Experimental Workflow

TRACER-IL24 gene and Deliver gene were synthesized by Jiangsu Genecefe Biotechnology. in accordance with the Biobrick standard. Among these, the TRACER-IL24 gene was obtained as a linear fragment via PCR; the pET-28a(+) plasmid was amplified using the DH5α strain for high-copy replication and subsequently isolated via plasmid miniprep.

Constant Temperature Water Bath, Shaker Incubator, Centrifuge, Micropipette, Erlenmeyer Flask (150 mL), 1.5 mL Microcentrifuge Tube, Plasmid Miniprep Kit, Chemically Competent E. coli DH5α Cells, Solid LB Agar Medium supplemented with Kanamycin, Kanamycin Sulfate Solution (100 mg/mL), LB Medium.

1. Transformation

2. Plasmid Miniprep

Thermocycler, 0.2 mL PCR tubes, Micropipettes, Incubator, 2× PCR mix, Forward primer, Reverse primer, Sterile water, DNA template, pET-28a(+) plasmid (miniprep product), Rcut Smart Buffer (10X), HindIII restriction enzyme, EcoRI restriction enzyme.

PCR

Two PCR tubes were prepared with the reaction components as specified (total volume 25 μL per tube).

PCR system	Volume
2× PCR Mix	12.5 μL
Template DNA	1.5 μL
Forward Primer	1 μL
Reverse Primer	1 μL
H₂O	9 μL
Total	25 μL

• The tubes were placed in a thermocycler and subjected to the following program: initial denaturation at 94°C for 120 seconds; 30 cycles of denaturation at 94°C for 30 seconds, annealing at 56°C for 30 seconds, and extension at 72°C for 60 seconds; followed by a final extension at 72°C for 5 minutes.
• The PCR products were stored at -20°C.

Enzyme digestion

The restriction digestion was set up as a 20 μL reaction system.

Digestion system	Volume
Rcut smart	2 μL
DNA strain	17 μL
Hind III	0.5 μL
EcoR I	0.5 μL
Total	20 μL

• All microcentrifuge tubes were subsequently incubated in a constant temperature incubator at 37°C for 2 hours.

Constant temperature water bath, electronic balance, electrophoresis system, gel imaging analysis system, centrifuge, micropipettes, glass flask, microcentrifuge tubes (1.5 mL, 2 mL), agarose, electrophoresis buffer, fluorescent nucleic acid dye, UE DNA gel extraction kit, DNA loading buffer, nucleic acid standard marker.

Agarose Gel Electrophoresis

• 1.5% Agarose Gel Preparation: Measure 30 mL of 1x TAE electrophoresis buffer and add it to a glass bottle. Weigh 0.45 g of agarose and add it to the bottle to prepare a 1.5% agarose solution. Heat the mixture in a microwave oven until boiling. Allow the solution to cool at room temperature to approximately 60°C, then add 3 μL of fluorescent dye and mix thoroughly. Retrieve the gel casting tray and insert the comb. Pour the prepared gel solution into the tray and allow it to solidify at room temperature.
• Place the cooled and solidified agarose gel into the electrophoresis tank. Mix the restriction digestion product with DNA loading buffer at a 5:1 ratio, and load the mixture into the wells. Turn on the electrophoresis power supply and set the voltage to 110 V. Run the gel for 40 minutes.
• Remove the agarose gel. Analyze and document the electrophoresis results using a gel imaging analysis system.

