1) Chloramphenicol stock solution: Weigh 0.5g of chloramphenicol powder, dissolve it in 10 mL of anhydrous ethanol to form a stock solution with a final concentration of 0.05g/mL; then filter it through a 0.22 um membrane into a sterile container, dispense it into 1.5 mL of EP, and store it under the condition of -20℃; the above operations were carried out in the ultra-clean workbench.

Masterbatch concentration: 50mg/mL; working concentration: 100μg/mL.

2) Kanamycin stock solution: Weigh 0.5g of kanamycin powder, dissolve it in 10 mL of sterile water to form a stock solution with a final concentration of 0.05g/mL; then filter it through a 0.22 um membrane into a sterile container, dispense it into 1.5mL of EP, and store it under the condition of -20℃; the above operations were carried out in the ultra-clean workbench.

Masterbatch concentration: 50mg/mL; working concentration: 100μg/mL.

3) 50% glycerol solution:50 ml of glycerol was added to 50 ml of ddH2O to obtain 50% glycerol solution .Autoclave for 20 min at 121℃ to sterilize the solution.

4) TAE (1×) buffer: 80 mL of 50× TAE buffer was added to 920 mL of ddH2O to obtain 1× TAE buffer.

1) LB liquid medium: The solution was weighed according to the formulation in the table1 and finally use ddH2O to adjust the volume to the required volume. Autoclave for 20 min at 121℃ to sterilize the solution.

Component	Mass and Volumes
Tryptone	10g/L
Yeast extract	5g/L
NaCl	10g/L

Table1: LB liquid medium components

2) LB plate: The solution was weighed according to the formulation in the table2 and finally use ddH2O to adjust the volume to the required volume. Autoclave for 20 min at 121℃ to sterilize the solution. When the medium was cooled to about 55 °C, the appropriate concentration of antibiotics was added to the medium. Then, the medium was poured into 90 mm plates of about 20 mL each and solidified on a clean bench for use.

Component	Mass and Volumes
Tryptone	10g/L
Yeast extract	5g/L
NaCl	10g/L
Agar powder	15g/L

Table2: LB plate components

In the cloning process, we used E. coli Nissle 1917 electrocompetent cells (ZOMANBIO®). In order to improve the ease of operation and transformation efficiency, we used the electrotransformation method for bacterial transformation.

1) Electrocompetent cells were initially removed from the -80°C refrigerator and thawed on ice for 10mins;

2) Add 1ul of purified plasmid (cooled and free of salt ions) to 50ul of prepared receptor cells, mix the two and incubate on ice for 5mins;

3) Place the electrode cups in an ice bath and pre-cool to 4°C; transfer the plasmid/cell mixture to the cooled 2mm electrode cups, avoiding air bubbles, and ice bath for 10mins;

4) Dry the surface of the electrode cup, put it into the electroconverter and press the button to select the corresponding program;

5) Electroshock conditions: electroporation of the hole mixture at an electric field strength of C = 25 μF, PC = 200 Ω, V = 2.4 kv, removal after 2 min, and rapid insertion into ice;

6) Rapidly transfer the recipient bacteria to an EP tube containing 800ul LB in an ultra-clean bench, mix well and transfer to a 50mL shaker tube, replenish LB medium to 5mL into the shaker tube. 37°C, 225rpm recovery for 60min;

7) Centrifuge at 5000 rpm for one minute, discard the supernatant, resuspend the bacteria in 2 mL LB medium, mix well, and take 1.1 μL of the bacterial solution for gradient dilution (10^-2 to 10^-6), and spread it on plates containing the corresponding antibiotics. The plates should then be incubated upside down at 37 °C for 13 to 17 hours.

1) Observe the coated plate after electrotransformation; pick a single colony on the plate with the tip of a pipette gun and transfer it to a 1.5 ml EP tube filled with 10 ul of sterilized ddH2O (colony selection: isolated, rounded);

2) Take 1ul suspension of bacteria as PCR reaction template;

The PCR reaction components were mixed homogeneously according to Table 3, and if any droplets were hung on the wall of the tube, rapid centrifugation was performed.

