iGEM 2025 — Team bnu-china

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Notebook

Scroll down to see our Wet Lab Notebook.

Notebook
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Notebook

Browse our Wet Lab notebooks to see the full record of experiments of our project. Each PDF includes detailed methods, results, and key observations that guided our work.

## *In Vitro* Assembly Notebook

# *In Vivo* Assembly Notebook

# Enzyme Activity Assay Notebook

# Ligation and product Assay Notebook

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Engineering

Design, Bulid, Test and Learn.

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Figure 1. Wet Lab DBTL cycles for V-CHARGEs development.

The engineering of V-CHARGEs followed five iterative DBTL cycles: protein expression and purification, P22-VLP self-assembly optimization, enzyme stability testing,
enzyme coupling, and product detection. These steps together established a robust and modular ATP regeneration platform.

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Wet Lab1

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Wet Lab2

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Wet Lab3

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Wet Lab4

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Wet Lab5

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Dry lab

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HP1

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HP2

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HP3

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Experiments

Protocols, Equipments, Reagent Formulations and References.

Experiments
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Abstract

In this section, we systematically document the equipment, reagent sources, experimental procedures and reagent formulations required for our project. We carried out **a series of workflows** ranging from DNA manipulation (plasmid extraction, transformation, PCR, etc.) to protein expression, purification, and detection, supplemented by multiple analytical methods (electrophoresis, Western Blot, enzyme activity assays, etc.) to validate our results.

To ensure reproducibility and reliability, we provide **detailed operation guidelines** for instruments, list the sources of key reagents and kits, and include standard formulations for culture media and buffers. Together, these elements establish a complete, transparent, and reproducible experimental framework.

# Protocol

## DNA Lab

▼

### Plasmid Extraction

▼

**Materials:**

Plasmid MiniPrep Kit

Overnight bacterial culture

Double-distilled water

1.5 mL and 2 mL Eppendorf tubes

**Equipment:**

Bench-top microcentrifuge

NanoDrop spectrophotometer

**Procedure:**

1. Harvest the overnight bacterial culture (~5 mL) in an Eppendorf tube and centrifuge at 8000 × g for 2 min. Discard the supernatant.

2. Resuspend the bacterial pellet in 250 μL Buffer RB (colorless, containing RNase A). Transfer the suspension into two 2 mL Eppendorf tubes.

3. Add 250 μL Buffer LB (blue) to each tube and gently invert several times to lyse the cells.

4. Add 350 μL Buffer NB (yellow) and invert gently until a tight yellow precipitate forms. Incubate at room temperature for ~2 min.

5. Centrifuge at 12,000 × g for 5 min. Carefully transfer the supernatant to a spin column.

6. Centrifuge at 12,000 × g for 1 min. Discard the flow-through and repeat once.

7. Add 650 μL Buffer WB to the spin column, centrifuge at 12,000 × g for 1 min, and repeat once.

8. Centrifuge at 12,000 × g for 2 min to remove residual wash buffer. Leave the column open at room temperature for 5 min to dry.

9. Place the spin column in a new 1.5 mL tube (with cap removed). Add 30 μL preheated (70 °C) Buffer EB directly to the center of the membrane. Incubate at room temperature for 1 min.

10. Centrifuge at 12,000 × g for 1 min to elute the plasmid DNA.

11. Measure plasmid concentration and purity using a NanoDrop spectrophotometer (use EB as blank). Record dsDNA content.

**Notes:**

* Ensure all centrifugation steps are carried out at room temperature.
* For optimal elution, Buffer EB should be prewarmed.
* Avoid disturbing the pellet or precipitate during transfer steps to ensure plasmid purity.

### Plasmid Heat Shock Transformation

▼

**Materials:**

* Competent cells
* 1.5 mL Eppendorf tubes
* Plasmid DNA
* LB liquid medium
* LB agar plates

**Equipment:**

* Water bath or dry bath incubator
* Shaker incubator
* Incubator

**Procedure:**

1. Transfer ~50 μL of competent cells into a 1.5 mL Eppendorf tube.

2. Once thawed, add no more than 42.5 ng plasmid DNA to the cells and mix gently.

3. Incubate the mixture on ice for 30 min. For BL21(DE3) cells, place the tube in a 42 °C water bath (or dry bath) for 45 s, then immediately transfer back to ice for 2 min.

4. Add 800 μL LB liquid medium to the tube, seal with Parafilm, and mix gently.

5. Incubate the culture at 37 °C, ~200 rpm, for 1 h to allow recovery.

6. Take 50 μL of the culture, dilute with ddH₂O to a final volume of 100 μL, and spread 50 μL onto an LB agar plate.

7. Incubate the plates overnight at 37 °C in an inverted position.

**Notes:**

* Ensure competent cells remain on ice until heat shock to maintain transformation efficiency.
* Do not exceed the recommended plasmid DNA amount, as high concentrations may reduce efficiency.
* Plates should be completely dry before spreading to avoid colony smearing.

### Polymerase Chain Reaction (PCR)

▼

**Materials:**

* PCR tubes
* DNA template
* Forward and reverse primers
* ddH₂O
* High-fidelity DNA polymerase mix

**Equipment:**

* PCR thermocycler

**Procedure:**

1. Prepare the PCR reaction mix in a PCR tube as follows (depending on desired reaction volume):

• Template DNA: 1 μL

• Forward primer: 0.2 / 0.4 / 1 μL

• Reverse primer: 0.2 / 0.4 / 1 μL

• PCR Mix: 5 / 10 / 25 μL

• ddH₂O: add to a final volume of 10 / 20 / 50 μL

(Choose reaction volume based on experimental requirements).

2. Place the PCR tube(s) into the thermocycler.

3. Set up the PCR program:

• Denaturation: ~94–95 °C

• Annealing: set according to primer Tm value

• Extension: adjust based on fragment size (typically 1–2 kb/min at 72 °C)

• Number of cycles: 35

4. Start the run and ensure the program operates normally.

5. After completion, remove the PCR tube(s) for downstream applications.

**Notes:**

* Use nuclease-free reagents to prevent contamination.
* Always keep PCR reagents on ice before starting to minimize nonspecific amplification.
* Optimize annealing temperature and Mg²⁺ concentration if nonspecific bands occur.

### Colony PCR

▼

**Materials:**

* PCR tubes
* Bacterial suspension or single colony
* Forward and reverse primers
* ddH₂O
* High-fidelity DNA polymerase mix

**Equipment:**

* PCR thermocycler

**Procedure:**

1. Template preparation

• For bacterial suspension: Take 1 µL of suspension and dilute it in 9 µL ddH₂O.

• For single colony: Pick a single colony (obtained from streak plate or dilution plating) and resuspend in 10 µL ddH₂O.

2. Reaction setup

• Add the following components into a PCR tube (depending on desired reaction volume):

 - Diluted bacterial suspension: 1 μL

 - Forward primer: 0.2 / 0.4 / 1 μL

 - Reverse primer: 0.2 / 0.4 / 1 μL

 - PCR Mix: 5 / 10 / 25 μL

 - ddH₂O: to a final volume of 10 / 20 / 50 μL

3. Thermocycler loading

• Place the PCR tube into the thermocycler.

4. Program setup

• Initial denaturation: ~94–95 °C

• Annealing: set according to primer Tm value

• Extension: 1–2 kb/min at 72 °C, depending on amplicon size

• Number of cycles: 35

5. Run and completion

• Start the program and monitor normal operation;

• After completion, remove PCR tubes for subsequent analysis.

**Notes:**

* Ensure colonies are well-isolated to avoid cross-contamination.
* Use fresh colonies (12–24 h) for optimal DNA template quality.
* If PCR fails, perform a quick cell lysis (e.g., boiling for 5 min) before use as template.

### Agarose Gel Electrophoresis

▼

**Materials:**

* Agarose powder
* 1× TAE buffer
* GelStain (nucleic acid dye, 10,000× stock)
* Loading buffer
* Gel casting tray
* Comb
* DNA marker/ladder

**Equipment:**

* Electrophoresis apparatus
* BG-gdsUVIEW Ultraviolet analyzer (or equivalent UV transilluminator)

**Procedure:**

1. Gel preparation

• To prepare a 1.0% agarose gel (100 mL total volume), weigh 1.0 g agarose powder into an Erlenmeyer flask.

• Add 100 mL of 1× TAE buffer.

• Heat the mixture in a microwave until boiling, swirling intermittently until the agarose is completely dissolved.

• Add the appropriate volume of 10,000× GelStain, mix thoroughly.

• Allow the solution to cool to ~60 °C (warm to touch but not hot).

2. Casting the gel

• Clean and dry the gel casting tray.

• Pour the agarose solution into the tray and insert the comb at the designated slot.

• Let the gel solidify completely.

• After polymerization, carefully remove the comb vertically.

• Transfer the gel (with tray) into the electrophoresis tank and cover with 1× TAE buffer.

3. Sample loading

• Mix DNA samples with appropriate volume of loading buffer.

• Using a micropipette, carefully load the samples into the wells.

• Replace pipette tips between samples to avoid cross-contamination.

• Load DNA marker/ladder into the first well.

4. Electrophoresis

• Connect the gel tank to the power supply and run at 90 V.

• Stop electrophoresis when the bromophenol blue dye front has migrated to ~1 cm from the bottom of the gel (typically ~20 minutes).

5. Visualization and analysis

• Remove the gel and place it onto the UV transilluminator.

• Observe DNA bands under UV illumination.

• Compare migration distances with the DNA ladder to determine fragment sizes.

**Notes:**

* Ensure uniform gel casting to avoid uneven migration.
* Do not overload wells to prevent smearing.
* Always wear UV-protective goggles when observing gels under UV light.

### Infusion Cloning

▼

**Materials:**

* 1.5 mL microcentrifuge tubes
* DNA fragment(s)
* Linearized vector
* 2× In-Fusion enzyme mix
* ddH₂O

**Equipment:**

* Benchtop microcentrifuge
* Dry bath incubator

**Procedure:**

1. Reaction setup

• In a 1.5 mL microcentrifuge tube, assemble the reaction mixture according to the following proportions:

 - 10 μL of 2× In-Fusion enzyme mix

 - 50–100 ng of linearized vector

 - Insert DNA fragment at a molar ratio of 2–3× relative to the vector

 - Add ddH₂O to a final volume of 20 μL

2. Mixing

• Briefly centrifuge the reaction mixture in a benchtop microcentrifuge to collect contents at the bottom of the tube.

3. Incubation

• Incubate the mixture at 50 °C for 20 minutes in a dry bath incubator.

### Takara Restriction Enzyme Digestion

▼

**Materials:**

* Restriction enzyme(s)
* Corresponding reaction buffer
* DNA fragment
* ddH₂O

**Equipment:**

* Dry bath incubator or incubator

**Procedure:**

1. Enzyme and buffer selection

• Consult the product catalog to identify the appropriate buffer system and optimal reaction temperature for the chosen enzyme(s).

• For double digestion, confirm buffer compatibility and determine whether a simultaneous (co-digestion) or sequential digestion is required.

2. Reaction setup

• For a 20 μL reaction, add the following components to a PCR tube:

 - 1 μL restriction enzyme

 - 2 μL of the corresponding 10× buffer

 - 1 μg DNA fragment (adjust volume according to concentration)

 - ddH₂O to a final volume of 20 μL

3. Digestion

• Incubate the reaction mixture at the recommended temperature in a dry bath incubator or incubator for 6 hours.

4. Post-digestion treatment

• If DNA fragments are to be recovered: heat-inactivate the enzyme at ≥65 °C for 20 minutes, or alternatively proceed directly to DNA purification (e.g., column purification or agarose gel electrophoresis followed by gel extraction).

• If only verification is required: add loading buffer to a final concentration of 1× and analyze by agarose gel electrophoresis.

### Fast Restriction Enzyme Digestion

▼

**Materials:**

* Restriction enzyme(s)
* Corresponding reaction buffer
* DNA fragment
* ddH₂O

**Equipment:**

* Dry bath incubator or incubator

**Procedure:**

1. Enzyme and buffer selection

• Consult the product catalog to confirm the appropriate buffer system and optimal reaction temperature for the selected enzyme(s).

• For double digestion, determine buffer compatibility and decide whether to perform co-digestion or sequential digestion.

2. Reaction setup (20 μL system)

• Add the following components into a PCR tube:

 - 1 μL restriction enzyme

 - 2 μL of the corresponding 10× buffer

 - 1 μg DNA fragment (adjust volume according to DNA concentration)

 - ddH₂O to a final volume of 20 μL

3. Mixing

• Mix gently and briefly centrifuge to collect liquid at the bottom.

4. Incubation

• Incubate at 37 °C for 5–10 minutes.

5. Post-digestion treatment

• If DNA fragments are required: heat-inactivate the restriction enzyme at ≥65 °C for 20 minutes, or proceed directly with DNA purification (e.g., liquid recovery or agarose gel electrophoresis followed by gel extraction).

• If only verification is needed: add loading buffer to a final concentration of 1× and analyze by agarose gel electrophoresis.

### DNA Gel Extraction (SanPrep Column Kit)

▼

**Materials:**

* SanPrep Column DNA Gel Extraction Kit

**Equipment:**

* Water bath (50 °C)
* Benchtop microcentrifuge

**Procedure:**

1. Check whether Wash Solution has ethanol added; check if Buffer B2 contains precipitate; preheat the water bath to 50 °C.

2. Excise the agarose gel slice containing the target DNA fragment and weigh it.

3. Add 3–6 volumes of Buffer B2 relative to the gel weight. Incubate at 50 °C for 5–10 minutes until the gel is completely dissolved.

4. For DNA fragments <500 bp, add 1/3 volume of isopropanol relative to the volume of Buffer B2.

5. Transfer the dissolved gel solution into a spin column. Centrifuge at 8,000×g for 30 seconds, discard the flow-through.

6. Add 500 μL Wash Solution, centrifuge at 9,000×g for 30 seconds, discard the flow-through.

7. Repeat Step 6 once.

8. Dry-spin the empty column at 9,000×g for 1 minute to remove residual wash buffer.

9. Place the column into a clean 1.5 mL microcentrifuge tube, add 15–40 μL Elution Buffer directly to the center of the membrane, incubate at room temperature for 1 minute, then centrifuge for 1 minute to elute DNA. Collect and store the DNA solution.

### PCR Product Purification (SanPrep Column Kit)

▼

**Materials:**

* SanPrep Column PCR Product Purification Kit

**Equipment:**

* Benchtop microcentrifuge

**Procedure:**

1. Check whether Wash Solution has ethanol added; check if Buffer B3 contains precipitate; ensure Buffer B3 has isopropanol added.

2. Add 5 volumes of Buffer B3 to the PCR reaction mixture, mix thoroughly.

3. Load the mixture into the spin column, centrifuge at 8,000×g for 30 seconds, discard the flow-through.

4. Add 500 μL Wash Solution, centrifuge at 9,000×g for 30 seconds, discard the flow-through.

5. Repeat Step 4 once.

6. Dry-spin the empty column at 9,000×g for 1 minute to remove residual wash buffer.

7. Place the column into a clean 1.5 mL microcentrifuge tube, add 15–40 μL Elution Buffer directly to the center of the membrane, incubate at room temperature for 1 minute, then centrifuge for 1 minute to elute DNA. Collect and store the purified DNA solution.

## Protein Lab

▼

### Bacterial Protein Expression & Purification

▼

**Materials:**

* LB medium
* M9 medium
* IPTG reagent
* Wash buffer A
* Wash buffer B
* Wash buffer C (imidazole 100–500 mM gradient)

**Instruments:**

* V-5100 Visible Light Spectrophotometer
* High-speed centrifuge
* SONICS Ultrasonic Homogenizer
* HiTrap Desalting Column / AKTA Protein Purification System
* Microplate Reader

**Expression & Purification of CP-ST**

1. Inoculate *E. coli* BL21(DE3) strain into LB medium and culture overnight at 37°C, 180–200 rpm.

2. Dilute the overnight culture into fresh LB medium containing the appropriate antibiotic at a 1:100 ratio (inoculum : medium).

3. Grow at 37°C, 200 rpm for ~3 hours until the culture reaches OD₆₀₀ = 0.6–0.8.

4. Induce protein expression by adding IPTG stock solution (50 mg/mL) to a final concentration of 1 / 0.8 / 0.5 / 0.3 / 0.1 mM (4.76 / 3.808 / 2.38 / 1.428 / 0.476 mL per L medium). Continue incubation at 16°C, 180 rpm for 12–16 h.

5. Harvest cells by centrifugation at 6,000 rpm, 4°C for 10 min. Collect the pellet (pellets can be stored at 4°C if necessary).

6. Resuspend the pellet in 1/10 volume of buffer using a 5 mL pipette or dropper. Keep samples on ice.

7. Lyse cells by ultrasonication on ice: place the probe ~1 cm below the liquid surface. Use a pulse mode (3 s on / 6 s off) for ~10 min or longer as needed.

8. Centrifuge the lysate at 12,000 rpm, 4°C for 45 min. Discard pellet and retain supernatant.

9. Thaw protein samples on ice. Perform all subsequent purification steps at 4°C on ice.

10. Prepare a Ni-NTA affinity column with 600 µL Ni²⁺ resin beads.

11. Equilibrate the column with 3 mL Buffer B, incubate 3 min, collect flow-through; repeat 3 times.

12. Load protein supernatant in portions (~3 mL each). After each load, incubate 3 min, gently pipette up and down, incubate another 3 min, then collect flow-through. Repeat until all supernatant has been loaded.

13. Wash the column with 4 mL Buffer B, incubate 10 min, collect flow-through; repeat 3 times.

14. Elute bound protein sequentially: add 300 µL Buffer C1, incubate 3 min, collect eluate, repeat 3 times; then switch to Buffer C2, repeat the same steps.

### Ultracentrifugation

▼

**Materials:**

* Samples to be centrifuged
* Sucrose cushion (if required)

**Procedure:**

1. Pre-cooling

• Place the rotor in a 4 °C refrigerator overnight to ensure complete pre-cooling before use.

2. Power On

• After preparing the samples, switch on the ultracentrifuge (main power on the right side, ON).

• Wait for the instrument to complete self-checking, then open the sliding chamber lid.

3. Sample Loading and Balancing

• Transfer samples into thin-walled, uncapped Beckman ultracentrifuge tubes.

• Balance tubes to a precision of ±0.01 g.

• Insert tubes symmetrically into the rotor.

4. Rotor Installation

• Align the rotor with the centrifuge spindle and gently lower it vertically into place.

• Turn clockwise slightly to secure, then close the chamber lid.

5. Program Setup

• Input centrifugation parameters:

 - Speed (based on rotor and tube specifications, not exceeding their maximum rated speed).

 - Time.

 - Temperature.

• Confirm that settings comply with rotor and tube safety limits.

6. Running

• Start the vacuum pump; wait until vacuum pressure is below 70 µmHg.

• Press Start to initiate centrifugation.

• Remain nearby until the rotor reaches stable speed.

• If abnormal noise or vibration occurs, press STOP immediately to abort.

7. Completion

• After centrifugation finishes, wait for the rotor to come to a complete stop.

• Release vacuum pressure, then open the chamber lid.

• Carefully remove the rotor and retrieve all tubes and adapters.

8. Shutdown and Cleaning

• Remove the rotor, wipe off condensation, and rinse with tap water if any leakage occurred. Air-dry inverted to prevent corrosion.

• Wipe condensation inside the centrifuge chamber. Keep the lid open until the chamber returns to room temperature and is completely dry.

• Close the lid and switch off the power.

**Notes:**

* Always use the correct rotor, tubes, and adapters compatible with the instrument.
* Do not exceed the specified maximum speed of the rotor and tubes.
* Balancing precision is critical to prevent rotor damage and ensure operator safety.
* Operators must receive specific ultracentrifuge safety training.

### Dialysis

▼

**Materials:**

* Dialysis tubing (pre-activated and suitable MWCO)
* Dialysis buffer (target buffer for exchange)

**Procedure:**

1. Sample Loading

• Carefully transfer the prepared sample into the pre-activated dialysis tubing.

• Expel any trapped air and seal the tubing securely with clamps.

2. Dialysis Process

• Submerge the dialysis bag into a large volume of dialysis buffer.

• Gently stir or rock the buffer to maintain mixing, avoiding vigorous agitation that may damage the membrane or cause foaming.

3. Buffer Exchange

• Replace the external buffer multiple times according to the experimental design, ensuring sufficient buffer volume and exchange frequency to achieve the desired dialysis efficiency.

4. Sample Recovery

• After dialysis, carefully remove the tubing from the buffer.

• Recover the sample into a clean container.

• Record the final sample volume and concentration.

**Notes:**

* Select dialysis tubing with the appropriate molecular weight cutoff (MWCO) based on the sample size.
* Ensure a large buffer-to-sample volume ratio (commonly 100–1000×) for efficient exchange.
* Perform dialysis at 4 °C when working with proteins or other unstable biomolecules to maintain stability.
* Avoid prolonged dialysis that may lead to sample dilution or degradation.

### SDS-PAGE (Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis)

▼

**Materials:**

* Double distilled water (ddH₂O)
* SDS (sodium dodecyl sulfate)
* APS (ammonium persulfate)
* TEMED (tetramethylethylenediamine)
* Acrylamide/Bisacrylamide solution (Acr-Bis)
* Tris-HCl buffer
* SDS running buffer
* 5× loading buffer
* Protein marker (ladder)
* Coomassie Brilliant Blue solution (or alternative staining reagents such as silver stain, enzyme activity stain)

**Protocol:**

1. Gel Preparation

• Clean the glass plates and assemble the gel casting system.

• Prepare the separating gel solution (adding TEMed last), mix thoroughly, and pour into the gel cassette. Stop pouring ~3 cm below the top edge.

• Overlay with water to prevent evaporation and ensure a flat gel surface.

2. Stacking Gel Preparation

• Once the separating gel has polymerized, carefully remove the overlay water and blot dry with filter paper.

• Prepare the stacking gel solution, mix well, and pour on top of the separating gel. Insert the comb and allow the gel to polymerize for ~10 min.

• Remove the comb carefully after solidification.

3. Electrophoresis Setup

• Place the gel cassette in the electrophoresis tank and fill with SDS running buffer.

• Load samples into the wells:

 - 5 μL protein marker.

 - 25 μL of each protein sample (20 μL protein + 5 μL 5× loading buffer, boiled or denatured if required).

4. Electrophoresis Conditions

• Run the gel at 90 V for ~20 min (stacking gel phase).

• Increase to 150 V for ~45 min (resolving gel phase) until the dye front (green marker) reaches the bottom.

5. Staining

• Following electrophoresis, stain the gel with Coomassie Brilliant Blue or alternative methods (e.g., silver staining, activity staining) depending on sensitivity and downstream application.

**Notes:**

* Always add APS and TEMED immediately before pouring to ensure proper polymerization.
* Avoid introducing air bubbles when casting gels.
* Ensure equal sample loading for consistent band intensity.
* Use freshly prepared running buffer for best results.

### Native-PAGE (Non-denaturing Polyacrylamide Gel Electrophoresis)

▼

**Materials:**

* Double distilled water (ddH₂O)
* APS (ammonium persulfate)
* TEMED (tetramethylethylenediamine)
* Acrylamide/Bisacrylamide solution (Acr-Bis)
* Tris-HCl buffer
* Native running buffer
* 4× native loading buffer
* Protein marker (native ladder)
* Coomassie Brilliant Blue (or alternative staining reagents such as silver stain or activity stain)

**Protocol:**

1. Gel Casting

• Clean the glass plates and assemble the gel casting system.

• Prepare the separating gel according to the molecular weight of the target protein (without SDS). Add TEMED last, mix thoroughly, and immediately pour into the gel cassette. Stop pouring when ~3 cm from the top edge. Overlay with water to seal.

2. Stacking Gel Preparation

• Once the separating gel has polymerized, carefully remove the overlay water.

• Prepare the stacking gel (without SDS), add TEMED last, mix thoroughly, and pour on top of the separating gel. Insert the comb and allow to polymerize for ~10 min. Remove the comb gently once solidified.

3. Buffer Addition

• Fill the inner chamber of the electrophoresis tank with native running buffer.

• Add buffer to the outer chamber to ~1/3 full (too little may disrupt the circuit; too much may reduce efficiency).

4. Sample Preparation and Loading

• Keep all protein samples on ice. Do not boil or add denaturing agents (e.g., SDS, β-mercaptoethanol, DTT).

• Mix protein samples with 4× native loading buffer. Load ~10 μL per well.

• Load 10 μL of native protein marker into the first lane.

5. Electrophoresis

• Place the electrophoresis apparatus on ice to avoid heat generation, which may disrupt protein tertiary and quaternary structure.

• Run the gel at 90 V for ~20 min (stacking gel phase), then increase to 150 V for ~45 min (resolving gel phase) until the dye front (green marker) is visible near the bottom.

6. Staining

• Stain the gel with Coomassie Brilliant Blue, or use silver staining/enzyme activity staining depending on the sensitivity required.

**Notes:**

* Native-PAGE separates proteins by size, charge, and conformation, not purely by molecular weight.
* Avoid heat and chemical denaturants throughout the process.
* Always run at low temperature (on ice or in a cold room) to preserve protein activity and structure.

### Western Blot

▼

**Materials:**

* PVDF membrane
* TBST (TBS + 0.1% Tween-20)
* Primary antibody (1° Ab)
* Secondary antibody (2° Ab)
* Blocking buffer (e.g., 5% non-fat dry milk in TBST)

**Protocol:**

1. SDS-PAGE

• Perform SDS-PAGE to separate proteins on an unstained gel (do not stain the gel).

2. Membrane Preparation

• Cut the PVDF membrane to match the size of the gel.

• Soak the PVDF membrane and filter papers in transfer buffer until fully equilibrated.

3. Protein Transfer

• Assemble the transfer "sandwich" according to the system used (gel in direct contact with the PVDF membrane, avoiding air bubbles).

• Perform transfer under constant current conditions. Adjust transfer time depending on protein molecular weight.

4. Blocking

• After transfer, incubate the PVDF membrane in 10 mL blocking buffer (e.g., 5% non-fat dry milk in TBST) with gentle shaking for 1 hour at room temperature.

5. Primary Antibody Incubation

• Wash the membrane in TBST three times, 10 min each.

• Incubate the membrane in 10 mL of primary antibody solution at 4°C overnight with gentle agitation.

6. Secondary Antibody Incubation

• Wash the membrane in TBST three times, 10 min each.

• Incubate the membrane in 10 mL of secondary antibody solution at room temperature for 1 hour.

7. Detection

• Wash the membrane in TBST three times, 10 min each.

• Add chemiluminescent substrate (ECL reagent) to cover the membrane surface and remove air bubbles.

• Detect signal using an imaging system.

**Notes:**

* For high background, increase washing time or use BSA instead of milk as blocking reagent.
* Transfer efficiency depends on protein size:

   - <30 kDa → short transfer (30–45 min)

   - 30–100 kDa → ~1 h

   - 100 kDa → extended transfer or wet transfer system
* Always handle the PVDF membrane with clean tweezers and avoid touching the surface.

### Agarose Gel Electrophoresis for Proteins

▼

**Materials:**

* Bromophenol blue
* Glycerol
* Agarose
* Tris-HCl
* Sodium acetate
* EDTA
* Coomassie Brilliant Blue (CBB)
* Methanol
* Lactic acid

**Protocol:**

1. Preparation of Bromophenol Blue Stock Solution

• Dissolve 0.02 g bromophenol blue in 2 mL ddH₂O to obtain a 0.2% bromophenol blue solution.

• Store at 4°C.

2. Preparation of Loading Buffer

• Mix 800 μL glycerol with 1 mL 0.2% bromophenol blue solution.

• Adjust the final volume to 2 mL with ddH₂O.

• The final loading buffer contains 40% glycerol + 0.1% bromophenol blue.

• Store at 4°C.

3. Preparation of Agarose Gel

• Prepare a 0.5% agarose gel in modified TAE buffer (without nucleic acid stain).

4. Sample Preparation and Loading

• Mix protein sample with loading buffer at a 1:1 ratio.

• Load 20 μL per well.

• Running buffer: modified low-acetate TAE (40 mM Tris, 5 mM sodium acetate, 1 mM EDTA).

5. Electrophoresis

• Run the gel at a constant voltage of 10 V for ~10 h.

6. Staining

• Stain the gel overnight at 4°C with Coomassie Brilliant Blue.

7. Destaining

• Destain the gel in a solution containing 40% methanol + 7% lactic acid until clear background is obtained.

**Notes:**

* This protocol is suitable for native protein separation under mild conditions (non-denaturing).
* Lower acetate concentration in the buffer minimizes overheating during long electrophoresis.
* Ensure complete destaining for sharp visualization of protein bands.