DNA Fragment Gel Extraction

• Under UV illumination, excise the agarose gel slice containing the target DNA band. Blot the surface dry with laboratory tissue and mince the gel. Determine the gel weight (the weight of a 1.5 mL microcentrifuge tube should be tared in advance), treating this weight as the gel volume (where 100 mg = 100 μL).
• Add three gel volumes of Buffer DE-A to the minced gel and mix thoroughly. Incubate at 75°C, vortexing intermittently every 2-3 minutes until the gel is completely dissolved.
• Add 0.5 volume of Buffer DE-B relative to the volume of Buffer DE-A used, and mix thoroughly.
• Transfer the mixture to a DNA preparation column (assembled in a 2 mL collection tube). Centrifuge at 12,000 rpm for 1 minute. Discard the flow-through. Reassemble the column with the collection tube, add 500 μL of Buffer W1, and centrifuge at 12,000 rpm for 30 seconds. Discard the flow-through.
• Place the column back into the collection tube, add 700 μL of Buffer W2, and centrifuge at 12,000 × g for 30 seconds. Discard the flow-through. Repeat the washing step with an additional 700 μL of Buffer W2, followed by centrifugation at 12,000 rpm for 1 minute.
• Reinsert the column into the collection tube and perform a final centrifugation at 12,000 rpm for 1 minute to remove residual wash buffer.
• Transfer the column to a clean 1.5 mL microcentrifuge tube. Apply 25-30 μL of Elution Buffer directly to the center of the column membrane. Allow it to stand at room temperature for 1 minute, then centrifuge at 12,000 rpm for 1 minute to elute the DNA.
• Store the final product at -20°C.

PCR tubes, tube floats, foam rack, vector backbone, insert fragment, 10X DNA Ligase Buffer, nuclease-free water, T4 DNA Ligase.

Ligation system	Volume
Vector fragment	3 μL
Target fragment	12 μL
DNA ligase buffer 10x	2.5 μL
Nuclease-free Water	6.5 μL
T4 DNA Ligase	1 μL
Total	25 μL

Following the pipetting scheme outlined in the table above, the reaction mixtures in PCR tubes were placed in an ice-water bath for a 24-hour ligation reaction.

Constant temperature water bath, shaker incubator, centrifuge, micropipettes, 150 mL Erlenmeyer flasks, 1.5 mL microcentrifuge tubes, pET-28a(+)-TRACER plasmid solution, E. coli BL21(DE3) competent cells, kanamycin-resistant solid LB agar medium, kanamycin sulfate solution (100 mg/mL), LB medium, 50% glycerol solution.

• Thaw a 100 μL of commercially available competent cells on ice. Pre-sterilize the biological safety cabinet by UV irradiation for 15 minutes. Add plasmid solution to each tube at a 10:1 volume ratio (competent cells : plasmid solution).
• Place the microcentrifuge tubes containing the plasmid-cell mixtures on ice for 30 minutes. Preheat a water bath to 42°C. Following ice incubation, subject the tubes to a 42°C heat shock for 45 seconds, then immediately return them to ice for 2 minutes to complete the transformation.
• Retrieve pre-aliquoted antibiotic-free LB liquid medium. Add 500 μL of the medium to each heat-shocked microcentrifuge tube. Incubate the tubes in a shaker incubator at 37°C and 180 rpm for 1 hour for bacterial recovery.
• Centrifuge the tubes at 7,000 rpm for 30 seconds after incubation. Aseptically aspirate and discard approximately 400 μL of supernatant near an alcohol burner flame. Resuspend the cell pellet in the remaining medium. Retrieve solid LB agar medium supplemented with the appropriate antibiotic corresponding to the plasmid resistance marker. Aseptically spread 100 μL of the resuspended culture evenly onto the solid medium surface using a sterile spreader near the alcohol burner flame. Replace the Petri dish lid, invert the plate, and incubate at 37°C for 12 hours. Select well-isolated single colonies to inoculate 30 mL of LB liquid medium supplemented with 30 μL kanamycin sulfate solution, followed by overnight culture in a shaker incubator at 37°C and 180 rpm.
• Preserve bacterial strains at -20°C using the glycerol stock method by mixing 500 μL of bacterial culture with an equal volume of 50% glycerol solution per cryovial.