After mixing, the colony PCR program will be performed based on the cycling conditions in Table 4.

Reagent	Volume
2×EasyTaq PCR Super Mix	12.5uL
F-primer	1 uL
R-primer	1 uL
Bacterial suspenion	1 uL
ddH O2	To 25 uL

Table3:components of PCR mixture

Step	Temperature	Time
Stage 1/1	94°C	2-5min
Stage2/35	94°C 30sec 50-60°C 30sec 72°C 1kb/min
Stage 3/1	72°C	5min
Stage 4/1	16°C	Forever

Table4: PCR program

Gel preparation (1.5%):

1) Accurately weigh 0.9 g of agarose and then add 60 ml of 1 x TAE buffer;

2) Heat to boiling at least 3 times in the microwave to completely dissolve the agarose;

3) Prepare a gel manufacturing mold and select a suitable gel preparation comb;

4) Allow the melted gel to cool slightly and then add 6ul of nucleic acid dye, swirling gently to mix the gel solution well;

5) Pour the agarose solution into the gel-making mold and insert the comb (make sure there are no air bubbles under or between the teeth of the comb);

6) After 30~40min at room temperature, wait for the gel solution to completely set and carefully pull out the comb.

Sample loading and electrophoresis:

1) Add 1x TAE buffer to the nucleic acid electrophoresis tank and place the gel into the tank, making sure it can be submerged by the TAE buffer;

2) Mix the DNA sample with the corresponding volume of up-sampling buffer;

3) Using a pipette gun tilted at an angle, slowly add the DNA molecular mass standard and sample mixture to the upper sample well while recording the position of the sample;

4) Cover the electrophoresis tank lid, connect the electrode plug, turn on the electrophoresis instrument and connect it to the nucleic acid electrophoresis tank;

5) Set the voltage to 100 V and start electrophoresis;

6) When the DNA sample has migrated a sufficient distance in the gel, the power is turned off and electrophoresis is complete.

Gel imaging:

1) Remove the post-electrophoresis gel and put disposable gloves on your hands;

2) Turn on the computer and the gel imager. Open the software of the gel imager;

3) Place the gel in the imager. Set the exposure mode and capture an image of the gel;

4) Find the target strip and save the image.

Plasmid extraction

1) Prepare the bacterial suspension to be used by collecting the appropriate amount of bacterial suspension from the medium, ensuring that the concentration is suitable for plasmid extraction;

2) Select the appropriate plasmid extraction kit according to the experimental requirements and strictly follow the manual that comes with the kit;

3) The concentration as well as the purity of the extracted plasmid DNA was determined;

4) Deliver plasmid samples that meet quality requirements to the company for sequencing.

Additional protocols used:
(M5 HiPer Multi-color Plasmid Miniprep Kit (Mei5bio®), product number: MF031-plus-01)

We electrotransformed the plasmid into chassis cells Nissle 1917, induced it with thiosulfate solutions at different concentration gradients, and detected the fluorescence intensity with a fluorescent enzyme marker to determine the response.

Induction culture

1) Pick a single clone from the LB agar plate from the transformation step. It was inoculated onto chloramphenicol plates and incubated overnight. After that, they were inoculated in LB liquid medium at 37°C and 220 rpm and incubated with shaking for 12 h;

2) Take 100 μL of cultured bacterial solution and inoculate it in LB medium containing 5 mL of sodium thiosulfate in different concentration gradients (note: this refers to inoculation in LB medium containing antibiotics as well as different concentrations of sodium thiosulfate (0, 0.01 mmol/L, 0.1 mmol/L, 1.0 mmol/L 2.5 mmol/L, 5.0 mmol/L; for bacteria containing Ths S+Ths R)；

3) After 12 h of shaking incubation in a shaker at 37°C and 1000 rpm, 200 uL of bacterial solution from different concentration cultures were removed and transferred to labeled centrifuge tubes respectively. Centrifugation was performed at 37°C and 12000 g for 2 min to remove the supernatant; the precipitate was suspended in 400 uL of phosphate buffered saline (PBS);

Fluorescence intensity measurement

1) 200 μL of the sample was taken and added to the enzyme labeling strips for subsequent detection.