## Assembly Lab

▼

### Enzyme Encapsulation

▼

**Materials:**

* Dialysis membranes (MWCO 100 kDa and 3 kDa)
* 2% (w/v) sodium bicarbonate
* 1 mmol/L EDTA (pH 8.0)
* Distilled water
* Dialysis buffer

**Protocol:**

(1) Activation of Dialysis Membranes

1. Cut the dialysis tubing into small pieces of appropriate length.

2. Boil the dialysis membranes in a large volume of 2% (w/v) sodium bicarbonate and 1 mmol/L EDTA (pH 8.0) solution for 10 min.

3. Wash thoroughly with distilled water.

4. Boil again in 1 mmol/L EDTA (pH 8.0) solution for 10 min.

5. After cooling, wearing clean gloves, fill the membrane with water and then discard it to remove residual reagents. Rinse thoroughly.

(2) Enzyme Encapsulation Procedure

1. Mix 6 M guanidine hydrochloride (GuHCl) with 0.5 mg/mL CP-ST at a 1:1 volume ratio to obtain a final 3 M GuHCl solution. Incubate on ice for 1 h.

2. Add an appropriate volume of SlPPK-SP to the mixture so that the final GuHCl concentration is 1.5 M, with a molar ratio of SlPPK-SP : CP-ST = 1:2.

3. Transfer the mixture into the pre-activated 3 kDa dialysis tubing, and dialyze against 100× volume dialysis buffer at 4°C for 12–18 h.

4. Transfer the dialyzed solution into the pre-activated 100 kDa dialysis tubing, and dialyze against 100× volume dialysis buffer at 4°C for 12–18 h to remove unassembled free proteins.

**Notes:**

* Activation of dialysis tubing ensures removal of preservatives and improves permeability.
* Stepwise reduction of GuHCl concentration promotes proper folding and assembly of enzyme complexes.
* The second dialysis step with higher MWCO tubing allows removal of free unassembled proteins while retaining encapsulated complexes.

### Size-Exclusion Chromatography (SEC)

▼

**Materials:**

* Purified water
* Buffer B
* Absolute ethanol
* Protein sample solution

**Protocol:**

1. Gel Selection

• Select a gel matrix with an appropriate fractionation range based on the molecular weight, resolution requirements of the target protein and impurities, and sample stability.

2. Gel Swelling

• Dry gel must be fully swollen before use (gel : water = 1:10).

• Add purified water or Buffer B to the dry gel in a beaker, stir thoroughly, and let it stand.

• Discard the supernatant suspension, remove gel fragments floating on the surface, and repeat this process until the supernatant becomes clear.

3. Column Dimensions

• Determine the required bed volume, length, diameter, and aspect ratio of the gel column based on the sample volume, sample properties, and desired separation resolution.

4. Column Packing

• Close the outlet of the column and add ~1/3 of the total bed volume of water or buffer.

• Resuspend the pre-swollen gel in buffer, and gently pour the slurry into the column in one continuous step along the column wall.

• When the gel settles to ~5 cm in height, open the column outlet and adjust the flow rate to ~1 mL/min to allow uniform packing.

5. Sample Loading

• Use an appropriate buffer as the mobile phase.

• The sample volume should generally be 1–5% of the total column bed volume.

• Prior to loading, filter (0.22 µm) or centrifuge the sample to remove particulates that could clog the column.

6. Elution

• Elute with either a single buffer system or a buffer containing salt, depending on experimental requirements.

• Maintain a stable operating pressure and ensure the flow rate does not exceed 0.5–3.0 mL/min.

**Notes:**

* Avoid air bubbles in the column; always degas the buffer before use;
* Never allow the column bed to dry, keep it constantly hydrated;
* Control sample concentration and injection volume to prevent overloading and loss of resolution;
* Monitor system pressure during operation to avoid column damage due to overpressure.

### High-Performance Liquid Chromatography (HPLC)

▼

**Materials:**

* Potassium dihydrogen phosphate (KH₂PO₄)
* Methanol (HPLC grade)
* Ultrapure water

**Instruments:**

* HPLC system equipped with a UV detector
* C18 reversed-phase column (4.6 × 150 mm, Wondasil, Shimadzu GL Sciences)
* Dionex Chromeleon software for data acquisition and analysis

**Protocol:**

1. Chromatographic Conditions

• For general nucleotide detection:

 - Mobile phase: 50 mM potassium dihydrogen phosphate : methanol (v/v 97:3)

 - Flow rate: 0.6 mL/min

 - Column temperature: 30 °C

 - Detection wavelength: 270 nm

• For adenosine detection:

 - Mobile phase: 50 mM potassium dihydrogen phosphate : methanol (v/v 90:10)

 - Flow rate: 0.8 mL/min

 - Column temperature: 30 °C

 - Detection wavelength: 270 nm

2. Sample Preparation

• Dilute samples with ultrapure water.

• Filter through a 0.22 μm hydrophilic PTFE syringe filter (Anpel, Shanghai).

• Inject 10 μL of the filtrate for HPLC analysis.

3. Data Analysis

• Chromatographic data were recorded and processed using Dionex Chromeleon software.

**Notes:**

* All solvents must be freshly prepared and filtered through a 0.22 μm membrane to avoid baseline noise and column clogging.
* Equilibrate the column with the mobile phase for at least 20 minutes before sample injection.
* Use an appropriate standard curve for quantification of analytes.

### Transmission Electron Microscopy (TEM)

▼

**Materials:**

* Prepared samples
* 300-mesh carbon support film
* TEM instrument with operation software

**Protocol:**

1. Turn on the TEM system sequentially: main power → vacuum pump → cooling system → high-voltage power supply. Wait until the self-check process is completed, then enter the operation software.

2. Carefully mount the prepared sample on a 300-mesh carbon support film, fix it onto the loading rod, and insert into the TEM sample holder.

3. Set appropriate imaging parameters, including acceleration voltage, magnification, and focusing mode.

4. Activate the electron beam. Adjust the stage position, focusing rings, and apertures until a clear image is obtained. Acquire images and transfer the data to a computer for analysis.

5. After imaging, sequentially switch off the electron beam, high-voltage power supply, vacuum pump, cooling system, and main power supply.

**Notes:**

* Samples should be properly prepared, dried, and evenly distributed on the carbon film to avoid charging effects.
* Avoid prolonged exposure of the same area to the electron beam to minimize radiation damage.
* Ensure stable vacuum conditions throughout operation for optimal imaging quality.
* Only trained personnel should operate the TEM, due to high-voltage and vacuum safety concerns.

### Dynamic Light Scattering (DLS)

▼

**Materials:**

* Pure/deionized water (same solvent as sample)
* Disposable plastic or quartz cuvettes
* Protein, nanoparticle, or colloid samples

**Protocol:**

1. Turn on the DLS instrument, allowing the laser and detector to preheat and stabilize (typically 30 min).

2. Clean the sample chamber and cuvette with pure solvent. Avoid fingerprints and dust; if necessary, blow dry with filtered nitrogen.

3. If reusing cuvettes, wash with detergent, rinse thoroughly with deionized water, followed by filtered water, then rinse with solvent and dry with lint-free paper or air-dry.

4. Prepare the sample: dissolve in compatible buffer/solvent to obtain a clear solution. Typical protein concentration is 0.1–2 mg/mL; nanoparticles should follow manufacturer's or pre-experimental guidelines.

5. Remove aggregates and bubbles: centrifuge samples at 12,000–20,000 × g for 5–15 min or filter through 0.22 μm membranes (if compatible with particle size).

6. Load 1–2 mL of sample into a clean cuvette, avoiding bubbles. Seal with a cap.

7. Place the cuvette into the instrument and ensure proper alignment. For temperature-controlled measurements, set the desired temperature and wait 2–5 min for equilibration.

8. Input sample parameters into the software, including solvent refractive index, viscosity, and measurement wavelength (or allow software auto-calculation).

9. Configure measurement settings:

• Number of runs: 3–12 repeats (commonly 5)

• Acquisition time: 10–60 s per run

• Ensure the count rate falls within the optimal detection range.

10. Start data acquisition and perform multiple repeats per sample to ensure reproducibility.

11. Save results, analyze data, and plot size distribution (log10 particle diameter in nm vs. intensity).

**Notes:**

* Samples must be transparent and free of dust or bubbles to avoid scattering artifacts.
* Protein and nanoparticle concentrations should be optimized to prevent multiple scattering or low signal.
* Always use solvent-matched blanks for calibration.
* Avoid vibration or strong light interference during measurement.

### Pull-down Assay

▼

**Materials:**

* Protein A/G Magnetic Magnetic Beads
* Binding Buffer
* Elution Buffer
* FLAG antibody (or other tag-specific antibody)
* 1.5 mL microcentrifuge tubes

**Protocol:**

1. Vortex the Protein A/G Magnetic Beads for 30 s to ensure they are well resuspended.

2. Transfer 50 μL of beads into a 1.5 mL microcentrifuge tube and place on a magnetic stand to separate.

3. Add 150 μL of binding buffer, gently resuspend, and separate using the magnet. Repeat this washing step twice.

4. Dilute 10 μL of antibody in 40 μL binding buffer.

5. Incubate the diluted antibody with the magnetic beads at room temperature for 1 h on a rotator.

6. After incubation, gently resuspend and then separate using the magnet.

7. Wash beads with 150 μL binding buffer, gently resuspend, and separate with the magnet.

8. Add 100 μL of protein sample to the beads, gently resuspend, and incubate at room temperature for 10 min.

9. After incubation, gently resuspend and separate with the magnet.

10. Wash the beads with 300 μL binding buffer, separate with the magnet, and repeat the washing step three times.

11. Add 150 μL binding buffer, transfer the beads to a new microcentrifuge tube, and separate with the magnet.

12. Elute bound proteins by adding 100 μL of elution buffer, resuspending, and separating. Collect the eluate for downstream analysis (e.g., SDS-PAGE, Western blot, or mass spectrometry).

**Notes:**

* Always keep beads in suspension by gentle mixing to maximize binding efficiency.
* Avoid introducing bubbles during pipetting, which may reduce reproducibility.
* Use freshly prepared binding and elution buffers for optimal results.
* For weak interactions, consider longer incubation times or lower stringency wash buffers.
* Work quickly at low temperature (e.g., 4 °C) if target proteins are unstable.

### Agarose-based ELISA

▼

**Materials:**

* Buffer B
* 75% ethanol
* Agarose (1% solution prepared fresh)
* 5% skim milk (blocking solution)
* Primary antibody
* Secondary antibody
* ECL substrate (chemiluminescent detection reagent)
* Glass slides
* Agarose beads (400 μL per assay)

**Protocol:**

1. Clean glass slides with 75% ethanol, allow to air dry.

2. Take 400 μL beads and wash 3 times with Buffer B.

3. Prepare 15 mL of 1% agarose with Buffer B, maintain in a melted state.

4. Mix 100 μL melted agarose with 100 μL beads immediately, and spread evenly on the slide using a pipette tip. The agarose layer should form a continuous thin coating on the slide surface, without breaks or aggregates.

5. Place slides at 4 °C to allow agarose to solidify.

6. Wash slides 3 times with Buffer B.

7. Apply 100 μL of sample to the agarose-coated slide, incubate at room temperature for 30 min.

8. Wash slides 3 times with Buffer B.

9. Block the slide by immersing it in 5% skim milk for 30 min at room temperature.

10. Wash slides 3 times with Buffer B.

11. Incubate slides with primary antibody solution for 1 h at room temperature.

12. Wash slides 3 times with Buffer B.

13. Incubate slides with secondary antibody for 1 h at room temperature.

14. Wash slides 3 times with Buffer B.

15. Develop signal using ECL substrate, and detect chemiluminescence with an imaging system.

**Notes:**

* The agarose coating must be continuous and uniform; uneven or cracked gels will cause inconsistent signal.
* Keep agarose solution melted before mixing with beads; solidification during handling will reduce reproducibility.
* Blocking with 5% skim milk is critical to minimize non-specific binding.
* All washes should be performed gently to avoid detaching the agarose layer.
* Perform ECL development in the dark and image promptly to prevent signal decay.

### Dot Blotting

▼

**Materials:**

* Methanol
* Transfer buffer
* TBST (Tris-buffered saline with 0.1% Tween-20)
* 5% skim milk (blocking buffer)
* Primary antibody
* Secondary antibody
* ECL substrate

**Protocol:**

1. Membrane activation

• Immerse the PVDF membrane in methanol for 1 min.

• Wash thoroughly in transfer buffer to remove methanol.

• Place the membrane on a flat surface and allow residual water to air-dry naturally (do not over-dry).

2. Sample loading

• Spot 1–3 μL of sample solution directly onto the membrane surface.

• Allow the membrane to absorb the sample completely (avoid forced air-drying).

3. Washing & blocking

• Wash the membrane twice with TBST for 5 min each.

• Block with 5% skim milk in TBST at room temperature with gentle shaking for 40 min.

4. Primary antibody incubation

• Wash the membrane 3 times with TBST (10 min each).

• Incubate the membrane with diluted primary antibody at 4 °C overnight with gentle shaking.

5. Secondary antibody incubation

• Wash the membrane 3 times with TBST (10 min each).

• Incubate with the corresponding secondary antibody for 1 h at room temperature.

6. Detection

• Wash the membrane with TBST (3 × 10 min).

• Add ECL substrate evenly over the spotted areas, avoid bubbles.

• Detect the chemiluminescent signal using an imaging system.

**Notes:**

* Avoid applying excessive sample volume, as it can cause diffusion and blurred spots.
* Ensure equal sample volume for each spot to enable semi-quantitative comparison.
* If background is high, extend washing steps or use BSA as an alternative blocking agent.
* Membrane should not be allowed to dry completely after sample spotting until blocking is finished.

### GSH ELISA

▼

**Materials:**

* Methanol
* Transfer buffer
* TBST (Tris-buffered saline with 0.1% Tween-20)
* 5% skim milk (blocking buffer)
* Primary antibody
* Secondary antibody
* ECL substrate

**Protocol:**

1. Membrane activation

• Immerse the PVDF membrane in methanol for 1 min.

• Wash thoroughly in transfer buffer to remove methanol.

• Place the membrane on a flat surface and allow residual water to air-dry naturally (do not over-dry).

2. Sample loading

• Spot 1–3 μL of sample solution directly onto the membrane surface.

• Allow the membrane to absorb the sample completely (avoid forced air-drying).

3. Washing & blocking

• Wash the membrane twice with TBST for 5 min each.

• Block with 5% skim milk in TBST at room temperature with gentle shaking for 40 min.

4. Primary antibody incubation

• Wash the membrane 3 times with TBST (10 min each).

• Incubate the membrane with diluted primary antibody at 4 °C overnight with gentle shaking.

5. Secondary antibody incubation

• Wash the membrane 3 times with TBST (10 min each).

• Incubate with the corresponding secondary antibody for 1 h at room temperature.

6. Detection

• Wash the membrane with TBST (3 × 10 min).

• Add ECL substrate evenly over the spotted areas, avoid bubbles.

• Detect the chemiluminescent signal using an imaging system.

**Notes:**

* Avoid applying excessive sample volume, as it can cause diffusion and blurred spots.
* Ensure equal sample volume for each spot to enable semi-quantitative comparison.
* If background is high, extend washing steps or use BSA as an alternative blocking agent.
* Membrane should not be allowed to dry completely after sample spotting until blocking is finished.

### Temperature-Dependent Assay

▼

**Materials:**

* ATP detection reagent
* Dilution buffer
* Standard substrate solution
* Distilled water
* Enzyme sample
* Assembled protein/sample mixture
* Microplate reader
* 96-well microplate

**Protocol:**

1. Preparation of ATP working solution

• Mix 10 μL ATP detection reagent with 90 μL dilution buffer.

• Prepare 100 μL of ATP working solution per reaction group.

2. Loading substrate

• Add 100 μL ATP working solution to each well of the microplate.

• Incubate at room temperature for 2 min.

• Add 20 μL standard substrate solution to each well.

3. Enzyme treatment

• Pre-incubate enzyme samples under the desired temperature conditions.

• Heat in a metal bath for 10 min, then immediately transfer.

• Add 10 μL pre-treated enzyme sample to each well.

4. Bioluminescence measurement

• Mix briefly in the microplate reader (2 s).

• Record bioluminescence intensity every 20 s for a total of 15 min.

• Export data and plot bioluminescence vs. time curves for analysis.

**Notes:**

* Ensure all reagents are equilibrated to room temperature before use.
* Maintain consistent pipetting speed and timing to minimize variability between wells.
* Optimize excitation/emission wavelengths of the plate reader according to the ATP detection kit specifications.
* For comparative assays, process all samples in parallel to ensure uniform incubation times.

### pH-Modulation Assay

▼

**Materials:**

* ATP detection reagent
* Dilution buffer
* Standard substrate solution
* 1 M HCl (for pH adjustment)
* Distilled water
* Enzyme sample
* Assembled protein/sample mixture
* Microplate reader
* 96-well microplate

**Protocol:**

1. Preparation of ATP working solution

• Mix 10 μL ATP detection reagent with 90 μL dilution buffer.

• Prepare 100 μL of ATP working solution per reaction group.

2. Loading substrate

• Add 100 μL ATP working solution to each well of the microplate.

• Incubate at room temperature for 2 min.

• Add 20 μL standard substrate solution to each well.

3. Enzyme and pH gradient treatment

• Add 10 μL enzyme sample to each well and mix gently by pipetting.

• Immediately add 30 μL of pre-prepared gradient pH buffer B solution to adjust reaction conditions.

4. Bioluminescence measurement

• Mix in the microplate reader for 5 s.

• Record bioluminescence intensity every 30 s for a total of 15 min.

• Save raw data and generate plots of bioluminescence vs. time under different pH conditions.

**Notes:**

* Ensure all pH buffer solutions are freshly prepared and equilibrated to room temperature before use.
* Handle 1 M HCl with caution; always wear appropriate PPE.
* To minimize variability, prepare all pH gradient conditions in parallel.
* Optimize excitation/emission wavelengths of the plate reader according to the ATP detection kit specifications.

### Phosphate Substrate Inhibition Assay

▼

**Materials:**

* ATP detection reagent
* Standard substrate solution
* Dilution buffer
* Phosphate solution (prepared at different gradient concentrations)
* Distilled water
* Enzyme sample
* Assembled protein/sample mixture
* Microplate reader
* 96-well microplate

**Protocol:**

1. Preparation of ATP working solution

• Mix 10 μL ATP detection reagent with 90 μL dilution buffer.

• Prepare 100 μL ATP working solution per reaction group.

2. Loading substrate

• Add 100 μL ATP working solution to each well of the microplate.

• Incubate at room temperature for 2 min.

• Add 20 μL standard substrate solution to each well.

3. Enzyme and phosphate gradient treatment

• Add 10 μL enzyme sample to each well and mix gently by pipetting.

• Immediately add phosphate solution at different gradient concentrations, corresponding to 10% of the total reaction volume.

4. Bioluminescence measurement

• Mix in the microplate reader for 5 s.

• Record Bioluminescence intensity every 30 s for a total of 15 min.

• Save raw data and generate inhibition curves by plotting relative Bioluminescence vs. phosphate concentration.

**Notes:**

* Phosphate solutions should be freshly prepared to avoid precipitation and pH drift.
* Optimize excitation/emission wavelengths of the plate reader according to the ATP detection kit specifications.
* Perform at least three biological replicates for reliable kinetic analysis.

# Equipment

### Use of Micropipette

▼

**Forward Pipetting (Press 1 – Release 2)**

1. Attach the appropriate tip to the pipette.

2. Adjust the pipette volume to the required value.

3. Hold the pipette in the air and press it down to the first stop.

4. Immerse the pipette tip below the surface of the liquid to be aspirated.

5. Slowly release the plunger to the original position.

6. Move the pipette to the target container.

7. Press the plunger down to the second stop to dispense all the liquid.

8. Press the tip ejector button to discard the tip.

**Reverse Pipetting (Press 2 – Release 1)**

1. Attach the appropriate tip to the pipette.

2. Adjust the pipette volume to the required value.

3. Hold the pipette in the air and press it down to the second stop.

4. Immerse the pipette tip below the surface of the liquid to be aspirated.

5. Slowly release the plunger to the original position.

6. Move the pipette to the target container.

7. Press the plunger down to the first stop to dispense the required volume of liquid.

8. Press the tip ejector button to discard the tip.

**Notes:**

1. Each pipette corresponds to specific tips. Different volume ranges require different tips, which are often color-coded.

2. Always use the pipette within its specified range; do not go below the minimum volume.

3. When attaching the tip, apply firm pressure and gently rock side to side to ensure it is securely fitted.

4. Forward pipetting is used in most pipetting situations. Reverse pipetting is suitable for viscous liquids (where forward pipetting may leave residue and cause inaccurate pipetting) or when bubbles must be avoided (since forward pipetting can easily generate bubblesbubbles).

5. When using reverse pipetting, aspirate the liquid slowly; otherwise, due to inertia, the liquid may surge upwards and contaminate the pipette.

6. Do not place the pipette horizontally or upside down after aspirating liquid, to prevent contamination.

7. After use, set the pipette to its maximum volume and place it back in its holder.

8. Tips should be sterilized using autoclaving at 121°C for 20 minutes. Secure the tip box with rubber bands, and wrap in kraft paper or newspaper if necessary.

### Tray Balance

▼

**Purpose:**

Used to weigh solid or liquid samples required for experiments, ensuring accurate measurement.

**Materials:**

* Sample to be weighed
* Weighing paper or weighing boat

**Protocol:**

1. Before use, check whether the balance is level and adjust the leveling screws if necessary;

2. Ensure the sliding weight is set to zero and the pointer aligns with the zero mark on the scale;

3. Place the weighing paper or weighing boat on the pan and re-balance to zero;

4. Gradually add the sample to the weighing paper or boat until it approaches the desired mass;

5. Move the sliding weight to fine-tune until the pointer returns to zero, then record the reading;

6. After weighing, clean the weighing paper and remove any residual sample to keep the balance clean;

7. When finished, return the sliding weight to zero and cover the balance with the dust cover.

### Electronic Balance

▼

**Purpose:**

Used for rapid and precise weighing of solid or liquid samples required for experiments.

**Materials:**

* Sample to be weighed
* Weighing paper or weighing boat

**Protocol:**

1. Before use, check that the electronic balance is placed in a stable environment free of vibration and airflow;

2. Turn on the power and allow the balance to preheat for a few minutes to ensure stable readings;

3. Place the weighing paper or weighing boat on the pan and press the TARE button to reset the display to zero;

4. Slowly add the sample to avoid spillage or overloading;

5. Read the mass value displayed on the screen and record it promptly;

6. After weighing, remove the sample and confirm the balance returns to zero;

7. After use, turn off the power and keep the balance clean to prevent residues from affecting accuracy.

### Electronic Analytical Balance

▼

**Purpose:**

Used for high-precision measurement of sample mass, suitable for scientific experiments that require high weighing accuracy.

**Materials:**

* Sample to be weighed
* Weighing paper or weighing boat
* Tweezers (for placing or removing samples to avoid contamination by hand contact)

**Protocol:**

1. Place the balance in a stable environment free of vibration, airflow, and with constant temperature. Ensure it is properly leveled;

2. Turn on the power and allow the balance to preheat for about 30 minutes to ensure measurement stability;

3. Open the draft shield, place the weighing paper or weighing boat on the pan, close the draft shield, and press the TARE button to reset to zero;

4. Use tweezers to gently place the sample into the weighing paper or boat, close the draft shield, and wait for the reading to stabilize;

5. Record the mass value displayed on the screen promptly;

6. For multiple measurements, press the TARE button to zero before each weighing;

7. After finishing, remove the weighing paper or boat, close the draft shield, turn off the power, and keep the balance clean;

8. Do not overload the balance to avoid damaging the sensor.

### Mini Centrifuge

▼

**Purpose:**

Used for rapid sedimentation or separation of small-volume samples (such as PCR tubes or microcentrifuge tubes).

**Materials:**

* Microcentrifuge tubes or PCR tubes
* Sample solution

**Protocol:**

1. Before use, check that the power supply is normal and ensure the centrifuge is placed on a stable laboratory bench;

2. Add the sample into the centrifuge tube, ensuring that the number of tubes used is symmetrical and the mass is balanced;

3. Open the centrifuge lid, place the tubes into the rotor symmetrically;

4. Close the lid securely and start centrifugation. Do not open the lid during operation. Remove the samples only after the rotor has completely stopped;

5. After use, turn off the power, wipe the surface of the centrifuge, and keep it clean;

6. Regularly check the rotor for cracks or wear to avoid potential safety hazards.

### Mini Benchtop Centrifuge

▼

**Purpose:**

Used for sedimentation and separation of small-volume samples (such as cells, proteins, or nucleic acid solutions).

**Materials:**

* Microcentrifuge tubes or compatible sample tubes
* Sample solution to be separated

**Protocol:**

1. Before use, ensure the centrifuge is placed on a stable and level laboratory bench;

2. Add samples into centrifuge tubes, ensuring strict balance between tubes;

3. Open the centrifuge lid and place the tubes symmetrically into the rotor;

4. Close the lid securely and set the appropriate speed and centrifugation time;

5. Start the centrifuge. Do not open the lid during operation;

6. Wait until the rotor has completely stopped before opening the lid and removing samples;

7. After use, turn off the power, wipe the inner chamber and outer surface of the centrifuge to remove condensation or residues;

8. Regularly inspect the rotor and tube adapters for cracksss or damage to avoid safety risksss.

### High-Speed Centrifuge

▼

**Purpose:**

Used to collect cells or insoluble fractions after cell lysis by centrifugation.

**Materials:**

* Bacterial solution after induction of expression

**Protocol:**

1. Strictly ensure balance!

2. The centrifuge rotor must be used together with the corresponding lid and centrifuge containers;

3. The rotor and lid must be tightened securely;

4. Centrifugation parameters must comply with the rotor’s usage requirements;

5. The loading volume must not exceed 2/3 of the container capacity;

6. After use, wipe the centrifuge to remove condensation, and store the rotor inverted in a 4 °C refrigerator.

### Ultracentrifuge

▼

**Purpose:**

Used for high-speed separation of proteins, nucleic acids, viral particles, organelles, and other biomolecules. It is suitable for ultrahigh-speed sedimentation and separation of substances with small molecular weight or close density.

**Materials:**

* Sample solution to be separated
* Specialized ultracentrifuge tubes
* Matching rotor and rotor lid

**Protocol:**

1. Strictly balance samples: Centrifuge tubes must be placed in pairs with weight differences controlled within 0.001 g;

2. Use specialized tubes: Ensure centrifuge tubes match the rotor model to prevent rupture under high centrifugal force;

3. Sample loading: Fill centrifuge tubes completely to avoid cavitation. Use sealing caps or sealing film if necessary;

4. Install rotor: Place the rotor into the chamber, ensure it is stable, and tighten the rotor lid securely;

5. Set parameters: Adjust speed and time according to experimental requirements, strictly following the rotor’s maximum speed and temperature limits;

6. Safety check: Close the centrifuge chamber lid, ensure the chamber is properly vacuum-sealed before starting;

7. Do not open lid during operation: The centrifuge must not be opened while running. The operator should stay in the lab and monitor the process;

8. Finishing operation: Wait until the rotor has completely stopped before opening the lid and removing samples. Do not force stop the centrifuge;

9. Maintenance: After use, wipe condensation from the chamber, store the rotor inverted in a 4 °C refrigerator to prevent corrosion and damage;

10. Notes: Only trained personnel are allowed to operate the ultracentrifuge. Untrained individuals are strictly prohibited from using it.

### Vortex Mixer

▼

**Purpose:**

Used for rapid mixingiing of small-volume liquid sampleses, such as solutions, suspensionss, and reagents.

**Materials:**

* Sample tubes (e.g., EP tubes, centrifuge tubeses)
* Samples to be mixed

**Protocol:**

1. Before use, check that the instrument is placed stably on the laboratory bench;

2. Turn on the power switch and select the appropriate mode (touch mode or continuous mode);

3. Hold the bottom of the sample tube vertically and press it gently onto the mixingiing head, ensuring full contact;

4. Adjust the speed control knob to select the appropriate vortexing speed as required;

5. Avoid applying excessive force during mixingiing to prevent tube damage or liquid splashing;

6. After mixingiing, remove the sample tube and turn off the power;

7. After use, keep the instrument clean and avoid reagent residues on the mixiing head.

### Magnetic Stirrer

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**Purpose:**

Used for uniform stirring and mixing of liquid samples, commonly applied in dissolving, reactions, or maintaining solution homogeneity.