Erlenmeyer flasks (150 mL), Shaker incubator, Centrifuge, Micropipettes, 1.5 mL microcentrifuge tubes, Ultrasonic cell disruptor, E. coli BL21(DE3) strain harboring the pET-28a(+)-Tracer plasmid, Double-distilled water (ddH₂O), Kanamycin sulfate solution (100 mg/mL), LB medium, IPTG solution (100 mg/mL), PBS buffer (pH 7.4), Protease inhibitor cocktail (50×), Ammonium bicarbonate, Urea.

• Lysis Buffer Preparation: 50 mM Ammonium Bicarbonate, 8 M Urea, 1:50 Protease Inhibitor Cocktail.
• Retrieve the preserved bacterial strain and thaw it on ice. Add 30 μL of Kanamycin sulfate solution to each 30 mL culture medium bottle, and inoculate with 0.3 mL of the bacterial culture. Incubate in a shaker at 37°C, 180 rpm until the OD600 reaches between 0.4 and 0.6. Under a sterile workbench, add 30 μL of 100 mg/mL IPTG solution to each bottle. Subsequently, incubate the cultures in a shaker at 25°C, 180 rpm for 16 hours.
• Collect the bacterial culture and aliquot it into 15 mL centrifuge tubes. Centrifuge at 3500 rpm for 10 minutes.
• Discard the supernatant. Resuspend the cell pellet in 1 mL of PBS solution, then transfer the suspension to a 1.5 mL centrifuge tube. Centrifuge at 5000 rpm for 10 minutes.
• Repeat the previous step (PBS wash and centrifugation) once more. After the second wash, resuspend the final cell pellet in 1 mL of Lysis Buffer.
• Initiate the Ultrasonic Cell Disruptor. Disrupt the cell suspension using a probe sonicator on an ice-water bath with the following parameters: 2-second pulse ON, 2-second pulse OFF for 12 minutes until the bacterial suspension becomes clear and translucent. After disruption, centrifuge the lysate at 4°C, 12000 rpm for 20 minutes to separate the supernatant and pellet.
• Store the resulting products at -20°C.

Gravity flow column with upper and lower frits, Micropipettes, Centrifuge tubes, Retort stand, Ni-NTA resin, 10× TBS buffer, Imidazole, Urea, Triton X-100, Sample for purification.

Preparations

• Assemble the gravity flow column. Insert the bottom frit, and wash the column with 1 column volume (CV) of purified water. Add 2–4 mL of pre-mixed Ni-NTA resin slurry to the column. Open the bottom outlet and allow the storage solution to drain completely. Wash the resin with 5 CV of purified water, then insert the top frit. Equilibrate the prepared column with 5 CV of 1× TBS buffer.

Supernatant Protein Purification

The compositions of all solutions used for the purification process are detailed in the table below.

Solutions	Tris	NaCl	Imidazole
Equilibration Buffer	50 mM	0.15 M	20 mM
Elution Buffer Ⅰ	50 mM	0.15 M	50 mM
Elution Buffer Ⅱ	50 mM	0.15 M	100 mM
Elution Buffer Ⅲ	50 mM	0.15 M	300 mM

• All solutions mentioned above were prepared using 1× TBS buffer, pH 7.6.
• The sample was recirculated through the column more than 5 times. The column was then washed with 10-20 column volumes (CV) of Equilibration Buffer, and the final column volume of wash effluent was collected and retained for later analysis. Subsequently, the target protein was eluted using a gradient with 5 CV each of Elution Buffer I, II, and III, and the eluates were collected.

