Protocol

Plasmid Construction

Two plasmids were constructed using molecular cloning, one containing the T4 phage expression genes and the other encoding genes for the α-MSH antagonistic peptide, TGF-β mimetic peptide, and cell-penetrating peptide.

Plasmid Selection and Primer Design:

This experiment employed a dual-plasmid system. The genes for TGF-Beta protein and Melan protein were cloned into the pET28a(+) plasmid, while the T4 holin and T4 endolysin genes were cloned into pBAD30. SnapGene was used to design the plasmids and primers, which were subsequently synthesized by a biotechnology company. The constructed plasmids were named pET-28a(+)_Melan-SD and pBAD30-T4 lysis.

Molecular experiment

• Plasmid Extraction
  
  1. Column Equilibration: Add 500 μL of Buffer BL to the Bind DNA Mini Column. Centrifuge at 10,000 g for 1 minute. Discard the flow-through from the Collection Tube. Place the Bind DNA Mini Column back into the Collection Tube.
  2. Bacterial Cell Harvesting: Transfer 1-5 mL of fresh overnight bacterial culture into a centrifuge tube. Centrifuge at 10,000 g for 1 minute. Carefully aspirate and discard the supernatant.
  3. Resuspension: Add 250 μL of Buffer S1 to the pellet containing the bacterial cells. Resuspend the pellet completely by pipetting or vortexing. Incubate at room temperature for 5 minutes.
  4. Lysis: Add 250 μL of Buffer S2 to the tube. Mix thoroughly by inverting the tube 4-6 times until the solution becomes clear and viscous. Do not vortex. Ensure complete lysis.
  5. Neutralization: Add 350 μL of Buffer S3 to the tube. Mix immediately and thoroughly by inverting the tube several times until a fluffy white precipitate forms. Centrifuge at 13,000 g for 10-15 minutes. Carefully transfer the supernatant (cleared lysate) to the prepared Bind DNA Mini Column (from step 1). Centrifuge at 10,000 g for 1 minute. Discard the flow-through from the Collection Tube. Place the Bind DNA Mini Column back into the Collection Tube.
  6. Wash 1: Add 500 μL of Buffer PD to the Bind DNA Mini Column. Centrifuge at 10,000 g for 1 minute. Discard the flow-through from the Collection Tube. Place the Bind DNA Mini Column back into the Collection Tube.
  7. Wash 2: Add 700 μL of Buffer PW (ensure ethanol has been added beforehand) to the Bind DNA Mini Column. Centrifuge at 10,000 g for 1 minute. Discard the flow-through from the Collection Tube. Place the Bind DNA Mini Column back into the Collection Tube. Repeat this wash step once (total of two washes with Buffer PW).
  8. Dry Column: Centrifuge the empty Bind DNA Mini Column (in the Collection Tube) at 10,000 g for 2 minutes to remove residual ethanol. Place the Bind DNA Mini Column into a new, labeled 1.5 mL nuclease-free centrifuge tube. Let it stand at room temperature for 5 minutes to allow any residual ethanol to evaporate.
  9. Elution: Add 50-100 μL of Elution Buffer or Nuclease-free Water directly onto the center of the silica membrane in the Bind DNA Mini Column, without touching the membrane. Let it stand at room temperature for 2 minutes. Centrifuge at 10,000 g for 2 minutes to collect the purified DNA solution in the clean centrifuge tube.
• Agarose Gel Electrophoresis
  

  This process enables DNA fragments of different lengths moving through an agarose gel at different speeds to cause them to separate. This step allows us to isolate and examine the target gene and test whether the target gene has been successfully transferred into the recipient cell.

  Agarose Gel Preparation:

  This process enables DNA fragments of different lengths moving through an agarose gel at different speeds to cause them to separate. This step allows us to isolate and examine the target gene and test whether the target gene has been successfully transferred into the recipient cell.

  1. Add 0.3g agarose and 30 mL 1 x TAE to the conical bottle.
  2. Heat the agarose until it dissolves.
  3. After the agarose solution has cooled down slightly, add 3μl of Ultra GelRed and shake well.
  4. Pour agarose gel into gel caster and install the comb into the gel caster.
  5. Wait until the agarose gel has solidified.

  Sample Loading and Running:

  1. Place agarose gel in the electrophoresis chamber.
  2. Add 1 x TAE to the electrophoresis chamber until it covers the agarose gel.
  3. Load the sample and 10μL DNA marker to the wells of agarose gel.
  4. Set the voltage and running time of electrophoresis.
  5. Start electrophoresis.
  6. After electrophoresis, remove the agarose gel from the horizontal electrophoresis.
  7. Put agarose gel under ultraviolet light, observe and take pictures.
     
  8. Cut off the agarose gel containing the desired gene.
• Preparation of Competent Cells — Rubidium Chloride Method
  
  1. Inoculate ClearColi BL21(DE3) into 100 ml of LB medium at a 1% inoculation volume.
  2. Culture with shaking at 37°C until OD600 reaches 0.3–0.4.
  3. Rapidly cool the bacterial culture on ice.
  4. Centrifuge at 4500 g and 4°C for 10 minutes, then discard the supernatant.
  5. Resuspend the pellet in Solution I and place on ice for 5 minutes.
  6. Centrifuge at 4500 g and 4°C for 10 minutes, then discard the supernatant.
  7. Resuspend the pellet in 4 ml of Solution II and incubate on ice for 2 hours.
  8. Aliquot 200 μl per tube and flash-freeze in liquid nitrogen.
  9. Store at -80°C.

  Solution I

  

  Adjust the pH to 5.8 using 0.2M acetic acid, followed by filter sterilization.

  Solution II

  

  Then adjust the pH to 6.5 with KOH, and perform filter sterilization again.

• Plasmid Transformation
  
  1. Thaw the ClearColi BL21(DE3) competent cells on ice.
  2. Add the plasmid DNA and mix gently, then incubate on ice for 15-45 minutes.
  3. Heat-shock at 42°C for 45 seconds, then immediately place on ice for 2 minutes.Add 0.9 ml of LB medium and incubate at 37°C for 40-60 minutes.
  4. Centrifuge at 3000 g for 15 seconds, and remove the supernatant until approximately 200 μl remains.
  5. Spread the cells onto solid medium containing the appropriate antibiotic(s) (Amp for pBAD30, Kan for pET-28a(+), and both for dual plasmids) and incubate overnight at 37°C.

Expression Validation of the Functional Peptide Plasmid

• Cell Compatibility Test (CCK-8)
  
  1. Seed 100 μL of cells at a density of 3,000–7,000 per well in a 96-well plate and culture for 24 hours under conditions of 37°C, 5% CO₂, and 90% humidity.
  2. Prepare sample solutions with concentration gradients (300, 100, 33.3, 11.1, 3.7, 1.2, 0.4, 0.14 μM) and add to the 96-well plate with three replicate wells per concentration. Incubate for an appropriate duration under conditions of 37°C, 5% CO₂, and 90% humidity.
  3. Thaw and centrifuge the CCK-8 reagent at room temperature before use.
  4. Add 10 μL of CCK-8 solution to each well. Incubate for 0.5–4 hours under conditions of 37°C, 5% CO₂, and 90% humidity.
  5. Measure the absorbance at 450 nm using a microplate reader.
     
• Quantitative Real-time polymerase chain reaction (mechanism verification)
  

  It integrates PCR technology with real-time fluorescence detection, enabling real-time monitoring of DNA amplification during the PCR process and precise quantification of target gene transcription levels.

  Extraction of Sample RNA

  1. Thaw the frozen lysed cells and allow them to stand at room temperature for 5 minutes to dissolve completely.
  2. Phase Separation: Add 0.2 ml of chloroform per 1 ml of TRIZOL reagent used for lysing the sample. Tightly cap the tube and shake vigorously by hand for 15 seconds. Incubate at 15–30°C for 2–3 minutes. Centrifuge at 12,000 rpm for 15 minutes at 4°C. After centrifugation, the mixture will separate into a lower red phenol-chloroform phase, an interphase, and a colorless upper aqueous phase. The RNA is entirely contained in the aqueous phase, whose volume is approximately 60% of the volume of TRIZOL reagent used for homogenization.
  3. RNA Precipitation: Transfer the upper aqueous phase to a clean RNase-free centrifuge tube. Add an equal volume of isopropanol to precipitate the RNA. Mix well and incubate at 15–30°C for 10 minutes. Centrifuge at 12,000 rpm for 10 minutes at 4°C. The previously invisible RNA precipitate will form a gel-like pellet at the bottom and sides of the tube.
  4. RNA Washing: Remove the supernatant. Add at least 1 ml of 75% ethanol (prepared with DEPC-H₂O) per 1 ml of TRIZOL reagent used for lysing the sample to wash the RNA pellet. Mix well and centrifuge at 7,000 rpm for 5 minutes at 4°C.
  5. RNA Drying: Carefully remove most of the ethanol solution, and allow the RNA pellet to air-dry at room temperature for 5–10 minutes.
  6. Dissolving the RNA Pellet: To dissolve the RNA, add 40 μl of RNase-free water and pipette repeatedly several times until completely dissolved. The obtained RNA solution should be stored at –80°C for future use.

  RNA Quality AssessmentUltraviolet Absorption Measurement

  First, zero the spectrophotometer using the TE dilution buffer. Then, dilute a small amount of the RNA solution with TE (1:100) and measure the absorbance at 260 nm and 280 nm to determine the concentration and purity of the RNA solution.

  1. Concentration Measurement
     An A260 reading of 1 corresponds to 40 μg RNA/ml. The RNA concentration (μg/ml) is calculated as: A260 × dilution factor × 40 μg/ml. Specific calculation example:RNA is dissolved in 40 μl of DEPC water; 5 μl is taken and diluted 1:100 into 495 μl of TE buffer, giving an A260 = 0.21.RNA concentration = 0.21 × 100 × 40 μg/ml = 840 μg/ml = 0.84 μg/μl.After taking 5 μl for measurement, the remaining RNA sample volume is 35 μl, and the total remaining RNA amount is:35 μl × 0.84 μg/μl = 29.4 μg.
  2. Purity Measurement
     The ratio of A260/A280 of the RNA solution indicates its purity. The ratio should be between 1.8 and 2.1.

  Synthesis of Sample cDNA

  Reaction System

  

  Gently flick the tube bottom to mix the solution and briefly centrifuge at 6000 rpm.

  1. Heat the mixture at 70°C in a dry bath for 3 minutes before adding the reverse transcriptase MMLV. Immediately after removal, place it in an ice-water bath until the internal and external temperatures of the tube equalize. Then add 0.5 μl of reverse transcriptase and incubate in a 37°C water bath for 60 minutes.
  2. Immediately after removal, heat at 95°C in a dry bath for 3 minutes. The resulting final reverse transcription solution is the cDNA solution, which should be stored at -80°C for future use.

  Real-Time Quantitative PCR

  1. Preparation of Standard Gradient for β-actin Positive TemplateThe concentration of the positive template is 10^11. Before the reaction, take 3 μl and perform a 10-fold serial dilution (add 27 μl of water and mix thoroughly) to obtain 10^10, then sequentially dilute to 10^9, 10^8, 10^7, 10^6, 10^5, and 10^4 for later use.
  2. Reaction System

     Standard Reaction System

     

     Gently flick the tube bottom to mix the solution and briefly centrifuge at 6000 rpm.

     House-keeping Gene Reaction System

     

     Gently flick the tube bottom to mix the solution and briefly centrifuge at 6000 rpm.

  3. Load the prepared positive standards and test samples simultaneously. Reaction conditions: 93°C for 2 minutes, followed by 40 cycles of 93°C for 1 minute and 55°C for 2 minutes.

  Preparation of DNA Template for Gradient Dilution Standard Curve

  1. For each target gene to be measured, select a cDNA template confirmed to express the gene and perform PCR amplification.
     
     

     Gently flick the tube bottom to mix the solution and briefly centrifuge at 6000 rpm.
     Perform 35 PCR cycles (94°C for 1 minute; 55°C for 1 minute; 72°C for 1 minute), followed by a final extension at 72°C for 5 minutes.

  2. Analyze the PCR product and DNA ladder by 2% agarose gel electrophoresis with ethidium bromide staining to confirm whether the PCR product is a single specific band.
  3. Perform 10-fold serial dilutions of the PCR product: set the PCR product concentration as 1×10^10, and sequentially dilute to 10^9, 10^8, 10^7, 10^6, 10^5, and 10^4.

  Real-Time Quantitative PCR for Target Genes in Test Samples

  1. Prepare real-time PCR reaction systems for all cDNA samples.
     

     Reaction System

     
     

     Gently flick the tube bottom to mix the solution and briefly centrifuge at 6000 rpm.

  2. Place the prepared PCR reaction solution in a real-time PCR instrument for amplification. （Reaction conditions: pre-denaturation at 93°C for 2 minutes, followed by 40 cycles of 93°C for 1 minute, 55°C for 1 minute, and 72°C for 1 minute, with a final extension at 72°C for 7 minutes.）

  Perform real-time PCR for the target genes and housekeeping genes of each sample. Analyze the PCR products and DNA ladder by 2% agarose gel electrophoresis with GoldView staining to confirm whether the PCR products are single specific amplification bands.

  
• SDS-PAGE (Expression verification)
  