**Materials:**

* Solution to be stirred
* Beaker or Erlenmeyer flask
* Stir bar (magnetic bar)

**Protocol:**

1. Before use, ensure the stirrer is placed on a level and stable laboratory bench;

2. Place an appropriately sized stir bar into the container, then add the solution to be stirred;

3. Position the container at the center of the magnetic stirrer, aligning it with the stir bar;

4. Turn on the power and slowly adjust the speed control knob, gradually reaching the desired stirring speed to avoid stir bar jumping or liquid splashing;

5. If heating is required, turn on the heating function and set the desired temperature, taking care to avoid overheating;

6. Monitor the process to prevent excessive evaporation or container movement;

7. After stirring, reduce the speed to zero before turning off the power;

8. Remove the stir bar, clean the container and the instrument, and keep the device in good condition.

### Water Bath

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**Purpose:**

Used for constant temperature heating of samples, enzyme reactions, incubation of cells or proteins, and other laboratory temperature control operations.

**Materials:**

* Samples to be heated or kept at constant temperature
* Suitable containers (e.g., centrifuge tubes, culture flasks)
* Distilled water or deionized water (for filling the water bath)

**Protocol:**

1. Before use, check whether there is sufficient water in the bath. The water level should be above the heating element to avoid dry heating;

2. Connect the power supply and turn on the power switch;

3. Set the required temperature and wait until the water bath reaches and stabilizes at the set temperature;

4. Place the samples in appropriate containers and immerse them in the water bath, ensuring proper sealing to avoid water vapor contamination;

5. During the experiment, regularly check whether the temperature is stable and the water level is sufficient;

6. After the experiment, remove the samples and turn off the power switch;

7. If not used for a long time, drain the water from the bath and wipe it clean to keep the equipment dry and tidy.

### Dry Bath

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**Purpose:**

Used for constant temperature heating of samples, enzyme reactions, nucleic acid denaturation/renaturation, protein incubation, and other experimental operations. Commonly used as a liquid-free alternative to a water bath.

**Materials:**

* Samples to be heated
* Dedicated centrifuge tubes or PCR tubes
* Corresponding metal heating blocks (different apertures match different tube types)

**Protocol:**

1. Before use, ensure the dry bath is placed stably and the power supply is properly connected;

2. Install the appropriate metal block (ensure it matcheshes the type of tube used in the experiment);

3. Turn on the power switch, set the required temperature and time, and wait until the bath reaches and stabilizes at the set value;

4. Place the sample tubeses into the holeses of the metal block, ensuring close contact for uniform heat transfer;

5. During heating, avoid frequent opening of the cover or moving the sampleses to maintain temperature stability;

6. After the experiment, remove the sampleses and turn off the power;

7. Once cooled, wipe the surface of the metal block to keep it clean and avoid corrosion or contamination.

### SONICS Ultrasonic Cell Disruptor

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**Purpose:**

Used to disrupt bacteria for protein extraction.

**Materials:**

* Bacterial suspension resuspended in protein buffer
* Ultrapure water

**Protocol:**

1. Avoid running without load (the ultrasonic probe must be inserted into the sample solution before starting ultrasound);

2. The insertion depth of the ultrasonic probe should be 1/3–2/3 of the liquid level. Insertion that is too deep will reduce disruption efficiency. The probe should be positioned in the center of the liquid and must not touch the container wall;

3. Different probes correspond to different ultrasonic power levels;

4. Set the amplitude using the “AMPL” button;

5. Set the pulse duration for each ultrasound using the “PULSER” button (Pulse on = ultrasound; Pulse off = pause);

6. Set the total ultrasound time using the “TIMER” button:

o Total time = time per pulse × number of pulses.

### Alcohol Burner

▼

**Protocol:**

1. Open the alcohol burner cover and light the burner using a lighter or a match.

2. To extinguish the alcohol burner, cover it twice with the cap to ensure the flame is completely out.

**Notes:**

1. Before using the alcohol burner, check whether there is sufficient alcohol inside to avoid burning the wick when empty.

2. The alcohol burner should be filled with 95% ethanol.

3. Before lighting, make sure there is no alcohol or other flammable liquid on gloves or the workbench.

4. When sterilizing instruments (such as inoculation loops or needles), use the outer flame of the alcohol lamp.

### Autoclave

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**Protocol:**

1. Check the external water level gauge of the autoclave to ensure the water level is neither too high nor too low. Check the pressure gauge to confirm normal pressure. If the water level is too high, use the drain pipe at the bottom to release water until the level is appropriate.

2. Turn on the autoclave power and switch, and after confirming safety, open the lid.

3. Check the water level inside the autoclave to ensure it is not too high or too low. If too low, add distilled water.

4. Place the items to be sterilized in heat-resistant containers.

5. Close the lid, confirm it is sealed properly, then set the parameters and start sterilization.

6. After sterilization, wait until the pressure is safe and the temperature drops below 60 °C before opening the lid to remove sterilized items.

7. Tighten the lid of the autoclave and turn off the switch and power supply.

**Notes:**

1. When opening the lid, always press down on the center of the lid with one hand. Do not press the left or right side.

2. Once the lid is opened, hold it with both hands. When closing, also support the lid with both hands.

3. Do not add tap water or other ion-rich water, as it can cause scale buildup and localized overheating. Only distilled water should be added.

4. Sterilized items should not be stacked too high or touch the autoclave lid.

5. For LB medium, typically sterilize at 121 °C for 20 minutes. For glucose-containing medium, do not sterilize at 121 °C (to avoid caramelization). Instead, filter-sterilize glucose solutions. Antibiotics decompose at high temperature and should also be sterilized by filtration. For reagentsss that require sterilization, heating, or dissolution, select the corresponding program.

6. For glassware, do not seal completely. Use sealing film to loosely cover openings. For test tubeses, do not screw caps on tightly. For reagent bottles, also avoid completely tightening caps.

7. For pipette tip sterilization, secure the tip boxesess with rubber bands, and wrap them with newspaper or kraft paper if necessary.

8. For glassware, pipette tips, waste liquidsss, and used culture media, select 121 °C for 20 minutes.

9. Used culture media should be placed in plastic trash bags before sterilization.

10. After starting sterilization, you must sign the log sheet. Ensure the log sheet corresponds exactly to the specific autoclave being used.

11. After sterilization, only open the lid when the temperature has dropped below 60 °C, and be careful of steam when opening.

12. After using the autoclave, check whether any items remain inside. Confirm all have been removed before closing the lid securely.

### Laminar Flow Clean Bench

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**Purpose:**

Provides a sterile, dust-free localized clean environment for sample handling. Commonly used in cell culture, aseptic operations, and molecular biology experiments.

**Materials:**

* Experimental samples to be handled
* Sterile consumables (e.g., pipettes, tips, Petri dishes, centrifuge tubes)
* 75% ethanol or other disinfectants

**Protocol:**

1. Preparation before use

o Turn on the clean bench power and fan, and let it run for 15–30 minutes to establish stable laminar flow;

o Wipe the work surface and item surfaces with 75% ethanol to maintain sterility.

2. Arrangement of items

o Place required items in the bench according to the order of use, avoiding blockage of airflow;

o During operation, minimize crossing movements of hands and items inside the bench to avoid disturbing laminar flow.

3. Experimental operation

o Operators must wear lab coats, masks, and disposable gloves;

o Perform all manipulations in the central area of the airflow;

o Avoid sudden or large movements to maintain laminar flow stability.

4. Post-use procedure

o Remove samples and consumables after the experiment;

o Wipe the work surface again with 75% ethanol;

o Turn off the fan and power. If conditions allow, turn on the UV lamp for 15–30 minutes for disinfection.

5. Notes

o A laminar flow clean bench only protects the sample from contamination, but does not protect the operator. For work involving pathogens or hazardous gases, a biosafety cabinet must be used instead.

### Incubator

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**Purpose:**

Used for constant-temperature culture of cells, microorganisms, or tissue samples, providing a stable temperature and environmental conditions.

**Materials:**

* Samples to be cultured (e.g., cell culture flasks, Petri dishes, culture media)
* Suitable culture containers
* Disinfectants (e.g., 75% ethanol)

**Protocol:**

1. Preparation before use

o Check that the incubator is placed stably and that the power connection is normal;

o Wipe the inside of the incubator with 75% ethanol to maintain cleanliness;

o Set the temperature according to experimental requirements (e.g., 37 °C), and wait until it stabilizes.

2. Placing samples

o Place culture samples in the incubator, avoiding overcrowding of containers to ensure even airflow and temperature distribution;

o For CO₂ incubators, confirm that gas supply is functioning properly, and maintain appropriate humidity to prevent evaporation of culture medium.

3. During incubation

o Minimize the frequency and duration of opening the incubator door to avoid fluctuations in temperature, humidity, and gas concentration;

o Regularly monitor the culture status, and if necessary, perform handling under aseptic conditions.

4. After use

o Remove samples and clean up any remaining containers after the experiment;

o Regularly disinfect and clean the incubator to prevent microbial contamination;

o If not in use for a long time, turn off the power and keep the incubator dry.

5. Notes

o Different types of incubation require suitable incubators (e.g., standard constant-temperature incubator, CO₂ incubator, shaking incubator);

o Do not place flammable, volatile, or toxic substances inside.

### Shaker

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**Purpose:**

Used for mixing and culturing liquid samples, commonly applied in cell culture, microbial culture, and accelerating solution reactions.

**Materials:**

* Samples to be cultured or mixed (e.g., Erlenmeyer flasks, culture flasks, centrifuge tubes)
* Culture medium or reaction solution

**Protocol:**

1. Preparation before use

o Check that the shaker is placed stably and the power connection is normal;

o Ensure that clamps or the platform are securely installed to prevent samples from sliding during operation.

2. Placing samples

o Place sample containers evenly on the shaker platform to maintain balance and avoid uneven loading;

o Use appropriate clamps or rubber bands to secure containers and prevent them from falling during operation.

3. Setting parameters

o Set the shaking speed (rpm) and running time according to experimental requirements;

o For temperature-controlled shakers, also set the required temperature and wait until stabilization before starting the experiment.

4. During operation

o Start the shaker and ensure smooth operation without abnormal noise;

o Avoid opening the lid or moving samples during operation;

o Regularly monitor the culture or mixing process.

5. After use

o Stop the shaker, and wait until it has completely come to rest before removing samples;

o Turn off the power and clean the shaker platform to keep the equipment tidy.

6. Notes

o Do not overload the shaker or place containers that are too large;

o For cell or microbial culture, disinfect the shaker regularly to prevent contamination.

### NanoDrop Spectrophotometer

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**Purpose:**

Used for rapid determination of the concentration and purity of nucleic acids, proteins, or other samples.

**Materials:**

* Samples to be measured (e.g., DNA, RNA, or protein solutions)
* Deionized water or buffer (for blank calibration)
* Lint-free tissue (e.g., Kimwipes, for wiping the measurement pedestal)

**Protocol:**

1. Turn on the instrument, launch the NanoDrop software, and select the appropriate measurement mode (e.g., Nucleic Acid, Protein, UV-Vis, etc.);

2. Open the measurement arm, pipette 1–2 µL of buffer or deionized water onto the lower pedestal, close the arm, and perform blank calibration (Blank);

3. Open the arm, wipe the upper and lower pedestals with lint-free tissue to keep them clean;

4. Pipette 1–2 µL of the sample onto the pedestal, close the arm, and click Measure to start detection;

5. Record the sample’s concentration (ng/µL or mg/mL), purity ratios (e.g., A260/A280), and other relevant data;

6. After measurement, wipe the pedestal with lint-free tissue, and optionally add deionized water again to clean;

7. After use, close the software and turn off the instrument.

**Notes:**

* Sample volume should be limited to 1–2 µL to avoid contamination and waste;
* Clean the pedestal after each measurement to prevent residues from affecting subsequent samples;
* Regularly calibrate and maintain the instrument to ensure measurement accuracy.

### Spectrophotometer

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**Purpose:**

Used to measure the light absorption of samples at specific wavelengths, thereby analyzing their concentration, purity, or chemical properties.

**Materials:**

* Sample solution to be measured
* Cuvettes (quartz or glass, depending on detection wavelength)
* Blank control solution (e.g., deionized water or buffer)

**Protocol:**

1. Turn on the instrument and allow it to preheat for a few minutes until the light source stabilizes;

2. Select and set the detection wavelength according to experimental requirements;

3. Rinse the cuvette with deionized water or buffer, dry the outer walls, and avoid fingerprints or water droplets that may affect light transmission;

4. Fill the cuvette with the blank control solution, place it into the cuvette holder, close the lid, and press the Blank button to perform blank calibration;

5. Remove the blank cuvette and replace it with the sample-filled cuvette, ensuring consistent placement orientation (transparent sides facing the light path);

6. Record the absorbance (Abs) or transmittance (T%) of the sample at the set wavelength;

7. If a standard curve is required, measure a series of standard samples with concentration gradients;

8. After measurement, clean the cuvettes and turn off the instrument.

**Notes:**

* The outer walls of cuvettes must remain clean and transparent, free of bubbles or residual liquid;
* Quartz cuvettes must be used when detecting in the UV range (200–350 nm);
* Sample volume should be sufficient to fill the cuvette’s light path, typically 2/3–3/4 full.

### Electrophoresis Apparatus and Power Supply

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**Purpose:**

Used for the separation and detection of nucleic acids (DNA/RNA) or protein samples.

**Materials:**

* Agarose or polyacrylamide gels
* Electrophoresis buffer (e.g., TAE, TBE, SDS-PAGE buffer, etc.)
* Samples to be tested and loading buffer
* Electrophoresis tank and power supply
* DNA/RNA stain or protein staining reagents

**Protocol:**

1. Gel preparation

o Prepare agarose or polyacrylamide gel solution according to experimental requirements. Add appropriate dye (e.g., EB or a safer alternative), pour into the gel mold, and insert the comb. After solidification, carefully remove the comb.

2. Sample loading preparation

o Place the solidified gel into the electrophoresis tank and add sufficient electrophoresis buffer to cover the gel surface;

o Mix samples with loading buffer and carefully pipette into the gel wells. Load a molecular weight marker (ladder) in one well.

3. Running electrophoresis

o Cover the electrophoresis tank and connect it to the power supply, ensuring correct electrode orientation (DNA/RNA migrates toward the positive electrode);

o Set appropriate voltage/current (typically 80–150 V for agarose gels; SDS-PAGE usually starts at 80–120 V, then increases to 120–180 V);

o Start the power supply to begin electrophoresis.

4. Completion and processing

o When the tracking dye has migrated to the desired position, turn off the power and disconnect the electrodes;

o Carefully remove the gel for UV visualization (nucleic acids) or staining/destaining procedures (proteins).

5. Notes

o Ensure sufficient electrophoresis buffer to prevent the gel from drying out during the run;

o Load samples gently to avoid breaking wells or causing diffusion;

o Always wear protective goggles and shields when using UV light to avoid UV damage;

o Never touch electrodes during electrophoresis. Observe proper electrical safety.

### Semi-dry Transfer Apparatus

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**Purpose:**

Used after protein electrophoresis (SDS-PAGE) to rapidly transfer proteins from the gel onto PVDF or NC membranes for subsequent Western Blot analysis.

**Materials:**

* Gel after protein electrophoresis
* PVDF or NC membrane (membrane requires pretreatment, e.g., PVDF needs methanol activation)
* Filter paper (cut to the same size as the gel)
* Transfer buffer
* Semi-dry transfer apparatus and power supply

**Protocol:**

1. Membrane and gel preparation

o Immerse the PVDF membrane in methanol for 1 minute, then equilibrate it in transfer buffer;

o NC membranes can be directly equilibrated in transfer buffer;

o After electrophoresis, remove the gel, trim off excess parts, and equilibrate the gel in transfer buffer.

2. Assembly of transfer sandwich (from bottom to top)

o Filter paper (pre-soaked in transfer buffer)

o PVDF/NC membrane

o Gel

o Filter paper (pre-soaked in transfer buffer)

o Carefully remove any air bubbles using a roller to ensure even contact.

3. Transfer operation

o Place the assembled sandwich into the semi-dry transfer apparatus with the membrane on the anode side and the gel on the cathode side;

o Cover with the upper electrode plate, ensuring good contact;

o Connect the power supply and set the appropriate current/voltage (common conditions: current 0.8–1 mA/cm², transfer time 30–90 minutes, adjusted according to gel thickness and protein size);

o Start the transfer program.

4. Completion of transfer

o Turn off the power, disassemble the sandwich, and remove the membrane and gel;

o Immediately mark the membrane orientation (e.g., cut the top-left corner) and proceed with blocking and immunoblotting as required.

5. Notes

o All filter papers must be fully soaked to prevent dry spots that cause uneven transfer;

o Avoid air bubbles during assembly, as they will lead to blank spots on the membrane;

o Do not apply excessive current for long durations, as overheating can inactivate proteins.

### Polymerase Chain Reaction (PCR) Thermocycler

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**Purpose:**

Used for *in vitro* amplification of specific DNA fragments to facilitate molecular biology studies such as cloning, gene detection, and mutation analysis.

**Materials:**

* ddH₂O
* PCR buffer
* dNTPs
* Template DNA
* Forward and reverse primers
* DNA polymerase (e.g., Taq polymerase)
* PCR tubes

**Protocol:**

1. Prepare the reaction mixture

o On ice, prepare the PCR mixture (ddH₂O, buffer, dNTPs, primers, template DNA, DNA polymerase). Mix thoroughly and dispense into PCR tubes.

2. Power on and program setup

o Turn on the PCR thermocycler;

o Set the amplification program on the control panel or software, including:

 Initial denaturation (e.g., 94–95 °C, 2–5 minutes);

 Cycling parameters:

 Denaturation (94–95 °C, 30 seconds);

 Annealing (50–65 °C, 30 seconds);

 Extension (72 °C, 30 seconds–2 minutes, depending on fragment size);

 Number of cycles (typically 25–40);

 Final extension (72 °C, 5–10 minutes);

 Hold (4 °C).

3. Load samples

o Open the lid of the thermocycler and place the PCR tubes into the sample block, ensuring tight contact with the metal module;

o Close and lock the lid.

4. Run the program

o Confirm the program settings, then start the run;

o The thermocycler will automatically perform the programmed amplification cycles.

5. Completion

o After amplification, remove the PCR tubes;

o Turn off the power and clean the workspace.

**Notes:**

* PCR tubes must be compatible with the instrument model to avoid uneven heating;
* Always prepare the reaction mixture on ice to minimize nonspecific amplification;
* Ensure the thermocycler is properly calibrated and temperature control is accurate before use.

### Estain L1 Protein Staining Instrument

▼

**Purpose:**

Used for rapid fixation and staining of protein gels after electrophoresis, commonly applied in protein detection and analysis following SDS-PAGE.

**Materials:**

* Protein gel after electrophoresis
* Distilled water
* Filter paper
* Gel fixing clamp (with mesh)
* Estain L1 protein staining instrument

**Protocol:**

1. Add distilled water into the tray;

2. Carefully remove the gel after electrophoresis and immerse it in the tray for about 1 minute (Note: If using Life Tech. GEL, trim off any excess protrusions);

3. Open the gel fixing clamp and place it flat on the lab bench;

4. Place the pre-soaked gel onto the mesh surface of the clamp, ensuring the gel wells face the central axis and are positioned close to it (but not beyond the mesh surface);

5. Take a piece of filter paper, soak it in the tray, and place it flat on top of the gel;

6. Close the gel fixing clamp securely;

7. Insert the gel fixing clamp into the instrument channel, ensuring the correct orientation (mesh surface facing the operator);

8. Press the START B button for the corresponding channel to begin operation. The screen will display a countdown, and the START B button will flash;

9. Before the operation ends, the instrument will emit a beep signal;

10. After completion, the instrument will continue beeping. Press the START B button again to stop, and the screen will return to the default display.

**Notes:**

* Ensure the gel is fully soaked before operation to avoid cracking or uneven staining;
* Always check orientation when inserting the gel fixing clamp to avoid incorrect operation;
* After use, clean the tray and fixing clamp promptly to keep the instrument in good condition.

### BG-gdsUVIEW Ultraviolet Analyzer

▼

**Purpose:**

Used to observe and analyze nucleic acid electrophoresis gel results and perform gel excision, commonly applied in DNA recovery and subsequent molecular cloning experiments.

**Materials:**

* Nucleic acid gel after electrophoresis
* Gel cutter or scalpel blade
* Sample tray
* Camera or imaging system (optional)

**Protocol:**

1. Startup preparation

o Connect the instrument to a 220V power supply and turn on the main power switch;

o Pull out the drawer-style light box and place the sample on the tray.

2. Sample observation

o Turn on the light source switch;

o Lift the protective cover of the observation window to directly view the electrophoresis bands;

o If photography is required, close the protective cover of the observation window, open the lens cap, insert the camera lens into the lens port, and align with the sample for imaging.

3. Gel excision

o Open the side observation windows;

o Hold the gel cutter with both hands and insert them into the dark chamber to operate;

o Simultaneously observe the electrophoresis bands through the observation window and cut out the target band according to the desired size.

4. Completion

o Remove the excised gel block and sample;

o Clean the inside of the dark chamber to maintain instrument cleanliness;

o Turn off the light source and the main power switch.

**Notes:**

* Ultraviolet light is harmful to eyes and skin. Always wear protective goggles or use the observation window’s protective device during operation;
* Perform gel cutting gently to avoid damaging the target band;
* Clean the sample tray and gel cutter promptly to prevent cross-contamination;
* Disconnect the power supply and cover the instrument with a dust cover when not in use for extended periods.

### Microplate Reader

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**Purpose:**

Used to detect optical signals such as absorbance (OD), fluorescence, or luminescence in 96-well or 384-well microplates. Commonly applied in ELISA, enzyme activity assays, and cell-based experiments.

**Materials:**

* Prepared 96-well or 384-well microplate
* Samples and controls required for the experiment (standards, positive/negative controls)
* Microplate reader and supporting software

**Protocol:**

1. Startup and preparation

o Turn on the microplate reader and launch the software;

o Select the appropriate detection mode (e.g., absorbance, fluorescence, luminescence) based on experimental requirements.

2. Sample preparation

o Add samples, standards, and controls into the designated wells of the microplate;

o If incubation or color development is required, ensure these steps are completed before measurement.

3. Detection settings

o Place the microplate into the reader tray, ensuring the correct orientation;

o Set detection parameters in the software, such as wavelength (commonly 450 nm, 570 nm, 600 nm, etc.), reading mode (single-point or multi-point), and plate reading sequence.

4. Running and data acquisition

o Start the detection program. The instrument will automatically read each well;

o Once reading is completed, results will be displayed in the software interface;

o Export data in Excel, PDF, or other formats for further analysis.

5. Completion

o Remove the microplate and properly dispose of waste solutions;

o Close the software and power off the instrument;

o Clean the tray and workspace to maintain instrument hygiene.

**Notes:**

* The bottom of the microplate must be clean and free of bubbles, as these may affect accuracy;
* Select the appropriate detection mode and wavelength depending on the experiment;
* Regular wavelength calibration and optical path maintenance are required to ensure measurement accuracy.

### Gel Filtration Chromatography / Size-Exclusion Chromatography (SEC, Molecular Sieve)

▼

**Purpose:**

Used to separate biomacromolecules such as proteins, peptides, and polysaccharides based on molecular size. Common applications include sample purification, buffer exchange, and detection of protein aggregation states.

**Materials:**

* Chromatography column (packed with size-exclusion media such as Sephadex, Superdex, or Sephacryl)
* Sample solution to be separated
* Chromatography buffer (filtered and degassed)
* Chromatography system (including pump, injection valve, detector, and fraction collector)
* Collection tubes

**Protocol:**

1. System preparation

o Check all connections in the chromatography system to ensure there are no leaks;

o Equilibrate the column with filtered and degassed buffer (typically 1–2 column volumes, CV);

o Set an appropriate flow rate (commonly 0.5–1.0 mL/min, depending on column specifications).

2. Sample preparation

o Clarify samples by centrifugation or filtration (0.22 µm) to remove particulates or precipitates;

o Sample volume should not exceed 2–5% of the column volume to maintain resolution.

3. Sample loading and separation

o Inject the sample via the sample loop or injection port, then switch to load mode;

o Start buffer pumping to initiate separation;

o Monitor UV absorbance in real time (typically at 280 nm) or other detection signals.

4. Fraction collection

o Collect fractions based on chromatogram peak profiles, labeling tube numbers accordingly;

o Further analyze fractions for purity if required (e.g., SDS-PAGE, Western blot).

5. Shutdown and storage

o After separation, wash the column with 1–2 CV of buffer;

o For long-term storage, wash the column with storage solution (e.g., 20% ethanol) and seal properly;

o Turn off the system power and clean the sample loop and tubing.

**Notes:**

* Avoid air bubbles in the column; always degas the buffer before use;
* Never allow the column bed to dry, keep it constantly hydrated;
* Control sample concentration and injection volume to prevent overloading and loss of resolution;
* Monitor system pressure during operation to avoid column damage due to overpressure.

### Optical Microscope

▼

**Purpose:**

Used to observe the morphology and structure of cells, tissue sections, microorganisms, or other transparent/semi-transparent samples.

**Materials:**

* Prepared specimen slides (e.g., tissue sections, cell smears)
* Cover slips
* Tweezers, dropper (as needed)

**Protocol:**

1. Preparation before use

o Place the microscope on a stable workbench and connect the power supply;

o Turn on the light source and adjust the brightness to a suitable level;

o Check whether the objective lenses and eyepieces are clean; if dusty, wipe gently with lens paper.

2. Placing the sample

o Place the specimen slide at the center of the stage and secure it with stage clips;

o Select the lowest magnification objective (usually 4× or 10×), and rotate the coarse focus knob to bring the objective close to the sample.

3. Adjustment and observation

o Observe through the eyepiece, slowly rotate the coarse focus knob backward until the sample comes into clear focus;

o Use the fine focus knob to improve sharpness;

o Switch gradually to higher magnification objectives (40×, 100× oil immersion, etc.) as needed, and use fine focus after each change.

4. After use

o Return to the low-power objective and lower the stage;

o Turn off the light source and unplug the power;

o Wipe the lenses and stage to keep the microscope clean;

o Cover with a dust-proof cover and store in a dry environment.

**Notes:**

* When using the oil immersion lens (100×), add cedar oil and clean it immediately after use;
* Avoid touching the lenses directly with fingers;
* Do not use the coarse focus knob with high-power objectives to prevent damage to the slide or lens.

### Scanning Electron Microscope (SEM)

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**Purpose:**

Used to observe the microscopic morphology and structural features of sample surfaces. Commonly applied in materials science, biology, and nanotechnology research.

**Materials:**

* Samples to be tested (solid samples or biological samples that have been fixed and dried)
* Conductive adhesive or sample holder (stub)
* Metal coating materials (e.g., gold, platinum; for sputter-coating non-conductive samples)
* SEM instrument and control software

**Protocol:**

1. Sample preparation

o Solid samples: cut to an appropriate size and mount onto the sample holder;

o Biological or non-conductive samples: fix, dehydrate, dry, and sputter-coat with metal to enhance conductivity.

2. Sample loading

o Attach the sample to the stub using conductive adhesive;

o Place the sample holder into the SEM chamber and close the chamber door.

3. Startup and settings

o Turn on the SEM power; sequentially start the vacuum system, cooling system, and electron gun high voltage;

o Wait until the chamber reaches the required vacuum level;

o Set parameters in the control software, including accelerating voltage, magnification, and detector mode (secondary electron or backscattered electron imaging).

4. Imaging and adjustment

o Activate the electron beam and move the stage to locate the target area;

o Adjust focus, brightness, contrast, and working distance until the image is clear and stable;

o Observe multiple regions and magnifications as needed, and acquire images.

5. After use

o Turn off the electron beam and high-voltage power supply;

o Sequentially shut down the vacuum pump, cooling system, and main power;

o Remove the sample and clean the sample stage.

**Notes:**

* Samples must be dry and conductive; otherwise, charging artifacts may occur and distort the image;
* Avoid rapid switching of high voltage or frequent on/off of the electron gun to prevent damage;
* Do not focus at high magnification on the same region for extended periods to reduce electron beam damage;
* Only trained personnel should operate SEM, with familiarity in vacuum and high-voltage procedures.

### Talos 200S Transmission Electron Microscope (TEM)

▼

**Purpose:**

Used to observe ultrastructures of cells or proteins, such as nanodiscs.

**Materials:**

* Prepared samples
* 300-mesh carbon support grids

**Protocol:**

1. Startup

o Sequentially turn on the TEM main power, vacuum pump, cooling system, and high-voltage power supply;

o Wait for the instrument to complete self-checks and then enter the operation software interface.

2. Sample loading

o Carefully mount the prepared sample onto the TEM loading rod;

o Ensure the sample is positioned accurately and securely fixed.

