The human breast cancer cell line MCF-7, the multidrug-resistant human breast cancer cell line MCF-7/MDR, the cisplatin-resistant human ovarian cancer cell line SKOV3/CDDP and the cisplatin-resistant human ovarian cancer cell line A2780/CDDP were provided by The Key Laboratory of Pathobiology of the Ministry of Education, Jilin University.
Cell line revival
1. Remove the RPMl-1640 medium containing 10% fetal bovine serum and 1% penicilin-streptomycin from the 4°C refrigerator. Allow it to reach room temperature. Before the experiment, sterilize the laminar flow hood, 10 cm culture dishes pipette tips, and EP tubes by irradiating with a UV lamp. Clean the surface of the laminar flow hood with a cotton ball soaked in 75% alcohol.
2. Retrieve the frozen cells from liquid nitrogen and quickly thaw them in a 37°C water bath. Ignite the alcohol lamp in the laminar flow hood, then add the culture medium to the frozen cells in the laminar flow hood. After mixing, transfer the mixture to a centrifuge and spin at 1050 rpm for 5 minutes.
3. Discard the supernatant and resuspend the pellet in the culture medium. Transfer the cells to a 10 cm culture dish containing 6-7 mL of culture medium. Label the culture dish with the cell line and revival date, and place it in a cell incubator at 5% CO2 and 37°C for cultivation.
Cell media exchange
1. Prepare as mentioned above.
2. When the cells have not yet fully covered the culture dish, transfer the culture dish from the cell incubator to the laminar flow hood. Use a pipette to remove the old culture medium and wash the culture dish with 1-2 mL of 0.01M PBS buffer. After removing the buffer, add 6-7 ml of fresh culture medium to the dish, and transfer it back to the cell incubator for cultivation.
Cell passing
1. Prepare as mentioned above.
2. When the cell confluence reaches 80%-90%, wash the culture dish with 1-2 mL of 0.01M PBS buffer. After removing the buffer, add 1 mL of 0.25% trypsin digestion solution to the dish for cell digestion. Under the microscope, observe until 60%-70% of the cells detach from the dish. Add 1 mL of culture medium to stop digestion, and use a pipette to gently detach the cells from the dish. Collect the cell suspension and centrifuge at 1050 rpm for 5 minutes.
3. Discard the supernatant and resuspend the cells in 1 mL of fresh culture medium. Transfer an appropriate volume of the cell suspension to a new culture dish containing culture medium. Place the dish in the cell incubator for further experiments, or take an appropriate amount of cells for cell cryopreservation.
Cell Cryopreservation
1. Prepare as mentioned above.
2. Take the cells in logarithmic growth phase as mentioned above. Digest and collect them by centrifugation, discard the supernatant, add a certain amount of cell cryopreservation solution, store in cryovials, seal with plastic wrap and medical adhesive tape, label the type of cryopreservation, date, and user, and store at -80°C freezer or in liquid nitrogen.
The plasmid was designed with the following key components:
1. pnirB::HlyA::HA tag::terminator: Hypoxia-induced expression of the hlyA (encoding listeriolysin O) fused with an HA tag under the control of the nirB promoter, allowing selective expression in tumor microenvironments.
2. T7 promoter::shRNA::terminator: The T7 promoter drives expression of shRNA targeting the CLDN6 gene (sh-CLDN6). The shRNA sequence (caGTGCAAGGTGTACGACTCATTCAAGAGATGAGTCGTACACCTTGCACtg) was based on a previously validated sequence used in the lab. ShRNA sequences with no homology to human genes were used as controls.
3. plac(ΔlacO)::Lpp’::OmpA(46-159)-RGD::His tag: The plac(ΔlacO) promoter, a constitutive version with the lac operator binding site deleted, ensures continuous expression of the Lpp’::OmpA(46-159)-RGD fusion protein. This protein contains an RGD peptide for tumor targeting and a His tag for detection and purification.
4. araC PBAD-murA-asd: Essential for survival of the delayed lysis strain χ11802, this system is controlled by the araC pBAD promoter, driving expression of the murA and asd genes.
Figure 1. pSliencer-CLDN6
Precautions:
1. The plasmid carries a kanamycin resistance marker (KanR) for selection.
2. The plasmid was synthesized by Yunzhou Biosciences (Guangzhou) Co., Ltd, based on the design described above. The sequence was verified by the company through sequencing, and the final construct was delivered in lyophilized form.
3. The bacterial strain used for the experiments was the attenuated regulated delayed lysis Salmonella enterica subspecies Serovar Typhimurium strain χ11802. The plasmids were electroporated into the Salmonella before use.
1. Inoculate bacteria (ΔmsbB-pRE112 χ7213; χ11802) into 5 mL LB culture medium and culture on a shaker at 200 rpm, 37℃ for 16 h.
2. Mix 400 μl of recipient strain with 800 μl of donor strain in a 1.5 mL Eppendorf tube by pipetting. (Note: Recipient and donor strains are mixed in a 1:2 ratio)
3. Transfer the entire mixed culture onto a DAP-LB plate. Gently swirl (no need to spread with a spreader), and incubate in the incubator at 37℃ for 20 h. (Note: The plate should be incubated upright. Due to excess liquid, it cannot be inverted for incubation)
4. From the bacterial mat formed on the DAP-LB plate in step 3, take a small amount of the bacterial mat with a pipette tip and perform a three-zone streak on the Cm+-LB plate, then incubate in the incubator at 37℃ for 20 hours.
