Molecular Cloning

Primer synthesis service is provided by Sangon Biotech (Shanghai) Co., Ltd. Gene synthesis service is provided by Genscript.

Preparation: The simple fragment amplification system is generally 50 μL, and the components of the non-50 μL system can be increased or decreased in proportion. Using different DNA polymerases, the optimal amount of template and primer may vary. 2X Phanta Max Master Mix is commonly used in our laboratory. The following is the general reaction system for PCR using this enzyme.

Two-fragment recombination is performed using ClonExpression® II One Step Cloning Kit from Vazyme biotech co., ltd and following the provided protocol.

Plasmid Extraction is performed using Plasmid Mini Kit I from Omega Bio-Tek biotech co., ltd and following the provided protocol.

Gel Extraction is performed using SanPrep Column DNA Gel Extraction Kit from Sangon Biotech (Shanghai) Co., Ltd and following the provided protocol.

Preparation of competent E. coli Nissle 1917

1. EcN was cultured in LB medium in a shaker at 200 rpm at 37 °C overnight.
2. Transfer 1/100 to growth medium for culture at 200 rpm on a shaking table at 37 °C;
3. Culture to OD₆₀₀ about 0.5.
4. Ice cooling for 20-30 min (to stop the growth of bacteria, try not to be too long).
5. Centrifugal force 5000 rpm (large centrifuge) at 4 °C for 10 min.
6. Wash three times with wash buffer.
7. Resuspension in the washing buffer at 1/100 of the original culture volume.
8. Pack 120 μL per centrifuge tube into 1.5 mL centrifuge tube, freeze liquid nitrogen and store in -80 °C refrigerator.

Wash buffer:

Transformation

1. Remove the competent cells from -80 °C and immediately melt them in an ice water bath for 5 min.
2. Add the plasmid DNA into 100 μL competent cells, gently stir well, and incubate on ice for 30 min.
3. Place the centrifuge tube in the 42 °C water bath without shaking. After the heat shock of 90 s, immediately place it in the ice water bath for 2-3 min.
4. Add 900 μL LB medium preheated at room temperature or 37 °C to the centrifuge tube, and then put it in a 37 °C shaker for 45 min.
5. Take different volumes of transformation products, spread them on the correct resistant plates with sterile coating sticks, and incubate them overnight at 37 °C in an incubator.

Cell Lysis

1. First, dilute 0.5 mL of 10× Cell Lysis Buffer with deionized water to prepare 5 mL of 1× Cell Lysis Buffer.
2. Centrifuge the host bacteria expressing the target protein at 8000 rpm (5700 ×g) for 10 min. Discard the supernatant and wash the pellet with 1X PBS buffer. Resuspend the bacterial pellet in 4 mL of 1× Cell Lysis Buffer per 100 mL of original culture (at OD₆₀₀=1).
3. Add 40 μL of PMSF and 80 μL of Lysozyme to the 1× Cell Lysis Buffer. Incubate the bacterial suspension at 37 °C for 60 min.
4. Lysate the cells via sonication (300W, 10 sec pulse on / 10 sec pulse off cycles) until the bacterial suspension becomes clear.
5. Add 20 μL of DNase/RNase and continue incubating on a shaker at 37 °C for 10 min.
6. Transfer the lysate to a refrigerated centrifuge and centrifuge at 4 °C, 5000 rpm (3000 ×g) for 30 min. Discard the pellet (insoluble debris) and collect the protein supernatant (soluble fraction) into a clean tube.

Protein Purification

1. Take a Ni-NTA pre-packed column of appropriate size based on the required binding capacity. Allow the storage buffer to drain out by gravity flow.
2. Equilibrate the column with two column volumes (CV) of Binding/Wash Buffer. Use a flow rate of 0.5-1 mL/min, allowing the buffer to pass through the resin slowly.
3. Mix the protein extract with Binding/Wash Buffer at a 1:1 ratio to prepare the sample solution. The total volume of the sample solution should be approximately two times the column volume.
4. Load the sample solution onto the column and collect the flow-through in a centrifuge tube. If excess sample remains, reload it onto the column. Passing the sample through the resin again can increase binding efficiency.
5. Wash the column with two CV of Binding/Wash Buffer and collect the flow-through. Repeat this step using a new collection tube until the absorbance of the flow-through at 280 nm approaches the baseline.
6. Elute the His-tagged protein from the column using two CV of Elution Buffer. Repeat this elution step twice, collecting the eluate fractions separately each time. Continue until the absorbance of the eluate at 280 nm approaches the baseline.
7. Column Post-processing: Wash the column resin with 5 CV of Elution Buffer, followed by equilibration with 5 CV of Binding/Wash Buffer. Finally, rinse with 5 CV of ddH₂O. Preserve the column by adding 20% ethanol and store at 2-8 °C. Do not freeze.