3. Parameter setup

o In the software interface, set appropriate parameters such as accelerating voltage, magnification, and focusing mode.

4. Imaging and analysis

o Activate the electron beam;

o Adjust the sample stage position, focusing rings, and apertures until the sample image is clearly displayed on the screen;

o Import recorded images or data into the computer for further processing and analysis.

5. Shutdown

o Turn off the electron beam and high-voltage power supply;

o Sequentially shut down the vacuum pump, cooling system, and main power supply.

**Notes:**

* Ensure samples are properly prepared and mounted to avoid beam damage or instability;
* Always follow correct vacuum and high-voltage operating procedures;
* Only trained personnel should perform TEM operations.

### Dynamic Light Scattering (DLS) Instrument

▼

**Purpose:**

Used to measure the particle size distribution, average hydrodynamic diameter, and aggregation state of nanoparticles, proteins, or colloids. It is commonly applied to assess the homogeneity and stability of samples.

**Materials:**

* Sample solution (proteins, nanoparticles, or colloids)
* Filtered buffer or solvent (for dilution and blank control)
* Disposable or quartz cuvettes (compatible with the DLS instrument)
* Micropipette and sterile tips

**Protocol:**

1. Sample preparation

o Centrifuge or filter the sample (e.g., through a 0.22 μm filter) to remove dust and large particles;

o Dilute the sample to an appropriate concentration to ensure scattering intensity is within the measurable range.

2. Instrument startup and settings

o Turn on the DLS instrument and launch the operating software;

o Select the desired measurement mode (particle size distribution, Zeta potential, etc.);

o Set the experimental parameters such as temperature, detection angle, and acquisition time.

3. Sample loading

o Use a pipette to transfer the sample into the cuvette, avoiding air bubbles;

o Place the cuvette into the sample holder, ensuring correct positioning.

4. Measurement process

o Click Measure to start data collection;

o The instrument analyzes fluctuations in scattered light caused by Brownian motion to calculate particle size;

o The software generates results automatically, including average hydrodynamic diameter and PDI (polydispersity index).

5. Experiment completion

o Record and export the measurement results (Excel or PDF format);

o Remove the cuvette, clean it immediately, and let it dry;

o Shut down the software and power off the instrument;

o Keep the instrument clean and dust-free.

**Notes:**

* Samples must be clear and free of bubbles, as these can cause significant artifacts;
* The concentration should be optimal: too high leads to multiple scattering, while too low results in weak signals;
* Ensure correct input of solvent parameters such as refractive index and viscosity for accurate calculation;
* Avoid vibrations and external light interference during measurement.

### pH Meter

▼

**Purpose:**

Used to measure the acidity or alkalinity (pH value) of solutions. Widely applied in biological experiments, chemical experiments, and culture medium preparation.

**Materials:**

* pH meter main unit
* Combination electrode or glass electrode
* Standard buffer solutions (pH 4.00, 7.00, 10.00)
* Sample solution
* Distilled or deionized water (for electrode rinsing)
* Soft lint-free tissue

**Protocol:**

1. Instrument preparation

o Turn on the power and allow the instrument to warm up for a few minutes;

o Check the electrode condition: the glass bulb should be filled with solution and not dry.

2. Calibration

o Rinse the electrode with distilled water and gently wipe dry;

o Immerse the electrode in the pH 7.00 standard buffer, press the Calibrate button;

o Follow software prompts to calibrate with pH 4.00 and pH 10.00 buffers in sequence to ensure measurement accuracy.

3. Sample measurement

o Rinse the electrode and gently dry;

o Immerse the electrode in the test solution, stir lightly, then let it stand until the reading stabilizes;

o Record the pH value.

4. End of operation

o After use, rinse the electrode with distilled water and wipe dry;

o For long-term storage, immerse the electrode in special storage solution (not dry);

o Turn off the power and keep the instrument and electrode clean.

**Notes:**

* The electrode should not touch hard surfaces or be over-wiped to avoid damaging the glass bulb;
* Always use fresh standard buffers for calibration;
* Between different samples, the electrode must be thoroughly rinsed to avoid cross-contamination;
* For long-term storage, keep the electrode in electrode storage solution or KCl solution.

### High-Performance Liquid Chromatography (HPLC)

▼

**Purpose:**

Used for separation, identification, and quantitative analysis of compounds in samples. Widely applied in pharmaceutical testing, protein purification, metabolite analysis, and other fields.

**Materials:**

* Prepared and filtered sample solution
* Mobile phase (filtered and degassed)
* Chromatography column (e.g., C18, ion-exchange, size-exclusion, depending on experiment)
* HPLC system (pump, injector, detector, and data acquisition system)
* Sample vials and injection syringe

**Protocol:**

1. System preparation

o Switch on the HPLC power, start the pump and detector;

o Flush the system with filtered and degassed mobile phase until the baseline stabilizes;

o Equilibrate the chromatography column (typically 10–20 minutes).

2. Sample preparation

o Filter the sample through a 0.22 µm or 0.45 µm membrane to remove particulates;

o Transfer to sample vials, place in autosampler tray or prepare for manual injection.

3. Chromatographic run

o Set detection parameters: mobile phase composition, flow rate, column temperature, detection wavelength, etc.;

o Program gradient elution if required;

o Inject the sample and start the run, recording chromatogram.

4. Data acquisition and analysis

o Use software to record signals and generate chromatograms;

o Perform quantitative analysis based on peak area or height;

o Identify compounds by comparing retention times with reference standards.

5. End of operation

o Flush the system with appropriate solvent (e.g., 100% water or methanol) to clean the column;

o For long-term storage, keep the column in the recommended storage solution;

o Turn off the pump, detector, and main power.

**Notes:**

* The mobile phase must be filtered and degassed to prevent bubbles interfering with detection;
* Samples must be clear and particle-free to avoid clogging the column;
* System pressure should remain within the recommended operating range of the column to prevent damage;
* Regularly clean the injector, sample loop, and flow path to maintain system performance.

# Reagent Formulation

## Culture Media

▼

### LB Medium (Luria-Bertani Medium)

▼

**LB Broth
Formula:**

* Tryptone 0.01 g/mL (10 g/L)
* Yeast extract 0.005 g/mL (5 g/L)
* Sodium chloride 0.01 g/mL (10 g/L)
* Deionized water
  OR
* Pre-mixed LB powder 0.025 g/mL (25 g/L)
* Deionized water

**Common preparation volumes:**

* 100 mL
  Tryptone 1 g, Yeast extract 0.5 g, NaCl 1 g, Deionized water to 100 mL OR Pre-mixed LB powder 2.5 g, Deionized water to 100 mL
* 200 mL
  Tryptone 2 g, Yeast extract 1 g, NaCl 2 g, Deionized water to 200 mL OR Pre-mixed LB powder 5 g, Deionized water to 200 mL
* 500 mL
  Tryptone 5 g, Yeast extract 2.5 g, NaCl 5 g, Deionized water to 500 mL OR Pre-mixed LB powder 12.5 g, Deionized water to 500 mL
* 1 L
  Tryptone 10 g, Yeast extract 5 g, NaCl 10 g, Deionized water to 1 L OR Pre-mixed LB powder 25 g, Deionized water to 1 L
* 2 L
  Tryptone 20 g, Yeast extract 10 g, NaCl 20 g, Deionized water to 2 L OR Pre-mixed LB powder 50 g, Deionized water to 2 L
* 3 L
  Tryptone 30 g, Yeast extract 15 g, NaCl 30 g, Deionized water to 3 L OR Pre-mixed LB powder 75 g, Deionized water to 3 L
* 4 L
  Tryptone 40 g, Yeast extract 20 g, NaCl 40 g, Deionized water to 4 L OR Pre-mixed LB powder 100 g, Deionized water to 4 L
* 5 L
  Tryptone 50 g, Yeast extract 25 g, NaCl 50 g, Deionized water to 5 L OR Pre-mixed LB powder 125 g, Deionized water to 5 L

**Sterilization:**

Autoclave at 121 °C for 20 min.

**Storage:**

Room temperature.

**Notes:**

1.Agar is not added when preparing LB broth.

2.Antibiotics are not added before autoclaving, since they are heat-labile. Add the appropriate antibiotic only after sterilization and cooling, depending on experimental requirements.

**LB Semi-Solid Medium**
**Formula:**

* Tryptone 0.01 g/mL (10 g/L)
* Yeast extract 0.005 g/mL (5 g/L)
* Sodium chloride 0.01 g/mL (10 g/L)
* Agar powder 0.3%–0.7%
* Deionized water
  OR
* Pre-mixed LB powder 0.025 g/mL (25 g/L)
* Agar powder 0.3%–0.7%
* Deionized water

**Common preparation volumes:**

* 100 mL
  Tryptone 1 g, Yeast extract 0.5 g, NaCl 1 g, Agar 0.3–0.7 g, Deionized water to 100 mL OR Pre-mixed LB powder 2.5 g, Agar 0.3–0.7 g, Deionized water to 100 mL
* 200 mL
  Tryptone 2 g, Yeast extract 1 g, NaCl 2 g, Agar 0.6–1.4 g, Deionized water to 200 mL OR Pre-mixed LB powder 5 g, Agar 0.6–1.4 g, Deionized water to 200 mL
* 500 mL
  Tryptone 5 g, Yeast extract 2.5 g, NaCl 5 g, Agar 1.5–3.5 g, Deionized water to 500 mL OR Pre-mixed LB powder 12.5 g, Agar 1.5–3.5 g, Deionized water to 500 mL
* 1 L
  Tryptone 10 g, Yeast extract 5 g, NaCl 10 g, Agar 3–7 g, Deionized water to 1 L OR Pre-mixed LB powder 25 g, Agar 3–7 g, Deionized water to 1 L
* 2 L
  Tryptone 20 g, Yeast extract 10 g, NaCl 20 g, Agar 6–14 g, Deionized water to 2 L OR Pre-mixed LB powder 50 g, Agar 6–14 g, Deionized water to 2 L
* 3 L
  Tryptone 30 g, Yeast extract 15 g, NaCl 30 g, Agar 9–21 g, Deionized water to 3 L OR Pre-mixed LB powder 75 g, Agar 9–21 g, Deionized water to 3 L
* 4 L
  Tryptone 40 g, Yeast extract 20 g, NaCl 40 g, Agar 12–28 g, Deionized water to 4 L OR Pre-mixed LB powder 100 g, Agar 12–28 g, Deionized water to 4 L
* 5 L
  Tryptone 50 g, Yeast extract 25 g, NaCl 50 g, Agar 15–35 g, Deionized water to 5 L OR Pre-mixed LB powder 125 g, Agar 15–35 g, Deionized water to 5

**Sterilization:**

Autoclave at 121 °C for 20 minutes.

**Storage:**

Room temperature.

**Notes:**

1.Agar must be added when preparing LB semi-solid medium, and it should be sterilized by autoclaving.

2.Do not add antibiotics before autoclaving, as they are heat-labile. Add antibiotics after sterilization and cooling, according to experimental requirements.

**LB Solid Medium**
**Formula:**

* Tryptone 0.01 g/mL (10 g/L)
* Yeast extract 0.005 g/mL (5 g/L)
* Sodium chloride 0.01 g/mL (10 g/L)
* Agar powder 1.5%–2%
* Deionized water
  OR
* Pre-mixed LB powder 0.025 g/mL (25 g/L)
* Agar powder 1.5%–2%
* Deionized water

**Common preparation volumes:**

* 100 mL
  Tryptone 1 g, Yeast extract 0.5 g, NaCl 1 g, Agar 1.5–2 g, Deionized water to 100 mL OR Pre-mixed LB powder 2.5 g, Agar 1.5–2 g, Deionized water to 100 mL
* 200 mL
  Tryptone 2 g, Yeast extract 1 g, NaCl 2 g, Agar 3–4 g, Deionized water to 200 mL OR Pre-mixed LB powder 5 g, Agar 3–4 g, Deionized water to 200 mL
* 500 mL
  Tryptone 5 g, Yeast extract 2.5 g, NaCl 5 g, Agar 7.5–10 g, Deionized water to 500 mL OR Pre-mixed LB powder 12.5 g, Agar 7.5–10 g, Deionized water to 500 mL
* 1 L
  Tryptone 10 g, Yeast extract 5 g, NaCl 10 g, Agar 15–20 g, Deionized water to 1 L OR Pre-mixed LB powder 25 g, Agar 15–20 g, Deionized water to 1 L
* 2 L
  Tryptone 20 g, Yeast extract 10 g, NaCl 20 g, Agar 30–40 g, Deionized water to 2 L OR Pre-mixed LB powder 50 g, Agar 30–40 g, Deionized water to 2 L
* 3 L
  Tryptone 30 g, Yeast extract 15 g, NaCl 30 g, Agar 45–60 g, Deionized water to 3 L OR Pre-mixed LB powder 75 g, Agar 45–60 g, Deionized water to 3 L
* 4 L
  Tryptone 40 g, Yeast extract 20 g, NaCl 40 g, Agar 60–80 g, Deionized water to 4 L OR Pre-mixed LB powder 100 g, Agar 60–80 g, Deionized water to 4 L
* 5 L
  Tryptone 50 g, Yeast extract 25 g, NaCl 50 g, Agar 75–100 g, Deionized water to 5 L OR Pre-mixed LB powder 125 g, Agar 75–100 g, Deionized water to 5 L
**Sterilization:**

Autoclave at 121 °C for 20 minutes.

**Storage:**

Room temperature.

**Notes:**

1.Agar must be added when preparing LB solid medium, and it should be sterilized by autoclaving.

2.Do not add antibiotics before autoclaving, as they are heat-labile. Add antibiotics after sterilization and cooling, according to experimental requirements.

3.If LB solid medium is to be poured into plates or dispensed into tubes, re-melt the medium using a microwave or heating block after sterilization. Once fully liquefied, pour into Petri dishes or tubes under aseptic conditions.

### M9 Minimal Medium (Liquid)

▼

**Formula (per 1 mL):**

* KH₂PO₄ 0.003 g/mL (0.3 g/L)
* Na₂HPO₄ 0.00678 g/mL (6.78 g/L) or Na₂HPO₄·12H₂O 0.0171 g/mL (17.1 g/L)
* NaCl 0.0005 g/mL (0.5 g/L)
* NH₄Cl 0.001 g/mL (1 g/L)
* MgSO₄ 0.000241 g/mL (0.241 g/L) or MgSO₄·7H₂O 0.000493 g/mL (0.493 g/L)
* CaCl₂ 0.000011 g/mL (0.011 g/L) or CaCl₂·2H₂O 0.0000147 g/mL (0.0147 g/L)
* Glucose 0.004 g/mL (4 g/L)
* Deionized water

**Common preparation volumes:**

* 100 mL
  KH₂PO₄ 0.03 g, Na₂HPO₄ 0.678 g or Na₂HPO₄·12H₂O 1.71 g, NaCl 0.05 g, NH₄Cl 0.1 g, MgSO₄ 0.0241 g or MgSO₄·7H₂O 0.0493 g, CaCl₂ 0.0011 g or CaCl₂·2H₂O 0.00147 g, Glucose 0.4 g, dH₂O to 100 mL
* 200 mL
  KH₂PO₄ 0.06 g, Na₂HPO₄ 1.356 g or Na₂HPO₄·12H₂O 3.42 g, NaCl 0.1 g, NH₄Cl 0.2 g, MgSO₄ 0.0482 g or MgSO₄·7H₂O 0.0986 g, CaCl₂ 0.0022 g or CaCl₂·2H₂O 0.00294 g, Glucose 0.8 g, dH₂O to 200 mL
* 500 mL
  KH₂PO₄ 0.15 g, Na₂HPO₄ 3.39 g or Na₂HPO₄·12H₂O 8.55 g, NaCl 0.25 g, NH₄Cl 0.5 g, MgSO₄ 0.1205 g or MgSO₄·7H₂O 0.2465 g, CaCl₂ 0.0055 g or CaCl₂·2H₂O 0.00735 g, Glucose 2 g, dH₂O to 500 mL
* 1 L
  KH₂PO₄ 0.3 g, Na₂HPO₄ 6.78 g or Na₂HPO₄·12H₂O 17.1 g, NaCl 0.5 g, NH₄Cl 1 g, MgSO₄ 0.241 g or MgSO₄·7H₂O 0.493 g, CaCl₂ 0.011 g or CaCl₂·2H₂O 0.0147 g, Glucose 4 g, dH₂O to 1 L
* 2 L
  KH₂PO₄ 0.6 g, Na₂HPO₄ 13.56 g or Na₂HPO₄·12H₂O 34.2 g, NaCl 1 g, NH₄Cl 2 g, MgSO₄ 0.482 g or MgSO₄·7H₂O 0.986 g, CaCl₂ 0.022 g or CaCl₂·2H₂O 0.0294 g, Glucose 8 g, dH₂O to 2 L
* 5 L
  KH₂PO₄ 1.5 g, Na₂HPO₄ 33.9 g or Na₂HPO₄·12H₂O 85.5 g, NaCl 2.5 g, NH₄Cl 5 g, MgSO₄ 1.205 g or MgSO₄·7H₂O 2.465 g, CaCl₂ 0.055 g or CaCl₂·2H₂O 0.0735 g, Glucose 20 g, dH₂O to 5 L

**Sterilization:**

* Ideally, autoclave all components except glucose at 121 °C for 20 minutes.
* Prepare glucose separately as a sterile stock solution (filter sterilized) and add aseptically after autoclaving.
* To simplify, M9 medium can also be autoclaved at 115 °C for 20 minutes, but never at 121 °C with glucose due to caramelization.

**Storage:**

Room temperature.

**Notes:**

1.M9 minimal medium does not contain agar (for liquid culture).

2.Antibiotics should not be added before sterilization (they are heat-labile). Add them after autoclaving, as required.

3.Glucose must not be autoclaved at 121 °C; it caramelizes and becomes unusable. Always sterilize separately by filtration.

4.Due to the very small amounts of MgSO₄ and CaCl₂ required, prepare concentrated sterile stock solutions for accuracy and add by volume. These can be sterilized separately and mixed with the base medium under aseptic conditions.

5.Only prepare 1× M9. Do not prepare concentrated stock solutions for later dilution.

## Antibiotics

▼

### Ampicillin (Amp)

▼

**Working concentration:**

50 μg/mL

**Stock concentration:**

50 μg/mL

**Addition ratio:**

1:1000

**Common preparation volumes:**

* 5 mL
  250 mg Ampicillin, add dH₂O to 5 mL
* 10 mL
  500 mg Ampicillin, add dH₂O to 10 mL
* 15 mL
  750 mg Ampicillin, add dH₂O to 15 mL
* 20 mL
  1000 mg Ampicillin, add dH₂O to 20 mL

**Sterilization:**

Filter sterilization (0.22 μm membrane)

**Storage:**

4 °C, protected from light

**Notes:**

1.Antibiotics are heat-labile and must not be autoclaved; filter sterilization is required.

2.Do not add antibiotics to freshly autoclaved media while still hot. Always wait until the medium has cooled sufficiently (≈50 °C) before adding.
Ampicillin is light-sensitive and degrades under UV exposure; avoid exposing solutions to UV lamps in laminar flow hoods.

### Kanamycin (Kan)

▼

**Working concentration:**

50 μg/mL

**Stock concentration:**

50 mg/mL

**Addition ratio:**

1:1000

**Common preparation volumes:**

* 5 mL
  250 mg Kanamycin, add dH₂O to 5 mL
* 10 mL
  500 mg Kanamycin, add dH₂O to 10 mL
* 15 mL
  750 mg Kanamycin, add dH₂O to 15 mL
* 20 mL
  1000 mg Kanamycin, add dH₂O to 20 mL

**Sterilization:**

Filter sterilization (0.22 μm membrane)

**Storage:**

4 °C, protected from light

**Note:**

1.Kanamycin is heat-sensitive and must not be autoclaved; sterilization should be performed by filtration.

2.Do not add Kanamycin into freshly autoclaved media while still hot. Allow the medium to cool adequately (~50 °C) before addition.

3.Kanamycin is degraded by UV light; avoid exposing solutions to UV in laminar flow hoods.

### Chloramphenicol (Cm)

▼

**Working concentration:**

25 μg/mL

**Stock concentration:**

5 mg/mL

**Addition ratio:**

1:200

**Common preparation volumes:**

* 5 mL
  25 mg Chloramphenicol, add dH₂O to 5 mL
* 10 mL
  50 mg Chloramphenicol, add dH₂O to 10 mL
* 15 mL
  75 mg Chloramphenicol, add dH₂O to 15 mL
* 20 mL
  100 mg Chloramphenicol, add dH₂O to 20 mL

**Sterilization:**

Filter sterilization (0.22 μm membrane)

**Storage:**

4 °C, protected from light

**Notes:**

1.Chloramphenicol is heat-sensitive and must not be autoclaved; sterilization should be performed by filtration.

2.Do not add Chloramphenicol into freshly autoclaved media while still hot. Allow the medium to cool adequately (~50 °C) before addition.

3.Chloramphenicol is degraded by UV light; avoid exposing solutions to UV in laminar flow hoods.

### Gentamicin (Gm)

▼

**Working concentration:**

40 μg/mL

**Stock concentration:**

40 mg/mL

**Addition ratio:**

1:1000

**Common preparation volumes:**

* 5 mL
  200 mg Gentamicin, add dH₂O to 5 mL
* 10 mL
  400 mg Gentamicin, add dH₂O to 10 mL
* 15 mL
  600 mg Gentamicin, add dH₂O to 15 mL
* 20 mL
  800 mg Gentamicin, add dH₂O to 20 mL

**Sterilization:**

Filter sterilization (0.22 μm membrane)

**Storage:**

4 °C, protected from light

**Notes:**

1.Gentamicin is heat-sensitive and must not be autoclaved; sterilization should be performed by filtration.

2.Do not add Gentamicin to freshly autoclaved media while still hot. Allow the medium to cool (~50 °C) before addition.

3.Gentamicin is degraded by UV light; avoid exposing solutions to UV in laminar flow hoods.

## Agarose Gel and Related Reagents

▼

### Agarose Gel

▼

**Composition:** Agarose powder, 1× TAE buffer, 10,000× GelStain
**Typical preparation:**

* 1.0% agarose gel (100 mL total volume)
* 1.0 g agarose powder
* 100 mL 1× TAE buffer
* 10 μL 10,000× GelStain

**Sterilization:**

Not required

**Storage:**

Prepare fresh for use; store solidified gels at 4 °C, wrapped in plastic wrap or sealed bag, up to several days.

### Running Buffer (TAE buffer)

▼

**Composition (50× stock):**

* 2 mol/L Tris-acetate
* 50 mmol/L EDTA

**Working solution:**

Dilute to 1× before use

**Sterilization:**

Not required

**Storage:**

4 °C

### Loading Buffer

▼

**Composition:**

Typically glycerol, tracking dye (e.g., bromophenol blue, xylene cyanol) in suitable buffer (commercially available or lab-prepared)

**Working concentration:**

5× or 6×, diluted to 1× in sample

**Sterilization:**

Not required

**Storage:**

4 °C

### DNA Ladder/Marker (DNA molecular weight standard)

▼

**Composition:**

Mixture of DNA fragments of defined lengths, supplied by manufacturer

**Usage:**

Typically 5–10 μL per lane, depending on ladder concentration

**Storage:**

4 °C for short-term, –20 °C for long-term

## Polyacrylamide Gels and Related Reagents

▼

### SDS-PAGE Gel (Sodium Dodecyl Sulfate–Polyacrylamide Gel)

▼

**Composition:**

Acrylamide (Acr), Methylene Bisacrylamide (Bis), SDS, Ammonium Persulfate (APS), TEMED, buffer, dH₂O

**Typical preparation:**

* Separating gel (7 mL per gel, concentration depending on protein size range)
* Stacking gel (3 mL per gel)
* Example (10 mL separating gel, 10%): 4.17 mL H₂O, 3.33 mL 30%
  Acr-Bis (29:1), 2.5 mL 4× Separating Buffer, 100 μL 10% APS, 4 μL TEMED
* Example (4 mL stacking gel): 2.28 mL H₂O, 0.68 mL 30% Acr-Bis (29:1), 1 mL 4× Stacking Buffer, 40 μL 10% APS, 4 μL TEMED

**Sterilization:**

Not required (freshly prepared before use)

**Storage:**

* APS at –20 °C
* Other reagents at 4 °C
* Prepared gels stored in dH₂O at 4 °C (short-term use)

### Native Polyacrylamide Gel

▼

**Composition:**

Acrylamide (Acr), Methylene Bisacrylamide (Bis), APS, TEMED, buffer, dH₂O

**Sterilization:**

Not required

**Storage:**

APS at –20 °C, other reagents at 4 °C; prepared gels stored in dH₂O at 4 °C

### Running Buffer (SDS-PAGE Running Buffer)

▼

**Composition (per 1 L, 1×):**

3.03 g Tris, 17.99 g Glycine, 1 g SDS, add dH₂O to 1 L

**Sterilization:**

Not required

**Storage:**

Room temperature

### Loading Buffer (Sample Buffer)

▼

**Composition:**

Typically contains Tris-HCl, glycerol, SDS, DTT/β-mercaptoethanol, and tracking dyes (bromophenol blue, xylene cyanol); commercially available or lab-prepared

**Working concentration:**

2×, 5×, or 6×; dilute to 1× in protein sample before loading

**Sterilization:**

Not required

**Storage:**

–20 °C

### Protein Ladder/Marker (Molecular Weight Standard)

▼

**Composition:**

Mixture of proteins of defined molecular weights, supplied by manufacturer (prestained or unstained)

**Usage:**

Typically 5–10 μL per lane, depending on concentration

**Storage:**

–20 °C

## Protein Induction, Purification, and Quantification Reagents

▼

### IPTG (Isopropyl β-D-1-thiogalactopyranoside)

▼

**Stock concentration:**

200 mg/mL in dH₂O

**Working concentration (typical):**

0.05–1.0 mM (added to bacterial culture for protein induction)

**Typical additions (for 200 mg/mL stock):**

* 0.05 mM: 6–24 μL per 100–400 mL culture
* 0.1 mM: 12–48 μL per 100–400 mL culture
* 0.2 mM: 24–96 μL per 100–400 mL culture
* 0.4 mM: 48–191 μL per 100–400 mL culture
* 1.0 mM: 119–477 μL per 100–400 mL culture

**Sterilization:**

Filter sterilization (0.22 μm membrane)

**Storage:**

4 °C, protected from light

**Notes:**

Heat- and UV-sensitive; do not autoclave.

### Buffer A (Lysis/Binding buffer)

▼

**Composition:**

50 mM Tris-HCl (pH 8.0), 100 mM NaCl, 5% glycerol, 2 mM β-mercaptoethanol, dH₂O

**Sterilization:**

Filter sterilization recommended

**Storage:**

4 °C

### Buffer B (Binding buffer, low imidazole)

▼

**Composition:**

50 mM Tris-HCl (pH 8.0), 20 mM imidazole, 400 mM NaCl, 5% glycerol, 2 mM β-mercaptoethanol, dH₂O

**Sterilization:**

Filter sterilization recommended

**Storage:**

4 °C

### Buffer C1 (Wash buffer, medium imidazole)

▼

**Composition:**

50 mM Tris-HCl (pH 8.0), 100 mM imidazole, 400 mM NaCl, 5% glycerol, 2 mM β-mercaptoethanol, dH₂O

**Sterilization:**

Filter sterilization recommended

**Storage:**

4 °C

### Buffer C2 (Wash buffer, high imidazole)

▼

**Composition:**

50 mM Tris-HCl (pH 8.0), 300 mM imidazole, 400 mM NaCl, 5% glycerol, 2 mM β-mercaptoethanol, dH₂O

**Sterilization:**

Filter sterilization recommended

**Storage:**

4 °C

### Buffer C3 (Elution buffer, very high imidazole)

▼

**Composition:**

50 mM Tris-HCl (pH 8.0), 500 mM imidazole, 400 mM NaCl, 5% glycerol, 2 mM β-mercaptoethanol, dH₂O

**Sterilization:**

Filter sterilization recommended

**Storage:**

4 °C

### Bradford Reagent (Protein quantification dye)

▼

**Composition:**

Coomassie Brilliant Blue G-250 dye in acidic solution (commercial kit, 5× concentrate)

**Working solution:**

Dilute 5× stock to 1× with dH₂O before use

**Sterilization:**

Not required

**Storage:**

4 °C

## Dialysis Buffers

▼

### Inclusion Body Refolding Dialysis Buffer

▼

**Composition:**

* 4 mM GSH (reduced glutathione)
* 0.4 mM GSSG (oxidized glutathione)
* 50 mM Tris-HCl
* 25 mM NaCl
* 2 mM EDTA
* 3 mM β-mercaptoethanol
* 15% glycerol

**Sterilization:**

Filter sterilization (0.22 μm membrane) recommended

**Storage:**

4 °C

### Assembly Dialysis Buffer

▼

**Composition:**

* 50 mM Tris-HCl
* 25 mM NaCl
* 2 mM EDTA
* 3 mM β-mercaptoethanol
* 10% glycerol

**Sterilization:**

Filter sterilization (0.22 μm membrane) recommended

**Storage:**

4 °C

## Enzyme Activity Assay Reagents

▼

### Substrate Solution

▼

**Composition:**

* 6 mM PolyP
* 10 mM ADP
* 20 mM MgCl₂
* 100 mM Tris-HCl (pH 8.0)

**Sterilization:**

Filter sterilization (0.22 μm membrane) recommended

**Storage:**

4 °C

### ATP Detection Solution

▼

**Composition:**

ATP assay reagent : Dilution buffer = 1 : 9

**Sterilization:**

Not required

**Storage:**

Prepare fresh before use; short-term storage at 4 °C possible

### ATP Detection Solution

▼

**Composition:**

1 M KH₂PO₃ / K₂HPO₃

**Sterilization:**

Filter sterilization (0.22 μm membrane) recommended

**Storage:**

4 °C

## Pull-down Buffers

▼

### Binding Buffer

▼

**Composition:**

* 50 mM Tris-HCl (pH 8.0)
* 150 mM NaCl
* 0.1% Triton X-100
* dH₂O

**Typical preparation (10 mL):**

* 250 μL Tris-HCl (pH 8.0)
* 0.088 g NaCl
* 10 μL Triton X-100
* Add dH₂O to 10 mL final volume

**Sterilization:**

Not required

**Storage:**

Room temperature

**Notes:**

* pH adjustment is not necessary since Tris-HCl stock is already pH 8.0.
* Due to the high viscosity of Triton X-100, use reverse pipetting or immerse the pipette tip directly into the reagent to improve accuracy.

### Elution Buffer

▼

**Composition:**

* 0.1 M Glycine
* 0.1% Triton X-100
* dH₂O
* Adjust pH to 2.5–3.1 with HCl

**Typical preparation (10 mL):**

* 0.075 g Glycine
* 10 μL Triton X-100
* Add dH₂O to 10 mL final volume
* Adjust pH to 2.5–3.1 with HCl

**Sterilization:**

Not required

**Storage:**

Room temperature

**Notes:**

* Accurate pH adjustment (2.5–3.1) is critical for efficient protein elution; calibrate pH meter carefully before use.
* Triton X-100 is viscous; use reverse pipetting or immerse tip directly for accurate measurement.

## Western Blot & Dot Blot Reagents

▼

### Transfer Buffer

▼

**Composition:**

* 25 mM Tris
* 192 mM Glycine
* 20% (v/v) Methanol
* dH₂O

**Typical preparation (1 L):**

* 3.03 g Tris
* 14.4 g Glycine
* 200 mL Methanol
* Add dH₂O to 1 L final volume

**Sterilization:**

Not required

**Storage:**

Room temperature

**Notes:**

Methanol is volatile, toxic, and has a pungent odor; avoid inhalation and ensure good ventilation.

### TBST Buffer

▼

**Composition:**

* 20 mM Tris-HCl (pH 7.4–7.6)
* 150 mM NaCl
* 0.1% (v/v) Tween-20
* dH₂O

**Typical preparation (1 L):**

* 10 mL of 2 M Tris-HCl stock (pH 7.4–7.6)
* 8.77 g NaCl
* 1 mL Tween-20
* Add dH₂O to 1 L final volume and adjust pH if necessary

**Sterilization:**

Not required

**Storage:**

Room temperature

**Notes:**

* Tween-20 is highly viscous; use reverse pipetting or immerse pipette tip directly into the reagent.
* Store Tween-20 properly; avoid contamination when retrieving.

### Blocking Solution

▼

**Composition:**

5% (w/v) Skim milk powder in TBST (alternative: BSA in TBST)

**Typical preparation (1 L):
50 g Skim milk powder
Dissolve in 1 L TBST
oSterilization:**

Not required

**Storage:**

Room temperature

**Notes:**

Skim milk is strictly for laboratory use; not suitable for consumption.

### Primary Antibody Solution

▼

**Composition:**

Dilute antibody to appropriate working concentration in blocking solution (skim milk/TBST or BSA/TBST, depending on protocol).

**Storage:**

4 °C (short-term, in-use)

**Notes:**

Antibodies are expensive and sensitive; use minimal volumes and store promptly.

### Secondary Antibody Solution

▼

**Composition:**

Dilute antibody to appropriate working concentration in blocking solution.

**Storage:**

4 °C (short-term, in-use)

**Notes:**

Handle carefully; store properly to avoid degradation.

### CL Detection Reagent

▼

**Composition:**

Mix equal volumes of Solution A (contains luminol) and Solution B (contains hydrogen peroxide) immediately before use.

**Sterilization:**

Not required

**Storage:**

Not storable once mixed; prepare fresh each time

**Notes:**

Always prepare freshly; do not store mixed ECL solution as it rapidly loses activity.

## Silver Staining Reagents

▼

### Fixing Solution

▼

**Composition:**

* 30% (v/v) Ethanol
* 12% (v/v) Acetic acid
* 0.018% (v/v) 37% Formaldehyde
* dH₂O

**Typical preparation (100 mL):**

* 30 mL Ethanol
* 12 mL Acetic acid
* 50 μL 37% Formaldehyde
* Add dH₂O to 100 mL final volume

**Sterilization:**

Not required

**Storage:**

Room temperature

### Silver Nitrate Staining Solution

▼

**Composition:**

* 0.02% (w/v) AgNO₃
* 0.028% (v/v) 37% Formaldehyde
* dH₂O

**Typical preparation (100 mL):**

* 20 mg AgNO₃
* 75 μL 37% Formaldehyde
* Add dH₂O to 100 mL final volume

**Sterilization:**

Not required

**Storage:**

Prepare fresh; do not store

### Developing Solution

▼

**Composition:**

* 6% (w/v) Sodium carbonate (Na₂CO₃)
* 0.0004% (w/v) Sodium thiosulfate (Na₂S₂O₃)
* 0.028% (v/v) 37% Formaldehyde
* (Optional: add 2 mL Sensitizing solution per 100 mL)
* dH₂O

**Typical preparation (100 mL):**

* 6 g Sodium carbonate
* 0.4 mg Sodium thiosulfate
* 75 μL 37% Formaldehyde
* (Optional: add 2 mL Sensitizing solution)
* Add dH₂O to 100 mL final volume

**Sterilization:**

Not required

**Storage:**

Prepare fresh; do not store

### Stopping (Quenching) Solution

▼

**Composition:**

* 1% (v/v) Acetic acid
* dH₂O

**Typical preparation (100 mL):**

* 1 mL Acetic acid
* 99 mL dH₂O

**Sterilization:**

Not required

**Storage:**

Room temperature

### Sensitizing Solution

▼

**Composition:**

* 0.02% (w/v) Sodium thiosulfate (Na₂S₂O₃)
* dH₂O

**Typical preparation (100 mL):**

* 20 mg Sodium thiosulfate
* Add dH₂O to 100 mL final volume

**Sterilization:**

Not required

**Storage:**

Prepare fresh; do not store

## Sucrose Gradient Solutions

▼

### Composition

▼

Buffer A (pre-prepared)

Sucrose (analytical grade)

### Typical gradient preparations (w/v):

▼

**20% Sucrose Solution:**

20 g sucrose dissolved in 100 mL Buffer A

**35% Sucrose Solution:**

35 g sucrose dissolved in 100 mL Buffer A

**45% Sucrose Solution:**

45 g sucrose dissolved in 100 mL Buffer A

**50% Sucrose Solution:**

50 g sucrose dissolved in 100 mL Buffer A

**60% Sucrose Solution:**

60 g sucrose dissolved in 100 mL Buffer A

### Sterilization

▼

Filter sterilization (0.22 μm membrane) recommended if solutions are used for sensitive protein or cell experiments.

### Storage

▼

4 °C, protected from microbial contamination. Solutions are stable for 1–2 weeks.

## Major Reagents and Kits Sources

▼

### Major Reagents Sources

▼

Ni-NTA Beads 6FF (Nickel resin): LabLead

β-Mercaptoethanol: Macklin

Imidazole: Sangon Biotech

Guanidine Hydrochloride(GuHCl): Sangon Biotech

Aspartic Acid: Sangon Biotech

Sodium Hexametaphosphate: Sangon Biotech

*E. coli* BW25113 strain: Sangon Biotech

ArcticExpress strain: Beyotime

ADP: Mei5bio

Gentamicin Sulfate: Mei5bio

Kanamycin Sulfate: Mei5bio

Immunoprecipitation Magnetic Beads: Mei5bio

FLAG Monoclonal Antibody: Mei5bio

Neutral Protease: Mei5bio

Cysteine: Mei5bio

CMP (Cytidine Monophosphate): Mei5bio

Cytidine: Mei5bio

L-Arabinose: Mei5bio

### Major Kits Sources

▼

Native-PAGE Kit: Sangon Biotech

Glutathione ELISA Kit: Sangon Biotech

ATP Assay Kit: Beyotime

SDS-PAGE Kit: Mei5bio

BCA Protein Quantification Kit: Mei5bio

Reduced Glutathione Assay Kit: Mei5bio

### Acknowledgments

▼

We thank Sangon Biotech for sequencing services and oligonucleotide synthesis. We also acknowledge the College of Chemistry and the College of Physics and Astronomy, Beijing Normal University, the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Servicebio company for providing transmission electron microscopy (TEM) services.

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[87] Chang, L., Cui, H., Li, F., Zhang, Y., & Zhang, L. (2024). ATP regeneration by ATPases for *in vitro* biotransformation.. Biotechnology advances, 108377 .

[88] Yue, K., Chen, J., Li, Y., & Kai, L. (2023). Advancing synthetic biology through cell-free protein synthesis. Computational and Structural Biotechnology Journal.

[89] Sharma, J., & Douglas, T. (2019). Tuning the catalytic properties of P22 nanoreactors through compositional control. Nanoscale.

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Results

Our Purposes, Hypotheses, Data and Conclusions.

Results
![to-left](https://static.igem.wiki/teams/5872/submatl/to-left.svg)

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# Overview

![engi-top](https://static.igem.wiki/teams/5872/results/result-main-newnewnew.webp)

Figure I. Schematic Diagram of Our Results.

Our Wet Lab consists of four main sections: *In Vitro* Assembly of V-CHARGEs, *In Vivo* Assembly of V-CHARGEs, Enzyme Activity Validation and Product Determination.

Our **V-CHARGEs** system not only enables efficient ATP regeneration but also establishes a novel **expandable multi-enzyme catalytic platform** through P22-VLP encapsulation and external enzyme coupling.

We first assembled V-CHARGEs *in vitro*, then developed a simpler and more cost-effective *in vivo* assembly and separation method. Through rigorous testing, we confirmed the system's **high efficiency and stability**, and further validated its **platform potential** by coupling external enzymes.

Overall, V-CHARGEs serve as a **typical representative of functional protein nanoparticles**, applicable to high-value molecule production, artificial metabolic networks, and novel catalytic system development.

# 1.*In Vitro* Assembly

![engi-top](https://static.igem.wiki/teams/5872/results/vitro-main.svg)

Figure II. Schematic of V-CHARGEs *In Vitro* Assembly.

Figure shows the purification of fusion proteins (SlPPK-SP and CP-SpyTag) and subsequent assembly under optimized conditions.

* **We successfully expressed and purified SlPPK-SP and CP-SpyTag portein after extensive optimization.**
* **We explored the optimal conditions for *in vitro* assembly.**
* **We characterized the V-CHARGEs particles obtained from *in vitro* assembly.**

**Introduction and Objectives**

This section aims to verify the feasibility of utilizing P22 Virus-Like Particles (P22-VLPs)
for
the
*in vitro*
assembly and encapsulation of SIPPK to construct *in vitro* assembled V-CHARGEs, and to
characterize the assembly products structurally and
functionally. We conducted work based on commonly used *in vitro* assembly protocols
from
the
literature and performed extensive
optimization and repeated experiments in three aspects: expression/purification, *in
vitro*
assembly, and characterization (SEC, DLS, TEM).
**Ultimately, we successfully obtained *in vitro* assembled V-CHARGEs particles
with
observable SIPPK encapsulation and verified their catalytic activity.**
However, we also identified and quantified bottlenecks in assembly efficiency and product
homogeneity.

## 1.1 Plasmid Construction for SIPPK-SP and CP-SpyTag

▼

We selected *Escherichia coli* (*E. coli*) BL21 (DE3) for the heterologous expression of SIPPK-SP and
CP-SpyTag proteins. We constructed the following genetic pathways
(Figure 1) and integrated the genes encoding the fusion proteins into plasmid backbones. We
chose
the pET-28a vector backbone. This T7 promoter-based vector
contains a kanamycin resistance gene as a selectable marker and features an inducible lac operon
(including the lacI repressor), ensuring that IPTG
(IUPAC: Propan-2-yl-1-thio-β-D-galactopyranoside) can induce the expression of the fusion
proteins.
In the constructed plasmids, the C-terminus of the target gene
(SIPPK-SP or CP-SpyTag) was fused with a His-tag to facilitate subsequent purification via
nickel
affinity chromatography. We used SnapGene software to design the
plasmids (Figure 2),amplified the target gene fragments using primers synthesized by Sangon
Biotech,
and used In-Fusion Cloning technology to insert the gene fragments into linearized vectors.

![img](https://static.igem.wiki/teams/5872/results/1-2.webp)

**Figure 1. Genetic pathway
construction.
diagrams for *in vitro* assembly of SlPPK-SP and CP-SpyTag.**

**(A)** shows the genetic pathway construction for *in
vitro* assembly of SIPPK-SP. The pathway in **(A)** includes the T7 promoter,
6×His, SIPPK, GS linker, SP, and terminator.

**(B)** shows the genetic pathway
construction
for *in vitro* assembly of CP-SpyTag.
The pathway in **(B)** includes the T7 promoter, 6×His, CP, GS linker, SpyTag, and
terminator.

![img](https://static.igem.wiki/teams/5872/results/2-2.webp)

**Figure 2. Plasmid maps of
*pET-28a::SlPPK-SP-FLAG-His* and *pET-28a::CP-SpyTag-His*.**

The plasmids contain elements such as the T7 promoter
(white),
SIPPK-SP gene (purple), CP-Spytag gene (blue),
and kanamycin resistance gene (green).

Plasmids were extracted from *E. coli* DH5α and transformed into *E. coli* BL21(DE3) via heat shock. After
transformation, the cells were inoculated on LB
medium containing kanamycin for selection. After overnight incubation, we observed distinct
bacterial colonies, indicating successful transformation.
Finally, we identified the target genes by Polymerase Chain Reaction (PCR) and agarose gel
electrophoresis. The gel electrophoresis image showed
that the lengths of both SIPPK-SP and CP-SpyTag were between 1000 bp and 2000 bp (Figure 3),
consistent with their actual sizes.The successfully
transformed plasmids were sent to Sangon Biotech for sequencing, and the sequencing results
confirmed that the target gene fragments were successfully
inserted into the plasmids.

![img](https://static.igem.wiki/teams/5872/results/3.webp)

**Figure 3. Agarose gel electrophoresis for
PCR verification of SIPPK-SP and CP-SpyTag target gene insertion.**

From left to right: Lane 1 contains M5 HiClear DL2000 plus
DNA
marker, Lane 2 contains water, Lane 3 contains PCR product using SlPPK-SP as template,
Lane 4 contains PCR product using CP-SpyTag as template.

**Summary**

We successfully constructed the recombinant plasmids *pET-28a::SlPPK-SP-FLAG-His* and
*pET-28a::CP-SpyTag-His* for expressing SIPPK-SP and CP-SpyTag,
and transformed them into the expression strain BL21(DE3), laying the foundation for the
subsequent
heterologous expression and purification of the two proteins.

## 1.2 Expression and Purification of SIPPK-SP and CP-SpyTag

▼

The heterologous expression of SIPPK-SP and CP-SpyTag proteins was particularly challenging
compared
to other proteins, as we faced significant
issues with inclusion body formation. The expression of SIPPK-SP was highly prone to forming
inclusion bodies, which is closely related to SIPPK's
natural tetrameric state, large molecular weight (monomer ~50 kDa, tetramer ~200 kDa), and its
folding/oligomerization requirements. After attempting
inclusion body refolding, optimizing induction conditions, and improving protein purification
procedures **[(Engineering Wet Lab1),](https://2025.igem.wiki/bnu-china/engineering)** we successfully
developed a set of induction conditions and a mature protein purification method to purify both
proteins.

The overnight-cultured seed culture was inoculated into fresh LB medium at a ratio of 1:100, and
cultured at 37 °C with shaking at 200 rpm until the OD₆₀₀
reached 0.6–0.8. Then, 0.1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) was added to induce
protein
expression, and cultivation continued at 16°C, 200
rpm for 16 h. Bacterial cells were collected by centrifugation at 6000 rpm for 10 min,
resuspended
in Buffer A (50 mM Tris-HCl, pH 8.0, 300 mM NaCl, 3 mM
β-mercaptoethanol), and subjected to ultrasonic disruption (cycle of 3 s sonication / 6 s
interval,
total 10 min). The lysate was centrifuged at 12000 rpm
for 45 min, and the supernatant and pellet were collected separately for subsequent
purification.

We used nickel column affinity chromatography to isolate and purify the proteins from the
supernatant. First, proteins were eluted stepwise using imidazole
gradients ranging from 100 mM to 500 mM. Then, SDS-polyacrylamide gel electrophoresis (SDS-PAGE)
was
performed on the protein solution, flow-through,
and eluates from different imidazole concentrations to analyze protein expression. To quantify
the
concentration of the extracted proteins,
we used the Bradford method to determine the concentration of the purified proteins.

From the SDS-PAGE results of SIPPK-SP and CP-SpyTag (Figure 4), it can be observed that 500 mM
imidazole eluted the SIPPK-SP protein from the column,
and 300 mM imidazole eluted the CP-SpyTag protein from the column. At these concentrations, the
purified proteins were relatively pure with minimal contaminating proteins. And we superisely observe that
the molecular weight of SIPPK-SP and CP-SpyTag is too close to seperate, which makes a little trouble in later experiments.

![img](https://static.igem.wiki/teams/5872/results/4.webp)

**Figure 4.
SDS polyacrylamide gel electrophoresis (SDS-PAGE) of SIPPK-SP and CP-SpyTag.**

**(A)** **(B)** are SDS-PAGE of SIPPK-SP and CP-SpyTag,
respectively. Lanes 1–6 (from left to right): M5 Prestained Protein Ladder
(10-180 kDa) with 45 kDa band highlighted, Lysate (supernatant after centrifugation),
Flow-through, Eluate with 100 mM imidazole, Eluate with 300 mM imidazole,
Eluate with 500 mM imidazole. Bands between 45–65 kDa can be observed in the rightmost
two
lanes, consistent with the sizes of SIPPK-SP and CP-SpyTag.

We also characterized the particle size of the CP-SpyTag self-assembly products by Dynamic Light
Scattering (DLS) and characterized their shape and particle size using
Transmission Electron Microscopy (TEM) (Figure 5). We found that most CP-SpyTag could
self-assemble
into VLPs with particle sizes ranging from 50 to 500 nm. This demonstrates
that we successfully expressed and purified CP-SpyTag, and also proves that CP-SpyTag can
self-assemble into spherical structures *in vitro*, increasing the feasibility of
subsequently
encapsulating SIPPK-SP using VLPs.

![img](https://static.igem.wiki/teams/5872/results/5.webp)

**Figure 5. DLS and TEM analysis
results
for CP SpyTag self-assembly products.**

**(A)** is the average DLS intensity distribution of
CP-SpyTag
self-assembly products. This graph shows the relationship between the intensity
distribution function G(d) of the sample's hydrodynamic diameter and the particle size
d.
The horizontal axis represents particle size (nm) on a logarithmic scale.
The distribution curve shows peaks around 50 nm, 100 nm, and 300 nm, indicating a
multimodal
size distribution for the CP-SpyTag self-assembly product particles.

**(B) (C)** are Transmission Electron Microscope images of CP-SpyTag self-assembly
products. The white spherical particles in the images are the CP-SpyTag self-assembly
products.
The scale bar in B is 100 nm, and in C is 500 nm.

DLS measures the hydrodynamic diameter, which includes the hydration layer and possible aggregates,
while TEM shows the dry core size. Therefore, DLS values appear larger than TEM results.

**Summary**

By optimizing expression conditions and purification methods, we successfully obtained highly
pure,
soluble SIPPK-SP and CP-SpyTag proteins from *E. coli*, providing the key materials for
the *in vitro* assembly of SIPPK-SP and CP-SpyTag. We also successfully characterized the
self-assembly products of CP-SpyTag, strengthening our confidence in achieving VLP encapsulation
of
enzymes.

## 1.3 Assembly and Characterization

▼

After obtaining the two proteins, we performed *in vitro* assembly of SlPPK-SP and
CP-SpyTag. Equal volumes of guanidine hydrochloride and CP-SpyTag protein solution were mixed and
incubated on ice for 30 minutes.
Then, SlPPK-SP was added at a specific ratio and mixed thoroughly. Considering that the SP:CP
ratio
varies in different literatures, we tested different ratios and finally determined that a
concentration
ratio of 1:2 yielded the best assembly results, producing *in vitro* assembled V-CHARGE
particles. Two rounds of dialysis were performed to remove most of the unassembled protein. The
specific assembly principle is shown in Figure 6.
To further improve the purity of the *in vitro* assembled V-CHARGEs, we used size exclusion
chromatography to purify the assembly mixture and analyzed the obtained, higher-purity V-CHARGEs
by
SDS-PAGE (Figure 7).

![img](https://static.igem.wiki/teams/5872/results/vitro-main.svg)

**Figure 6. Assembly Principle Diagram.**

![img](https://static.igem.wiki/teams/5872/results/7.webp)

**Figure 7. Purification and
identification
of *in vitro* assembled V-CHARGEs.**

**(A)** is the SEC chromatogram showing the absorption
peaks
from the purification of *in vitro* assembled V-CHARGEs. Two absorption peaks were
obtained before the
salt peak (absorption peaks at 38 mL and 43 mL).

**(B)** is the SDS-PAGE analysis of the
SEC
results for the *in vitro* assembled V-CHARGEs. The six lanes from left to right
contain: M5 Prestained
Protein Ladder (10-180 kDa) with 45 kDa band highlighted, CP-SpyTag eluate with 300 mM
imidazole, SIPPK-SP eluate with 500 mM imidazole (condition indicated by the red box in
the
figure),
the product after *in vitro* assembly, the solution obtained from the first SEC
peak
(38 ml) after assembly, and the solution obtained from the second SEC peak (43 ml) after
assembly.

We characterized the particle size of the *in vitro* assembled V-CHARGEs by Dynamic Light
Scattering (DLS) and used Transmission Electron Microscopy (TEM) to characterize the assembly
status,
including shape and particle size (Figure 8), comparing these results with the characterization
results of VLPs assembled solely from CP.

DLS and TEM analysis results showed that the particle size of the *in vitro* assembled
V-CHARGEs
was around 200 nm. However, there were many small particles (size < 10 nm) present in the
system, likely indicating that a significant amount of SIPPK-SP was not encapsulated into
the spheres assembled by CP-SpyTag but remained dispersed in the system, suggesting
suboptimal *in
vitro* assembly efficiency.

![img](https://static.igem.wiki/teams/5872/results/8.webp)

**Figure 8. DLS and TEM Analysis
results of
*in vitro* Assembled V-CHARGEs.**

**(A)** is the average DLS intensity distribution of *in
vitro* assembled V-CHARGEs. This graph shows the relationship between the
intensity
distribution function G(d) of the
sample's hydrodynamic diameter and the particle size d. The horizontal axis represents
particle size (nm) on a logarithmic scale. The distribution curve shows peaks around
0.2-0.5
nm, 1-2 nm, 5-10 nm,
and 200 nm, indicating a multimodal particle size distribution for the *in vitro*
assembled V-CHARGEs particles.

**(B) (C)** are Transmission Electron Microscope
images of
the *in vitro* assembly products.
The larger, dark spherical particles (>50 nm) in the images are the *in vitro*
assembled V-CHARGEs, while the smaller, flocculent dark particles (<50 nm) are
unsuccessfully assembled SIPPK-SP and CP-SpyTag.
The scale bars in B and C are 50 nm.

DLS measures the hydrodynamic diameter, which includes the hydration layer and possible aggregates,
while TEM shows the dry core size. Therefore, DLS values appear larger than TEM results.

To achieve better assembly results, we further explored the assembly conditions in detail **[(Engineering Wet Lab2)](https://2025.igem.wiki/bnu-china/engineering)**. However, we found that regardless of optimization efforts, we could not
achieve efficient assembly
to obtain large quantities of *in vitro* assembled V-CHARGEs particles.

**Summary**

Using *in vitro* co-assembly (mixing SIPPK-SP and CP-SpyTag at different ratios, incubating
under appropriate buffer and temperature conditions), we were able to observe the target *in
vitro* assembled V-CHARGEs peak and collect the corresponding
fractions via SEC. Transmission Electron Microscopy (TEM) showed recognizable hollow or solid
nanoparticles, and density (dark regions) could be detected inside some particles, inferred to
be
successful encapsulation of SIPPK.
However, the assembly products exhibited high polydispersity: a large number of small particles
coexisted with unassembled proteins, resulting in a relatively overall low assembly yield.

## 1.4 Discussion

**Summary and Reflection**

In this part of the study, we successfully constructed and expressed the SIPPK-SP and
CP-SpyTag
fusion proteins. By optimizing induction conditions and purification processes, we
effectively
addressed the severe inclusion
body problem, establishing a unique expression and purification strategy suitable for
large-molecular-weight proteins. Through nickel affinity chromatography, size exclusion
chromatography and other steps, we obtained target
proteins with relatively high purity and completed the *in vitro* assembly and
characterization
of V-CHARGEs and their characterization. Dynamic Light Scattering and Transmission Electron
Microscopy results demonstrated
that SIPPK-SP could be successfully encapsulated within P22-VLPs. This is the first time
that a
tetrameric enzyme with a molecular weight of up to 200 kDa has been encapsulated in the
P22-VLP
system, providing experimental
evidence for expanding the application scope of this system.

However, we also identified a clear issue of insufficient
assembly
efficiency: despite exploring
different assembly ratios and conditions, the system still contained numerous small
particles or
incompletely assembled structures,
indicating significant steric hindrance limitations for large, multimeric proteins during
the
*in
vitro* assembly process. This result contrasts sharply with the encapsulation
efficiency
reported in the literature for small to
medium-sized monomeric enzymes like GFP or alcohol dehydrogenase, suggesting that we need to
overcome the limitations of traditional *in vitro* assembly strategies when
constructing
more
complex enzyme systems.

**Possible Mechanisms Underlying Low Yield and Polydispersity**

**Causes of Low Yield and Polydispersity**

**1. Steric Hindrance:** SIPPK has a high molecular
weight
and functions as a tetramer. The volume of a single SIPPK-SP and the space occupied by the
formed tetramer might exceed the capacity of the P22 typical internal
cavity under conditions without additional expansion. This could prevent some SP-SIPPK from
being correctly incorporated or lead to misassembly. During normal P22 assembly,
approximately
420 CP subunits and about 100–300 SP subunits
cooperatively form a capsid with a diameter of about 58 nm; when the cargo volume approaches
the
cavity limit or the cargo is a rigid tetramer, the assembly pathway and product distribution
can
be significantly altered (naturally
tending to produce unassembled proteins or aberrant particles). [1]

**2.Multimer Kinetics and Folding:** If SIPPK pre-forms
tetramers or misfolds *in vitro*, the localization signal at the SP end might be
masked,
thereby reducing loading efficiency.