  1. Gel Preparation:
     Secure the gel casting plates and confirm there are no leaks by adding water. Prepare the gel according to the kit instructions. Pour the resolving gel into the plate, leaving 1-2 cm at the top, and then add the stacking gel. Insert the comb and allow it to solidify for 20 minutes.
  2. Sample Preparation:
     Set the third slot of the metal bath to 95°C for 10 minutes, and prepare the centrifuge at 4°C, 13,000 rpm, for 1 minute. For the samples that need to be run, centrifuge at 13,000 rpm for 1 minute to remove the supernatant. Resuspend the pre-induction samples in 500 uL of PBS, and the post-induction samples in 1 mL of PBS.Break the samples using ice-cooled sonication (75% power, 2 seconds on, 10 seconds off, for a total of 5 minutes).
     For each sample, aliquot 40 uL into a 1.5 mL centrifuge tube and add 10 uL of 5*loading dye. For the remaining samples, centrifuge at 15,000g for 20 minutes at 4°C, and then take 40 uL of supernatant, adding 10uL of 5*loading dye. Heat the prepared samples at 95°C in a metal bath for 10 minutes, then cool to room temperature before loading.
  3. Running the Gel:
     Mount the prepared gel in the electrophoresis apparatus and add running buffer, ensuring the inner chamber does not leak. Remove the comb from the precast gel and fill the outer chamber with MOPS-SDS running buffer (for precast gels) or SDS running buffer (for freshly made gels) up to the fill line. Load 20 uL of each sample and 15 uL of the marker. Connect the electrodes, set the voltage to 180V, and run the gel until the dye front exits the bottom of the gel.
     After electrophoresis, remove the gel from the plates and place it in a disposable container. Pour in staining buffer, heat in the microwave for 1 minute, and gently shake for 10 minutes. Decant the staining solution and add destaining buffer. Heat again and gently agitate until the bands become visible. Observe the protein bands for analysis.
     
     
• Immunofluorescence Staining (Functional verification)
  1. Discard the culture medium and wash the cells three times with PBS for 5 minutes each time. Then fix the cells with 4% PFA for 20 minutes.
  2. Wash the cells three times with PBS for 5 minutes each, followed by permeabilization with 0.5% Triton X-100 for 20 minutes on a shaker.
  3. Wash the cells three times with PBS for 5 minutes each. Add 5% BSA to cover all cells and block at room temperature on a shaker for 1 hour.
  4. Dilute the primary antibody (an antibody protein specifically binding to TRP1) in a dilution buffer, add the primary antibody, and incubate overnight at 4°C on a shaker.
  5. The next day, recover the primary antibody and wash the cells three times with PBS for 5 minutes each. Dilute the secondary antibody and incubate at room temperature for 2 hours protected from light.
  6. Add DAPI and incubate at room temperature for 15 minutes to stain the nuclei. Finally, wash the cells three times with PBS for 5 minutes each. After staining, observe the cells under a microscope. The target protein will appear fluorescent green.
     

Expression Validation of the Inducible Suicide Switch Plasmid

• Time-kill Curve
  

  The suicide switch plasmid utilizes an arabinose-inducible promoter. Therefore, a bacterial killing curve experiment was conducted during the functional validation phase to dynamically observe the lytic effect of the suicide switch element on bacteria under induced conditions.

  1. A 50 μL aliquot of the original bacterial culture in the logarithmic growth phase (OD600 ≈ 0.5) was taken and diluted 100-fold by adding 4950 μL of liquid medium to serve as the test bacterial suspension.
  2. Arabinose working solutions were prepared at concentrations of 0.25×, 0.5×, 1×, 2×, 4×, 8×, 16×, and 32× MIC (by dissolving arabinose into 25×, 50×, 100×, 200×, 400×, 800×, 1600×, and 3200× MIC stock solutions, then diluting 3 μL of each stock solution with 297 μL of the test bacterial suspension to achieve a 100-fold dilution).
  3. A 250 μL aliquot of the above drug-containing bacterial suspension was transferred into a 96-well plate. A 250 μL aliquot of the test bacterial suspension without arabinose was added to another well as a blank control. The plate was carefully placed in a 37°C conventional incubator for incubation.
  4. Samples were taken at incubation time points of 0 h, 1 h, 2 h, 4 h, 6 h, 8 h, and 24 h. After sampling, the bacterial suspensions were serially diluted 10-fold up to 10⁻⁸. A 100 μL aliquot of each diluted suspension was spread onto solid medium plates. After 24 hours, colony counts were performed, and the results were expressed as CFU/mL.
  5. For data processing, a time-killing curve was plotted with Lg CFU/mL as the vertical axis and time as the horizontal axis.
     
• Live-Dead Cell Staining
  

  It allows for intuitive discrimination between live and dead cells, enabling visualization of the lytic effect mediated by pBAD30.

  1. When the cells reach 90% confluency, seed them into a 96-well plate.
  2. After the cells have adhered for 24 hours, treat them with arabinose.24 hours after adding the drug, aspirate and discard the culture medium, and gently wash the cells 1-2 times with pre-warmed PBS buffer.
  3. Dilute the Calcein-AM and PI staining solutions to a 1X concentration, and add 100 μL of the staining solution to each well of the 96-well plate.
  4. Incubate the plate in a 37°C incubator for 30 minutes, then observe the staining results using a fluorescence microscope.Calcein exhibits green fluorescence (indicating live cells), while PI exhibits red fluorescence (indicating dead cells).