SDS-PAGE Analysis and Western Blot

1. Sample processing: Labeled, 200 μL culture medium supernatant was removed and added with 50 μl 5× Loading Buffer, the rest was discarded, and the precipitate was resuspended with 200 μL 1x Loading Buffer. The supernatant was vortexed and mixed, and the precipitate was blown and mixed. They were divided into whole bacteria, supernatant and precipitate. The samples were boiled at 95-100 °C for 10 min, and stored at -20 °C for a short time. The samples were removed before protein electrophoresis and centrifuged at 12000 rpm and 25 °C for 5-10 min.
2. According to the instructions of the electrophoresis device, install a clean and dry glass plate, and detect leakage.
3. The separation gel (12%) was prepared, the ingredients were quickly vortexed and mixed after addition, and carefully injected into the prepared glass plate gap (about 5 mL) with a pipettor, leaving enough space for the concentrated gel. A thin layer of isopropanol is gently added to the top layer to remove air bubbles, flatten the separation gel, and prevent the oxygen in the air from inhibiting the polymerization of the gel. After about 1 h, the gel polymerization was completed, the isopropyl alcohol was poured off, the residual isopropyl alcohol at the top was blotted by filter paper, and the glass plate was placed upside down on the paper to drain. Prepare the comb.
4. Prepare concentrated gel (5%), the pH of buffer used in the two gel is different, and each component is quickly vortexed and mixed after adding, and it is infused into the separation gel with a pipettor (about 2 mL). After filling, it is quickly and carefully inserted into the sampling comb, first inserted on one side and then on the other side to avoid bubbles as much as possible.
5. After the concentrated gel solidifies (about 30min), carefully pull out the comb.
6. The gel was fixed on the electrophoresis device, and enough electrophoresis buffer was added, and 10 μL of each sample was added to the sampling well.
7. When the sample is electrophorezed in the concentrated gel, the voltage of 80 V can be used and the time can be set to 6 h in order to stop and pause manually. Then the power supply was turned off and the electrophoresis buffer was recovered.
8. Remove the gel, immerse it in at least 5 times the volume of Coomassie Brilliant Blue R-250 staining solution, place it on a horizontal shaker for staining at room temperature for at least 4 h, then remove the stained gel and withdraw the staining solution for re-use, immerse the gel in Coomassie Brilliant blue decolorization solution, decolorize it for 4-8 h on a horizontal shaker, and remove the dye solution for further use. During this period, the decolorization solution was changed 3-4 times until the gel was decolorized until the band was clear. The results were observed and recorded and photographed.