**3.Kinetics and Concentration Effects:** Assembly is a
nucleation-and-growth process. The relative concentrations of CP and SP, as well as the
total
protein concentration,
affect the nucleation rate and competition with correct assembly pathways. Excessively high
protein concentrations might lead to "multiple nucleation" or condensate formation (which
has been demonstrated to occur in studies of other VLP assemblies). [2]

**Comparison with Literature**

Previous studies have successfully encapsulated enzyme cascade systems inside P22 VLPs and
demonstrated protection of enzyme activity and proximity effects. However, these successes
mostly involved monomeric or smaller enzymes (or mild multimers);
encapsulating large tetramers with volumes approaching or exceeding the internal cavity
capacity
presents new challenges. The pore and molecular diffusion characteristics of P22 also
determine
the entry and exit of small molecules and the permeation
behavior of inhibitors (like Pi), which significantly impact the subsequent ATP/ADP cycle
(the
pore and molecular sieve effects of P22 have been quantitatively studied in the literature).
[3]

**Outlook**

This phenomenon suggests that traditional *in vitro* assembly methods are inadequate
for
the
efficient loading of large, multimeric enzymes. To break through this bottleneck, we
plan to
shift
to an *in vivo* assembly strategy.
Leveraging the natural folding and assembly processes within the cellular environment,
we
expect
to
achieve higher loading efficiency, more uniform particle distribution, and, based on
this,
conduct
more in-depth enzyme activity studies.

# 2. *In Vivo* Assembly

![engi-top](https://static.igem.wiki/teams/5872/results/vivo-main.svg)

Figure III. Schematic Diagram of V-CHARGEs *In Vivo* Assembly.

We co-express SlPPK-SP and CP-SpyTag in *E. coli* to construct the *in vivo* assembled V-CHARGEs, establishing a simple and low-cost production method.

* **We co-expressed SP and CP proteins in the same *E. coli* strain to produce V-CHARGEs particles.**
* **We tested various purification methods and established a simple, low-cost production process.**
* **We characterized the V-CHARGEs particles obtained from *in vivo* assembly.**

**Introduction and Objectives**

At the early stage of our project, we attempted to assemble V-CHARGEs particles ***in
vitro***. However, during the experiments, we encountered several challenges. On
the
one
hand, protein extraction and purification inevitably caused significant loss,
leading to low overall yield. On the other hand, the assembly efficiency was unsatisfactory:
results
from DLS and TEM revealed that most particles were incomplete with heterogeneous size
distribution,
which resulted in insufficient enzymatic activity.
These difficulties are consistent with previous reports on the challenges of ***in
vitro
assembly*** of P22-VLPs, suggesting that this approach has inherent limitations
when
dealing with multi-enzyme complexes.

Based on these findings, we proposed an *in vivo* assembly strategy to address the above
issues.
Specifically, we co-expressed SlPPK-SP and CP-SpyTag in *E. coli* BL21(DE3), allowing them to
spontaneously assemble into V-CHARGEs particles within the cell.
Compared with *in vitro* assembly, *in vivo* assembly offers several potential
advantages:

**1. Higher assembly efficiency** — Proteins can
interact
as
they are synthesized, reducing the accumulation of unassembled monomers.

**2. Structures closer to the native state** — Folding
and
spatial arrangement in the cellular environment may help maintain enzymatic activity and
stability.

**3. Simplified workflow** — Avoiding repeated *in
vitro*
purification and assembly steps, target particles can be obtained directly through affinity
chromatography or ultracentrifugation.

**4. Greater scalability** — This strategy may provide
a
foundation for large-scale production and future industrial applications.

In subsequent experiments, we constructed both dual-plasmid and single-plasmid expression
systems,
and optimized induction conditions (stepwise induction with arabinose and IPTG). By
combining
Ni-affinity chromatography, sucrose density gradient
ultracentrifugation, and size-exclusion purification, we successfully obtained relatively
pure
*in
vivo* assembled V-CHARGEs particles from *E. coli* lysates. Further characterization by
DLS
and
TEM confirmed particle formation, demonstrating the feasibility and advantages
of the *in vivo* assembly strategy.

**Conception of the Idea**

During our experiments, we found that *in vitro* assembly of V-CHARGEs faced several
obstacles.
First, protein purification inevitably led to protein loss and contamination by non-specific
proteins; even after repeated purification with
size-exclusion and Ni-affinity chromatography, it was difficult to obtain highly pure assembly
products. Second, the assembly efficiency was low: DLS and TEM revealed mostly incomplete or
size-heterogeneous substructures, resulting in limited enzymatic activity.
Similar issues have been reported in studies of *in vitro* assembly of P22-VLPs
[4,5], suggesting that *in vitro* assembly inherently struggles with large
macromolecular complexes or multi-enzyme systems.

To overcome these challenges, we explored an alternative strategy: co-expressing SlPPK-SP and
CP-SpyTag in *E. coli* BL21(DE3), enabling spontaneous *in vivo* assembly of V-CHARGEs
particles.
This approach avoids protein loss and inefficiency associated with *in vitro* assembly,
achieving “on-site” assembly of the particles. We then aimed to isolate and enrich *in
vivo*
assembled V-CHARGEs directly from cell lysates via Ni-affinity chromatography or sucrose
gradient
ultracentrifugation.

Literature review revealed that *in vivo* assembly has been previously employed in
constructing
P22-VLP complexes, with reported advantages. For example, P22 coat protein can interact with
scaffold protein and assemble directly within *E. coli* cells [6]; similarly,
certain engineered fusion proteins can achieve spontaneous encapsulation upon co-expression
[7]. These findings indicate that *in vivo* assembly, by allowing proteins to
interact immediately upon synthesis in the same environment, significantly
improves assembly efficiency and reduces unassembled intermediates. Moreover, the resulting
particles are more likely to adopt native-like folding and conformations, potentially enhancing
enzymatic stability and activity. Most importantly, this method eliminates multiple purification
and assembly steps, simplifying the workflow and offering better scalability and industrial
potential.

In summary, we hypothesize that *in vivo* assembly not only overcomes the low yield and
purification difficulties of *in vitro* assembly, but may also offer unique advantages in
maintaining enzymatic activity and enabling scalable production.
In future work, we will continue optimizing the *in vivo* co-expression system and validate
its
performance through systematic biochemical and structural characterization.

## 2.1 *In Vivo* Assembly Plasmid Construction

▼

We constructed the following genetic pathway (Figure 9) and integrated the genes encoding fusion
proteins into the plasmid backbone. In order to sequentially induce the expression of SlPPK-SP
and
CP-SpyTag carried by the dual-plasmid system
in the bacterial cells, we additionally constructed an arabinose-inducible plasmid containing
SlPPK-SP (Figure 10A). We chose the pBAD33 vector backbone, which is based on the arabinose
promoter
and contains a chloramphenicol resistance gene
as a selection marker. It is also equipped with an arabinose operon to ensure that arabinose
(IUPAC:
L-(+)-Arabinose) can induce the expression of the fusion protein. For CP-SpyTag, we still used
the
*pET-28a::CP-Spytag-His* plasmid from the in
vitro assembly, induced its expression by adding IPTG, and used kanamycin for selection.

At the same time, we also attempted to introduce SlPPK-SP and CP-SpyTag into a single plasmid
(Figure
10B). We chose the pACYCDuet-1 vector backbone, which is based on the T7 promoter and contains a
chloramphenicol resistance gene as a
selection marker. It is also equipped with an inducible lac operon (including the lacI
repressor) to
ensure that IPTG can induce the expression of
the
fusion protein. However, due to time constraints,
at the time of Wiki submission, we only successfully constructed the plasmid but were unable to
carry out protein induction expression and related experiments, which will be continued later.

We used SnapGene software to design plasmids (Figure 10), amplified target gene fragments with
primers synthesized by Sangon Biotech, and inserted gene fragments into linearized vectors using
seamless cloning technology.

![img](https://static.igem.wiki/teams/5872/results/9-2.webp)

**Figure 9. *In vivo* assembly
pathway
construction diagram of SlPPK-SP, CP-SpyTag, and SlPPK-SP-CP-SpyTag.**

**(A)** and **(B)** show the genetic pathway constructions of
SlPPK-SP and CP-SpyTag in the dual-plasmid system for *in vivo* assembly. The
pathway
in **(A)** contains the araBAD promoter, SlPPK, GS linker, SP, and terminator.
The pathway in **(B)** contains the T7 promoter, 6×His, CP, GS linker, SpyTag, and
terminator. **(C)** shows the genetic pathway construction of SlPPK-SP-CP-SpyTag in the
single-plasmid system for *in vivo* assembly. The pathway in **(C)** contains
the T7 promoter, CP, GS linker, SpyTag, 6×His, SlPPK, GS linker, SP, and
terminator.

![img](https://static.igem.wiki/teams/5872/results/10-2.webp)

**Figure 10. Plasmid maps of
*pBAD33::SlPPK-SP-FLAG*, *pACYCDuet-1::SlPPK-SP-CP-SpyTag-His.***

The *pBAD33::SlPPK-SP-FLAG* plasmid contains the araBAD
promoter
(white), SlPPK-SP gene (purple), chloramphenicol resistance gene (light green),
and other elements. The *pACYCDuet-1::SlPPK-SP-CP-SpyTag-His* plasmid
contains the T7 promoter (white), SlPPK-SP gene (purple), chloramphenicol resistance
gene
(light green), CP-SpyTag (dark blue), and other elements.

We prepared competent *E. coli* BL21(DE3) containing *CP-SpyTag-His* and transformed the obtained
*pBAD33::SlPPK-SP-FLAG* plasmid into the prepared competent cells using the heat shock method.
After
transformation, the strains were inoculated onto LB agar plates containing
chloramphenicol and kanamycin for selection. Since the plasmid carries the chloramphenicol
resistance gene and the competent cells contain the kanamycin resistance gene, only those *E.
coli*
cells that successfully acquired the plasmid could grow on these plates.
After overnight incubation, we observed distinct bacterial colonies, indicating successful
transformation. Finally, we identified the target gene by Polymerase Chain Reaction (PCR) and
agarose gel electrophoresis. The gel electrophoresis results showed that the lengths
of SlPPK-SP and CP-SpyTag were both between 1000 bp and 2000 bp (Figure 11), consistent with
their
actual sizes. We submitted the successfully transformed plasmids to Sangon Biotech for
sequencing,
and the results confirmed that the target gene fragments were successfully and correctly
inserted.

![img](https://static.igem.wiki/teams/5872/results/11.webp)

**Figure 11. Agarose gel electrophoresis for PCR verification of SlPPK-SP(pBAD/pACYC) and CP-SpyTag(pACYC) target gene insertion.**

From left to right, the first lane was loaded with M5 HiClear DL2000 plus DNA marker,
the second lane was loaded with the PCR product using SlPPK-SP as a template from the *pBAD33::SlPPK-SP-FLAG* plasmid, the third lane was loaded with the PCR product using CP-SpyTag as a template
from the *pACYCDuet-1::SlPPK-SP-CP-SpyTag-His* plasmid, and the fourth lane was loaded with the PCR product using SlPPK-SP as a template from the *pACYCDuet-1::SlPPK-SP-CP-SpyTag-His* plasmid.

**Summary**

We successfully constructed dual-plasmid and single-plasmid systems expressing the two proteins
SlPPK-SP and CP-SpyTag. Through resistance screening and PCR verification, we ensured the
correct
insertion of the genes, laying the foundation for the subsequent expression of these two
proteins
and the purification of products after *in vivo* assembly.

## 2.2 *In Vivo* Expression and Purification

▼

In this part, we first adopted the method of sequential induction with arabinose + IPTG to induce
the
expression of SP-SlPPK and CP-SpyTag proteins. By exploring the induction expression conditions
(including induction time, induction temperature, IPTG concentration added during induction
**[(Engineering Wet Lab2)](https://2025.igem.wiki/bnu-china/engineering)**, we obtained good expression results. Subsequently, we used
Ni-bead affinity chromatography purification, ultracentrifugation purification, and sucrose
gradient)
ultracentrifugation to directly isolate and enrich *in vivo* assembled V-CHARGEs from cell
lysates. Then, we characterized *in vivo* assembled V-CHARGEs
particles using SEM, DLS, and TEM.

For the double-plasmid system, the overnight seed culture was inoculated into fresh LB medium at
a
ratio of 1:100, and incubated at 37 ℃, 200 rpm until OD600 reached 0.3–0.5. Subsequently, 0.2%
arabinose was added to induce SlPPK-SP expression, and incubation was continued at 28 ℃, 200 rpm
until OD600 reached 1.2–1.5.
Then, 0.1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) was added to induce CP-SpyTag
expression,
and incubation was continued at 16 ℃, 200 rpm for 12 h.

We hypothesize that under this sequential induction condition, SP-SlPPK proteins are first fully
expressed in the cytoplasm, providing anchors and templates for the subsequent CP-SpyTag self-assembly.
Once CP-SpyTag is induced, it gradually assembles with SP-SlPPK inside the cell to form complete
V-CHARGEs particles (Figure 12).

![img](https://static.igem.wiki/teams/5872/results/vivo-main.svg)

**Figure 12. Schematic diagram of *in vivo* assembly.**

To confirm the successful induction expression and self-assembly of V-CHARGEs within host cells,
the
cell samples of the double-plasmid system after induction expression were sent to ServiceBio for
ultrathin sectioning and transmission electron microscopy (TEM) detection. TEM results showed
that a
large number of
uniform spherical particles with a diameter of about 50 nm could be observed in the cytoplasmic
region of cell sections (Figure 13), whose morphology was highly consistent with the *in
vivo*
assembled V-CHARGEs nanoparticles subsequently obtained by purification. This result indicated
that
after sequential induction by arabinose and IPTG,
SlPPK-SP and CP-SpyTag could effectively complete assembly in cells to directly form the target
nanoparticle structure. This result provided intuitive evidence for the success of the *in
vivo* assembly strategy.

![img](https://static.igem.wiki/teams/5872/results/13.webp)

**Figure 13. TEM observation of *in
vivo* assembled V-CHARGEs in bacterial sections.**

**(A)** TEM image of bacterial cell section, scale bar:
500 nm
per 10 grids.

**(B)** Enlarged TEM image of bacterial cell section, spherical particles
with a diameter of about 50 nm can be observed (indicated by red arrows), whose size and
morphology are consistent with
purified *in vivo* assembled V-CHARGEs, indicating that V-CHARGEs had completed
assembly inside cells. Scale bar: 200 nm per 10 grids.

After induction expression, the cells were collected by centrifugation at 6000 rpm for 10 min,
resuspended in Buffer A (50 mM Tris-HCl, pH 8.0, 300 mM NaCl, 3 mM β-mercaptoethanol), and
disrupted
by ultrasonication (3 s on/6 s off cycle, for a total of 10 min). The lysate was centrifuged at
12000 rpm for 45 min,
and the supernatant and precipitate were separately collected for subsequent purification.

We first used Ni-column affinity chromatography to separate and purify the proteins in the
supernatant. First, proteins were eluted with 300 mM imidazole, and then the eluted fraction was
analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE).
The protein expression status was analyzed through gel electrophoresis results. Meanwhile,
Western
blotting was also performed to verify the correctness of the *in vivo* assembly product
structure. Size-exclusion chromatography was further used to purify the protein,
and the results are shown in Figure 14. Combined with WB results, we found that a large amount
of
SlPPK-SP was still present in the eluted fraction obtained after Ni-column affinity
purification,
while size-exclusion chromatography could effectively remove free SlPPK-SP.

![img](https://static.igem.wiki/teams/5872/results/14.webp)

**Figure 14. Purification and
identification of *in vivo* assembled V-CHARGEs.**

**(A)** Size-exclusion chromatography purification
chromatogram of *in vivo* assembled V-CHARGEs. Three absorption peaks were obtained
before the salt peak (Peak 1 at 44 mL, Peak 2 at 55 mL, and Peak 3 at 66 mL).

**(B)** SDS-PAGE analysis of size-exclusion chromatography purification results of
*in
vivo* assembled V-CHARGEs. Based on the molecular weight data of
V-CHARGEs,
Peak 1 corresponds to V-CHARGEs (< 600nm), indicating that the protein eluted at Peak 1
was
successfully assembled and purified V-CHARGEs.

**(C):** Western blot analysis showed that the Peak 1 fraction
exhibited a FLAG band consistent with the size of SlPPK-SP-FLAG-CP-SpyTag, confirming that the protein
eluted at Peak 1 was the successfully *in vivo* assembled and purified V-CHARGEs.

To further verify whether *in vivo* assembled V-CHARGEs differ from *in vitro*
assembled
ones and empty shell CP, we performed native agarose gel electrophoresis of different protein
samples under optimized conditions (upper 0.5% separating gel, lower 1% supporting gel, 8–10 V
voltage,
10–15 h electrophoresis, run on ice). Clear protein migration bands were successfully obtained.

The results showed that the migration rates of *in vivo* assembled particles, *in
vitro*
assembled particles, and empty shell CP were different: *in vitro* assembled particles
migrated
slower than empty shell CP, while *in vivo* assembled particles migrated faster than empty
shell CP (Figure 15).
This difference indicated that SP-SlPPK successfully assembled with CP both *in vivo* and
*in
vitro*, but the assembly modes might be different.

![img](https://static.igem.wiki/teams/5872/results/15.webp)

**Figure 15. Native agarose gel
electrophoresis results showing migration rate differences among *in vivo*
assembled,
*in vitro* assembled, and empty shell CP.**

From left to right: Lane 1, SlPPK-SP, diffuse band; Lane 2
and
Lane 5, CP-SpyTag self-assembled shells, stable bands; Lane 3, *in vitro* assembled
V-CHARGEs (SlPPK-SP-CP-SpyTag), slightly slower migration than CP shell; Lane 4, *in
vivo* assembled V-CHARGEs
(SlPPK-SP-CP-SpyTag); Lane 6, *in vivo* assembled V-CHARGEs concentrated by
ultrafiltration, both showing slightly faster migration than CP shell.

To facilitate the extraction of *in vivo* assembled V-CHARGEs in industrial production, we
referred to published methods of ultracentrifugation separation of Virus-Like Particles (VLPs)
(Vicente et al., 2011; Peyret, 2015; Bakhshizadeh Gashti et al., 2024), and optimized them
accordingly. Specifically,
the induced cells were collected by centrifugation, disrupted by ultrasonication in Buffer A,
centrifuged again to remove debris, and the obtained supernatant was subjected to sucrose
density
gradient ultracentrifugation (gradient from top to bottom: 20%, 35%, 45%, 50%, 60%, 5 mL each
layer;
150,000 × g, 4 ℃, 6 h), as shown in Figure 16.

![img](https://static.igem.wiki/teams/5872/results/16.webp)

**Figure 16. Schematic diagram of
ultracentrifugation operation.**

After ultracentrifugation, we obtained an obvious protein band in the 45% sucrose layer. The
actual
photograph and schematic diagram of the protein band are shown in Figure 17. We aspirated and
retained the protein supernatant from the top layer after ultracentrifugation as sample No. 0,
and then separated the remaining sucrose cushion into samples by aspirating 1 mL from top to
bottom
sequentially, numbered 1–26. All the obtained samples were subjected to SDS-PAGE to investigate
the
separation effect of ultracentrifugation. Based on the gel electrophoresis results,
we also used ImageJ to quantitatively analyze the relative protein concentrations of the protein
supernatants in different layers after ultracentrifugation.

![img](https://static.igem.wiki/teams/5872/results/17.webp)

**Figure 17. Actual photograph and
schematic diagram of protein band after ultracentrifugation.**

According to the SDS-PAGE results (Figure 18), we obtained strong CP and SP-SlPPK bands at the
position corresponding to the protein band (No. 16), with very few impurities. In the
surrounding
samples, CP and SP-SlPPK bands gradually weakened; at the bottom of the sucrose cushion (No.
26),
we obtained very strong CP and SP-SlPPK bands, along with some impurities.

![img](https://static.igem.wiki/teams/5872/results/18-2.webp)

**Figure 18. SDS-PAGE gel
electrophoresis
after ultracentrifugation and relative protein content analysis.**

**(A)** In the left picture, from left to right: the first lane was loaded
with
M5 Prestained Protein Ladder (10–180 kDa) with 45 kDa, the second lane was the
supernatant
after ultracentrifugation, and lanes 3–13 were the 1–11 mL fractions of the sucrose cushion aspirated
sequentially
from the top to bottom after ultracentrifugation. In the right picture, from left to
right:
the first lane was loaded with M5 Prestained Protein Ladder (10–180 kDa) with 45 kDa,
and
lanes 2–14 were the 12–24 mL fractions of the sucrose cushion aspirated sequentially from the top to bottom
after
ultracentrifugation.

**(B)** shows the relative concentrations of target protein bands in each lane obtained
through ImageJ analysis, indicating that around sample No. 16 there was a higher
concentration of target protein, while sample No. 26 (at the bottom) showed a very high
concentration of target protein.

To confirm which of the different protein samples obtained after ultracentrifugation represented
the
best assembled product with complete assembly and almost no free SP-SlPPK, we used Dot Blotting
for
verification. The specific operation and principle are shown in Figure 19. Water (blank
control),
CP empty shell, purified SP-SlPPK, ultracentrifugation sample No. 16, and sample No. 26 were
diluted
to the same concentration (1 ng/μL), and 1 μL and 3 μL were spotted onto the PVDF membrane as
replicates.

![img](https://static.igem.wiki/teams/5872/results/19.webp)

**Figure 19. Schematic diagram of Dot
Blotting operation and principle.**

The detection results showed that the SP-SlPPK control exhibited a strong signal, confirming the
validity of the detection system; CP and the blank control showed no signal. Comparing samples
16
and 26, sample 16 showed a very weak signal, while sample 26 showed a stronger signal (Figure
20),
indicating that sample 16 contained little free or exposed SP-SlPPK, with higher particle
assembly
integrity; while sample 26 still contained a certain amount of exposed SP-SlPPK. Based on this
result, we selected sample 16 for subsequent size-exclusion chromatography purification and
further
functional verification.

![img](https://static.igem.wiki/teams/5872/results/20-2.webp)

**Figure 20. Dot Blotting detection of
SP-SlPPK presence in different samples.**

Water (blank control), CP empty shell, purified SP-SlPPK,
ultracentrifugation sample No. 16, and sample No. 26 were diluted to 1 ng/μL, and 1 μL
and 3
μL were spotted onto the PVDF membrane in two rows, corresponding to 1 ng and 3 ng
loading
amounts. In the third column,
SP-SlPPK showed a strong signal; in the first and second columns, CP empty shell and
water
control showed no signal. In the fourth column, sample 16 showed a very weak signal; in
the
fifth column, sample 26 showed a strong signal.

To further compare the enzymatic properties of different fractions obtained from
ultracentrifugation,
we measured the specific enzyme activities of samples 16 and 26 (Figure 21). The results showed
that
although sample 26 had a higher protein concentration, its specific enzyme activity
(enzyme activity per unit protein content) was significantly lower than that of sample 16;
conversely, sample 16 showed higher specific enzyme activity, indicating that the conformation
of
SlPPK within its particles was more intact and its function better preserved.

![img](https://static.igem.wiki/teams/5872/results/21.webp)

**Figure 21. Comparison of specific
enzyme
activities between sample 16 and sample 26.**

Using protein content as the standard, the enzyme
activities of
the two samples were compared. The results showed that sample 16 had significantly
higher
specific enzyme activity than sample 26, indicating higher assembly integrity and better
functional preservation.

Combining the results of SDS-PAGE (Figure 18), Dot Blotting (Figure 20), and specific enzyme
activity
measurement (Figure 21), it can be seen that: in sample 16, CP and SP-SlPPK bands were clear and
with few impurities; the Dot Blotting signal was extremely weak,
indicating almost no free or exposed SP-SlPPK. In terms of assembly integrity and enzymatic
performance, sample 16 was superior to sample 26. Therefore, we selected sample 16 as the
representative assembled product of *in vivo* assembled V-CHARGEs for subsequent
size-exclusion
chromatography purification and functional verification experiments.

We further purified the *in vivo* assembled V-CHARGEs obtained after ultracentrifugation
using
size-exclusion chromatography. Based on the SDS-PAGE, Dot Blotting, and preliminary enzyme
activity
measurement results, we selected the protein band sample at the protein ring (sample No. 16),
which
had high target protein content,
very few impurities, high particle assembly integrity, and high enzymatic activity, for further
size-exclusion chromatography purification. The size-exclusion chromatography results are shown
in
Figure 22. The results indicated that the sample obtained at the protein band after
ultracentrifugation eluted outside the column
volume of the size-exclusion chromatography column, suggesting that it was successfully *in
vivo* assembled V-CHARGEs. Furthermore, the sample showed very high purity with almost no
impurity peaks.

![img](https://static.igem.wiki/teams/5872/results/22.webp)

**Figure 22. Purification and
identification of *in vivo* assembled V-CHARGEs after ultracentrifugation.**

Figure 22 shows the size-exclusion chromatography
purification
elution profile of *in vivo* assembled V-CHARGEs after ultracentrifugation. Before
the
salt peak, one absorption peak was obtained (Peak at 45 mL).

**Summary**

In this part, V-CHARGEs were efficiently expressed and assembled *in vivo* using the
double-plasmid system. Through TEM observation of ultrathin sections of optimized induced
samples, a
large number of uniform spherical particles with a diameter of about 50 nm
were clearly captured in the cytoplasmic region of host cells, whose morphology was highly
consistent with purified *in vivo* assembled V-CHARGEs.

Through preliminary Ni-column affinity chromatography and size-exclusion chromatography
purification,
we successfully obtained correctly *in vivo* assembled V-CHARGEs particles. We further
confirmed through native gel electrophoresis that the migration rate of *in vivo* assembled
products differed from *in vitro* assembled ones and CP empty shells,
suggesting possible differences in assembly mode. By ultracentrifugation combined with sucrose
density gradient separation, we obtained a large amount of *in vivo* assembled V-CHARGEs in
a
convenient manner. The protein band at fraction 16 corresponded to high purity, nearly no free
SP-SlPPK, and significantly higher specific activity
than the sediment fraction 26, indicating better assembly integrity and functional preservation.
Finally, after further purification by size-exclusion chromatography, fraction 16 exhibited a
single
and highly pure peak, confirming it as successfully assembled and high-purity *in vivo*
assembled V-CHARGEs. Overall, we established a stable and effective
preparation process of V-CHARGEs from *in vivo* expression to purification, obtaining
functional and high-purity representative assembled products, laying a solid foundation for
subsequent functional studies.

## 2.3 Characterization

▼

We characterized the particle size of *in vivo* assembled V-CHARGEs before and after
ultracentrifugation by Dynamic Light Scattering (DLS), and characterized the assembly of *in
vivo* assembled proteins including shape and particle size by Transmission Electron
Microscope (TEM). DLS results showed that the particle size of *in vivo* assembled
V-CHARGEs was about 150 nm. TEM images clearly showed approximately spherical nanoparticles with
diameters similar to VLPs without encapsulated SlPPK. At the same time, compared with before
ultracentrifugation, the particle size range of the samples after ultracentrifugation was
smaller
and the homogeneity was better, indicating that the
*in vivo* assembled samples obtained after ultracentrifugation were relatively pure and the
assembly effect was good.

![img](https://static.igem.wiki/teams/5872/results/23.webp)

**Figure 23. Results of dynamic light
scattering and transmission electron microscopy analysis of V-CHARGEs before and after
ultracentrifugation *in vivo* assembly.**

**(A) (B)** are the average DLS intensity distribution
graphs of *in vivo* assembled V-CHARGEs before and after ultracentrifugation *in
vivo* assembly. The graph shows the variation of the intensity distribution
function
G(d) of the sample hydrated hydrodynamic diameter with the particle size d.
The horizontal axis is the particle size (nm) with logarithmic scale. The unimodal
distribution curve peaks at 150 nm.

**(C) (D)** are TEM images of V-CHARGEs before
and
after ultracentrifugation *in vivo* assembly. The circular spherical particles (~50
nm)
in the images are *in vivo* assembled V-CHARGEs.
The scale bars of **(C) (D)** are 100 nm.

DLS measures the hydrodynamic diameter, which includes the hydration layer and possible aggregates,
while TEM shows the dry core size. Therefore, DLS values appear larger than TEM results.

**Summary**

DLS and TEM characterization confirmed the formation of *in vivo* assembled V-CHARGEs
particles,
with uniform particle size and complete structure, and better effect after ultracentrifugation.
These results are consistent with expectations, indicating that the *in vivo* assembly
strategy
successfully produced functional nanoparticles,
providing high-quality core materials for subsequent experiments to detect the enzymatic
activity of
SlPPK-SP encapsulated in *in vivo* assembled V-CHARGEs particles.

## 2.4 Discussion

**Summary and Reflection**

In this study, we successfully achieved the ***in vivo* assembly** of
V-CHARGEs
particles and established a complete purification and identification process. Compared with
*in
vitro* assembly, *in vivo* assembly shows the following significant advantages:

**1. Higher assembly efficiency:** SlPPK-SP and CP-SpyTag were co-expressed in
cells and
spontaneously assembled, reducing the accumulation of unassembled monomers;

**2. Higher purity and stability:** Through the combined application of affinity
chromatography, sucrose density gradient ultracentrifugation, and size-exclusion
chromatography, we
were able to stably obtain high-purity particles with significantly reduced contaminating
proteins;

**3. Closer to natural conformation:** DLS and TEM results showed that *in
vivo*
assembled particles had uniform particle size and complete spherical structures, indicating that
this
strategy could maintain correct folding and spatial conformation in the cellular environment.