Precipitation Protein Purification

• Resuspend the pellet in Lysis Buffer (pH 8.0, 50 mM Tris, 2 mM EDTA, 150 mM NaCl, 20 mM imidazole, 1% Triton X-100). Disrupt the cell suspension using a probe sonicator on an ice-water bath with the following parameters: 3-second pulse ON, 8-second pulse OFF, for a total duration of 10 minutes.
• Centrifuge the lysate at 13,000 rpm for 20 minutes at 4°C. Collect the insoluble pellet.
• Solubilize the pellet obtained from centrifugation by first adding 1 mL of Wash Buffer (20 mM imidazole, 20 mM Tris, pH 8.0) per gram of pellet. Then, add 4-5 mL of Denaturation Buffer (50 mM Tris buffer, pH 8.0, containing 0.15 M NaCl and 8 M urea) per gram of pellet.
• Subject the mixture to probe sonication on an ice-water bath using the following cycle: 3-second pulse ON, 8-second pulse OFF until the suspension becomes clear and translucent.
• Centrifuge the sonicated lysate at 12,000 rpm for 10 minutes at 4°C. Collect the supernatant and subject it to a second centrifugation under the same conditions (12,000 rpm, 10 minutes, 4°C). Collect the final supernatant.

The compositions of all solutions used for the purification process are detailed in the table below.

Solutions	Tris	NaCl	Urea	Imidazole
Equilibration Buffer	50 mM	0.15 M	8 M	20 mM
Elution Buffer Ⅰ	50 mM	0.15 M	8 M	50 mM
Elution Buffer Ⅱ	50 mM	0.15 M	8 M	100 mM
Elution Buffer Ⅲ	50 mM	0.15 M	8 M	300 mM
Elution Buffer Ⅳ	50 mM	0.15 M	8 M	500 mM

• All solutions mentioned above were prepared using 1× TBS buffer, pH 8.0.
• The sample was passed through the column at least 5 times, followed by washing with 20 CV of Equilibration Buffer.
• Elution Buffers I, II, III, and IV were applied sequentially, each using 5 CV. The eluates containing the target protein were collected.
• All collected fractions were stored frozen at -20°C.

Constant temperature water bath, centrifuge, micropipettes, 15 mL centrifuge tubes, 1.5 mL microcentrifuge tubes, vacuum freeze-dryer, purified protein solution, saturated ammonium sulfate solution, 1× TBS buffer.

Water Bath Concentration

The purified protein solution was placed in a preheated thermostatic water bath at 40°C. The tube caps were opened, and the tubes were tilted and heated until the remaining volume in each tube was approximately 1.5 mL.

Salting-out Concentration

• For each 12 mL tube of purified protein solution, slowly add a volume of saturated ammonium sulfate solution corresponding to a 3:2 ratio (ammonium sulfate solution : protein solution) with gentle mixing, and repeat this process. Incubate the mixture on an ice bath for 30 minutes.
• Aliquot the solution into 1.5 mL centrifuge tubes. Centrifuge at 4°C, 10,000 rpm for 10-15 minutes. Carefully discard the supernatant.
• Add 1-3 mL of saturated ammonium sulfate solution to resuspend the pellet. Centrifuge again at 4°C, 10,000 rpm for 5 minutes.
• Discard the supernatant, and resuspend the final pellet in 1× TBS Solution.

Lyophilization Concentration

• The caps of each tube containing the purified protein solution were loosened, the openings were covered with punctured paraffin film.
• The tubes were then placed in a pre-cooled vacuum freeze-dryer. The vacuum pump was activated, and lyophilization concentration was performed under conditions of -40°C and a vacuum pressure of 500 Pa for 10-12 hours.

Thermostatic incubator (37°C), microplate reader, BCA protein assay kit, test samples.

Prepare the BSA Standard Sample

• Serially dilute the BSA standard (2 mg/mL) to generate standard curve samples with final concentrations as specified in the table below.

Number	BSA Concentration (μg/mL)
A	2000
B	1500
C	1000
D	750
E	500
F	250
G	125
H	25
I	0

• Prepare the BCA working reagent by thoroughly mixing BCA Reagent A and BCA Reagent B at a 50:1 volume ratio.
• Pipette 10 μL of each standard and unknown sample into individual wells of a 96-well plate. Subsequently, add 200 μL of the prepared BCA working reagent to each well. Incubate the plate at 37°C for 30 minutes, protected from light.
• Cool the plate to room temperature. Measure the absorbance of each well at a wavelength of 562 nm using a microplate reader. Perform blank subtraction on the acquired data.
• Generate a standard curve by plotting the blank-corrected mean absorbance values at 562 nm of the BSA standards against their corresponding concentrations (μg/mL).
• Determine the protein concentration of the samples by interpolating their blank-corrected absorbance values from the standard curve.