1. Sample processing: Labeled, 200 μL supernatant was removed and added to 50 μL of 5× Loading Buffer, the rest was discarded, and the precipitate was resuspended in 200 μL of 1× Loading Buffer. The supernatant was vortexed to mix and the precipitate was blown to mix. They were divided into whole bacteria, supernatant and precipitate. The sample was boiled at 95-100 °C for 10 min. It could be stored at -20 °C for a short time, removed before protein electrophoresis, and centrifuged at 12000 rpm and 25 °C for 5-10 min.
2. According to the instructions of the electrophoresis device, install a clean and dry glass plate to detect leakage.
3. The separation gel (12%) was prepared, the ingredients were quickly vortexed and mixed after addition, and carefully injected into the prepared glass plate gap (about 5 mL) with a pipettor, leaving enough space for the concentrated gel. A thin layer of isopropanol is gently added to the top layer to remove air bubbles, flatten the separation gel, and prevent the oxygen in the air from inhibiting the polymerization of the gel. After about 1 h, the gel polymerization was completed, the isopropyl alcohol was poured off, the residual isopropyl alcohol at the top was blotted by filter paper, and the glass plate was placed upside down on the paper to drain. Prepare comb.
4. Prepare concentrated gel (5%), the buffer pH used in the two gel is different, and the components are quickly vortexed and mixed after adding, and are infused into the separation gel with a pipet (about 2 mL). After filling, quickly and carefully insert the sampling comb, first insert one side and then insert the other side to avoid bubbles as much as possible.
5. The gel was fixed on the electrophoresis device, and enough 1×Gly electrophoresis buffer was added, and 10 μL of each sample was added to the sampling well.
6. Load sample.
7. Prepare the membrane transfer buffer, prepare the transfer tank, enamel plate, sponge pad, etc.
8. After turning off the power, cut off the concentrated gel, pry open the glass, cut off the unpointed belt, and keep the gel moist with the membrane buffer during the process.
9. Measure the size of the gel, cut out a PVDF membrane slightly larger than the gel, and put the membrane into the methyl alcohol 20 s.
10. Place the membrane and gel between sponge pads, ensuring the black side faces the cathode and the white side faces the anode before inserting into the transfer tank
11. Electrophoresis at 60 V (400 mA) for 1.5h and put ice box ice bath in the tank.
12. Block with bovine serum albumin (BSA) for 1h by shaking on a shaker.
13. Incubate with primary antibodies and eluted three times with EZ-Buffers H 10X TBST Buffer.
14. Incubate with secondary antibodies and eluted three times with EZ-Buffers H 10X TBST Buffer.
15. Developed using an ECL substrate.

Cell experiment protocol

To enter the cell room, laboratory personnel need to wear a lab coat and disposable gloves, and spray hands and cuffs with 75% alcohol. Before starting the cell experiment operation, all the consumables need to be irradiated and sterilized by ultraviolet radiation for 30min and make sure that the required consumables, reagents, culture media, etc. are ready and reduce the number of times you leave the ultra-clean bench.

1. Prepare complete culture medium and PBS,and preheat in a 37 °C water bath for 15min
2. Transfer the cells to the bench, use the pipette tip to carefully aspirate the supernatant, and add the preheated PBS to aspirate the PBS, after shakingand add fresh complete culture medium (99% Endothelial Basal Medium Cat.no: STM-CM-0516+1%Pen-Strep Cat.no: BL505A).

1. Carefully aspirate the supernatant and add preheat PBS and shake to aspirate the PBS.
2. Add appropriate amount of trypsin(Cat.no: 25200-072) and put it back into the incubator for three minutes. Re-transfer to the platform and add more than lml of medium to neutralize the trypsin.
3. Centrifuge at 900 rpm for 5min, resuspend cells with complete medium.
4. Add the cell suspension according to the amount of medium needed for each culture dish or plate, and make 8 strokes on the countertop to make the cells evenly distributed in the culture dish.

1. Make sure the cell confluence is 50%-80%, the medium is clarified and the cells are not contaminated.
2. Prepare freezing solution(50% complete culture medium+40% FBS Cat.no: FSP500 +10%DMSO Cat.no:219605580), PBS and other reagents in advance.
3. Use a pipette tip to carefully aspirate the supernatant, and add preheat PBS to shake and then aspirate the PBS.
4. Add appropriate amount of trypsin (according to the bottom area of the petri dish) and put it back into the incubator for a few minutes (according to the cell type); re-transfer to the platform and add more than l ml of medium to neutralize the trypsin.
5. Resuspend by adding cryopreservation solution, transfer to pre-labeled cryopreservation tubes and tighten, transfer to cryoboxes at room temperature (make sure cryoboxes have isopropyl alcohol) and transfer to -80 °C refrigerator for slow freezing, and transfer to liquid nitrogen the next day.

1. Prepare the consumables and culture medium for cell recovery in advance.
2. Remove the needed cryopreservation tubes from the liquid nitrogen tank quickly to reduce the exposure time of other cells/tubes/cassettes at room temperature.
3. Place the cryotubes into the 37 °C water bath for 2-3min.
4. Transfer to 5 times volume of medium (1ml frozen cells + 4ml medium), centrifuge (900 rpm, 5min), remove supernatant, resuspend with fresh medium and add to cell culture dish, put into incubator.