These results verified the feasibility of *in vivo* assembly and also indicated its
greater
potential compared with *in vitro* assembly when dealing with complex multi-enzyme
systems.
Meanwhile, the process of combining ultracentrifugation and size-exclusion chromatography
provides a
technical basis for subsequent large-scale applications.

**Outlook**

In vivo assembly not only provided us with high-purity and complete V-CHARGEs particles,
but
more
importantly, laid a reliable foundation for **functional detection**. Our
next
research
will focus on the **enzymatic properties** of SlPPK-SP inside the
particles.
Through
enzymatic property experiments, we will verify the enhancement
effect of CP shell encapsulation of SlPPK on its activity and stability, and further
reveal
the
application potential of V-CHARGEs as a multi-enzyme complex platform.

# 3. Enzyme Activity Validation

![engi-top](https://static.igem.wiki/teams/5872/results/enzyme-main2.webp)

Figure IV. Schematic Diagram of Enzyme Activity Validation of V-CHARGEs.

We confirm the stability of V-CHARGEs against challenging environments like acidic pH, high phosphate levels, and protease degradation.

* **We verified that V-CHARGEs can tolerate acidic environments,
  high phosphate concentrations, and protease degradation.**

**Introduction and Objectives**

In the *in vivo* assembly section, we successfully isolated and purified high-purity,
intact
*in vivo* assembled V-CHARGEs particles,
laying a reliable foundation for functional assays. In this part, we systematically compared
the
ATP
production capacity of free SlPPK and *in vivo* assembled V-CHARGEs under prolonged
reaction
times and various stress conditions (pH, Pi, protease).

This section utilized a firefly luciferase-based ATP detection kit to measure ATP levels
under
different experimental conditions, thereby reflecting the activity of the relevant enzymes.
First, a
standard curve correlating ATP concentration with luminescence
intensity was established to ensure the accuracy and reliability of ATP quantification.
Based on
this, the activities of free SlPPK and *in vivo* assembled V-CHARGEs were compared, and
the
effects of different protease concentrations, pH, and phosphate levels on ATP generation
were
further investigated.

The results indicated that SlPPK encapsulated within *in vivo* assembled V-CHARGEs
exhibited
significantly higher ATP levels compared to free SlPPK under high phosphate concentrations
and
protease treatment, demonstrating their protective effect on the enzyme. Furthermore, under
acidic
conditions (pH 3.0~5.5), the ATP levels of *in vivo* assembled V-CHARGEs
were also superior to those of free SlPPK, highlighting their stability advantage against
acid
stress. Concurrently, control experiments showed that firefly luciferase activity was
largely
unaffected by different pH, phosphate, and protease conditions, further confirming the
reliability
of the experimental results.

## 3.1 ATP Detection Method

▼

* **Procedure:** A standard curve for ATP was established using the firefly
  luciferase bioluminescence assay, based on which the ATP concentrations in samples were
  calculated to characterize enzyme activity.

* **Conclusion:** The standard curve proved reliable and is suitable for
  subsequent
  comparative analysis of ATP yield and enzyme activity.

We used a firefly luciferase-based ATP detection kit for ATP concentration measurement.
Luminescence
intensity was measured using a FlexStation®3 multi-mode microplate reader, and data were
analyzed
with SoftMax Pro software. The ATP concentration in samples was calculated based on the
luminescence
intensity-ATP concentration standard curve,
subsequently reflecting enzyme activity through the ATP concentration. The specific detection
method
is illustrated in Figure 24.

![img](https://static.igem.wiki/teams/5872/results/24-2.webp)

**Figure 24. Schematic of the firefly
luciferase ATP detection method.**

ADP, PolyP, Tris-HCl buffer, Mg²⁺, firefly luciferase
(FLuc),
and the sample to be tested are combined in the reaction system. The generated ATP can
be
converted by firefly luciferase into a detectable luminescence signal through a
catalyzed
light-emitting reaction.
By performing the reaction in a 96-well plate and detecting the luminescence intensity
in a
multi-mode microplate reader, the ATP concentration in the sample can be indirectly
characterized.

To calibrate the relationship between ATP concentration and luminescence intensity, we used 20μL
of
ATP standard solutions at concentrations of 0.1μM, 0.3μM, 1μM, 3μM, and 10μM as samples.Each
sample
was mixed with 100 μL of ATP detection solution for bioluminescence intensity measurement. After
data
analysis,
a bioluminescence intensity-ATP concentration standard curve was obtained(Figure 25).

![img](https://static.igem.wiki/teams/5872/results/25.webp)

**Figure 25. ATP standard
curve.**

Using ATP standard solutions of different concentrations
(0.1
μM, 0.3 μM, 1 μM, 3 μM, 10 μM) as test samples, the luminescence intensity was measured
and
linearly fitted, yielding the equation y=27965x+1207 (R2=0.9971). This indicates a good
linear relationship
between luminescence intensity and ATP concentration, which can be used to estimate
sample
ATP concentration and evaluate enzyme activity.

## 3.2 Determining the Ratio of SlPPK-SP to CP-SpyTag in *in vivo* assembled V-CHARGEs

▼

* **Procedure:** Integrating modeling results with experimental data, the ratio
  of
  SlPPK-SP to CP-SpyTag within *in vivo* assembled V-CHARGEs was deduced and validated to
  ensure rigor in subsequent enzyme activity comparisons.
**[(Link to Dry Lab Part2)](https://2025.igem.wiki/bnu-china/model)**

* **Conclusion:** A nearly equivalent ratio of SlPPK-SP to CP-SpyTag within *in
  vivo* assembled V-CHARGEs can be achieved by maintaining the total protein
  concentration
  at 25%–30% of that used for free SlPPK-SP, thereby ensuring comparability between the
  assembled
  system and free enzyme controls in enzyme activity assays.

In the preliminary experiments, based on modeling, we theoretically estimated the encapsulation
ratio
of SlPPK-SP within *in vivo* assembled V-CHARGEs. It was determined that when the protein
concentration of the *in vivo* assembled V-CHARGEs sample is controlled at 25%~30% of that
of
the free SlPPK protein sample, the absolute amount of
encapsulated SlPPK-SP is roughly equivalent to that in the free control group. This minimizes
interference from differences in protein dosage, ensuring that differences in ATP generation
experiments primarily stem from the "free" versus "assembled" states rather than inconsistencies
in
protein quantity.

**Summary**

According to the modeling results, to ensure that the amount of SlPPK-SP encapsulated in *in
vivo* assembled V-CHARGEs is essentially the same as that of the free SlPPK-SP control,
in
subsequent experiments, we controlled the protein concentration of the assembled protein sample
to
be 25%~30% of that of the free protein sample.

## 3.3 Prolonged Reaction

▼

* **Procedure:** The ATP production and reaction rates of free SlPPK-SP and
  V-CHARGEs
  were compared over extended reaction periods.

* **Conclusion:** V-CHARGEs demonstrated a higher and more sustained ATP
  production
  capacity during prolonged reactions.

We first measured the changes in ATP content within the system over 6 hours and calculated the
rate
of ATP content change. The results are shown in Figure 26.

![img](https://static.igem.wiki/teams/5872/results/26.webp)

**Figure 26. ATP generation and rate
changes of SlPPK-SP and *in vivo* assembled V-CHARGEs during prolonged
reaction.**

**(A)** ATP concentration changes over 6h for SlPPK-SP
group
(red) and *in vivo* assembled V-CHARGEs group (blue). Thick lines represent actual
ATP
concentrations calculated from the standard curve, thin lines represent fitted
concentration
change curves.

**(B)** Rate of ATP content
change over 6h for SlPPK-SP group (red) and *in vivo* assembled V-CHARGEs group
(blue).

From the ATP generation curve of the prolonged reaction (Figure 26A), the ATP concentration of
the
control group (free SlPPK) increased faster in the initial phase of the reaction (0~100 min),
while
the *in vivo* assembled V-CHARGEs group showed a slightly slower increase rate due to the
presence of the assembled system.
However, as time progressed (>150 min), the ATP level of the control group plateaued,
stabilizing at
approximately 1.5 mg/mL, whereas the *in vivo* assembled V-CHARGEs group continued to rise,
eventually maintaining a higher ATP concentration (close to 2.0 mg/mL). This result indicates
that
although the ATP generation
efficiency of the assembled system was slightly lower than the free control in the early stage,
it
exhibited stronger sustainability and stability under prolonged reaction conditions. Further
analysis of the reaction rate changes (Figure 26B) showed that the slope of the control group's
curve was
significantly higher than that of *in vivo* assembled V-CHARGEs in the early reaction
stage,
indicating a faster ATP generation rate. However, over time, the control group's rate decreased
rapidly and approached zero, showing gradual decay of its catalytic activity. In contrast, the
*in
vivo* assembled V-CHARGEs group, although starting with
a slightly lower rate, exhibited a more gradual decline trend, maintaining a certain level of
catalytic activity throughout the entire reaction process. This result suggests that *in
vivo*
assembled V-CHARGEs can effectively delay the loss of enzyme activity, enabling it to maintain
higher ATP yield in prolonged reaction systems.

**Summary**

The prolonged reaction experiment results show that the initial ATP generation rate of *in
vivo*
assembled V-CHARGEs was slightly lower than that of free SlPPK. However, as the reaction time
extended, the catalytic activity of the free enzyme rapidly decayed and tended towards
inactivation,
while *in vivo* assembled V-CHARGEs still maintained
sustained ATP generation capacity. Ultimately, the ATP yield of *in vivo* assembled
V-CHARGEs
was higher than that of the control group, and its catalytic rate decline trend was more
gradual.
This indicates that *in vivo* assembled V-CHARGEs not only enhance the stability of SlPPK
but
also extend its functional lifespan in prolonged reaction systems,
offering potential advantages for applications in continuous energy supply and long-term
industrial
reactions.

In practical applications, maintaining enzyme activity over long periods is a key challenge in
cell-free synthesis systems and industrial continuous reactions. Free enzymes often rapidly
inactivate under prolonged reaction conditions, leading to limited product accumulation and
reduced
production efficiency. *In vivo* assembled V-CHARGEs, capable of maintaining stable ATP
generation capacity over extended periods, not only provide continuous energy assurance for
cell-free synthesis systems but also offer a more reliable power source for industrial
fermentation
and synthetic reactions requiring long-term operation. This characteristic
significantly enhances the potential value of SlPPK in complex environments and large-scale
applications.

## 3.4 Influence of Firefly Luciferase

▼

* **Procedure:** The influence of varying pH levels, phosphate concentrations,
  and
  protease conditions on the luminescence intensity of firefly luciferase in the presence of
  equivalent
  ATP was assessed.

* **Conclusion:** Firefly Luciferase activity remained largely unaffected by changes in
  pH
  and phosphate levels, with only minor interference from protease, confirming the robustness
  of
  the detection system.

To subsequently assess the effects of factors like pH, phosphate content, and protease content on
*in vivo* assembled V-CHARGEs and free SlPPK, aiming to demonstrate the advantages *in
vivo* assembled of V-CHARGEs under adverse conditions, we first needed to exclude the
potential influence of these adverse factors on firefly luciferase
activity to ensure experimental rigor. We tested the luminescence intensity generated by equal
amounts of ATP activating firefly luciferase under different pH, phosphate concentrations, and protease
concentrations, plotted scatter diagrams, and performed linear fitting on the results (Figure
27).
We found that firefly luciferase activity was almost unaffected by pH and phosphate, and only slightly
affected by protease, validating the reliability of the detection system.

![img](https://static.igem.wiki/teams/5872/results/27.webp)

**Figure 27. Luminescence intensity
generated by equal amounts of ATP activating firefly luciferase under different pH, phosphate
concentrations, and protease concentrations.**

**(A)** Luminescence intensity under different pH. Blue
data
points are measured values, red dashed line is linear fit: Y = -478.7\*X + 6818,
R²=0.6796.

**(B)** Luminescence intensity under different phosphate concentrations (mM). Blue data
points, red dashed line: Y = -66.87\*X + 21069, R²=0.1278.

**(C):** Luminescence intensity under different protease amounts (μg). Blue data points,
red
dashed line: Y = -219.7\*X + 82409, R²=0.8986.

**Summary**

We found that the luminescence intensity generated by firefly luciferase was essentially the same
under different conditions, meaning that firefly luciferase activity was largely unaffected by
phosphate concentration and protease, and only slightly affected by pH. However, this minor
effect
could be mitigated in subsequent calculations through differential methods. This proves the
reliability of our experimental results, indicating that the firefly luciferase ATP detection
method
can be used to assess the effects of pH, phosphate, and protease on SlPPK activity.

## 3.5 Advantage Assessment

▼

* **Procedure:** ATP production levels of *in vivo* assembled V-CHARGEs and
  free
  SlPPK were compared under different conditions of pH, phosphate, and protease exposure.

* **Conclusion:** *In vivo* assembled V-CHARGEs effectively protected SlPPK
  from
  inhibition and degradation, maintaining higher enzymatic activity under various adverse
  conditions.

To demonstrate the advantages and value of our *in vivo* assembled V-CHARGEs under
unfavorable
conditions, we compared the ATP generation levels of *in vivo* assembled V-CHARGEs and free
SlPPK under different phosphate, protease and pH conditions. We found that *in vivo*
assembled
V-CHARGEs effectively protected SlPPK from inhibition and degradation,
maintaining higher enzyme activity under various adverse conditions.

To investigate the effects of phosphate concentration, protease concentration, and pH on enzyme
activity, using free SlPPK as a control, we respectively examined the luminescence intensity
after a
15-minute reaction catalyzed by *in vivo* assembled V-CHARGEs with sodium hexametaphosphate
and
ADP
under different phosphate concentrations, protease concentrations, and pH levels, and generated
bar
charts for each.

### 3.5.1 Phosphate

To investigate whether *in vivo* assembled V-CHARGEs encapsulation could enhance SlPPK's
resistance to phosphate inhibition, we detected the relative ATP levels of free SlPPK and
*in
vivo* assembled V-CHARGEs-encapsulated SlPPK under different phosphate concentrations
(0–20
mM). As shown in Figure 28, under low phosphate concentrations
(0–7.5 mM), the ATP generation levels of the two groups were similar, indicating weak
phosphate
inhibition at this stage. However, under higher phosphate concentrations (≥12.5 mM), the
activity of
free SlPPK decreased significantly, with relative ATP levels below 0.2. In contrast, SlPPK
encapsulated
within *in vivo* assembled V-CHARGEs maintained levels around 0.6–0.8, exhibiting
significantly
higher residual activity. That is, at phosphate concentrations of 12.5 mM and above, the ATP
production level of *in vivo* assembled V-CHARGEs was significantly higher than that of
the
control group. These results indicate that *in vivo* assembled V-CHARGEs
encapsulation effectively attenuates the inhibitory effect of phosphate on SlPPK, thereby
significantly improving the enzyme's functional stability under adverse conditions.

![img](https://static.igem.wiki/teams/5872/results/28.webp)

**Figure 28. Effect of phosphate
concentration on *in vivo* assembled V-CHARGE.**

Figure 28 evaluates the inhibitory effect of phosphate
on
SlPPK
activity by detecting the relative ATP levels produced by enzyme catalysis under
different
phosphate concentrations (0-20 mM). Compared with the control group (free SlPPK,
red),
SlPPK
encapsulated
in V-CHARGEs (blue) still maintained significantly higher ATP production under high
phosphate concentrations (12.5-20 mM). Data are presented as mean ± standard
deviation
(n=3). Statistical analysis was performed using two-way analysis of variance,
\*\*\*\* indicates p<0.0001.

**Summary**

The results investigating the effect of different phosphate concentrations on the ATP level
of
*in
vivo* assembled V-CHARGEs showed that the ATP production activity of *in vivo*
assembled
V-CHARGEs under high phosphate treatment was significantly higher than that of the free
SlPPK
control group, highlighting the superiority of *in vivo* assembled V-CHARGEs in
protecting
SlPPK against phosphate.

In practical applications, the presence of high concentrations of inorganic phosphate is a
common
challenge in many biocatalytic processes. For example, in cell-free synthesis systems, ATP
regeneration cycles often lead to the continuous accumulation of phosphate by-products,
severely
inhibiting the
activity of various metabolic enzymes. In industrial-scale fermentation and production
processes,
buffers and culture media commonly contain high concentrations of phosphate, greatly
limiting
enzyme
stability and reaction efficiency.

*In vivo* assembled V-CHARGEs encapsulation provides a practical solution to this
long-standing
bottleneck problem. By protecting SlPPK from phosphate inhibition, *in vivo* assembled
V-CHARGEs not only extend the enzyme's operational lifespan but also maintain high catalytic
efficiency in high-phosphate environments, thereby demonstrating higher application
value in large-scale production and complex environmental conditions.

### 3.5.2 Protease

To evaluate whether *in vivo* assembled V-CHARGEs can protect SlPPK from protease
degradation,
we compared the residual activity of free SlPPK and *in vivo* assembled
V-CHARGEs-encapsulated
SlPPK after treatment with different protease concentrations. As shown in Figure 29, under
low
concentration protease treatment (20 μg), there was no significant difference in ATP levels
between the two groups, indicating that mild protease action was not sufficient to
significantly
affect enzyme activity. However, as the protease concentration increased, the activity of
free
SlPPK
decreased rapidly, showing a significant reduction in the 40~160 μg range, with less than
40%
activity remaining at 160 μg. In contrast,
the activity decline of *in vivo* assembled V-CHARGEs-encapsulated SlPPK was slower,
maintaining about 60% activity even under high concentration protease treatment (160 μg).
Overall,
*in vivo* assembled V-CHARGEs encapsulation significantly enhanced the stability of
SlPPK
under
protease treatment conditions, further demonstrating its protective effect.

![img](https://static.igem.wiki/teams/5872/results/29.webp)

**Figure 29. Effect of Protease
Treatment
on *in vivo* assembled V-CHARGEs.**

Figure 29 shows the relative ATP levels (relative to
their
respective untreated groups) of *in vivo* assembled V-CHARGEs containing SlPPK
(blue
bars) and the free SlPPK control group (red bars) after treatment with different
concentrations of protease (20, 40, 80, 100, 120, 160 μg). Error bars represent mean
±
SEM
(n=3). Statistical analysis was performed using two-way ANOVA; ns indicates no
significant
difference, \* indicates p < 0.05, \*\* indicates p < 0.01.

**Summary**

The results investigating the effect of different protease concentrations on the ATP level of
*in
vivo* assembled V-CHARGEs showed that the ATP production activity of *in vivo*
assembled
V-CHARGEs under protease treatment was significantly higher than that of the free SlPPK
control
group, highlighting the superiority of *in vivo* assembled V-CHARGEs in protecting
SlPPK
from
protease degradation.

In practical applications, exogenous enzymes often reside in complex environments containing
proteases, which is almost a "common problem" in industrial applications. Proteases commonly
present
in industrial fermentation systems can rapidly diminish the activity of added enzymes during
scaled-up production, causing a sharp drop in catalytic efficiency. Therefore, resistance to
protease degradation is not merely a "tolerance improvement" but addresses a common
bottleneck
problem spanning industrial,
agricultural, and medical biotechnology sectors.

### 3.5.3 pH

To investigate the effect of acidic environments on SlPPK, we compared the residual activity
of
free
SlPPK and *in vivo* assembled V-CHARGEs-encapsulated SlPPK under different pH
conditions.
As
shown in Figure 30, at pH 6.5, the ATP generation levels of both groups decreased, with no
significant difference observed between them. As the pH decreased to 5.5, the activity of
free
SlPPK
significantly dropped to less than 10%, while *in vivo* assembled
V-CHARGEs-encapsulated
SlPPK
retained about 20% activity.
When the pH was further reduced to 3.5 and 3, the activity of free SlPPK was almost
completely
lost,
whereas *in vivo* assembled V-CHARGEs-encapsulated SlPPK still maintained levels around
25~30%,
displaying significantly higher residual activity. The results show that under different pH
conditions, the ATP levels of *in vivo* assembled V-CHARGEs were generally higher than
those of
the free SlPPK control group,
especially under strongly acidic conditions of pH 3.0 to 3.5, where *in vivo* assembled
V-CHARGEs showed a significant advantage in ATP production activity (\*\*\*p < 0.001). The
results
indicate that *in vivo* assembled V-CHARGEs can effectively protect SlPPK under acidic
conditions, significantly enhancing its stability and tolerance.

![img](https://static.igem.wiki/teams/5872/results/30.webp)

**Figure 30. Effect of pH
adjustment on
*in vivo* assembled V-CHARGEs.**

By adjusting the reaction system pH to 6.5, 5.5, 3.5,
and
3,
the relative ATP levels (relative to their respective untreated groups) of *in
vivo*
assembled V-CHARGEs-encapsulated SlPPK (blue) and free SlPPK (red) were determined.
Error
bars represent mean ± SEM (n=3). Statistical analysis was performed using two-way
ANOVA;
ns
indicates no significant difference, \* indicates p < 0.05, \*\*\*\* indicates p <
0.0001.

**Summary**

The results investigating the effect of different pH on the ATP level of *in vivo*
assembled
V-CHARGEs showed that the ATP production activity of *in vivo* assembled V-CHARGEs
under
acidic
condition treatment was significantly higher than that of the free SlPPK control group,
highlighting
the superiority of *in vivo* assembled V-CHARGEs in protecting SlPPK against acidic
environments.

In practical applications, extreme pH conditions are a common challenge to enzyme stability
and
activity. In industrial fermentation and organic acid production processes, systems often
become
acidic due to the accumulation of metabolic by-products. In food processing, environments
like
fruit
juices and fermented beverages are naturally at low pH. In these scenarios, traditional
enzymes
often rapidly inactivate, limiting their catalytic applicability.

*In vivo* assembled V-CHARGEs encapsulation provides an effective solution to this
problem.
By
enhancing the stability and residual activity of SlPPK in acidic environments, *in
vivo*
assembled V-CHARGEs not only reduce the need for additional neutralization and regulation
steps,
thereby lowering production costs, but also broaden the application potential of enzymes in
the
food industry and environmental remediation, offering a more feasible and sustainable
strategy
for
biocatalysis under extreme conditions.

**Overall Summary**

Integrating the above results, *in vivo* assembled V-CHARGEs demonstrated significant
protective
effects on SlPPK under multiple adverse environmental factors. Whether facing substrate
inhibition
effects from high phosphate concentrations, degradation risks caused by exogenous proteases, or
inactivation issues under acidic conditions, *in vivo* assembled V-CHARGEs-encapsulated
SlPPK
performed significantly better than the free enzyme, maintaining higher residual activity
and ATP generation capacity. This indicates that *in vivo* assembled V-CHARGEs not only
improve
enzyme stability under individual adverse conditions but also provide a universal protective
mechanism spanning multiple stress factors.

From a practical application perspective, this "multi-stress resistance" is crucial for the
deployment of exogenous enzymes in complex systems. Application scenarios such as industrial
fermentation, cell-free synthesis systems, food processing, and environmental remediation often
simultaneously present challenges like acidic environments, phosphate accumulation, and protease
interference. Traditional free enzymes often rapidly
inactivate under these conditions, struggling to maintain catalytic efficiency. In contrast,
through
its nano-scale encapsulation and protection, *in vivo* assembled V-CHARGEs not only
significantly extend the operational lifespan of SlPPK but also reduce the system's sensitivity
to
environmental conditions, providing a practical solution for the application of exogenous
enzymes
under large-scale, long-duration, and extreme conditions.

Overall, *in vivo* assembled V-CHARGEs exhibit characteristics of a universal platform
capable
of significantly enhancing enzyme stress resistance. This not only addresses common bottleneck
problems in multiple industrial applications but also provides new insights for the future
development of biocatalysts and functional materials adapted to extreme environments.

## 3.6 Discussion

**Summary and Reflection**

This section systematically compared the catalytic performance of free SlPPK and *in
vivo*
assembled V-CHARGEs under prolonged reaction and different stress conditions (phosphate,
protease,
acidic pH) using a firefly luciferase-based ATP detection kit. Experiments showed that
although
the
initial transient generation rate of the assembled samples was slightly lower than that of
the
free
enzyme, they maintained and
accumulated significantly higher ATP levels during prolonged reactions (> 150 min). Under
high
phosphate concentrations (≥12.5 mM), high protease levels, and acidic conditions (pH <
6.5),
*in vivo* assembled V-CHARGEs consistently exhibited significantly better resistance
compared
to free SlPPK (statistically significant). This advantage indicates that by providing a
protected
microenvironment for SlPPK, *in vivo* assembled V-CHARGEs significantly delay the loss
of
enzyme activity,
allowing it to maintain efficient catalysis even in harsh environments and during prolonged
operation. Control experiments with firefly luciferase indicated that the detection system's
response to equal amounts of ATP was largely consistent across the tested pH, Pi, and
protease
ranges, confirming the reliability of the measurements.

These results support the hypothesis that "V-CHARGEs provide a protected microenvironment for
SlPPK".
This protection may originate from:

1. Spatial segregation and physical protection of the particle shell against proteases,
reducing
enzyme degradation.

2. Buffering of the local environment (pH, ions) or restricted diffusion of
substrates/products
leading to slowed enzyme inactivation kinetics.

These findings possess significant scientific value and application prospects. From a
scientific
perspective, this study clearly demonstrates for the first time that encapsulating
polyphosphate
kinase using P22-VLP can significantly enhance its tolerance and sustained activity,
providing a
novel approach to solving the problem of enzyme instability in complex environments.
Compared to
traditional enzyme immobilization or chemical modification methods,
the *in vivo* assembled V-CHARGEs assembly strategy is more efficient, mild, and
biocompatible,
substantially improving environmental adaptability while maintaining the enzyme's native
activity.
From an application perspective, this system shows broad potential in areas requiring
long-term,
stable energy supply, such as cell-free synthesis systems, *in vitro* biomanufacturing,
and
energy metabolism simulation.

**Outlook**

The enzyme activity assays in this section show that *in vivo* assembled V-CHARGEs
can
continuously produce ATP, perform significantly better than free SlPPK under high Pi,
protease,
and
acidic conditions, and maintain higher energy output and stability during prolonged
reactions.

Based on these results, our next core objective is to utilize *in vivo* assembled
V-CHARGEs
as
an energy module, using the ATP they produce to drive enzymatic reactions located on the
outer
surface of the particle
– specifically, by conjugating exogenous enzymes (FLuc, UCK, γ-GCS, GS) to the surface
of
*in
vivo* assembled V-CHARGEs via SpyTag/SpyCatcher, to validate the feasibility and
efficiency
of "intra-particle energy supply → extra-particle product synthesis".

# 4. Product Determination

![engi-top](https://static.igem.wiki/teams/5872/results/product-main-gai.svg)

Figure V. Schematic Diagram of Product Validation.

We successfully construct the V-CHARGEs multi-enzyme cascade platform, demonstrating versatility and high efficiency by producing high-value molecules like 5'-CMP and GSH.

* **By coupling multiple enzymes on the outer surface of V-CHARGEs, we successfully produced 5'-CMP and GSH,
  demonstrating the platform versatility and high efficiency of V-CHARGEs.**

**Introduction and Objectives**

In the previous experiments, we have verified that **SlPPK inside *in vivo*
assembled
V-CHARGEs can synthesize ATP, and the product has good enzymatic activity.**
However,
merely proving that *in vivo* assembled V-CHARGEs can generate ATP inside the shell is
not sufficient to demonstrate its potential in practical applications. To truly make it
functional,
we need to further prove whether this ATP can be efficiently utilized and **drive
external
energy-consuming reactions
to produce the desired products.**

To this end, we selected three representative ATP-dependent reactions:

* **Firefly luciferase luminescence** (catalyzed by firefly luciferase)

* **5’-CMP synthesis** (catalyzed by Uridine-Cytidine Kinase, UCK)

* **Glutathione synthesis** (catalyzed in two steps by γ-Glutamylcysteine
  Synthetase,
  γ-GCS, and Glutathione Synthetase, GS).

Using SpyTag/SpyCatcher technology to connect them with *in vivo* assembled V-CHARGEs,
the
generation of products was detected to demonstrate that *in vivo* assembled V-CHARGEs
can
produce ATP to provide energy for external reactions.

