant 3	100 μL
   5	Mutant 4	100 μL
   6	Mutant 5	100 μL
   6	Water (negative control)	100 μL

2. Pre-incubation:
   Add 100 μL of Reagent II to tubes 1-6 (wild-type and mutants). Tube 7 (water) receives no Reagent II at this step. Incubate all tubes at 37°C for 5 minutes.
3. Reaction initiation:
   Add 100 μL of Reagent IV to tubes 1-6.
   For the negative control (tube 7), add 100 μL of Reagent II and 100 μL of Reagent IV.
   Mix all tubes thoroughly.
4. Centrifugation:
   Centrifuge all tubes at 12,000 rpm for 5 minutes.
5. Supernatant transfer:
   Transfer 50 μL of the supernatant from each tube to a new corresponding tube.
6. Color development:
   Add 100 μL of Reagent III to each new tube.
7. Add Reagent I as follows:
   Tubes 2-7: 780 μL
   Wild-type positive control tube (tube 1): 850 μL
   Mix thoroughly and allow to stand at room temperature for 2 minutes. Observe color development in assay tubes. The water control should show minimal color change.
8. Measurement:
   Using the water tube (negative control) as the blank, measure absorbance of each tube at 412 nm using a spectrophotometer. Record the results for comparison of enzymatic activity.

1. Prepare three conical flasks, each containing 50 mL of LB liquid medium.
2. Following the table below, add different volumes of ampicillin-resistant E. coli strain and ampicillin solution to each flask.

   Group	Medium (mL)	Ampicillin-resistant E. coli culture (μL)	Ampicillin (μL)
   1	50	5	5
   2	50	50	5
   3	50	50	50

3. Tighten the bottle caps or seal the openings with sterile sealing film, then shake each bottle vigorously to thoroughly mix the bacterial culture and antibiotics.
4. Place the three bottles in a 37°C constant-temperature shaking incubator, set to 260 rpm.
5. Allow the bacterial cultures to incubate continuously in the shaking incubator for approximately 19 hours (overnight).
6. Observe and record the growth of the bacterial cultures in all three bottles.

1. Reagent Preparation
   Thaw all components of the cell-free protein expression kit stored at -80°C completely on ice. After thawing, briefly centrifuge and collect the supernatant to the bottom of the tube.
2. Reaction System Preparation (Perform on ice)
   Prepare a 50 μL reaction system in a PCR tube according to the following proportions:

   Component	Sample Reaction (×48)	Negative Control (×1)	Positive Control (×1)
   Reaction Mix	17.2 μL	17.2 μL	17.2 μL
   Template DNA*	10 μL	-	10 μL
   E. coli Cell Extract	7.4 μL	7.4 μL	7.4 μL
   Nuclease-free Water	to 50 μL	to 50 μL	to 50 μL

3. Reaction Incubation
   Place the prepared reaction tubes in a PCR machine or constant-temperature metal bath and incubate at 30°C for 2–3 hours.
4. Product Analysis
   After the reaction, take 2-5 μL of the reaction mixture for SDS-PAGE electrophoresis analysis or use it for other downstream experiments.

1. Add 30 milliliters of E. coli-specific cell lysis buffer per gram of bacterial cells (adjustable ratio range: 1:10 to 1:50).
2. Thoroughly disperse and mix the bacterial cells in the lysis buffer.
3. Freeze the mixture in a freezer at -20°C or below until completely solidified.
4. When ready for lysis, remove the sample and allow it to thaw naturally.
5. Perform high-speed cryo-centrifugation on the thawed sample.
6. After centrifugation, collect the supernatant (lysis supernatant) and the pellet (cell pellet) separately.

1. Under UV light, quickly cut out the gel block containing the target DNA band.
2. Place the gel block into a centrifuge tube, weigh it, and record the weight.
3. Add 300 μL of binding buffer per 100 mg of gel.
4. Incubate the centrifuge tube in a 55°C water bath until the gel is completely dissolved.
5. Transfer the entire dissolved solution to the purification column and let it stand at room temperature for 5 minutes.
6. Centrifuge at 12,000 rpm for 1 minute, then discard the supernatant.
7. Add 700 μL of wash buffer to the purification column.
8. Centrifuge at 12,000 rpm for 1 minute and discard the supernatant.
9. Repeat steps 7 and 8 for one additional wash.
10. Centrifuge the empty column at 12,000 rpm for 2 minutes to remove residual ethanol.
11. Transfer the purification column to a new centrifuge tube and add 30-50 μL of elution buffer.
12. Incubate at room temperature for 5 minutes, then centrifuge at 12,000 rpm for 1 minute to collect the DNA solution.
13. Perform agarose gel electrophoresis analysis using 2 μL of the product.
14. Store the remaining DNA solution at -20°C.

1. Initialization/Blank Calibration: Wipe the detection platform clean with lint-free paper.
2. Use buffer solution as blank control: Pipette 1 μL onto the detection platform and click “Blank.”
3. After blank calibration, clean the detection platform.
4. Pipette 1 microliter of nucleic acid into the detection head for measurement.
5. After approximately several seconds, the instrument displays:
   - Nucleic acid concentration (ng/μl)
   - A260/A280: Protein contamination in nucleic acid
   - A260/A230: Ion contamination in nucleic acid

1. Prepare Digestion Reaction Mixture

   Component	Volume
   Plasmid DNA	90 μL
   10× Restriction Buffer	10 μL
   Restriction Enzyme (XhoI/XhoII)	1 μL
   Total Volume	101 μL

   Mix gently by pipetting and briefly centrifuge to collect liquid.
2. Enzyme Digestion Reaction
   Incubate the reaction tubes in a 37°C water bath for 1 hour.
3. Agarose Gel Electrophoresis
   Prepare agarose gel at an appropriate concentration (e.g., 1%).
   Add an appropriate volume of DNA loading buffer to the digested samples and mix thoroughly.
   Load samples and DNA molecular weight markers into separate lanes.
   Run electrophoresis at an appropriate voltage until bands are fully separated.
4. Gel Imaging and Gel Cutting
   Observe electrophoresis results under a gel imaging system.
   Identify target bands: Linearized plasmid bands migrate faster (lower position) than supercoiled plasmids.
   Precisely cut out the gel fragment containing the target band using a clean blade, removing excess gel as much as possible.
5. Gel Recovery and Purification
   Weigh the excised gel and add Binding Buffer proportionally. Incubate in a 55-65°C water bath, mixing intermittently until the gel is completely dissolved.
   Transfer the lysate to the collection column provided in the kit and let it stand at room temperature for 2 minutes.
   Centrifuge at 12,000 rpm for 1 minute and discard the supernatant.
   Add 700 μL Wash Buffer to the column, centrifuge at 12,000 rpm for 1 minute, and discard the supernatant.
   Repeat the washing step once. Centrifuge the empty column for 2 minutes to completely remove ethanol.
   Place the column in a clean centrifuge tube, add 30-50 μL Elution Buffer, and let stand for 2 minutes.
   Centrifuge at 12,000 rpm for 1 minute. Collect the eluate, which is purified linearized plasmid DNA.
6. Quality Control
   Determine the concentration and purity of the purified product using a nucleic acid concentration meter (record the A260/A280 ratio).
   Perform agarose gel electrophoresis verification with a small amount of product. Compare with uncut plasmid control to confirm band size and purity.

1. Prepare a sterile 1M IPTG stock solution and aliquot it for storage at -20°C.
2. When the OD₆₀₀ value of the E. coli culture reaches 0.5–0.6, prepare for induction.
3. Add the inducer at a ratio of 50 μL of 1M IPTG per 100 mL of culture.
4. Gently shake the culture flask to ensure uniform dispersion of IPTG throughout the bacterial suspension.
5. Return the flask to the shaking incubator and incubate overnight at 16–18°C (16–24 hours).
6. Following induction, harvest the bacterial cells for subsequent protein analysis or purification experiments.

1. Prepare several sterile shaking tubes and add 25 mL of LB liquid medium to each tube.
2. Based on the type of resistance, add 25 μL of the corresponding antibiotic to each tube (e.g., ampicillin to a final concentration of 100 μg/mL).
3. Using a sterile pipette tip, pick a morphologically regular, well-defined, and uniformly sized single colony from the transformation plate.
4. Directly transfer the tip bearing the colony into the shaking tube prepared in Step 1 containing medium and antibiotic.
5. Tightly cap the shaking tube and incubate it in a 37°C constant-temperature shaking incubator at 220 rpm for 12 to 16 hours (overnight).