2) The samples were detected using a fluorescence enzyme marker, and the fluorescence and optical density values were recorded. Fluo/ OD600 (fluorescence value) characterizes the intensity of the reporter gene; E. coli ECN without sfGFP plasmid was used as a negative control in this experiment. (Fluorescence values are excitation wavelength of 475 nm and emission wavelength of 550 nm; OD600 values are absorbance at 600 nm)

We introduced the dual plasmid into chassis cells Nissle 1917 and used the optimal response concentration of thiosulfate solution for induction based on the pre-induction results. Subsequently, we evaluated the expression secretion of drug proteins and the synergistic effect of the dual plasmid system by western blot.

Induction culture

Based on the previous experiments, the optimal conditions for induction culture have been determined.

1) cycle3: The recombinant bacteria were transferred to LB liquid medium containing 1.0 mM sodium thiosulfate and antibiotics, and incubated for 12 h at 37°C, 1000 rpm with shaking;

2) cycle4: The recombinant bacteria were transferred to LB liquid medium containing 1.0 mM sodium thiosulfate and antibiotics, and incubated for 12 h at 37°C, 1000 rpm, and ensured to be incubated for 12 h with shaking in an environment containing nitric oxide (NO).

Protein sample extraction preparation

Collect the above bacterial fluid, centrifuge to remove cellular residues; concentrate the protein; resuspend the protein with buffer for subsequent experiments.

Protein quantification

Quantification was performed using the BCA Protein Concentration Assay Kit (TIANGEN®), a BCA standard curve was plotted, and the concentration of the total protein solution extracted was calculated based on the standard curve and the number of dilutions of the samples to be tested in order to determine the amount of protein uptake.

Western Blot

SDS-PAGE preparation：

1) Configuration of separation adhesive: stack two glass panels neatly, fix them on the base with clamps, and perform leakage check;

2) Prepare the separation gel component according to the recipe in Table 5, mix thoroughly, and drop between two glass plates with a pipette until the liquid level reaches 1 cm from the lower edge of the comb;

3) Add distilled water slowly with a pipette and let it stand, pour off the excess water after the separating gel has polymerized;

4) Preparation of concentrated gel: prepare the components in a beaker according to the recipe in Table 6, mix well and immediately add the concentrated gel over the separating gel with a pipette;

5) Gently insert the comb and leave it to set to make a gel plate.

Separating Gel (12%)	Reagent Volume 30% Acr 6.0 mL 1.5M Tris-HCl 3.8 mL 10% SDS 150uL 10% APS 150 uL TEMED 6 uL water 4.9mL

Table 5: Ingredients of the separating gel

Concentrating Gel (5%)	Reagent Volume 30% Acr 1.3 mL 1.5M Tris-HCl 1.0 mL 10% SDS 80uL 10% APS 80 uL TEMED 8 uL water 5.5mL

Table 6: Ingredients of the concentrating gel

Sample loading and electrophoresis:

1) The prepared gel plate was fixed in the electrophoresis tank, the inner tank was filled with Tris-glycine electrophoresis buffer, the liquid level of the outer tank was lower than that of the inner tank by 5-10 mm, and both hands pressed the left and right parts of the comb to pull it out smoothly and slowly;

2) Dissolve 5× SDS-PAGE Sampling Buffer at room temperature and store at room temperature;

3) Mix the samples thoroughly at a ratio of 1 part of 5× SDS-PAGE Protein Sampling Buffer for every 4 protein samples;