## 4.1 Plasmid construction

## 4.1 Plasmid construction of FLuc, UCK, γ-GCS, GS

▼

In order to express Firefly Luciferase (FLuc), Uridine-Cytidine Kinase (UCK),
γ-Glutamylcysteine
Synthetase (γ-GCS), and Glutathione Synthetase (GS) in *Escherichia coli* BL21 (DE3),
we constructed the genetic pathway (Figure 31) and integrated the genes encoding fusion
proteins
into the plasmid backbone. We chose the pET-28a vector backbone. This T7 promoter-based
backbone
contains a kanamycin resistance gene as the selection marker and an inducible lac operon
(including
the lacI repressor), ensuring that IPTG
can induce the expression of fusion proteins. In addition, the backbone includes a histidine
tag
(His-tag) located at the C-terminus of the fusion protein. The presence of this His-tag
allows us to subsequently purify the proteins through nickel affinity chromatography. We
used
SnapGene software to design the plasmids. For *pET-28a::SC-UCK*, *pET-28a::SC-γ-GCS*,
*pET-28a::SC-GS*,
we used primers synthesized by Sangon Biotech to amplify the target gene fragments, and the
gene
fragments were inserted into the linearized vector using seamless cloning technology.
For the *pET-28a::SC-FLuc* plasmid, we entrusted Genewiz to synthesize and sequence it, and
the
results were verified to be correct.

![img](https://static.igem.wiki/teams/5872/results/31.webp)

**Figure 31. Construction diagram of
FLuc,
UCK, γ-GCS, and GS genetic pathways.**

**(A) (B) (C) (D)** are the genetic pathway construction
diagrams
of
FLuc, UCK, γ-GCS, and GS, respectively. The pathway contains T7 promoter, target
gene
(FLuc/UCK/γ-GCS/GS), GS linker, SpyCatcher, 6×His, and terminator.

![img](https://static.igem.wiki/teams/5872/results/32-2.webp)

**Figure 32. Plasmid design diagram
of
*pET-28a::SC-UCK*, *pET-28a::SC-FLuc*, *pET-28a::SC-γ-GCS*, *pET-28a::SC-GS*.**

We successfully constructed four pET-28a plasmids
containing
SC-UCK(orange), SC-FLuc (pink), SC-γ-GCS (blue), and SC-GS (green) genes,
respectively.
These plasmids possess key expression regulatory elements such as T7 promoter
(white),
kanamycin resistance gene (light green), and lac operon (purple).

We extracted the plasmids obtained from *E. coli* DH5α and transformed them into *E. coli* BL21(DE3)
using
the
heat-shock method. After transformation, the cells were plated on LB medium containing
kanamycin
for
selection. After overnight incubation, we observed different bacterial colonies, indicating
successful transformation.
Finally, we identified the target genes by Polymerase Chain Reaction (PCR) and agarose gel
electrophoresis. From the gel electrophoresis results, the bands corresponding to UCK,
γ-GCS,
and GS
were observed between 1000 bp and 2000 bp (Figure 33), which matched the actual sizes of the
gene
fragments,
proving that the gene fragments had been successfully inserted into the plasmids. We
submitted
the
successfully constructed plasmids to Sangon Biotech for sequencing, and the sequencing
results
confirmed that the target gene fragments had been successfully integrated into the plasmids.

![img](https://static.igem.wiki/teams/5872/results/33-2.webp)

**Figure 33. PCR identification gel
electrophoresis of the target genes, UCK, GS, γ-GCS.**

From left to right, the first lane was loaded with M5
HiClear
DL2000 plus DNA marker, the second lane was loaded with water, the third lane was
loaded
with the PCR product using UCK as the template, the fourth lane was loaded with the
PCR
product using GS as the template, and the fifth lane was loaded with the PCR
product
using γ-GCS as the template. The gel image showed that all the target gene lanes
presented
specific bands consistent with the expected sizes, and the negative control had no
nonspecific bands, indicating that the PCR amplification of UCK, γ-GCS, and GS was
successful.

**Summary**

In this part, we successfully completed the construction of four recombinant plasmid vectors:
*pET-28a::SC-FLuc*, *pET-28a::SC-UCK*, *pET-28a::SC-γ-GCS*, and *pET-28a::SC-GS*. The plasmids were
verified
by Polymerase Chain Reaction(PCR) and sequencing, laying a solid foundation for subsequent
induced
expression and functional studies of FLuc, UCK, γ-GCS, and GS proteins.

## 4.2 Expression and Purification

▼

To obtain the four proteins, we inoculated overnight seed culture into fresh LB medium at a
ratio
of
1:100, cultured at 37 ℃ and 200 rpm until OD600 reached 0.6–0.8. Subsequently, 0.1 mM
isopropyl-β-D-thiogalactopyranoside (IPTG) was added to induce protein expression, and
cultivation
was continued at 16 ℃ and 200 rpm for 16 h.
The cells were harvested by centrifugation at 6000 rpm for 10 min, resuspended in Buffer A
(50
mM
Tris-HCl, pH 8.0, 300 mM NaCl, 3 mM β-mercaptoethanol), and disrupted by ultrasonication (3
s
sonication/6 s interval cycle, total 10 min). The lysate was centrifuged at 12000 rpm for 45
min,
and the supernatant and precipitate were collected
separately for subsequent purification.

We used nickel affinity chromatography to separate and purify proteins in the supernatant.
First,
proteins were eluted with imidazole at different concentration gradients, and then the
eluates
were
subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Protein expression was
analyzed
through the gel electrophoresis image (Figure 34). In the lane of SpyCatcher-FLuc,
a band of about 100 kDa was observed, which matched the size of SpyCatcher-FLuc and showed
almost no
impurity bands, indicating that SpyCatcher-FLuc protein purification was successful. In the
lane
of
SpyCatcher-UCK, a band of about 45 kDa was observed, which matched the size of
SpyCatcher-UCK
and
showed almost no impurity bands, indicating that SpyCatcher-UCK protein purification was
successful.
In the lane of SpyCatcher-γ-GCS, a band of about 75 kDa was observed, which matched the size
of
SpyCatcher-γ-GCS and showed almost no impurity bands, indicating that SpyCatcher-γ-GCS
protein
purification was successful. In the lane of SpyCatcher-GS, a band of 45–65 kDa was observed,
which
matched the size of SpyCatcher-GS and showed almost no impurity bands, indicating that
SpyCatcher-GS
protein purification was successful.

![img](https://static.igem.wiki/teams/5872/results/34.webp)

**Figure 34. SDS-PAGE gel
electrophoresis
of FLuc, UCK, γ-GCS, and GS.**

**(A)** From left to right, the two lanes were
loaded
respectively with M5 Prestained Protein Ladder (10–180 kDa) with 45 kDa, and the
FLuc
eluate
at an imidazole concentration of 300 mM.

**(B)** From left to right, the three
lanes
were loaded respectively with M5 Prestained Protein Ladder
(10–180 kDa) with 45 kDa, 300 mM imidazole solution, UCK eluate at an imidazole
concentration of 300 mM, γ-GCS eluate at an imidazole concentration of 300 mM, and
GS
eluate
at an imidazole concentration of 300 mM.

**Summary**

In this part, we successfully induced the expression and purified the three proteins FLuc,
UCK,
γ-GCS, and GS. Subsequently, we will use the SpyTag/SpyCatcher technology to link them with
*in
vivo* assembled V-CHARGEs, thereby testing the efficiency of ATP-dependent reactions
catalyzed by *in vivo* assembled V-CHARGEs with externally connected enzymes, and
demonstrating
the role of *in vivo* assembled V-CHARGEs in practical production.

## 4.3 Conjugation, Enzyme Activity Assay, and Characterization

▼

We mixed the purified SpyCatcher-enzyme with *in vivo* assembled V-CHARGES at a certain
ratio,
and placed the mixed sample on a shaker or vortex mixer for a certain period of time,
allowing
enzymes to be conjugated onto the outer surface of *in vivo* assembled V-CHARGEs. The
schematic
diagram of the procedure is shown in Figure 35.

![img](https://static.igem.wiki/teams/5872/results/35-2.webp)

**Figure 35. Conjugation schematic
diagram.**

### 4.3.1 FLuc

4.3.1 Firefly Luciferase (FLuc)

We mixed *in vivo* assembled V-CHARGES after ultracentrifugation with SpyCatcher-FLuc at
a
mass
ratio of 2:1, and shook the mixture at 37 °C for 1 h (specific operation details) to obtain
the
conjugation product. We performed SDS-PAGE on *in vivo* assembled V-CHARGES after
ultracentrifugation, SpyCatcher-FLuc, and the conjugation product,
and analyzed the conjugation through the gel electrophoresis results. The SDS-PAGE result is
shown
in Figure 36.

![img](https://static.igem.wiki/teams/5872/results/36.webp)

**Figure 36. SDS-PAGE gel electrophoresis of V-CHARGEs(FLuc)**

From left to right, the five lanes were loaded with M5 Prestained Protein Ladder (10–180 kDa)
with 45 kDa, H2O,SpyCatcher-FLuc, *in vivo* assembled V-CHARGEs, and the mixture after conjugation with FLuc. In the third lane, three bands were observed, corresponding
from bottom to top to the mixture of SlPPK-SP and CP-SpyTag, SpyCatcher-FLuc, and V-CHARGEs(FLuc).

Analysis of the gel image reveals that a portion of the V-CHARGEs assembled *in vivo* were successfully linked to FLuc. However, some residual FLuc remained in
the ligation products, indicating that the ligation efficiency was moderate. Therefore, we attempted to purify the ligation products using dialysis.

In order to verify that *in vivo* assembled V-CHARGEs can provide energy for enzymatic
reactions
outside the shell, we used Firefly Luciferase (FLuc) as a model enzyme and designed four
parallel
experiments as follows:

1. No V-CHARGEs and no FLuc group (negative control)

2. With V-CHARGEs but no FLuc group

3. No V-CHARGEs but with FLuc group

4. With V-CHARGEs and with FLuc group (complete system)

All reactions were carried out under the same buffer system and substrate conditions. We used
the
FlexStation®3 multifunctional microplate reader to detect the bioluminescence intensity
produced by
firefly luciferase in the four groups, and plotted a bar chart for analysis based on the obtained
data
(Figure 37).

The detection results showed that in the experimental group with both *in vivo*
assembled
V-CHARGEs and FLuc, the bioluminescence signal was significant, indicating that firefly luciferase
could
efficiently react under this condition. In contrast, the three groups containing only *in
vivo*
assembled V-CHARGEs, only FLuc, or the blank control all showed extremely weak bioluminescence
signals.

![img](https://static.igem.wiki/teams/5872/results/37.webp)

**Figure 37. Bar chart of
bioluminescence
intensity produced by firefly luciferase reactions in four parallel experiments.**

Figure 37 shows that the bioluminescence signal was
significant
in
the group with both *in vivo* assembled V-CHARGEs and FLuc, while the other
three
groups showed no obvious bioluminescence signal.

**Summary**

In this part, we successfully achieved efficient conjugation of SpyCatcher-FLuc with *in
vivo*
assembled V-CHARGEs, and obtained conjugates of relatively high purity through dialysis. The
bioluminescence detection results of firefly luciferase proved that *in vivo* assembled V-CHARGEs
can
provide energy for the external firefly luciferase reaction, thereby supporting its catalytic
process.
This laid an experimental foundation for subsequently using the energy of *in vivo*
assembled
V-CHARGEs to drive the reactions of UCK and γ-GCS/GS, further synthesizing the target
products.

### 4.3.2 UCK

4.3.2 Uridine-Cytidine Kinase (UCK)

We mixed *in vivo* assembled V-CHARGES after ultracentrifugation with SpyCatcher-UCK at
a
mass
ratio of 2:1, and shook the mixture at 37 °C for 1 h to obtain the conjugation product. We
performed
SDS-PAGE on *in vivo* assembled V-CHARGES after ultracentrifugation,
SpyCatcher-UCK, and the conjugation product, and analyzed the conjugation through the gel
electrophoresis results. The SDS-PAGE result is shown in Figure 38.

![img](https://static.igem.wiki/teams/5872/results/uck.webp)

**Figure 38. SDS-PAGE gel
electrophoresis
of V-CHARGEs(UCK).**

From left to right, the four lanes were loaded with M5
Prestained Protein Ladder (10–180 kDa) with 45 kDa, *in vivo* assembled
V-CHARGEs,
SpyCatcher-UCK, and the mixture after conjugation with UCK. In the fourth lane,
three
bands
were observed, corresponding from bottom to top to UCK, the mixture of SlPPK-SP and
CP-SpyTag, and V-CHARGEs(UCK).

Analysis of the gel showed that the band intensity of SpyCatcher-UCK was very close to that
of
the
conjugation product, and almost no extra bands were seen in the conjugation lane, indicating
that
SpyCatcher-UCK and *in vivo* assembled V-CHARGES had very high conjugation efficiency
and
the
product was of high purity, achieving the expected conjugation effect.

We characterized the conjugation product using Dynamic Light Scattering (DLS) and further
analyzed
the shape and particle size using Scanning Electron Microscopy (SEM) of V-CHARGEs(UCK)
(Figure
39).
DLS results showed two main size distribution peaks, around 20 nm and 100 nm, with particles
around
100 nm occupying
the dominant intensity proportion, indicating this was the main particle size distribution
of
the
conjugation product. TEM images showed that V-CHARGEs(UCK) presented uniform spherical
structures
with sizes around 100 nm, consistent with the DLS results.

![img](https://static.igem.wiki/teams/5872/results/39.webp)

**Figure 39. DLS and TEM analysis
results
of V-CHARGEs(UCK) conjugated with UCK.**

**(A)** shows the average DLS intensity distribution of
V-CHARGEs(UCK) conjugated with UCK, illustrating particle size (d, in nm, log10
scale)
and
corresponding intensity distribution G(d).

**(B) (C)** show TEM images of
V-CHARGEs(UCK).
The spherical particles (~50 nm) in the images are V-CHARGEs(UCK). Scale bar: 100
nm.

DLS measures the hydrodynamic diameter, which includes the hydration layer and possible aggregates,
while TEM shows the dry core size. Therefore, DLS values appear larger than TEM results.

To test catalytic efficiency, we used High Performance Liquid Chromatography (HPLC) to
quantitatively
analyze 5′-CMP and cytidine (CR) in the products. Standard curves (Figure 40) were generated
to
calibrate the peak areas at different concentrations. Figure 41 shows the HPLC chromatograms
of
the
measurement results. The earlier retention time peak corresponds to 5′-CMP, while the later
retention time peak corresponds to CR.

![img](https://static.igem.wiki/teams/5872/results/40.webp)

**Figure 40. HPLC standard curves
of
5′-CMP
and CR.**

**(A)** shows the 5′-CMP standard curve, with
concentration
(mg/mL) plotted against peak area response (AU·min). The fitted linear regression
equation
was Y = 7067626X + 314252, with R² = 0.9994.

**(B)** shows the CR standard curve,
with
concentration (mg/mL) plotted against peak area response (AU·min). The fitted linear
regression equation was Y = 12790923X + 469796, with R² = 0.9994.

![img](https://static.igem.wiki/teams/5872/results/41.webp)

**Figure 41. HPLC chromatograms of
5′-CMP
and CR measurement results.**

The first peak was 5′-CMP with a retention time of
5.207
min,
and the second peak was CR with a retention time of 13.412 min.

Based on the HPLC results, the conjugates successfully produced 5′-CMP with high catalytic
efficiency, indicating that our system had good production performance and could provide
sufficient
ATP to support efficient catalytic reactions for external enzymes.

To demonstrate that *in vivo* assembled V-CHARGEs could efficiently catalyze reactions
in
the
presence of UCK and play a key role in energy supply, we set up four parallel groups:

1.Without V-CHARGEs and without UCK (negative control)

2.With V-CHARGEs but without UCK

3.Without V-CHARGEs but with UCK

4.With V-CHARGEs and UCK (complete system)

All reactions were carried out under the same buffer system and substrate conditions, with
*in
vivo* assembled V-CHARGEs and SpyCatcher-UCK mixed at a mass ratio of 2:1. After the
reactions, 5'-CMP concentration was determined by HPLC.

The 5'-CMP yields of the four groups were plotted as a bar chart (Figure 42). Results showed
that
in
the absence of both *in vivo* assembled V-CHARGEs and UCK, the 5'-CMP concentration was
nearly
zero. When only *in vivo* assembled V-CHARGEs or only UCK was added, the 5'-CMP
concentration
was very low. However, when both *in vivo* assembled V-CHARGEs and UCK were present,
the 5'-CMP concentration significantly increased, reaching the highest value. This confirmed
that we
successfully synthesized 5'-CMP, and also demonstrated that *in vivo* assembled
V-CHARGEs
enhanced reaction efficiency, playing a catalytic role.

![img](https://static.igem.wiki/teams/5872/results/42-2.webp)

**Figure 42. Bar chart of the
effect of
*in vivo* assembled V-CHARGEs and UCK on 5′-CMP product concentration.**

The vertical axis indicates 5′-CMP concentration (mg/mL),
while
the
horizontal axis represents different experimental conditions. "+" means the condition was
present,
“−” means absent. In the absence of both UCK and *in vivo* assembled V-CHARGEs,
no
5′-CMP was produced; in systems with only UCK or only *in vivo* assembled
V-CHARGEs,
only small amounts of 5′-CMP were produced; in the complete system with both UCK and
*in
vivo* assembled V-CHARGEs, a large amount of 5′-CMP was obtained.

**Summary**

In this part, we successfully conjugated SpyCatcher-UCK with *in vivo* assembled
V-CHARGEs
with
high efficiency and obtained high-purity products. We characterized the products with DLS
and
TEM,
showing uniform spherical particles with expected size distribution. We analyzed the
products
with
HPLC and designed four parallel experiments, demonstrating the necessity of *in vivo*
assembled
V-CHARGEs in the reaction. The results showed that our system had high catalytic efficiency.
Overall,
our *in vivo* assembledV-CHARGEs successfully drove energy-consuming reactions after
conjugation with UCK and efficiently produced 5′-CMP, suggesting their potential for
industrial
application.

### 4.3.3 γ-GCS and GS

4.3.3 γ-Glutamylcysteine Synthetase (γ-GCS)
and glutathione
Synthetase (GS)

We mixed *in vivo* assembled V-CHARGES after ultracentrifugation with SpyCatcher-γ-GCS
and
SpyCatcher-GS at a mass ratio of 30:14:1, and vortexed for 20 min to obtain the conjugation
product.
We performed SDS-PAGE on *in vivo* assembled V-CHARGES after ultracentrifugation,
SpyCatcher-γ-GCS, SpyCatcher-GS, and the conjugation product, and analyzed the conjugation
through
the gel electrophoresis results (Figure 43).

![img](https://static.igem.wiki/teams/5872/results/gsh.webp)

**Figure 43. SDS-PAGE gel
electrophoresis
of V-CHARGEs(γ-GCS/GS).**

From left to right, the five lanes were loaded with M5
Prestained Protein Ladder (10–180 kDa) with 45 kDa, *in vivo* assembled sample
(V-CHARGEs), SpyCatcher-γ-GCS, SpyCatcher-GS, and the mixture after conjugation with
γ-GCS
and GS. In the fifth lane, four main bands were observed, corresponding from bottom
to
top
to GS, the mixture of SlPPK-SP and CP-SpyTag, γ-GCS, and the mixture after
conjugation
with
γ-GCS and GS.

Analysis of the gel showed that the conjugation lane had bands corresponding to conjugated
V-CHARGEs(γ-GCS/GS) (100–135 kDa), with some unbound γ-GCS and GS visible, and only a small
amount
of unbound *in vivo* assembled V-CHARGEs. This indicated that SpyCatcher-γ-GCS and
SpyCatcher-GS successfully conjugated with *in vivo* assembled V-CHARGES with high
efficiency,
but the mixing ratio could be optimized, as γ-GCS and GS were in excess. To obtain purified
products, we used dialysis to remove unbound enzymes.

We characterized the conjugation product using DLS and further analyzed the shape and
particle
size
using Transmission Electron Microscopy (TEM) of V-CHARGEs(γ-GCS/GS) (Figure 44). DLS results
showed
that the particle size distribution of the conjugates was mainly between 20–100 nm, with
particles
in the 50–100 nm range dominating. TEM results showed uniform granular structures with sizes
consistent with DLS, confirming good morphological characteristics.

![img](https://static.igem.wiki/teams/5872/results/44.webp)

**Figure 44. DLS and TEM analysis
results
of V-CHARGEs(γ-GCS/GS).**

**(A)** shows the average DLS intensity distribution of
V-CHARGEs(γ-GCS/GS), illustrating particle size (d, in nm, log10 scale) and
corresponding
intensity distribution G(d).

**(B) (C)** show TEM images of V-CHARGEs(γ-GCS/GS).
The
spherical particles (~50 nm) are V-CHARGEs(γ-GCS/GS). Scale bar: 100 nm.

DLS measures the hydrodynamic diameter, which includes the hydration layer and possible aggregates,
while TEM shows the dry core size. Therefore, DLS values appear larger than TEM results.

After completing the conjugation and characterization of V-CHARGEs(γ-GCS/GS), we further
tested
the
product using glutathione (GSH)-specific ELISA to verify the catalytic efficiency of the
conjugated
enzymes. To fully evaluate the key role of *in vivo* assembled V-CHARGEs in catalysis,
we
designed four parallel groups:

1.Without V-CHARGEs and without γ-GCS/GS (negative control)

2.With V-CHARGEs but without γ-GCS/GS

3.Without V-CHARGEs but with γ-GCS/GS

4.With V-CHARGEs and γ-GCS/GS (complete system)

All reactions were carried out under the same buffer system and substrate conditions, with
*in
vivo* assembled V-CHARGEs mixed with SpyCatcher-γ-GCS and SpyCatcher-GS at a mass
ratio
of
30:14:1. After the reactions, GSH concentration was determined by ELISA.

We first measured a series of GSH solutions with known concentrations using an ELISA kit and
generated a standard curve (Figure 45), obtaining the correlation between A450 OD values and
GSH
concentrations, which served as a reference for subsequent quantification.

![img](https://static.igem.wiki/teams/5872/results/45.webp)

**Figure 45. GSH ELISA standard
curve.**

A series of standards (100 μg/mL, 50 μg/mL, 25 μg/mL, 12.5 μg/mL, 6.25 μg/mL, 3.125 μg/mL,
1.5625
μg/mL) were measured by ELISA, and the A450 OD values were fitted with a four-parameter
logistics
equation to generate the GSH ELISA standard curve equation.

We plotted the ELISA results of the four groups using the GSH standard curve (Figure 46).
Results
showed that in the negative control without *in vivo* assembled V-CHARGEs and without
γ-GCS/GS,
the GSH concentration was close to zero; in groups with only V-CHARGEs or only γ-GCS/GS, the
GSH
concentration was very low, indicating that neither could
drive effective production alone. In the complete system with both *in vivo* assembled
V-CHARGEs and γ-GCS/GS, the GSH yield increased significantly, much higher than in the other
groups.
This demonstrated that only when the energy-supplying system (*in vivo* assembled
V-CHARGEs)
and the synthetic enzymes (γ-GCS and GS) were both present could glutathione synthesis be
efficiently driven, proving the essential role of *in vivo* assembled V-CHARGEs as an
energy
driver.

![img](https://static.igem.wiki/teams/5872/results/46-2.webp)

**Figure 46. Bar chart showing the
effect
of *in vivo* assembled V-CHARGEs with γ-GCS+GS on glutathione product
concentration.**

The vertical axis represents GSH concentration (μg/mL), and
the
horizontal axis
represents different experimental conditions. “+” means the condition was present,
“−”
means
absent. In the negative control without *in vivo* assembled V-CHARGEs and
γ-GCS/GS,
GSH
concentration was close to zero; in systems with only V-CHARGEs or only γ-GCS/GS,
the
GSH
concentration was low; in the complete system with both V-CHARGEs and γ-GCS/GS, GSH
concentration significantly increased.

**Summary**

In this experiment, we successfully conjugated SpyCatcher-γ-GCS and SpyCatcher-GS with *in
vivo*
assembled V-CHARGEs with high efficiency, and characterized the products with SDS-PAGE, DLS,
and
TEM. Results showed very high conjugation efficiency and uniform spherical particles with
size
distribution mainly in the 50–100 nm range. Further, by detecting GSH with ELISA and
analyzing
four
control groups, we clearly confirmed that *in vivo* assembled V-CHARGEs played an
indispensable
role in γ-GCS and GS-driven glutathione synthesis, providing continuous energy support for
efficient
catalysis.

**Overall Summary**

In this section, we successfully achieved external conjugation of UCK and γ-GCS&GS on *in
vivo*
assembled V-CHARGEs, and systematically characterized the structure and function of the
conjugates.
Results showed that UCK, γ-GCS, and GS all conjugated efficiently to the surface of *in
vivo*
assembled V-CHARGEs, and the products exhibited uniform and stable particle structures. By
detecting
products with HPLC and ELISA, we confirmed that both external enzymes catalyzed reactions
efficiently under *in vivo* assembled V-CHARGEs drive, producing target products
(5′-CMP
and
GSH). Control experiments clearly proved the essential energy-supplying role of *in
vivo*
assembled V-CHARGEs in these reactions.

These results demonstrate that our *in vivo* assembled V-CHARGEs can not only
efficiently
conjugate with multiple enzymes *in vitro*, but also provide continuous energy for extended
reactions,
driving complex multi-step metabolic processes. This lays a solid foundation for their
application
in multi-enzyme cascade reactions, cell-free synthesis systems, and industrial
biomanufacturing,
highlighting their broad potential in **cell factories and synthetic biology
applications.**

## 4.4 Discussion

**Summary and Reflection**

In this section of experiments, we have, for the first time, demonstrated that ***in
vivo*
assembled V-CHARGEs are not only capable of efficiently synthesizing ATP inside the
capsid,
but
these ATP molecules can also be directly utilized by energy-dependent enzymes attached
outside
the capsid.** By constructing, expressing, and purifying UCK, γ-GCS, and GS, we
achieved
their efficient connection
to *in vivo* assembled V-CHARGEs using the SpyTag/SpyCatcher system, and successfully
completed
the ***in vitro* synthesis of 5’-CMP** and **glutathione (GSH)**.
The
experimental results indicate:

1.In the **UCK experiments**, ATP generated by *in vivo* assembled
V-CHARGEs
was
able to drive UCK to catalyze the conversion of cytidine to 5’-CMP. HPLC quantitative
analysis
showed that in the complete system with both *in vivo* assembled V-CHARGEs and UCK, the
yield
of 5’-CMP was significantly higher than in the control group, demonstrating the decisive
role of
ATP
provided by *in vivo* assembled V-CHARGEs in the reaction.

2.In the **γ-GCS and GS experiments**, ELISA detection revealed that the
complete
system
produced substantial amounts of GSH, whereas the control groups yielded almost no detectable
product. This further confirms that *in vivo* assembled V-CHARGEs can stably supply
energy
in
multi-enzyme cascade reactions, powering complex metabolic pathways.

3.DLS and TEM characterization results both showed that *in vivo* assembled V-CHARGEs
maintained
good particle structure and size distribution after enzyme attachment, indicating that the
connection process did not significantly compromise their structural stability.

In summary, we successfully verified that *in vivo* assembled V-CHARGEs can operate in a
“energy
synthesized inside the capsid, reactions driven outside the capsid” mode, achieving
spatially
separated ATP transfer and utilization. These findings provide important evidence for the
potential
of *in vivo* assembled V-CHARGEs in **metabolic engineering and synthetic biology
applications.**

**Prospects**

In the future, we plan to further advance this work in the following directions:

**1.Expanding product types:** Test additional ATP-dependent reactions, such
as
peptide
synthesis, coenzyme regeneration, or production of small-molecule drug precursors, to
comprehensively demonstrate the versatility of *in vivo* assembled V-CHARGEs.

**2.Optimizing connection strategies:** Improve the efficiency and precision
of
multi-enzyme cascade reactions by adjusting the number and position of SpyTag/SpyCatcher
or
employing controllable connection methods.

**3.System stability and recyclability:** Explore the long-term stability
and
reusability of *in vivo* assembled V-CHARGEs, assessing their potential in
continuous
reactions
or industrial reactors

**4.Industrial application prospects:** Based on our experimental results,
*in
vivo* assembled V-CHARGEs could be envisioned as a programmable energy supply
platform
with
broad potential applications in biopharmaceuticals, green synthesis, and environmental
remediation.

# References

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[3]Patterson D. P. (2018). Encapsulation of Active Enzymes within
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[4]O'Neil, A., Reichhardt, C., Johnson, B., Prevelige, P. E., & Douglas, T.
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[5]Sharma, J., Uchida, M., Miettinen, H. M., & Douglas, T. (2017). Modular
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[6]Teschke, C. M., & Parent, K. N. (2010). 'Let the phage do the work': using
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[7]O'Neil, A., Reichhardt, C., Johnson, B., Prevelige, P. E., & Douglas, T.
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