1. Collect 1ml of supernatant from at least 3-day-old cell cultures with 100% confluence to ensure that the titer of mycoplasmas is above the detection level of the assay.
2. Spin 5min at the maximum speed. Aspirate excessive supernatant to approximately 100 µl (if the initial volume is less than 100 μl, add PBS to 100 μl), incubate in 95ºC for at least 30min.
3. Briefly spin down, take 1µl of the supernatant as PCR template.

1. Preparation of Nanobody Working Solutions: Dilute the nanobody stock solution with sterile PBS to generate a series of working solutions at 0.25, 0.5, 1, 2, and 5μg/mL.
2. Preparation of VEGF Working Solution: Dilute VEGF in PBS to a final working concentration of 50 ng/mL.
3. Preparation of MTT Solution: Prepare a 5 mg/mL stock solution by dissolving MTT powder in PBS. Filter-sterilize the solution through a 0.22 μm membrane and store protected from light at 4 °C.
4. Cell Seeding: Harvest HUVEC and HEK293 cells in the logarithmic growth phase by trypsinization. Resuspend the cells in complete medium and adjust the cell density of each type to 3.0-4.0 × 10⁴ cells/mL.
5. Plating and Experimental Groups: Seed 100 μL of cell suspension into each well of a 96-well plate (3,000-4,000 cells/well). Include the following groups in duplicate:
6. Blank Control: Complete medium only (no cells).
7. Negative Control: Cells + complete medium.
8. VEGF Stimulation Control: Cells + VEGF (50 ng/mL).
9. Experimental Groups: Cells + VEGF (50 ng/mL) + nanobodies (0.25, 0.5, 1, 2, or 5 μg/mL).
10. Cell Attachment: Gently shake the plate to distribute cells evenly and incubate at 37 °C in a 5% CO₂ incubator for 24 hours to allow for cell attachment.
11. Treatment: After the 24-hour attachment period, carefully aspirate the medium from all wells. Add 100 μL of serum-free medium containing the respective treatments (VEGF and/or nanobodies) to each well according to the experimental design.
12. Proliferation Assay: Incubate the plate under the same conditions (37 °C, 5% CO₂) for 24 or 48 hours.
13. MTT Assay: Following the treatment period, add 10-20 μL of MTT stock solution to each well. Incubate for 4 hours at 37 °C. After incubation, carefully aspirate the medium without disturbing the formed formazan crystals. Add 50 μL of DMSO or Anhydrous ethanol to each well to solubilize the crystals.
14. Absorbance Measurement: Gently shake the plate on a microplate shaker for 3 minutes to ensure complete dissolution. Measure the absorbance at a wavelength of 570 nm using a microplate reader.
15. Data Analysis: Calculate the percentage proliferation inhibition for HUVEC cells using the following formula:

Cell proliferation rate (%) =(Absorbance at 570 nm for antibody-treated cells/Absorbance at 570 nm for untreated cells) × 100%

ROS Biosensor Functional Verification

1. Inoculate single colonies with successful plasmid construction into LB medium.
2. After 12 h of cultivation, inoculate the bacterial liquid into fresh LB liquid medium at a ratio of 100:1, and add tert-Butyl hydroperoxide (tBHP) to make the concentration of tBHP in the medium 0 μM, 20 μM, 40 μM, 60 μM, 80 μM, 100 μM, 120 μM, 140 μM, 160 μM, 180 μM and 200 μM respectively.
3. Take 200 μL and add it to a 96-well plate, set up five parallel replicates for each concentration, and run the plate reader under conditions of 37 °C and 200 rpm for 4 hrs, measuring the fluorescence intensity at 486 nm every 20 mins.

IPTG induced bacterial expression experiment

1. Inoculate single colonies with successful plasmid construction into LB medium, and culture at 37 °C with shaking until the OD₆₀₀ of the bacterial solution reaches 0.4-0.6.
2. Add IPTG to the engineered bacteria to induce the expression of the toxin protein in the experimental group. If a control group is required, add an equal amount of sterile water for comparison.
3. Incubate the cultures at 37 °C with shaking at 200 rpm for 4 h, and measure OD₆₀₀ curves of the bacterial suspension over time using a Synergy H1 hybrid multimodal reader. In parallel, take samples every 30 min, dilute 10⁶-fold, and spread onto LB agar plates containing 50 μg/mL kanamycin, followed by overnight incubation at 37 °C. On the next day, count colonies and process the data to generate statistical graphs for comparison between groups.

Inhibitory riboswitch experiment

1. Inoculate single colonies carrying the inhibitory riboswitch construct into LB medium and culture at 37 °C with shaking until OD₆₀₀ reaches 0.4-0.6; harvest cells, wash with PBS, and resuspend in M9 minimal medium.
2. Allocate a 96-well microplate for riboswitch ligand gradients: assign wells in blocks (e.g., one block per concentration) to minimize cross-interference. Add 170 μL of the resuspended culture to each experimental well.
3. Add riboswitch ligand stock solution to achieve a 0-15 mM gradient in 1 mM increments (e.g., add 0-30.0 μL stock in 2.0 μL steps as per plate volume), and use equal volumes of sterile water for the control group.
4. Incubate the plate at 37 °C for 3-4 h.
5. Measure OD₆₀₀ and fluorescence using a Synergy H1 hybrid multimode reader: set fluorescence to Ex 453 nm / Em 486 nm; record both every 30 min (or endpoint at 4 h).
6. Normalize fluorescence by OD₆₀₀ to obtain relative fluorescence (RFU/OD₆₀₀); plot dose-response (0-15 mM) and/or time-course curves; compare groups statistically (e.g., t-test/ANOVA).

Outer membrane vesicle (OMV) extraction experiment

1. Inoculate glycerol stock into 200 mL LB medium (supplemented with antibiotic) and culture at 37 °C until OD₆₀₀ reaches 1.0.
2. Centrifuge the culture at 12,000 ×g for 20 min at 4 °C. Discard the bacterial pellet and collect the supernatant. Filter the supernatant through a 0.45 μm sterile filter to remove residual bacteria and cell debris, obtaining a sterile filtrate.
3. Transfer the sterile filtrate into a 100 kDa ultrafiltration tube and centrifuge at 4,000 rpm. Retain OMVs in the upper chamber of the ultrafiltration device. Repeat centrifugation, wash with PBS, and concentrate the sample to one-tenth of the original culture volume.
4. Ultracentrifuge the concentrated sample using a Beckman Optima XPN ultracentrifuge (SW 41Ti rotor) at 150,000 ×g and 4 °C for 4 h. After centrifugation, discard the supernatant, wash the pellet with 2 mL sterile PBS three times, resuspend, and store at -80 °C (avoiding repeated freeze-thaw cycles).

Expression quantification experiment

1. Inoculate single colonies carrying the successfully constructed plasmid into LB medium and culture at 37 °C with shaking until the OD₆₀₀ of the culture reaches 0.4-0.6.
2. Mix 1 mL of culture with 1 mL of glycerol to prepare a glycerol stock. Then inoculate 10 μL of the glycerol stock into 2 mL LB medium and mix thoroughly.
3. Measure fluorescence intensity (Ex: 453 nm, Em: 486 nm) and OD₆₀₀ continuously using an automatic microplate reader (Synergy H1 hybrid multimode reader). Incubate cultures at 37 °C to generate a one-step growth curve. At the end of detection, normalize fluorescence intensity by OD₆₀₀ to obtain relative fluorescence intensity, and analyze results by plotting to compare experimental and empty-plasmid control groups.

Expression visualization validation

1. Inoculate single colonies carrying the successfully constructed plasmid into LB medium supplemented with 50 μg/mL kanamycin and culture at 37 °C with shaking at 200 rpm for at least 12 h.
2. Once visible fluorescence is observed under ambient light, mix 10 mL of culture with 10 mL of glycerol to prepare a glycerol stock. Every 1 h, inoculate 100 μL of the glycerol stock into 10 mL M9 medium containing 50 μg/mL kanamycin and mix thoroughly.
3. Incubate at 16 °C with shaking at 200 rpm under white light. After 48 h, remove samples, observe, and photograph to compare the fluorescence of experimental and empty-plasmid control groups.