5. From the Cm+-LB plate in step 4, use a pipette tip to pick a single colony and perform purification culture on the Cm+-LB plate. Incubate at 37℃ for 20 hours.
6. Place 1 μl of Salmonella serum on a clean slide. Use a toothpick to pick a single colony from the Cm+-LB plate in step 5, smear it in the serum, gently mix and then observe.
7. If granular phenomena appear, which indicates Salmonella, take 200 μl of LB culture medium into a 1.5 mL Eppendorf tube, use a pipette tip to pick a verified purified colony from the Cm+-LB plate in step 6, and culture in a shaker at 200 rpm, 37℃ for 1 h. (Note: If a uniformly turbid phenomenon appears, it indicates Escherichia coli.)
8. Dilute the bacterial solution 1:103 and 1:104, and take 100 μl of the bacterial solution onto a 10% sucrose-LB plate, perform uniform plating, and incubate in the incubator at 37℃ for 20 h.
9. Use a toothpick to pick a single colony from the 10% sucrose-LB plate in step 8, and simultaneously perform corresponding spot inoculation on the Cm+-LB plate and LB plate, incubating in the incubator at 37℃ for 20 hours. (Note: First streak the Cm+-LB plate, then streak the LB plate.)
10. If no bacteria growth appears on the Cm+-LB plate, but bacteria grow at the corresponding position on the LB plate, it indicates the presence of target recombinant bacteria. Perform PCR on the target recombinant bacteria (primer: msbB homologous arm), and then identify by electrophoresis. (Note: Pick the target colony from the LB plate.)
11. If the identification in step 10 is successful, further purify the target colony on the LB plate. Incubate in the incubator at 37℃ for 20 hours, and perform PCR and electrophoresis identification (repeat step 10).
12. If step 11 is also successfully identified, incubate the target colony overnight and freeze it for storage at -80℃.
1. Take bacteria (:: Ptrc T7 RNA polymerase - pRE112 χ7213; ΔmsbB-χ11802 ) in 5 mL LB culture medium, cultured on a shaker at 200 rpm, 37℃ for 16 h.
2. Mix 400 μL of recipient strain with 800 μL of donor strain in a 1.5 mL Eppendorf tube by pipetting. (Note: The recipient and donor strains are mixed in a 1:2 ratio)
3. Transfer the entire mixed culture onto a DAP-LB plate. Gently swirl (no need to spread with a spreader), and incubate in the incubator at 37℃ for 20 h. (Note: The plate should be incubated upright. Due to excess liquid, it cannot be inverted for incubation)
4. From the bacterial mat formed on the DAP-LB plate in step 3, take a small amount of the bacterial mat with a pipette tip and perform a three-zone streak on the Cm+-LB plate, then incubate in the incubator at 37℃ for 20 hours.
5. From the Cm+-LB plate in step 4, use a pipette tip to pick a single colony and perform purification culture on the Cm+-LB plate, then incubate in the incubator at 37 ℃ for 20 hours.
6. Place 1 μL of Salmonella serum on a clean slide. Use a toothpick to pick a single colony from the Cm+-LB plate in step 5, smear it in the serum, gently mix and then observe.
7. If granular phenomena appear, which indicates Salmonella, take 200 μL of LB culture medium into a 1.5 mL Eppendorf tube, use a pipette tip to pick a verified purified colony from the Cm+-LB plate in step 6, and culture in a shaker at 200 rpm, 37℃ for 1 h. (Note: If a uniformly turbid phenomenon appears, it indicates Escherichia coli.)
8. Dilute the bacterial solution 1:103 and 1:104, and take 100 μL of the bacterial solution onto a 10% sucrose-LB plate, perform uniform plating, and incubate in the incubator at 37℃ for 20 h.
9. Use a toothpick to pick a single colony from the 10% sucrose-LB plate in step 8, and simultaneously perform corresponding spot inoculation on the Cm+-LB plate and LB plate, incubating in the incubator at 37℃ for 20 hours. (Note: First streak the Cm+-LB plate, then streak the LB plate.)
10. If no bacteria growth appears on the Cm+-LB plate, but bacteria grow at the corresponding position on the LB plate, then it is the target recombinant bacteria. Perform PCR on the target recombinant bacteria (primers: msbB-arms), and then identify by electrophoresis. (Note: The target colony is picked from the LB plate.)
11. Select the target colony based on the PCR results from step 10, and perform PCR again (primers: Ptrc T7 RNA polymerase), then identify by electrophoresis.
12. If the identification in step 11 is successful, further purify and culture the target colony on an LB plate, incubate at 37℃ for 20 hours, and repeat PCR and electrophoresis identification (steps 10 and 11).
13. If step 12 is also successfully identified, culture the target colony overnight and stored at -80℃ in the freezer.
14. Following the gene knockout of msbB and insertion of the T7 RNA polymerase, the modified strain was designated as χ11803, distinguishing it from its precursor strain, χ11802.
1. Primer treatment:
1.1 According to the relevant information of the primer wall, add buffer to the primer to a final concentration of 100 μM (μmol/L), and store it in a -20℃ refrigerator as a storage solution for storage. If used as simple cloning primers, quantitative primers, and primers for double-stranded interference, the stock primer can be diluted to 10 μM with ddH2O2 and stored as a working solution.
1.2 Befobre adding the solution, centrifuge the prime tube briefly at 3000-4000 rpm for about 1 mintue to collect the contents at the botton of the tube.
1.3 After centrifugation, open the tube cap slightly, insert the pipette tip, and add the solution. Deionized water or DNA-free water that has been autoclaved is acceptable, as is autoclaved ddH2O, but it must be autoclaved. The volume of water added should be 10 times the volume of the primer tube. For example, if the primer indicates 37uL of water to 100uM, we need to add 370uL of water to 10uM.
1.4 After adding the water, shake the primer for 10-20 minutes to ensure it is fully dissolved.
2. Prepare individual PCR systems.
3. Set up the PCR program on the machine.
3.1 Pre-denaturation: Denature double-stranded DNA to single-stranded DNA. This step is typically performed at 95°C for a specific duration, which can be found in the reagent instructions.
3.2 Denaturation: Separate double-stranded DNA into single-stranded DNA, preparing for primer annealing, and its usually placed at 95℃ for 30s or 15s (specific time can be referred to the reagent instructions).
3.3 Annealing: Anneal the primer to the template, typically setting annealing temperature based on the primer's Tm value. For example, if the primer Tm value is 60℃, the annealing temperature can initially be set to 60℃. If the result is not satisfactory, other temperatures can be explored.
3.4 Extension: Generally performed at 72℃, DNA polymerase extends from the binding site of the primer on the template, and synthesize a new DNA strand from the 5' end to the 3' end.
3.5 Complete extension: Extend the reaction at 72°C for 5-10 minutes to ensure that all DNA strands are fully synthesized, completing the extension of all double-stranded DNA.
Place the sample into the PCR instrument and set up the program. The specific settings should be based on the specific circumstances.
4. Agarose Gel Electrophoresis
4.1 Preparation of Electrophoresis buffer: 1xTAE (take 20 mL of 50xTAE and add ddH2O to make a final volume of 1L).
4.2 Preparation of agarose gel: Choose an appropriate gel concentration based on the desired size of the DNA fragment (or the amplified DNA fragment). Generally, prepare a 1% agarose gel. For example, dissolve 0.2g of agarose in 20 mL of 1xTAE by heating, and dissolve 0.4g of agarose in 40 mL of 1xTAE by heating. Heat the mixture in a microwave until boiling, then cool it down to a temperature that is not hot to touch.
4.3 Add nucleic acid dye:
a. For small wells, add 0.2g of agarose, 20 mL of 1xTAE, and 2μL of nucleic acid dye.
b. For medium wells, add 0.4g of agarose, 40 mL of 1xTAE, and 4μL of nucleic acid dye.
c. For large wells, add 0.8g of agarose, 80 mL of 1xTAE, and 8μL of nucleic acid dye.
4.4 Install combs and slide gently pour the gel solution into the wells from the bottom right, cooling it down. Try to avoid trapping air bubbles and wait for solidification (about 20 minutes). Remove the gel after solidification.
4.5 Loading samples: Place the gel with the plate into an appropriate electrophoresis chamber, then pour in 1xTAE buffer, ensuring that the gel is submerged by 1mm. Choose an appropriate sample volume:
PCR products: 2μL for a 20μL reaction system, and 4μL for a 50μL reaction system.
DNA extraction products: Mix 5μL of the product with 1μL of 6x loading buffer (to ensure a 1x concentration of loading buffer). Carefully pipette 2μL of the mixture into a sample well.
Marker: Choose a marker of approximately 100bp (within 1000bp) or 1000bp (within 10000bp) based on the size of the amplified products and add 1μL.
4.6 Run the gel: After covering the chamber and connecting the power supply (pay attention to the polarity), set the voltage to 60-100V and let it run for 20-40 minutes.
4.7 After completion, observe the gel bands and their positions using a gel imaging system to determine if the desired bands have been amplified.
Preparation of Electrocompetent Bacterial Cells
Day 1:
1. Using a pipette tip or inoculation loop, pick a single colony and inoculate it into a culture tube containing 5mL of LB liquid medium. Prepare two tubes to ensure a backup. Incubate overnight (14-16 hours) at 37°C with shaking at 220 RPM to allow for bacterial growth.
2. Sterilize large centrifuge bottles (250-500mL) and several 50mL centrifuge tubes for use the next day to collect the bacteria by centrifugation.
3.Prepare several bottles of sterilized water (note: use ultrapure water, total volume of about 1.5 liters), and store them in the freezer for use the next day to resuspend the bacterial cells at a lower temperature.
4. Prepare 10% glycerol using ultrapure water.
Day 2:
1. Take 2-5mL of overnight culture and inoculate into 500mL of LB (or 2XTV) liquid medium, controlling the initial OD600 to be between 0.03 and 0.05 for uniform growth. Incubate at 37°C with shaking at 220 RPM for 2-4 hours, measure the OD every half hour, and stop the culture when the OD value reaches 0.5.
2. Pre-chill the bacterial solution on ice for 30 minutes. At the same time, turn on the centrifuge and pre-cool it to 4°C to maintain low temperatures during centrifugation.
3. Transfer the bacterial solution into pre-chilled 250mL or 500mL centrifuge bottles and centrifuge at 4°C, 4000 RPM for 15 minutes.
4. Discard the supernatant. Firstly resuspend the pellet with a small amount (e.g., 20-50mL) of sterilized ice water. Then dilute with water to about 2/3 of the volume of the centrifuge bottle, fully resuspending the cells (can be done by shaking the bottle by hand). Centrifuge at 4°C, 4000 RPM for 15 minutes.
5. Discard the supernatant, add a small amount of sterilized water to resuspend the bacterial cells, then add water to suspend all cells in about 500mL of ice water. Centrifuge at 4°C, 4000 RPM for 15 minutes.
6. Discard the supernatant, add a small amount of sterilized 10% glycerol (pre-cooled) to the centrifuge tube, resuspend the bacterial cells. Then add 10% glycerol to a final volume of about 20mL. Centrifuge at 4°C, 4000 RPM for 10 minutes.
7. Carefully discard the supernatant (the pellet may be very loose), add 2mL of sterilized 10% glycerol (pre-cooled) to resuspend the cells.
8. Dispense the resuspended bacterial solution into 1.5mL EP centrifuge tubes at 200μL/tube, quickly freeze in liquid nitrogen, and then store at -70°C freezer.
9. To test the transformation efficiency, take 100μL of freshly prepared competent cells, add 0.01ng of known concentration plasmid DNA, electro-transform, and then plate 10μL (1%) and 100μL (10%) of the cells resuspended in 1mL of SOC medium to estimate the transformation efficiency.
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Brief Process for Preparing Competent Cells:
Collect cells, rinse with ice water twice, rinse with 10% glycerol once, resuspend cells in 10% glycerol, and dispense.
Electroporation
1. Remove competent cells from the -80°C freezer and thaw on ice.
2. Place the sterile electroporation cuvette on ice to pre-cool.
3. Transfer the thawed competent cells to a pre-cooled 1.5mL centrifuge tube at a volume of 40~100µL per tube, mix gently, and place on ice.
4. Take 1-2µL of purified plasmid or cloning ligation product into a 1.5mL centrifuge tube, and place on ice for 10 minutes. If the volume of added DNA is too large, the salt in it can cause electric sparks during electroporation!
5. Turn on the electroporator [BIO-RAD GENE PULSER SYSTEM], set to MANUAL, and adjust the parameters to 25µF, 200 Ohms, with a voltage of 2.0KV (these settings can be slightly adjusted based on experience and the specific protocol).
6. Transfer the mixture to the pre-cooled electroporation cuvette, gently tap the cuvette to ensure the mixture evenly enters the bottom of the electrode cuvette. Wipe the outer wall of the cuvette with a piece of absorbent paper to remove any condensation.
7. Push the cuvette into the electroporation apparatus, press the PULSE button, and after hearing the beep, quickly add 2X500µl of SOC liquid medium to the cuvette, resuspend the cells, and transfer to a 1.5mL centrifuge tube.
8. Resuscitation at 37°C, 220-250RPM for 1 hour.
9. Centrifuge, plate, incubate at 37°C overnight. Check the transformation results the next day.
Cleaning and storage of electroporation cuvettes
1. Rinse the used cuvettes with tap water, add 75% isopropyl alcohol to rinse the cuvettes.
2. Discard the alcohol, rinse 2~3 times with distilled water, and then use a 1mL pipette to repeatedly blow and suck the cuvette with ultrapure water more than 10 times.
3. Add 1-2mL of anhydrous ethanol to the cuvette, soak for 5 minutes.
4. Discard the anhydrous ethanol, and invert the cuvette on absorbent paper to allow the ethanol to fully evaporate.
5. Cover the cleaned and dried cuvette and store it for future use.
6. The RP4 plasmid is large, with a size of 60KPB, and the quality of the plasmid needs to be of a certain standard. It is best to desalt the extracted plasmid.
Validation of the Delayed Lysis System
1. Liquid Culture Growth:
1.1. Inoculate χ11803/pSilencer-CLDN6 (Salmonella strain χ11803 with plasmid pSilencer-CLDN6) into three sterile test tubes, one containing 5 mL of LB medium, one with 5 mL of ara medium (LB with arabinose), and one with 5 mL of carb medium (LB with carbenicillin).
1.2. Incubate the cultures at 37°C for 24 hours with shaking (220 RPM).
1.3. After 24 hours, visually inspect the turbidity of each tube to assess bacterial growth.
1.4. Record the growth observations for each medium.
2. Solid Culture Validation:
2.1. Prepare LB agar plates, ara plates (containing arabinose), and carb plates (containing carbenicillin).
2.2. Inoculate χ11803/pSilencer-CLDN6 onto the surface of each type of agar plate using the streak plate method.
2.3. Incubate the plates at 37°C for 24 hours.
2.4. After 24 hours, observe bacterial colony formation on each plate. Record which plates show growth and which do not.
Validation of Anaerobic-Induced hlyA Expression by Western Blot
Anaerobic Treatment
1. Bacterial Culture Preparation:
1.1. Add 5 mL of LB liquid medium and 50 μL of bacterial solution to a sterile 12 mL culture cube.
1.2. Incubate the culture for 5 hours at 37°C with shaking at 220 RPM.
2. Prepare Low-Oxygen Conditions:
2.1. Prepare a 100 g/L Na₂SO₃ solution using sterilized water, filter it through 0.2 μm membranes for sterilization.
2.2. Prepare 5 mL of LB liquid medium containing 1 g/L Na₂SO₃.
2.3. Pre-reduce the medium by placing it in an anaerobic tank with anaerobic bags at room temperature for at least 10 hours to absorb dissolved oxygen thoroughly.
3. Centrifugation:
3.1. After the initial culture period, centrifuge the bacterial culture at 3000×g at room temperature for 5 minutes.
3.2. Discard the supernatant.
4. Transfer Bacteria:
4.1. Resuspend the bacteria in the low-oxygen medium prepared in step 2.2.
4.2. Transfer the resuspended bacteria into the medium treated for hypoxia as quickly as possible.
5. Anaerobic Incubation:
5.1. Place the culture cube containing the resuspended bacteria in an anaerobic tank with anaerobic bags.
5.2. Incubate overnight at 37°C with shaking.
6. Sample Collection for Western Blot:
6.1. After incubation, collect 100 μL of the bacterial solution for Western blotting to validate hlyA gene expression.
1. Prepare TBST buffer
1.1 Take a sterile container and add 1L of 10X TBS (10X TBS buffer (pH 7.4): 1.37 M NaCl, 27 mM KCl, 100 mM Tris buffer)
1.2 Dilute the 10X PBS buffer to a 1X concentration using distilled water, and stir well.
1.3 Add 0.1% Tween-20 (the “0.1%” refers to the volume fraction, meaning add 1 mL of Tween-20 to 1L of 1X PBS buffer), continue stirring to mix thoroughly.
1.4 Filter or centrifuge the prepared TBST buffer to remove any particles or impurities that may be present.
1.5 Aliquot the buffer into small sterile bottles to avoid repeated freeze-thaw cycles, and store in a cool place at room temperature (or 4 degrees Celsius).
2. Sample preparation
Direct lysis method:
2.1. Pre-cool all reagents and consumables in an ice box.
2.2. Take out the culture dish with 80~90% cell confluence. Wash the cells three timeswith PBS, and use a pipette to remove the PBS, then add 0.5ml of protein lysis buffer containing PMSF. Transfer to an EP tube.
2.3. Incubate the EP tube on ice for 30 minutes, shaking every 5 minutes EP tube, then centrifuge at maximum speed for 15 minutes at 4℃.
2.4. Take the supernatant and transfer it to a new pre-chilled EP tube, label it, and prepare for BCA protein concentration measurement.
3. BCA protein concentration measurement
Perform protein concentration determination according to the instructions of the BCA protein quantification kit from Biotime.
3.1. Take the extracted protein and protein standards out of the refrigerator and place them in a pre-prepared ice box.
3.2. Dilute the thawed protein at a ratio of 1:10 (cells are 1:10), then centrifuge the diluted protein to mix.
3.3. Prepare the solution by mixing reagent A and reagent B from the BCA kit according to the ratio specified in the instructions.
3.4. Take out the 96-well plate and add 20μL to each well in the order of protein standards and the protein to be measured.
3.5. Add 200μL of the A and B mixed solution to each well, protect from light, and incubate at 37℃ for 30 minutes. Measure the OD value at 560nm using a microplate reader.
3.6. Create a standard curve and calculate the concentration of the protein to be measured based on the standard curve and the OD value of the sample.
Note: The optimal protein concentration is 3-5μg/μL
4. Protein denaturation
4.1. Take the extracted protein out of the refrigerator and place it in a freezing box to thaw.
4.2. Calculate the required amount of protein and lysis buffer based on the protein concentration, and add loading buffer for quantification.
4.3. Place the protein in a constant temperature device at 100℃ and boil for 5 minutes.
4.4. Allow the boiled protein to cool to room temperature, then place it on ice for later use.
5. Gel preparation
Prepare 12% separation gel or 5% concentration gel according to the table below, and pour into the electrophoresis solution. The sample loading amount should be suitable at 50~80μg;
5.1. After assembling the gel pouring device, water can be poured onto the glass plates to check for leaks, then invert and air dry for about 10 minutes.
If there is a gel leak: check if the device is tightly secured and if the gel strips are aged;
If the glass plates are not cleaned properly, it may lead to uneven gel or trailing bands.
5.2. Choose an appropriate concentration of separation gel based on the size of the proteins to be run.
5.3. With the long plate inside and the short plate outside, after adding the separation gel, add 1 ml of water or anhydrous ethanol to seal it. After 30 minutes, pour out the sealing liquid, and once the gel is dry, pour out the excess. After adding the concentration gel, wait for 30 minutes until the gel is completely solidified for subsequent electrophoresis.
6. Electrophoresis
6.1. Take the cooked protein sample out of the refrigerator and place it in a box to thaw.
6.2. Heat the thawed protein again at 100℃ for 5-10 minutes.
6.3. Add protein maker and protein samples into the SDS-PAGE gel.
6.4. 80V constant voltage electrophoresis, wait for the bromophenol blue to reach the interface of the two layers, then switch to 120V constant voltage electrophoresis.
7. Transfer membrane (PVDF membrane)
7.1. First, take out the gel and cut it as needed. Assemble the transfer device according to the filter paper-gel-membrane-filter paper sandwich structure.
7.2. Add pre-cooled transfer buffer, ice pack, and place it in the ice box.
7.3. For wet transfer, set the power supply to a constant voltage of 100V for 90 minutes. For semi-dry transfer, set the current to 2.5 times the membrane area (mA).
Tips: Pay attention to the assembly order of the transfer device and check whether the positive and negative power supply is correct; otherwise, it may result in no bands.
8. Milk blocking
8.1. Take out the PVDF membrane.
8.2. Place it in the incubation box pre-filled with milk, and slowly shake at room temperature for 1 hour.
8.3. Rinse the hybridization membrane with TBST buffer to remove residual blocking solution.
Tips: Common blocking agents: 5% non-fat milk, 3% BSA.
9. Antibody hybridization
9.1. Primary antibody hybridization: incubate at room temperature overnight at 4°C.
9.2. Discard the primary antibody or recover the primary antibody solution, add TBST to wash the membrane, 10 min each time, three times.
9.3. Secondary antibody hybridization: place the secondary antibody in the incubation box and incubate at room temperature for 1 hour.
10. Development
10.1. Discard the secondary antibody solution, wash the membrane with TBST three times, 10 min each time.
10.2. Remove excess liquid, place the membrane in the chemiluminescence imaging system, and evenly add ECL detection solution.
10.3. Analyze using Genesys analysis software.
Tips: Before using BST, ensure pH is between 7.4-7.6, and ECL luminescent solution must evenly cover the membrane surface.
Precautions:
1. After electrophoresis, the electrophoresis buffer in the tank can be recovered (be careful to distinguish it from new buffer).
2. After cleaning the gel plates, dry them and place them on the gel plate rack. Return the electrophoresis tank to its original position and turn off the electrophoresis apparatus.
3. The transfer tank should be cleaned promptly after use, and it can be returned to the drawer only after drying.
4. Dispose of staining solutions, gels, membranes, and waste promptly after the experiment.
5. After the experiment, return all items to their original positions and wipe off any water stains caused by the experiment.
1. Grow the Salmonella strain expressing RGD peptide in LB medium to an OD600 ≈ 0.8.
2. Take 1 mL of the bacterial culture (approx. 1.0 × 10⁹ CFU) and centrifuge at 4000 × g for 10 minutes to harvest the cells.
3. Wash the bacterial cells twice with PBS. Fix the cells with 4% paraformaldehyde for 15-20 minutes at room temperature.
4. After fixation, incubate the bacterial cells overnight at 4°C with a mouse anti-His tag monoclonal antibody (A00186, GenScript) diluted in PBA (PBS with 3% BSA).
5. Wash the cells thoroughly with PBS to remove the primary antibody. Resuspend the cells in PBA containing 3% BSA and incubate with goat anti-mouse IgG Alexa Fluor 488-conjugated antibody (A-11001, Invitrogen) for 1 hour at room temperature. Protect from light.
6. Wash the cells again with PBS and resuspend them in 500 μL of PBS for flow cytometry detection.
7. Detect the fluorescence signal using the FITC channel of the flow cytometer (NovoCyte 1030, Agilent, USA). Set the forward scatter (FSC) detection threshold to 1000 and record 100,000 events for analysis.
The plasmid pCLDN6-GFP was provided by The Key Laboratory of Pathobiology of the Ministry of Education, Jilin University.
Figure 2. pCLDN6-GFP
1. Cell Preparation
Plate the MCF-7 cells in a 24-well plate with 70%‐80% confluence.
2. Preparation of DNA Solution
Dilute the plasmid DNA with EP buffer to a concentration of 1 μg/μL. Then, take a sterile 1.5mL centrifuge tube, adding 30μL of plasmid DNA stock solution and 470μL of EP buffer to obtain a final volume of 500 μL of DNA dilution solution. Each well of cells will be transfected with 300 μL of DNA dilution solution.
3. Preparation and Disinfection of Electrodes
3.1 Take a clean 24-well plate and prepare 70% ethanol disinfectant in one well, leaving one well empty, and filling two wells with EP buffer.
3.2 Pre-UV irradiate the electrodes for 5-10 minutes in the workspace.
3.3 Immerse the electrodes in 70% ethanol disinfectant to disinfect and sterilize them.
3.4 Transfer the electrodes to an empty well to air dry.
3.5 Transfer the electrodes to EP buffer, and gently shake them to clean them from ethanol.
3.6 Transfer the electrodes to another well of EP buffer and repeat step 5 to clean off any remaining ethanol. Leave the electrodes in the well for later use.