Constant temperature water bath, Orbital shaker, Vertical slab electrophoresis system, Gel imaging and analysis system, DC regulated power supply, Gel casting cassette, Micropipettes, Tris-base, Glycine, SDS, SDS-PAGE gel preparation kit, Isopropanol, Protein loading buffer, Coomassie Brilliant Blue R250 staining solution (0.25 g Coomassie R250, 45 mL methanol, 10 mL glacial acetic acid, 45 mL deionized water), Gel destaining solution (250 mL methanol, 80 mL glacial acetic acid, brought to 1 L with deionized water), Test samples, Standard protein marker, Deionized water.

Sample Treatment

Combine the protein samples with 5× protein loading buffer at a ratio of 3:1 (sample : buffer). Denature the mixtures by heating at 100°C for 5 minutes in a heating block or water bath, then cool them to room temperature prior to loading.

Gel Preparation

Prepare the separating gel solution according to the formulations listed in the table below. Immediately after preparation, pipette the solution between the fixed glass plates and overlay with a layer of isopropanol to ensure a flat interface. After complete polymerization of the separating gel, pour off the isopropanol layer. Subsequently, prepare the stacking gel solution as specified in the table and promptly introduce it into the assembly. Carefully insert a comb into the stacking gel solution, avoiding air bubbles.

Component (Volume: mL)	12% SDS-PAGE separating gel	SDS-PAGE stacking gel
Distilled Water	1.000	1.4000
30% Acr-Bis Solution (29:1)	2.000	0.330
1M Tris, pH 8.8	1.900	0.250
10%SDS	0.050	0.020
10% Ammonium Persulfate (APS)	0.050	0.020
TEMED Substitute	0.002	0.002
Total	5.000	2.000

Prepare 1x electrophoresis running buffer according to the composition specified in the table below.

Component	Concentration
Tris	3.029 g/L
Glycine	14.413 g/L
SDS	1 g/L

• Secure the pre-cast gel within the electrophoresis unit, fill the buffer chambers with the prepared running buffer, remove the comb.
• Load the standard samples and test samples into the wells in sequence. Perform electrophoresis initially at 80 V for 30 minutes, until the samples are concentrated at the top of the separating gel. Then, increase the voltage to 130 V and continue electrophoresis for 40 minutes. Remove the gel and discard the stacking gel portion.
• Immerse the separating gel portion of the above gel in Coomassie Brilliant Blue R250 staining solution. Stain with constant shaking for 45 minutes.
• Transfer the stained gel into a container with destaining solution and agitate for 30 minutes, then replace the destaining solution. Repeat the destaining process 4-5 times. Observe and document the results using a gel imaging analysis system.

• Cells were seeded in 96-well plates at a density of 6,000 cells per well and cultured for 24 hours in a humidified atmosphere at 37°C with 5% CO₂ to allow cell adhesion.
• The original medium was removed and replaced with fresh medium containing varying concentrations of the TRACER protein. A blank control group containing only medium (without cells) was also established.
• After 48 hours of treatment, 10 μL of MTT solution (5 mg/mL in PBS) was added to each well, followed by incubation at 37°C protected from light for an additional 3.5 hours.
• The supernatant was carefully aspirated, and the resulting formazan crystals were dissolved in 100 μL of DMSO. The plates were subjected to gentle shaking for 10 minutes to ensure complete dissolution. The absorbance was measured at a wavelength of 570 nm using a multi-function microplate reader.
• Cell viability was calculated as follows: