Plasmid Extraction

1. E. coli solution containing pUC 18 plasmid was grown on LA medium;
2. Single colonies were picked up with sterile toothpicks, inoculated in LB medium containing Amp antibiotics, and incubated overnight in a 37℃ shaker —— 250 r / min;
3. Draw 1.5 ml bacteria, centrifuge at 12000 g for 2 minutes, collect bacteria and pour out bacteria; draw 1.5 ml bacteria, collect bacteria again and pour it as clean as possible;
4. Add 300 ml of solution I to beat well, re-suspend the cells, and shake well (note: thoroughly beat evenly precipitate or pieces);
5. Add 300 ml solution II, gently mix and well, place until clear, generally no more than 5 minutes;
6. Add 300 ml of solution III and mix well, place on ice for 10 minutes to make impurities fully precipitate;
7.1 Centrifuged at 2000 g for 10 minutes;
8. Draw 800 ml of supernatant (note: do not absorb floating impurities) into another Eppendorf tube, add 2 / 3 volume of isopropyl alcohol, and leave it at room temperature for 5 minutes;
9.12000 g at room temperature for 15 minutes;
10. Pour supernatant, add 75% ethanol to soak and remove salt (place for a moment or centrifugation for 3 minutes before pour the supernatant);
11. Dry DNA at room temperature or on an ultra-clean table;
12. Add 40 ml of sterilized ultrapure water or TE dissolution; 13. Quality detection of plasmid and BAC, stored at-20℃.

Culture of E. Coli Nissle 1917

1.Prepare the starting culture
Individual colonies were picked from freshly underlined plates and inoculated into 3 to 5 mL of medium to obtain starting cultures.
Incubation was done at 250 – 300rpm shake flasks for approximately 8 hours at 37 C * with the appropriate antibiotics.
The starting cultures were inoculated in the overnight cultures. The starting cultures were diluted with the appropriate volume of medium in large flasks 1:500 to 1:1000, and incubated at 250-300rpm for 12 to 16 hours at 37℃.

2.Suspension culture
Prepare LB medium: weigh the powder medium and add it to the water of the sterile bottle The broth was heat-autoclaved and cooled to room temperature
About 1 mL of E. coli cultures grown overnight were transferred to bottles in a laminar flow chamber
Seal the bottle mouth with sterile cotton (non-adsorption) plug, and do not tighten it Overnight incubation at 37℃ with continuous shaking

3.Vaccination with E. coli
Prepare LB agar: weigh the appropriate amount of powder medium, agar and water and add it to the sterile bottles
The medium was autoclaved and cooled to the bottle just not hot
About 25-30 mL of LB agar was poured into a sterile plate The plate was set in a laminar flow chamber and the lid slightly opened The surface of the frozen E. coli glycerol stock was gently scraped with a sterile inoculation ring
E. coli colonies were removed from the culture plates with a sterile inoculation ring 10 – 100 μ L of E. coli suspension culture was added and another LB agar plate was added
Wipe the inoculum ring on the LB agar plates
The cultures was spread over the entire culture plate using a sterile glass applicator
The plates were incubated overnight at 37℃

Transformation of E. Coli Nissle 1917

1. ulate a single colony on 5 ml of LB medium and incubated at 37℃ with gentle shaking for 5 h or overnight.

2. Add 2.5 ml culture to 2 L flask with 500 ml LB medium and grow to an OD 600 of 0.5~0.6.

3. Bacteria were cooled in an ice-water bath for 10 to 15 min and then transferred to a pre-cooled 1-liter centrifuge bottle. The precipitate was centrifuged at 5 000 g for 20 min and dissolved in 5 ml pre-cooled water for 6.

4. Add 500 ml of ice-cold water, mix well, repeat the centrifuge once in step 3, immediately pour out the supernatant, and resuspend the cells in residual liquid.

5. freshly made bacteria
(1) The suspension was added to a pre-cooled 50 ml polypropylene tube and centrifuged at 5 000 g for 10 min.
(2) Estimate the volume of cell precipitation, which was resuspended with an equal volume of ice-cold water.
(3) divide 50~300 ul into pre-cooled microcentrifuge tube.