1. Materials
   • Template DNA (e.g., plasmid)
   • Specific Forward & Reverse Primers
   • 2× Taq Master Mix (containing Taq DNA Polymerase, dNTPs, Mg²⁺, Reaction Buffer)
   • Sterile Nuclease-free Water
2. Reaction Setup

   Component	Volume	Final Concentration/Amount
   2× Taq Master Mix	50 μL	1×
   Forward Primer (10 μM)	2 μL	0.2 μM
   Reverse Primer (10 μM)	2 μL	0.2 μM
   Template DNA	1.5 μL	Variable (e.g., 10-100 ng)
   Nuclease-free Water	44.5 μL	-
   Total Volume	100 μL	-

3. Steps
   1. Prepare the PCR reaction mixture in an ice bath.
   2. Add the components in the following order to the sterile 0.2 mL PCR tube.
   3. Carefully move the liquid up and down to mix it evenly, avoiding the formation of bubbles.
   4. The brief centrifugation causes the liquid surface to concentrate at the bottom of the tube.
   5. Place the PCR tube in the thermal cycling instrument.
   6. Set the program for the thermal cycling instrument as follows:

   Step	Temperature	Time	Cycles
   Initial Denaturation	95°C	2 minutes	1
   Denaturation	95°C	30 seconds	30
   Annealing	65°C	40 seconds	30
   Extension	72°C	30 seconds	30
   Final Extension	72°C	1 minute	1
   Hold	4°C	∞	1

   7. Start the operation program of the thermal cycling instrument.
   8. After the program is completed, remove the test tube from the thermal cycling instrument.
   The PCR products are now ready for analysis or can be stored at -20°C.

1. Transfer 1.6 mL of overnight bacterial culture to a centrifuge tube. Centrifuge at 12,000 rpm for 1 minute, then aspirate the supernatant. If the bacterial volume is large, perform multiple centrifugations to collect the bacterial pellet into four centrifuge tubes.
2. Add 500 μl of Solution P1 to the centrifuge tube containing the bacterial pellet. Thoroughly resuspend the bacterial cells using a pipette or vortex mixer.
3. Add 500 μl of Solution P2 to the centrifuge tube. Gently invert the tube 6-8 times to ensure complete lysis of the bacterial cells.
4. Add 500 μl of Solution P4 to the tube. Immediately invert gently 6–8 times to mix thoroughly. A white flocculent precipitate will form. Incubate at room temperature for approximately 10 minutes, then centrifuge at 12,000 rpm for 10 minutes. A precipitate will form at the bottom of the tube.
5. Add the supernatant collected in the previous step in portions to the filtration column CS (with the column placed in the collection tube). Centrifuge at 12,000 rpm for 2 min. Collect the filtrate in a clean 2-mL centrifuge tube.
6. Add 600 μl of wash buffer PW to the CP4 adsorption column. Centrifuge at 12,000 rpm for 1 minute. Discard the waste liquid from the collection tube and place the CP4 adsorption column back into the collection tube.
7. Add 600 μl of wash buffer PW to the CP4 adsorption column. Centrifuge at 12,000 rpm for 1 minute. Discard the waste liquid from the collection tube.
8. Return the CP4 adsorption column to the collection tube and centrifuge at 12,000 rpm for 2 min.
9. Transfer the CP4 adsorption column to a clean centrifuge tube. Gently dispense 100–300 μl of elution buffer TB onto the center of the adsorption membrane. Incubate at room temperature for 2 minutes, then centrifuge at 12,000 rpm for 1 minute to collect the plasmid solution into the centrifuge tube.

1. Adsorption Column Pretreatment
   Add 500 μl of equilibration buffer BL to adsorption column CP4 (placed in the collection tube). Centrifuge at 12,000 rpm for 1 minute. Discard the supernatant and return the column to the collection tube. (Use columns processed on the same day.)
2. Cell Harvesting
   Take 5–15 ml of overnight bacterial culture, centrifuge at 12,000 rpm for 1 minute, and remove supernatant as thoroughly as possible.
   Note: For larger volumes, centrifuge in batches to collect cells. The cell volume should be sufficient for complete lysis.
3. Cell Resuspension
   Add 500 μl of Solution P1 to the cell pellet. Thoroughly resuspend the pellet using a pipette or vortex mixer.
   Note: Complete resuspension is essential. Unmixed cell clumps will impair lysis efficiency.
4. Alkaline Lysis
   Add 500 μl of Solution P2. Gently invert the tube 6-8 times to thoroughly lyse the cells.
   Note: Mix gently; avoid vigorous shaking. The solution should become clear and viscous. Do not exceed 5 minutes for this step.
5. Neutralization Reaction
   Add 500 μl of Solution P4. Immediately invert the tube gently 6-8 times to mix. Incubate at room temperature for 10 minutes, then centrifuge at 12,000 rpm for 10 minutes. Collect the supernatant.
   Note: Mix immediately after adding P4 to prevent localized precipitation. If precipitation remains in the supernatant, centrifuge again.
6. Filtration Purification
   Add the supernatant in portions to the filtration column CS. Centrifuge at 12,000 rpm for 2 minutes and collect the filtrate into a clean centrifuge tube.
7. Isopropanol Precipitation
   Add 0.3x volume of isopropanol to the filtrate. Mix thoroughly and transfer in aliquots to the CP4 adsorption column.
   Note: Excess isopropanol may cause RNA contamination. Do not exceed 700 μl per column loading.
8. Centrifugal Adsorption
   Centrifuge at 12,000 rpm for 1 minute. Discard the supernatant. Place the adsorption column into the recovery tube.
9. Protein Removal
   Add 500 μl protein removal solution PD. Centrifuge at 12,000 rpm for 1 minute. Discard the supernatant.
10. First Wash
    Add 600 μl wash buffer PW (containing ethanol). Centrifuge at 12,000 rpm for 1 minute. Discard the supernatant.
    Note: Allowing PW to stand at room temperature for 2-5 minutes enhances impurity removal.
11. Second Wash
    Add 600 μl wash buffer PW. Centrifuge at 12,000 rpm for 1 minute. Discard supernatant.
12. Residual Ethanol Removal
    Centrifuge empty column at 12,000 rpm for 2 minutes. Open cap and allow to air dry completely at room temperature for several minutes.
    Note: Residual ethanol may interfere with downstream enzymatic reactions.
13. Plasmid Elution
    Place the CP4 adsorption column into a new centrifuge tube. Add 100-300 μl of TB elution buffer by pipetting directly onto the center of the adsorption membrane. Incubate at room temperature for 2 minutes. Centrifuge at 12,000 rpm for 1 minute to collect the plasmid solution.

1. Weigh the quantities of NaCl, Tryptone, and Yeast Extract listed in the table above and place them in a 250 mL conical flask or beaker.
2. Add approximately 90 mL of deionized water and stir thoroughly until dissolved.
3. Monitoring with a pH meter, titrate with small amounts of 1 M NaOH or 1 M HCl solution to precisely adjust the pH to 7.0.
4. Adjust the volume of the solution to 100 mL and mix thoroughly.
5. Seal the container with aluminum foil or a lid and subject it to high-pressure steam sterilization (121°C, 20 minutes). Allow to cool to room temperature after sterilization before use.

1. Calculate quantities: Common formulas: n = c × V ; n = m / M
2. Accurately weigh components using a balance: Fold weighing paper four times, tare the balance, transfer to beaker after weighing, and add calculated amount of Tris.
3. Mix thoroughly with a magnetic stirrer until solute dissolves.
4. Adjust pH: Insert pH electrode into solution (keep electrode submerged but not touching bottom). Read pH after value stabilizes. Each time a small amount of acid or alkali is added, the addition should be stopped until the value stabilizes before proceeding with further additions.
   • If pH is low, slowly add NaOH dropwise.
   • If pH is high, slowly add HCl dropwise.
   • Typically adjust to 7.5 as needed for specific experiment requirements.
5. Dilution to Volume: Transfer solution to a graduated cylinder and add water to reach the required volume.
6. Filtration
7. Record composition, concentration, and date.

1. Carefully remove the gel from the glass plate after electrophoresis is complete.
2. Place the gel into a clean container.
3. Pour sufficient Coomassie Brilliant Blue staining solution into the container, ensuring the liquid completely covers the gel.
4. Place the container in the microwave and heat on high for 2 minutes.
5. Remove the container and place it on a shaker at room temperature for slow agitation during staining.
6. Drain the staining solution from the container.
7. Add sufficient volume of Coomassie Brilliant Blue destaining solution to the container, ensuring complete coverage of the gel.
8. Place the container on a shaker and destain at room temperature for 30 minutes.
9. Observe the results: protein bands should appear blue, with the gel background appearing nearly colorless.

1. Materials and Reagents:
   • 1.5 M Tris-HCl (pH 8.8) (for separation gel)
   • 1.0 M Tris-HCl (pH 6.8) (for concentration gel)
   • 30% Acr/Bis solution (29:1)
   • TEMED (crosslinking agent)
   • 10% APS (ammonium persulfate, prepare fresh)
   • 10% SDS (10% SDS solution)
   • Double-distilled water (ddH₂O)
   • Gel casting molds, combs, pipettes, beakers, etc.
2. Preparation of Separating Gel (10%) (Suitable for one mini gel, approximately 5 ml)

   Component	Volume (ml)	Final Concentration
   ddH₂O	1.9	-
   30% Acr/Bis (29:1)	1.7	10% Acr
   1.5 M Tris (pH 8.8)	1.3	375 mM Tris
   10% SDS	0.05	0.1% SDS
   Subtotal	4.95
   10% APS	0.05	0.1% APS
   TEMED	0.002	~0.04%