4) Placed in a metal bath at 100°C to fully denature the protein, after which it was cooled to room temperature and set aside;

5) The amount of sample was calculated based on the protein quantification results and the samples were taken sequentially;

6) The electrophoresis tank was covered and energized, initially using a low voltage of 80 V and running for 30 min to allow the sample to form a straight line at the interface of the separator and concentrator gels, after which the high voltage was raised to 120 V to continue electrophoresis until the blue dye (often bromophenol blue dye) reached the bottom end of the gel and electrophoresis was stopped;

7) When electrophoresis is complete, remove the adhesive plate and pry it apart; use a knife to gently cut the connection between the adhesive and the glass along the edge of the glass and remove the adhesive.

Transmembrane and Closure：

1) Prepare the electrotransfer buffer according to the table below;

2) Based on the size of the gel, 6 pieces of PVDF membrane and filter paper were cut, and the PVDF membrane was activated in pure methanol for 30 s, after which the membrane and filter paper were transferred to pre-cooled transfer buffer and equilibrated for 10 min;

3) Assemble the transfer "sandwich": sponge/3 layers of filter paper/glue/film/3 layers of filter paper/sponge, put each layer in place and carefully drive out the air bubbles;

4) Place the transfer bath in an ice bath, put in the "sandwich", the membrane near the positive pole, the glue near the negative pole, add the transfer buffer, plug in the electrode, constant current 300mA to transfer the membrane, for 95min;

5) At the end of the membrane transfer, cut off the power supply, remove the PVDF membrane and carefully cut off a corner at the upper left position to distinguish the direction;

6) The membrane was activated by submerging in methanol for 3 min, and then washed three times with TBST for 10 min each time ;

7) After that, it was immersed in Lichun red staining solution and placed on a shaker at room temperature for 10 min, after which it was washed several times with distilled water until protein bands were presented;

8) Wash again with TBST and repeat until the red dye fades from the PVDF membrane.

Antibody incubation：

1) The membranes were placed in 5% skim milk powder closure and incubated for 2 hours at room temperature;

2) Wash three times with TBST solution for 10 min each;

3) The membrane was placed in 10 ml of primary antibody dilution buffer, placed on a shaker, and incubated at 4 ℃ overnight; after completion, the membrane was washed with TBST solution three times for 10 min each;

4) The membrane was placed in secondary antibody blocking buffer and incubated on a shaker for 2 h at room temperature; subsequently, it was washed three times with TBST solution for 10 min each.

Color rendering：

1) While the membrane was washed for the last time, the luminescent working solution was freshly prepared according to the ECL Ultrasensitive Kit instructions;

2) The appropriate amount of luminescent solution was pipetted until the PVDF membrane was covered, and the ECL luminescent meter was exposed and the image was captured.

We introduced the double plasmid into the chassis cell Nissle 1917, removed the DETA-NO solution, and induced the culture in a NO-free environment to simulate the improved intestinal environment of IBD, in order to evaluate whether the suicide system is working.

Induction culture

The recombinant bacteria were transferred to LB liquid medium containing antibiotics and cultured with shaking at 37°C and 1000 rpm in the absence of NO. The bacterial liquid was extracted after 1 h, 2 h and 3 h of culture, respectively, for subsequent experiments.

Trypan Blue staining method

1) The cells to be stained were diluted to the desired concentration and the cells were counted;

2) Cell suspensions were stained with freshly prepared Trypan Blue staining solution for 3-5 min at room temperature;

3) For stained cell material, a drop of cell suspension was placed on a slide, covered with a coverslip and placed under high magnification;

4) Dead cells are light blue in color and expanded, without luster. Living cells are not colored and retain their normal shape and luster;

5) Count the number of live and dead cells;

6) Unstained cells are counted. Cell viability can be calculated according to the formula. (Cell viability = number of unstained cells/total number of cells observed x 100%)