1. Genetic engineering of bacteria for the expression of SIRPα that can specifically recognize CD47 protein on the surfaces of tumor cells.
2. Metabolomic and proteomic analysis of Escherichia coli Nissle 1917 (EcN) that live on lactic acid.
3. 3D modeling of TNBC tumor spheroids.
4. Chemical modification of bacteria with MnO₂@AuNPs nanoparticles.

实验耗材 Apparatus	厂商 Manufacturer
T25/T75 cell culture flask	Corning
FBS	Gibco
2mL RNase free EP	Eppendorf
DMEM	Gibco
PBS	Gibco
HS	Gibco
P/S	Gibco
0.25% Trypsin-EDTA	Beijing Solarbao Technology Co., Ltd.
Total RNA Extractor (Trizol)	Sangon Biotech (Shanghai) Co., Ltd.
MightyScript First Strand cDNA Synthesis Master Mix	Sangon Biotech (Shanghai) Co., Ltd.
LB	Sangon Biotech (Shanghai) Co., Ltd.
PCR Kit	Sangon Biotech (Shanghai) Co., Ltd.
Tris-HCL	Sangon Biotech (Shanghai) Co., Ltd.
L-Cys	Beijing Solarbao Technology Co., Ltd.
DTNB	Macklin
Na2HPO4（pH＝7.0）	Sangon Biotech (Shanghai) Co., Ltd.

仪器名称 Instrument	厂商 Manufacturer
Ultrasonic cell breaker	Ningbo Scientz Biotechnology Co.,Ltd
High-speed cryogenic centrifuge	Hunan Xiang Yi Laboratory Instrument Development Co., Ltd.
MILI-Q Water Purifier	Shanghai Canrex Analytic Instrument Co.,Ltd Pudong Shanghai China
Ultrasonic cleaner	Ningbo Scientz Biotechnology Co.,Ltd
Orbital Shaker	Kylin-Bell Lab Instruments Co.,Ltd.

1. Take a bottle of cells, open the cap and empty the medium, and wash twice with pbs solution with 1 mL of PBS solution.
2. Add 2 mL of transzol reagent to the culture bottle, blow it repeatedly with the gun until the cells are fully integrated into the solution, suck out the solution and put it into two 1.5 mL centrifuge tubes.
3. Stand at room temperature for 15 min.
4. For each 1 mL transzol solution, 0.2 mL RNA extraction was added, violently shaken for 15 s, and incubated at room temperature for 3 min.
5. 10000 Xg was centrifuged at 4°C for 15 min. At this time, the sample is divided into three layers, with the colorless water phase in the upper and middle layers, and the pink organic phase in the lower layer. The RNA is mainly in the aqueous phase, whose volume is about 60% of the transzol reagent used.
6. Transfer the colorless water phase to a centrifuge tube, using 1 mL of transzol reagent to add 0.5 mL of isopropanol, mix well and enrich for ten minutes at room temperature.
7. 10000 Xg was centrifuged at 4°C for 10 minutes and remove the supernatant. A gelatinous precipitate is formed at the tube side and bottom.
8. Add 1 mL of 75% ethanol (prepared with DEPC water) to a vigorous vortex.
9. 7500 Xg was centrifuged at 4 °C for 5 minutes.
10. Discard the supernatant and let dry at room temperature for about 10 minutes and dissolve in 75 μL of RNA dissolution solution.
11. 57°C for 10 min and samples were stored to -80°C refrigerator for long-term use.
12. RNA Purity and concentration detection: the concentration of RNA was measured by agarose gel electrophoresis

(1) After residual genomic DNA was removed, configuration mixture (15 μL), and incubated for 10 min in a 42°C water bath.

constituents	application amount
template DNA	2μL
5×gDNA digester Mix	3μL
RNase free ddH2O	10μL

(2) Configure the reverse recording reaction system.

constituents	application amount
Rereaction solution for the first step	15μL
4×III MLVRTMix	5μL

(3) Reverse transcription using a PCR instrument according to the following conditions.

temperature	hour
25°C	5 min
55°C	15 min
85°C	5 min

(4) Configuration of PCR system (50 μL)

constituents	application amount
ddH2O	25 μL
10*Buffer(PCR)	10 μL
Mix(2mM)	10 μL
CDNA	2 μL
primer（F/R）	1+1 μL
enzyme（polymerase）	1μL

(5) Using a PCR instrument, amplification.

temperature	hour	recurring number
95°C	3 min	1
98°C	30 s	30
64/66/68°C	30 s
72°C	90 s
72°C	10 min	1
4°C	limitless	1

1. Rubber blocks containing the target fragments were cut from the agarose gel and weighed.
2. Add 4 times Buffer B2 and 50 C water bath for 5 minutes.
3. The sol solution was moved into the adsorption column and centrifuged at 8000 Xg for 30s. Remove the liquid from the collection tube.
4. Add 500 μL Wash Solution and centrifuge at 9000 Xg for 30 seconds. Remove the liquid from the collection tube.
5. Repeat Step 4 once.
6. The empty adsorption column was centrifuged at 9000 Xg for 1 minute.
7. Put the adsorption column in a clean 1.5 mL centrifuge tube, add 30 μL Elution Buffer in the center of the adsorption membrane, stand at room temperature for 1 minute, centrifuge for 1 minute, and save the DNA solution in the tube

(1) Three bottles of 100 mL of LB liquid medium were prepared and sterilized.