6. Frozen storage of bacteria
(1) Add 40 ml of ice-cold 10% glycerol, mix, and then centrifuge according to step 5.
(2) The volume of precipitation was estimated, then an equal volume of ice-cold 10% glycerol was added and resuspension of the bacteria was performed.
(3) divide 50~300 ul into pre-cooled microcentrifuge tube.
(4) Frozen on dry ice and stored at-80℃.

7. Adjust the electrical converter to 2.5 kV, 25 uF, and the pulse controller to 200~400 Ω;

8. Add 1 ul of plasmid DNA to tubules containing freshly prepared bacteria or melted frozen bacteria and mix well.

9.The transfer mixture was transferred to a pre-cooled electroconversion cell, drained the outer surface of the pool, and then placed into the sample tank.

10. Pulse electroconversion, then remove the electroconversion pool, add 1 ml SOC of culture fluid immediately, and transfer to a sterile culture tube with a Pasteur pipette.

11 at 37℃ for 30 – 60 min.

12. Apply in small parts to LB plates containing antibiotics.

Fluorescence Observation

1.Sample preparation

2.(1) For adherent cells: The clean cover slips were soaked in 70% ethanol and then placed into the petri dish with clean, sterile forceps, and the residual ethanol was washed away with sterile PBS. After the cells are close to growth, the cover glass is removed and operated carefully to prevent the cells from desating.
(2)For suspended cells:
① A fixation step in suspension, then drop the cells on the slide, and dry the cells are close to the slide.
② Fixed and staining steps in suspension, eluted by centrifugation and then pipetted to cassette slides for subsequent staining steps.

3. Fixed (prevent diffusion of autolytic antigen in isolated tissue) Including: organic solvent (methanol, ethanol, acetone, etc.); crosslinker (4% PFA, 10% neutral formalin), the choice of fixative depends on the nature of the studied antigen and the characteristics of the antibody used, however, formaldehyde is the most, but for phosphorylated antibody, not suitable for formaldehyde, phosphoprotein transfer from the membrane surface to the cytosol, so should choose cold anhydrous methanol or absolute ethanol, and should pay attention to formaldehyde will volatilize, in 4-8 C for too long. Fixed time: depending on the size and type of the combined block, for most tissues, 18-24h is relatively ideal, the cell fixed time is short, generally 2% formaldehyde can be fixed at room temperature for 20min.
Take cell samples as an example: climbing slides were fixed with 4% paraformaldehyde for 15min, and slides were soaked in PBS for 3 times for 3min each.

4.permeability (to make the antibody into the cell) 0.5%Triton X-100 (a reducing agent, prepared in PBS) was permeable at room temperature for 20min (for intracellular antigen); except Triton X-100, acetone is also used as a permeable agent, and acetone does not need to be permeabilized.

5. Blocking (reducing the primary and secondary antibodies for binding to non-specific sites) After permeabilization, the slides were washed with PBS three times for 3 min, blotted paper, dripping goat serum on the slides, and blocked for 30min at room temperature. Commonly used blocking solutions include: serum from the same source as the secondary antibodies, BSA, or sheep serum. Start all steps from sealing, be sure to pay attention to the moisturizing of the sample and avoid drying the sample, otherwise it is easy to produce a high background. Aldehyde-fixed samples were blocked with a blocking solution containing 0.3M glycine before incubation with primary antibodies.

6. Incubation with primary antibody According to the instructions of the primary antibody, the primary antibody was diluted with primary antibody diluent according to the appropriate proportion, the blocking solution was aspirated with absorbent paper, and placed in a wet box and incubated overnight at 4℃. The primary antibody was recovered, PBST was added, and washed for 5min on slow shaking for a total of 3 times

7. and the incubation with the fluorescent secondary antibody Soak up the washing solution with absorbent paper and then add the diluted fluorescent secondary antibody, wet box

Elisa

The bacterial cultures were diluted to OD600 of 0.3 with tris-buffered saline (TBS). The specimens (200 μL) were transferred to a Multiscreen-GV 96-well filter plate, filtered, and washed with TBST buffer (TBS, 0.1% Tween-20). After blocking with with 1% bovine serum albumin (BSA) and 0.01% H2O2 in TBST for 1.5 h at 37 °C, and subsequent washing steps, 50 µL of anti-6xHis antibody-horseradish peroxidase (HRP) conjugate (1:200, Thermo Fisher, MA1-80218) was added to each well and incubated for 2 h at 25 °C. For the TFF3 antibody-binding assay, samples were incubated with 50 µL of anti-TFF3 primary antibody (1:450, Sigma, WH0007033M1) for 2 h at 25 °C and washed three times with TBST buffer, followed by incubation of 100 µL goat anti-mouse-HRP conjugated secondary antibody (1:5000, Thermo Fisher, 31430) for 1 h at 25 °C and three subsequent washes. After the wash steps, Ultra-TMB (3,3′,5,5′-tetramethylbenzidine) ELISA substrate (100 µL) was added to each well and incubated for 10 min at 25 °C. To stop the reaction, 50 µL of 2 M sulfuric acid was added to each well. One-hundred microliters of the final reaction was transferred to 96-well plate and measured the absorbance at 450 and 650 nm. The relative amount of displayed peptide was measured by subtracting absorbance at 450 nm with absorbance 650 nm.

RT-qPCR

1. The extraction of the RNA from No
(1) 50-100mg of tissue or (5-10) of 106 cells, add 1mL Trizol of reagent. For tissue, it is necessary to homogenizer for a few minutes until the tissue is completely broken. For cultured cells, cells can be blown up and down with a pipette or broken by a homogenizer.
(2) The sample was centrifuged at 4℃ and 12 000g for 10 min and the supernatant was transferred.
(3) Place the supernatant for 5min at room temperature, add 0.2 mL chloroform, vibrate 15S by hand or Votex, and place for 2~3min at room temperature.
(4) It was centrifuged at 4℃ and 11 000 g for 15 min to transfer the aqueous phase.
(5) Add 0.25 mL isopropyl alcohol and 0.25 mL high salt precipitate (0.8mol / L sodium citrate, 1.2 mol/L NaCl) in the aqueous phase and mix it at room temperature for 10min.
(6) The supernatant was centrifuged at 4℃ and 11000 g for 10 min.
(7) Add 1 mL of 75% ethanol, mix with Votex, centrifuge at 4℃ and 7000g for 5min, and remove the supernatant.
(8) The precipitate was dried in air for 5~10 min, dissolved in 100 μL DEPC water, sucked at the gun head several times, and placed in a 55℃ water bath for 10min.
(9) Electrophoresis and detection of OD value, check the amount and integrity of RNA. (10) The RNA was stored in-70℃.

2. Isolation of the mRNA from the A
(1) Test the amount of starting RNA (the amount of starting RNA should be less than or equal to 0.25mg). Total RNA was added to a RNase-free 1.5 mL Eppendorf tube, and water without RNase was added to a total volume of 250 μ L.
(2) Add 250 μ L of Buffer OBB and 15 μ L of Oligotex suspension and mix the solution completely.
(3) Put the sample solution in a 70℃ water bath for 3 min.
(4) Remove the sample solution from the water bath and leave it at room temperature for 10min.(This step allows the hybridization of the Oligo-dT 30 in the Oligotex particles and the poly-A tail of the mRNA)
(5) The sample solution was centrifuged for 2min at the maximum speed (14000~18000 g) to precipitate the Oligotex-mRNA complex, and the supernatant was carefully removed with a pipette.
(6) The Oligotex-mRNA complex precipitate was resuspended in 400 μ L or 600 μL Buffer OW2 with a vortex oscillator (Votex) or pipette and then added to a small centrifugal column (for 400 μ L suspension) or large centrifugal column (for 600 μ L suspension) in a 1.5 mL microcentrifuge tube and centrifuged at a maximum speed of 1 min.
(7) Transfer the centrifugal column to a new 1.5 mL microcentrifuge tube without RNA enzyme, then add 400 μ L or 600 μL Buffer OW2 to the centrifugal column, centrifugation for 1 min, and the centrifuge fluid is discarded.
(8) Transfer the centrifugal column to a new 1.5 mL microcentrifuge tube without RNA enzyme with 20-100μL Bulfer OEB .

Measurement of horizontal gene transfer

1.Donor and recipient strains were grown overnight in LB medium at 37 C and then collected by centrifugation at 5,500 rpm for 5 min at 4 C.

2.The ted cells were washed three times with PBS (pH = 7.4) and then resuspended in PBS at OD 6001.0.

3. The resuspended donor and recipient bacteria were mixed (v / v=1:1) with 4-NPO (7.7 μg/mL) and Acylase I. (75 μg/mL) added. Bacteria were shaken at 250rpm for 30 min.

4.The bacteria were then incubated at 37 C for 16 h of transfer.

5.Mixed bacteria were diluted with fresh LB medium and then inoculated into LB agar with antibiotics.

6. The combined transfer frequency was calculated by dividing the number of bacteria grown on the medium with four antibiotics (including tetracycline, kanamycin, ampicillin, and rifampicin) by the number of bacteria grown on rifampicin-only medium.

7.In addition, after 16 h incubation with donor and recipient bacteria, RNA was extracted using a bacterial RNA extraction kit (Sangon Biotech, China) and then immediately reverse transcribed into cDNA (TransGen Biotech, China). TOP Green qPCR Super Mix kit (TransGen Biotech, China) was used for RT-qPCR amplification. The relative expression of each gene was calculated using the formula, using 16-S rRNA as the reference for quantitative Ct (2-Ct).

Measurement of horizontal gene transfer

Bacterial cultures were induced for 48 h before being subjected to filtration through a 47 mm (dia.) polycarbonate membrane with 10 µm pores (EMD Millipore) using vacuum filtration.8 The concentrated biomass was rinsed three times on the filter membrane with 25 mL of sterile DI water. Next, the filtered biomass was incubated with 5 mL of 5% (m/v) SDS in water for 5 min, followed by vacuum filtration of the liquid and three rinses with 25 mL of DI water. Hydrogel that was formed on the filter membrane was collected by gently scraping with a spatula, and was stored wet at 4 °C or lyophilized for further studies. Cell density was determined by streaking the hydrogel on LB-agar plates for CFU counting.

SEM

Two-hundred microliters of the testing cultures were filtered onto Nucleopore Track-Etched membranes (0.22 μm pore size) under vacuum and placed in fixative solution (2% glutaraldehyde and 2% paraformaldehyde in 0.1 M sodium cacodylate buffer) for 2 h at room temperature. After fixation, the membranes were gently rinsed with water and subjected to an ethanol gradient (25, 50, 75, 100, and 100% (volume per volume)) with a 15 min incubation for each concentration. The samples were then transferred to a critical point dryer). The dried membranes were placed on Scanning Electron Microscopy sample holders with carbon adhesives and sputter coated with 80:20 Pt:Pd (5 nm-thick). A Zeiss Ultra 55 Field Emission Scanning Electron Microscope was used to image the samples.

Hydrogel Rheology

Small strain amplitude oscillatory rheology measurements were performed on a TA Instruments AR-G2 rheometer with plate-plate geometry. 8 mm plates were used for strain sweeps and frequency sweeps with a gap width of 500 µm and moisture trap. Strain sweep measurements were carried out from a strain amplitude of 0.1% to 25% at 25.0 °C (±0.1 °C) and a frequency of 1.0 Hz to determine small deformation linearity. Frequency sweeps were then performed from 0.1 to 100 Hz at 25.0 °C with a controlled strain amplitude of 1.0%, which was within the linear response range for all samples.

Quantitative Congo Red-binding assay

One milliliter of bacterial culture was pelleted at 8000 rpm for 10 min and resuspended in a 0.025 mM a solution of Congo Red (CR) in phosphate-buffered saline (PBS) for 10 min. After pelleting the cells again at 14,000 rpm for 10 min, the absorbance of the supernatant at 490 nm was measured using a microplate reader. Normalized curli fiber production was calculated by subtracting the measured absorbance value from that measured for 0.025 mM Congo Red in PBS and normalized by the OD600 of the original bacterial culture.

Determination of adhesion capacity

The 50 µL of cTFF2 was applied onto the tissue sample and cover with the second layer of tissue fixed to SEM pin stub. These layered tissue–hydrogel–tissue sandwiches were kept at room temperature for 30 min to achieve integration of the hydrogel with the gut mucosa. Finally, SEM pin stubs with hydrogel and tissue samples were loaded on the bottom stage of the mechanical tester (ADMET) and pulled off with a constant strain of 8 mm min−1. The applied force was monitored and the maximum force before full detachment of the patch was measured as the adhesion force. All the experiments were performed in triplicate.

Validation of non-icity

1.Cell culture
Caco-2 cells (C2BBe1 [clone of Caco-2] (ATCC® CRL2102™)) were maintained and passaged in Dulbecco’s modified Eagle medium (DMEM) supplied with 4.5 g L−1 glucose and glutamax, 15% fetal bovine serum (FBS) and 1% Penicillin–Streptomycin (Gibco) in a humidified incubator at 37 °C, 5% CO2. For the translocation assay, epithelial integrity and IL-8 production experiments, Caco-2 cells were grown to confluence on 3.0 μm semipermeable tissue culture inserts (24 wells, Transwell, Corning). After 14–21 days, the cell monolayers achieved a polarized, differentiated state and the transepithelial electrical resistance (TEER) reached 1000–1200 Ω cm−2.

2.Electron microscopy
Two-hundred microliters of the testing cultures were filtered onto Nucleopore Track-Etched membranes (0.22 μm pore size) under vacuum and placed in fixative solution (2% glutaraldehyde and 2% paraformaldehyde in 0.1 M sodium cacodylate buffer) for 2 h at room temperature. After fixation, the membranes were gently rinsed with water and subjected to an ethanol gradient (25, 50, 75, 100, and 100% (volume per volume)) with a 15 min incubation for each concentration. The samples were then transferred to a critical point dryer). The dried membranes were placed on Scanning Electron Microscopy sample holders with carbon adhesives and sputter coated with 80:20 Pt:Pd (5 nm-thick). A Zeiss Ultra 55 Field Emission Scanning Electron Microscope was used to image the samples.

3.Invasion assay
Caco-2 cells at passage 5–15 were plated in 24-well plates at a density of 105 cells per well in 500 μL of regular cell culture media and grown to 90% confluency. The bacterial cultures were pelleted, washed with PBS and diluted to an OD600 of 0.5 in DMEM with 1 g L−1 glucose and 1% FBS. The Caco-2 cells were rinsed twice with PBS to remove the antibiotic before addition of the bacteria (500 μL). Bacteria were incubated for 2 h with the Caco-2 cells and removed by aspiration. The Caco-2 cells were then washed twice with 500 μL PBS before receiving 500 μL of DMEM, 1 g L−1 glucose, 1% FBS, and 100 μg mL−1 gentamicin. After 1 h of incubation, the media were aspirated and replaced with 1 mL of 1% Triton-X. The cells were incubated with Triton-X at 37 °C for 10 min and repeatedly pipetted to homogenize. Each well was serially diluted and plated on kanamycin plates to count the colony-forming units (CFU) of bacteria that had invaded the Caco-2 cells.

4.Translocation assay
EcN variants and S. Typhimurium SL 1344 were grown and induced, if applicable, in LB media with appropriate antibiotics, pelleted, washed with PB,S and diluted to OD600 of 0.01 in DMEM with 1% glucose and 1% FBS. One day before the experiment, the culture media of polarized Caco-2 were switched to their corresponding non-antibiotic counterparts with 1% glucose and 1% FBS. Six-hundred microliters of diluted cultures were used to infect polarized Caco-2 apically. After 5 h of incubation, 100 μL of media were collected from apical and basolateral sides of the transwell, serially diluted and plated on antibiotic selective plates to enumerate percentage of translocated bacteria.