   Procedure:
   1. Add components in the order listed above (add TEMED and APS last), gently mixing to avoid creating bubbles.
   2. Swiftly pour the mixture into the gel casting mold, filling approximately two-thirds of the mold's height.
   3. Gently add a layer of isopropyl alcohol or anhydrous ethanol to the surface of the separating gel to level it. Allow it to stand for approximately 15–20 minutes until it solidifies.
   4. After solidification, pour off the isopropyl alcohol and use filter paper to absorb any residual liquid.
3. Preparation of Concentrated Gel (5%) (Suitable for one mini gel, approximately 2 ml)

   Component	Volume (ml)	Final Concentration
   ddH₂O	1.4	-
   30% Acr/Bis (29:1)	0.33	5% Acr
   1.0 M Tris (pH 6.8)	0.25	125 mM Tris
   10% SDS	0.02	0.1% SDS
   Subtotal	2.00
   10% APS	0.02	0.1% APS
   TEMED	0.002	~0.1%

   Procedure:
   1. Add components in sequence (add TEMED and APS last), gently mixing each addition.
   2. Pour the concentrated gel onto the solidified separating gel until level.
   3. Immediately insert the comb to prevent bubble formation.
   4. Allow to stand for approximately 10-15 minutes until the concentrated gel is fully solidified.

1. Immerse the coating rod in a 25% ethanol solution for 1 hour. Sterilize by flaming over an alcohol lamp before use. Allow to cool and set aside.
2. Take one tube of DH5α competent cells and slowly thaw at room temperature.
3. Add 1 μL of target gene plasmid DNA to the competent cells. Gently tap the tube wall to mix.
4. Let the mixture stand for 30 minutes.
5. Place the mixture in a 42°C water bath for 90 seconds of heat shock.
6. Immediately remove the tube and let it stand at room temperature for 2 minutes.
7. Add 1 mL of LB liquid medium (without antibiotics) to the tube.
8. Gently mix by pipetting up and down, then incubate the tube at 37°C for 1 hour.
9. Centrifuge the tube (4,000 rpm, 1 minute). Discard most of the supernatant, retaining approximately 100 μL of liquid to resuspend the bacterial cells.
10. Spread the entire bacterial suspension onto an antibiotic-containing LB agar plate using a spreader.
11. After the bacterial suspension is absorbed, invert the plate and incubate at 37°C for 12-16 hours.

The UV-vis absorption spectrum of samples was recorded using a UV1901PC spectrophotometer (Shanghai AOXi Scientific Instrument Co., Ltd., China). Measurements were performed at room temperature over a wavelength range of 190–1100 nm using quartz cuvettes, with deionized water as the reference.

1. Sample Preparation and Gel Electrophoresis: Proteins are solubilized and loaded onto a polyacrylamide gel to separate them by size.
2. Protein Transfer: The separated proteins are transferred from the gel to PVDF membrane.
3. Blocking: The membrane is blocked with 5% BSA to prevent non-specific binding of antibodies.
4. Primary Antibody Incubation: The membrane is incubated with a primary anti-His antibody(1:1000), which binds to the His-tag on AChE protein.
5. Secondary Antibody Incubation: Goat Anti-Mouse IgG-HRP (1:5000), conjugated to horseradish peroxidase, is added to bind to the primary antibody.
6. Detection: ECL is added to the membrane(1-2mins), which reacts with the enzyme to produce a detectable signal.

1. Reaction System Setup
   Prepare two sets of PCR reactions (Set A using Z1 high-fidelity DNA polymerase, Set B using Z2 ultra-high-fidelity DNA polymerase). Each set includes two parallel reactions (Reaction 1 and Reaction 2):

   Component	Reaction 1 (500 μL)	Reaction 2 (500 μL)
   PCR1 Reaction Mix (without polymerase)	475 μL	-
   PCR2 Reaction Mix (without polymerase)	-	475 μL
   Z1/Z2 DNA Polymerase	20 μL	20 μL
   DNA Template (>30 ng/μL)	5 μL	5 μL
   Nuclease-free Water	x μL (to 500 μL)	x μL (to 500 μL)

2. Thermal Cycling Conditions

   Step	Temperature	Time	Cycles
   Initial Denaturation	98°C	3 minutes	1
   Denaturation	98°C	20 seconds	30-33
   Annealing	52°C	15 seconds	30-33
   Extension	72°C	25 sec/kb (Z2) or 60 sec/kb (Z1)	30-33
   Final Extension	72°C	3 minutes	1
   Hold	4-10°C	∞	1