4. Pre-treatment of Culture Medium: Prior to the experiment, preheat EP buffer and culture medium to 37℃.
5. Electroporation Experiment
5.1 Wash the cells with EP buffer 1-2 times.
5.2 Remove the EP buffer from the targeted well and add 300 μL of DNA dilution solution.
5.3 Place the electrode at the bottom of the targeted well and stabilize it with your fingers.
5.4 Set the electroporation parameters, then measure and record the resistance. The conditions are 2500-3000 V, 200 Ω, 25 μF. and start the electroporation program.
5.5 Lift the electrode to separate it from the cells in the well, and rotate it 90°, then place it back in the well to increase the transfection area.
5.6 Measure and record the resistance again, and perform the electroporation program. Record the current, energy, and other parameters displayed by the instrument after the second electroporation program. These data can be used for troubleshooting. Collect and save the DNA dilution solution in the original 1.5mL centrifuge tube.
5.7 Perform electroporation for the next targeted well, following steps 1-7.
5.8 After the experiment, clean the electroporation cuvette: rinse with ddH2O first, then rinse with 75% ethanol, and finally soak in 75% ethanol. Typically, the electroporation cuvette is soaked in 75% ethanol. Before use, the electroporation cuvette needs to be exposed to UV irradiation for 15-30 minutes.
Bacterial Infection
1. Inoculate CLDN6-overexpressing MCF-7 cells in a culture dish and culture them until they reach an appropriate state and density (approximately 70-80% confluency).
2. Take 10 μL of χ11803/pSilencer-CLDN6 bacteria from glycerol preservation and add it to 5 mL of LB liquid medium. Incubate at 37°C with shaking at 220 rpm for 12 hours to allow bacterial expansion.
3. Transfer the bacterial solution to 50 mL LB liquid medium and grow until the OD600 reaches 0.5 (approximately 1.6×10⁸ CFU/mL).
4. At least 30 minutes before co-incubation, replace the cancer cell medium with DMEM basic culture medium containing 10% FBS but without antibiotics.
5. Take the appropriate amount of bacterial solution (based on the required MOI value), centrifuge the solution, and wash the bacteria twice with 1×PBS. Resuspend the bacteria in DMEM without antibiotics or serum.
6. Add the resuspended bacterial solution to the cancer cells at the desired MOI. Co-incubate the bacteria and cancer cells at 37°C for 2 hours.
7. After 2 hours of co-incubation, wash the cancer cells twice with 1× PBS. Wash once with DMEM culture medium containing antibiotics.
8. Continue to culture the cancer cells in a 37°C, 5% CO2 incubator with saturated humidity for 24 hours.
CLDN6 Protein Expression Analysis
After the 24-hour incubation, collect the cancer cells for Western Blot analysis.
Effects on MCF-7/MDR After CLDN6 Knockdown
Co-Culture of χ11803/pSilencer-CLDN6 and MCF-7/MDR Cells
1. Seed MCF-7/MDR cells in 24-well plates at a density of 5×10⁵ cells per well and incubate for 12 hours.
2. Take 10 μL of χ11803/pSilencer-CLDN6 bacteria from glycerol preservation and add to 5 mL LB liquid medium. Incubate at 30°C with shaking at 200 rpm for 12 hours.
3. Transfer the bacterial solution to 50 mL of LB liquid medium and grow until OD600 reaches 0.5.
4. Add the χ11803/pSilencer-CLDN6 bacteria to the MCF-7/MDR cell culture at a MOI (Multiplicity of Infection) of 1:150.
5. Co-culture the cells and bacteria for 5 hours. After 5 hours, wash the cells three times with sterile PBS.
6. Replace the culture medium with complete DMEM containing 5% fetal bovine serum (FBS) and 50 μg/mL gentamicin, and continue culturing the cells for an additional 24 hours.
CCK-8 Assay to Assess MCF-7/MDR Cells Sensitivity to Chemotherapy Drugs
1. Cell Preparation:
1.1 Prepare a cell suspension and perform cell counting.
1.2 Adjust the cell density to 3000 cells/100 μL.
1.3 Seed 100 μL of cell suspension per well into a 96-well plate.
1.4 Incubate the cells overnight in a 37°C incubator with 5% CO2.
2. Drug Treatment:
2.1 On the following day, add 100 μL of various concentrations of the chemotherapy drugs to each well.
2.2 Continue to incubate the cells for 48 hours at 37°C.
3. CCK-8 Reagent Addition:
3.1 After 48 hours of incubation, carefully remove the medium from each well.
3.2 Prepare the CCK-8 working solution by diluting CCK-8 reagent with serum-free medium at a ratio of 9:1.
3.3 Add 100 μL of the diluted CCK-8 working solution to each well.
4. Incubation and Measurement:
4.1 Incubate the plate for 1-2 hours at 37°C.
4.2 Measure the absorbance at 450 nm using a microplate reader.
5. Data Analysis:
5.1 Calculate the cell growth inhibition rate for each group.
5.2 Use GraphPad Prism software to calculate the IC50 value, which reflects the sensitivity of the cells to the drugs and indicates chemotherapy resistance.
Western Blot Analysis of Drug Resistance-Related Protein Expression
The expression of P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), and multidrug resistance-associated protein 1 (MRP-1) was validated through Western blot after co-culture. (The detailed procedure for Western blot has been described previously under the section 'Validation of Anaerobic-Induced hlyA Expression by Western Blot.' Please refer to that section for specific steps.)