Yeast Two Hybrid

1. Streak the frozen yeast stock on a YPDA plate. Incubate at 30 °C until colonies reach 2 mm in diameter (typically 3-5 days).
2. Inoculate several 2-3 mm colonies into 1 ml YPD medium. Vortex vigorously for 5 min to disperse cell clumps. Transfer to a conical flask containing 10 ml YPDA liquid medium. Incubate at 28 °C with 200 rpm shaking for 16-18 h until the stationary phase (OD₆₀₀ > 1.5). Optional: Preserve the strain by adding glycerol to 20% final concentration.
3. Dilute the overnight culture into 100 ml YPDA medium in a new flask. Adjust the OD₆₀₀ to 0.2-0.3 (typically 3-6 ml of overnight culture). Incubate at 28 °C with 230 rpm shaking for 3 h until OD₆₀₀ reaches 0.4-0.6.
4. Transfer the culture to a 50 ml centrifuge tube. Centrifuge at 1,000 ×g for 5 min at room temperature (RT). Discard the supernatant and resuspend the pellet in 25 ml sterile ddH₂O or TE buffer. Pool the suspension and centrifuge again (1,000 ×g, 5 min, RT). Discard the supernatant.
5. Add 8 ml of 1× TE/1× LiAc solution (prepared by mixing 2.5 ml 10× TE buffer, 2.5 ml 10× LiAc, and 25 ml ddH₂O). Gently resuspend the pellet and centrifuge (1,000 ×g, 5 min, RT). Discard the supernatant.
6. Resuspend the cells in 1× TE/1× LiAc solution. Transfer the suspension to a 1.5 ml microcentrifuge tube. Incubate at 28 °C with gentle shaking (100 rpm) for 30 min. Competent cells are now ready.
7. For each transformation: Mix 500 ng-1 μg each of AD and BD plasmids with 1 μl denatured salmon sperm DNA (boil for 10-15 min, then chill on ice) in a 1.5 ml tube. Add 0.1 ml competent cells and mix by vortexing.
8. Incubate at 28 °C with 200 rpm shaking for 30 min.
9. Add 0.6 ml of sterile 40% PEG4000/1× LiAc/1× TE solution (9.6 ml 50% PEG4000, 1.2 ml 10× LiAc, 1.2 ml 10× TE). Mix gently.
10. Incubate at 28 °C with 200 rpm shaking for 30 min. Add 70 μl DMSO and mix by inversion. Apply heat shock at 42 °C for 15 min, then immediately chill on ice for 1-2 min.
11. Centrifuge at 13,000 rpm for 5 s (RT). Discard the supernatant and resuspend the pellet in 0.3 ml sterile 1× TE buffer.
12. Spread the entire suspension onto an SD/-Leu/-Trp plate. Dry, seal the plate, and incubate at 28 °C for 2-3 days until colonies appear.
13. Pick 2-3 colonies from the SD/-Leu/-Trp plate and resuspend in 5 μl ddH₂O.
14. Spot 2.5 μl of the suspension onto both SD/-Leu/-Trp and SD/-Leu/-Trp/-His plates (10 spots per plate).
15. Incubate at 28 °C for 1-2 days. Positive controls should show growth on both plates.

In vitro Ubiquitination Assay

1. Prepare the reactions in total 30 μl, including eaction buffer(25 mM Tris-HCl, pH 8.0; 150 mM NaCl), 2 mM ATP, 5mM MgCl₂, 50 ng of E1, 200 ng of E2, 200-500 ng E3, 500 ng substrate proteins and 5 μg of ubiquitin. The reactions minus E1, minus E2 (and minus E3) respectively should be performed at the same time as control.
2. Incubate the reactions at 37 °C for 1, 2, 4, and 6 hours respectively to find the appropriate time point for the reaction to occur.
3. Split the reactions by adding 10 μl 4x SDS sample buffer (with DTT or β- mercaptoethanol) and boil the samples at 100 °C for 5 min.
4. The reaction products are separated with 8-12% SDS-PAGE gel and detected with anti-ubiquitin antibody or antibody for certain tag fused with substrate protein by Western blot.