(2) 1 mL of bacterial broth was added to each vial of medium and incubated for 3 hours at constant temperature.

(3) Measuring OD values

The first test	1.381	1.284	1.390
The first test	1.361	1.359	1.394
The first test	1.35	1.394	1.3769

Do sensory cells

（4）Plasmid enzyme was cut, and ligated

constituents	application amount
Pet-28 plasmid	1 μL
Hind 3	1 μL
Ecrol	1 μL
10*m Buffer	2 μL
DEPC water	15 μL

As for the 37 C water bath for 1 hour

(1) Add 4 μL of reaction solution to the prepared Nissle1917 sensory cells, flick several times, and incubate on ice for 30 min.

(2) Heat-excite the cells in a 42°C water bath for 90 seconds and then quickly put them on ice for 5 minutes.

(3) Add 500 μl of SOC liquid medium and incubate at 37°C for 45-60 minutes. d. Centrifuge at 4000 rpm for 3 minutes.

(4) Centrifuge at 4000 rpm for 3 minutes to collect the organisms and spread a certain amount of organisms evenly on the antibiotic-containing plate as needed.

(1) Pick a single colony and inoculate it into 100mL of LB medium containing 50ug/mL kanamycin, incubate it at 37°C, 220 rpm for 12-16h, and then incubate it for 12-16h.

(2) When the OD600 of the bacterial solution is about 0.4-0.6, add IPTG at a final concentration of 1 mM in an ultra-clean bench, add 50 μg/mL kanamycin, and incubate at 28°C, 160 rpm for 20h to induce large amounts of protein expression.

(1) Table 1 Absorbance of EcN at different time

Time (hour)	Absorbance 1	Absorbance 2	Absorbance 3	Average absorbance
0	0.885	0.884	0.888	0.886
1	0.894	0.898	0.896	0.896
2	0.893	0.898	0.895	0.895
3	0.892	0.896	0.892	0.893
4	0.909	0.906	0.901	0.905
5	0.941	0.930	0.969	0.947
6	1.115	1.111	1.105	1.110
7	1.412	1.410	1.421	1.414
8	1.462	1.467	1.473	1.467
9	1.509	1.512	1.520	1.514
10	1.543	1.547	1.554	1.548

(1) Prepar LB solid medium with lactic acid.

(2) Put EcN on LB solid media and grown at 37℃

(3) Gradual increase in lactic acid concentration to eventually obtain a strain (EcN-2.5) that grew stably at LB solid media with 2.5g/L lactic acid.

(4) Prepare LB liquid medium with 2.5g/L lactic acid, Expanded culture of EcN-2.5.

(5) Gradual increase in lactic acid concentration to eventually obtain a strain (EcN-3.0) that grew stably at LB liquid media with 3.0g/L lactic acid.

(1) Collect the bacterial solution, centrifuge it at 4000 xg for 10 min, collect the bacterial body in 2 mL centrifugal tube (weigh it in advance and calculate the bacterial weight).

(2) Wash three times with pre-cooling 0.01M (1X) PBS (pH 7.4) to remove supernatant.

(3) Add four times the volume of methanol to the precipitate

(4) fully oscillate and mix (about 5min)

(5) Centrifuge at 14,000 rpm for 15 min at 4 ℃ and collect the supernatant.

(1) Using a ACQUITY ULPC®HSS T3 1.8 μm(2.1X100 mm) chromatography column.

(2) The temperature of the autosampler is set to 10℃, with gradient elution at 0.1 mL/min flow rate, 40℃, column temperature and 2 μ L of injection.

(3) The mobile phase is positive ion: 0.1% formic acid water-0.1% formic acid acetonitrile; negative ion: 0.1% formic acid water-0.1% formic acid acetonitrile.

(4) The gradient elution procedure setting

0-1 min        2％B

1-9 min        2％-50％B

9-12 min       50％-98％B

12-13.5 min     98％B

13.5-14 min     98％-2％B

14-17min       2％B

The instrument uses an electrospray ion source (ESI), positive and negative ion ionization mode, positive ion spray voltage is 3.50 kV, negative ion spray voltage is 2.50 kV, sheath gas is 30 arb, auxiliary gas is 10 arb. The capillary temperature is 325 ℃, the full scan is performed at a resolution of 70,000, the scanning range is 81-1000, and the HCD is used for secondary cracking, the collision voltage is 30 eV, and the unnecessary MS/MS information is removed by dynamic exclusion.