Annexin V/PI Staining to Assess the Effect on Cell Apoptosis
1. Wash cells twice with cold PBS and then resuspend cells in 1X Binding Buffer at a concentration of 1 x 10^6 cells/ml.
2. Transfer 100 µl of the solution (1 x 10^5 cells) to a 5 ml culture tube.
3. Add 5 µl of FITC Annexin V and 5 µl PI.
4. Gently vortex the cells and incubate for 15 min at RT (25°C) in the dark.
5. Add 400 µl of 1X Binding Buffer to each tube. Analyze by flow cytometry within 1 hr.
Western Blot Analysis of Apoptosis-Related Protein Expression
The expression levels of cleaved-PARP and cleaved-caspase-9 were analyzed in MCF-7/MDR cells with different treatments using Western blot, following the steps previously described. (The detailed procedure for Western blot has been described previously under the section 'Validation of Anaerobic-Induced hlyA Expression by Western Blot.' Please refer to that section for specific steps.)
Effects on SKOV3/CDDP and A2780/CDDP After PGC1α Knockdown
To further validate the efficacy of our system, we selected two additional cisplatin-resistant ovarian cancer cell lines, SKOV3/CDDP and A2780/CDDP. We targeted the PGC1α gene (shRNA sequence: GTTATACCTGTGATGCTTT) to evaluate its role in chemotherapy sensitivity, drug resistance, and its effect on apoptosis in SKOV3/CDDP and A2780/CDDP cells. Chemotherapy sensitivity was assessed using the CCK-8 assay to measure cell viability after treatment with cisplatin. Apoptosis was evaluated using Annexin V/PI staining to quantify the apoptotic rate. Western blot analysis was performed to detect the expression of apoptosis-related proteins, including BAX, caspase-9, cleaved-caspase-3, and the anti-apoptotic protein BCL-2. The detailed experimental procedures have already been described in earlier sections.
Preparation of mPEG-PLGA-PLL
1. Preparation of mPEG-PLGA:
Vacuum heat-dry a heat-resistant glass tube, add a certain molar mass ratio of lactide / glycolide raw materials (ratios of 8:2, 7:3, 6:4, 5:5), then add PEG accounting for 1% to 20% of the total mass of the raw materials, with a molecular weight range of 350 to 5000, followed by the addition of a catalyst, nitrogen gas purging, heating to dissolve, vacuum extraction, cooling and solidifying, vacuum sealing the tube for 2 hours, and reacting at 120-150°C for 8-50 hours.
2. Preparation of mPEG-PLGA-Boc(Z):
A certain amount of mPEG-PLGA is dissolved in a dry organic solvent, stirred, and BOC-L-phenylalanine (1~15 eqv.) and N, N-dicyclohexylcarbodiimide (1~15 eqv.) are added dropwise at a slow rate while maintaining a temperature of 0~40℃. Afterward, 4-dimethylaminopyridine is added, nitrogen protection is applied, and the mixture is stirred at room temperature for 1~3 days. The solution is then filtered, washed with alkaline water (or dialyzed), concentrated, and the product is precipitated by adding ice methanol or ice ether, followed by filtration and vacuum drying.
3. Preparation of PEG-PLGA-NH2:
Dissolve mPEG-PLGA-Boc(Z) in a dry organic solvent under nitrogen protection, stir at 0℃ while adding dry trifluoroacetic acid dropwise over 10 to 60 minutes, continue the reaction for 1 to 3 hours, and remove the solvent and unreacted trifluoroacetic acid by rotary evaporation. Dissolve the residue in an organic solvent, precipitate with ice methanol or ice ether, filter, and dry under vacuum.
4. Preparation of mPEG-PLGA-PZLL:
Dissolve PEG-PLGA-NH2 in a dry organic solvent, add N-carboxyanhydride (NCA) (1 to 60 eqv.), protect under nitrogen, react at room temperature for 1 to 5 days, concentrate, precipitate with ice methanol or ice ether, filter, and dry under vacuum.
5. Preparation of mPEG-PLGA-PLL:
Dissolve mPEG-PLGA-PZLL in a measured amount of trifluoroacetic acid, add a small volume fraction of 33% hydrobromic acid (HBr) acetic solution, react at 0℃ for 0.5-8 hours, precipitate with ice methanol or ice ether, filter, and vacuum dry. Grafting of Arginine-Glycine-Aspartic acid (RGD): Dissolve mPEG-PLGA-PZLL in dimethyl sulfoxide, then add RGD and N,N'-carbonyldiimidazole (CDI), stir and react for 2-72 hours. After the reaction, dialyze the solution in a dialysis bag for 3-96 hours, then freeze-dry for storage.
Preparation of drug-loaded mPEG-PLGA-PLL nanoparticles
Prepared by the emulsification-evaporation method:
1. Dissolve the material mPEG-PLGA-PLL in a mixed solvent of dichloromethane or a mixture of dichloromethane and acetone, and add a solution of cisplatin.
2. After ultrasonic emulsification, add to a solution containing F68, and sonicate again.
3. Stir at room temperature for 0.5-5 hours to remove the organic phase, yielding mPEG-PLGA-PLL nanoparticles loaded with cisplatin.
4. ADM and 5-FU are encapsulated in the same manner.