(1) Cultivate MCF-7 in T25 cell culture flasks to 70%-80% adherence.

(1) Tris-HCL buffer (0.25 M): after accurate preparation by DDW, adjust pH 8.3 with hydrochloric acid.

(2) Cysteine standard solution (1 mM): accurately weigh 0.017563 g of L- cysteine (175.63), dissolve with 1 mL of formic acid, and then volume to 100 mL with DDW;

(3) DTNB (molecular weight: 396.35) standard solution (10 mM): accurately weigh 0.198175 g DTNB with 50 mM Na₂HPO₄ (pH = 7.0) to make 50 mL of solution, stored in a brown bottle in the dark at low temperature;

(4) DTNB analytical solution (0.1 mM): prepared from 1 volume of 10 mM DTNB standard solution plus 99 volume of 0.25 M Tris buffer, ready to use.

(1) Remove E. coli cultured for 12h, centrifuge at 5000xg for 10min, remove the supernatant. wash twice with PBS and resuspend with 987.5μL ice PBS. Add 12.5 μL 2-iminothiophane solution (2.5 μg/mL) to the suspension and incubate for 90min at room temperature.

(2) Thiolated bacteria (EcN@SH) were washed twice with PBS to eliminate residual 2-iminothiolane solution.

(3) Determination of the number of thiol groups. The number of thiol groups on the bacteria was determined after quantitative modification with DTNB reagent (Ellman's reagent). Bacteria were resuspended in 3 × 108 cfu /mL of PBS, and DTNB was added to a final working concentration of 0.1 mM, and the reaction was performed at room temperature for 2 h. After centrifugation to remove the precipitated bacteria (6000 × g, 5 min), 200 µL of the supernatant was transferred to a microtitre plate, and the absorbance was measured immediately at 412 nm. The amount of thiol fraction was calculated from the standard curve obtained with increasing concentration of L -cysteine hydrochloride hydrate.

(1) Prepare co-culture medium (without penicillin-streptomycin and containing 1.5 μg/mL gentamicin).

(2) Centrifuge the bacterial culture, determine the optical density OD600 value of the bacterial culture in a Nano Drop spectrophotometer, dilute the OD600 of the bacterial suspension with PBS to ~0.5. 2µL of bacterial solution was added to each well.

(3) At 4 h, 8 h and 12 h of co-culture, the co-culture plate was removed, and iodoacetamide solution was added to 1 well respectively, which reacted with the sulfhydryl group on the bacterial surface, and was excited to emit green fluorescence under UV irradiation at 335 nm, and the detection wavelength was 493 nm. According to the three fluorescence micrographs of the prolonged fluorescence observation with the time of co-cultivation, the fluorescence signals of the bacteria can be seen infiltrating into the center of the growing spheroplast and colonizing in the necrotic region. necrotic regions were colonized. (Iodoacetamide (IAM) decomposes easily in the presence of light, store at 4℃ in a brown bottle and use it now.)

(4) After 2d of co-culture, change the solution. Aspirate 60µL of sphere medium (tilt the tip of the gun at 45 degrees to prevent aspirating too many spheres), add 160µL of PBS to each well (the spheres will swell, add the liquid gently), aspirate 160µL of medium, and aspirate as much residual PBS as possible. 200µL of co-culture medium is added to remove bacteria from outside the spheres and to limit the growth of bacteria in the anoxic area of the tumor spheres.

(1) Preparation of raw material. Weigh 0.0115g of tetrachloroauric acid solid trihydrate in a 1.5 mL EP tube, add 600 microliters of ultrapure water and mix by blowing with a pipette gun. Weigh another 0.0061 g of sodium citrate dihydrate in a 1.5 mL EP tube, add 600 microliters of ultrapure water and mix by blowing with a pipette gun. Then 100 µl of 1 mg/mL of hollow mesoporous manganese dioxide nanoparticles and 3700 µl of ultrapure water were aspirated.

(2) Premixing. A premixed solution of 600 microliters of tetrachloroauric acid solution and 600 microliters of sodium citrate solution will be added sequentially to 3800 microliters of hollow mesoporous manganese dioxide nanoparticle solution.

(3) Construction of reaction conditions. 95 mL of ultrapure water was taken in a conical flask to which a magnetic stirrer was added and heated to boiling.

(4) The pre-mixed reaction solution was rapidly poured into the above conical flask, the heating gear was turned on to the highest setting, while the magnetic stirrer was turned on to the fifth setting, and the reaction was carried out with vigorous stirring for 60 minutes.

(5) Purification. The solution after 60 min of reaction was cooled at room temperature, transferred to a 50 mL EP tube, 12,000 rpm, centrifuged for 20 min, and the supernatant was decanted to leave a precipitate.

(6) Dissolution by sonication. Add 10 mL of ultrapure water to the above 50 mL EP tube and sonicate at 40 kHz for 15 minutes to dissolve almost all of the precipitate.