Lab Supplementary Materials

1. Plasmid extraction

Follow the instruction of DiaSpin Plasmid Mini-Preps Kit (Sangon, B110091-0050), for every 1ml of bacteria culture, we use 30ul ddH2O to elute.

2. Gel extraction

Follow the instruction of DiaSpin DNA Gel Extraction Kit (Sangon, B110092-0050), for all gel extraction, we use 30-50ul ddH2O pre heated to 65℃ to elute.

3. Restriction digest

We have applied specific restrictions enzymes to the plasmid backbones.Here, we use empty pET-28a backbone as a demonstration.

1. First calculate the volume of components that adds to 50 μl.

2. Then prepare 2000 to 3000 ug of the plasmid backbone we have amplified in previous PCRs and transfer them into a PCR tube.

3. Adding water and the mix to the PCR tube with the volume calculated earlier.

4. adding 1.5 μl of the restriction enzymes which we have used Xhol and Ncol with 1.5μ respectively.

5. Placing the PCR tube on a rack and transfer into 37°C environment for approximately 2 hours.

4. Gibson assembly

Follow the instructions of the product.

5. Regular PCR using Taq polymerase

Follow the instruction of the product, for all reaction, we always try with 56℃ annealing first.

6. Hi-Fi PCR

Follow the instructions of the product.

7. Protein electrolysis using SDS-PAGE

1. 2ml of bacteria culture centrifuge at 8,000×g for 2min at 4℃, the pallet was collected

2. 150ul one-step bacteria protein extraction reagent(beyotime, P0013Q-100ml）,1.5ul PMSF stock solution was added in and resuspend the cell, leave at room temperature for 15min.

3. Centrifuge at 12,000xg for 5min at 4℃，collect the pallet and supernatent.

4. Add 15ul 5X protein loading buffer to 60ul supernatent, add 100ul 1X protein loading buffer to the pallet, heat the sample at 98℃ for 10min

5. Load the sample to the well of the SDS-PAGE gel, use 150V to run the gel for 1.5h

6. Wash the gel in distilled water 3 times and stain with Fastblue protein stain(biosharp, BL607A) for 40min, wash away the stain and take picture of the gel.

8. Separation of OMV

1. bacteria were cultured as described above and IPTG (1M) was added to further induce protein expression when the optical density at 600nm (OD600) reached 0.6–0.8.

2. Bacteria were incubated overnight at 25 °C with shaking at 200 r.p.m or 4h at 37℃ 210rpm. and removed by centrifugation at 8,000×g for 10min at 4 °C.

3. The supernatant (200ml) was filtered through a 0.45μm polyvinylidene fluoride filter (Millipore, R8SA47939) to remove any cells and concentrated to 1ml using two 50 kDa ultrafiltration membrane (Millipore, UFC905008).

4. 1X PBS buffer was added into the upper column of the membrane, a final 500ul was thought to be concentrated OMV and was used for further tests.

9. Detection of OMV protein through western blot

1. The protein sample has been transformed from an SDS-PAGE gel to PVDF membrane (millipore, R1NB740V8) （soak in Methanol 3min before use) at 300V for 45min.(put the device in ice to cool down)

2. The membrane was blocked in 5% non-fat milk(dissolve in TBS buffer) for 1h at room temperature.

3. The membrane was washed in TSBT buffer for 10min

4. The membrane was incubated with anti-HA tag antibody(1:10000 in block buffer) and anti-β-actin antibody(internal reference) at 4℃ overnight.

5. The membrane was washed in TBST buffer for 15min *4 times

6. The membrane was incubated with Rabbit Anti-Mouse IgG H&L (HRP)(Abcam, ab6728)(1:5000in block buffer) for 1h at room temperature.

7. The membrane was washed in TBST buffer for 15min *4 times

8. The buffer A and buffer B of the ECL detection reagent(Beyotime,P0018S) was mixed and dropped on the membrane, signal was detected by the Tanon 5200 chemiluminescence system.

10. Transmission electron microscope image of OMV

1. bacteria were cultured as described above and IPTG (1M) was added to further induce protein expression when the optical density at 600nm (OD600) reached 0.6–0.8.

2. Bacteria were incubated overnight at 25 °C with shaking at 200 r.p.m or 4h at 37℃ 210rpm. and removed by centrifugation at 8,000×g for 10min at 4 °C.

3. The supernatant (50ml) was collected and transport with dry ice to Beyotime biotech and taking TEM image(service code: T5702)

11. NTA-Flow cell test of OMV

1. bacteria were cultured as described above and IPTG (1M) was added to further induce protein expression when the optical density at 600nm (OD600) reached 0.6–0.8.

2. Bacteria were incubated overnight at 16 °C with shaking at 160 r.p.m or 4h at 37℃ 210rpm. and removed by centrifugation at 8,000×g for 10min at 4 °C.

3. The supernatant (50ml) was collected and transport with dry ice to Beyotime biotech and run NTA-Flow cell test of OMV particle(service code: T5704)

12. Optimized IPTG concentration to maximize OMV protein expression

1. 10ul of glycerol stock bacteria was added into 5ml of LB medium containing kanamycin and culture overnight (Day1)

2. 1ml of the start up culture was added into 100ml LB medium containing kanamycin and culture at 37℃ 210rpm for 3-4h until the OD600 reach 0.6-0.8.

3. The shaking incubator will be set to 25℃ 200rpm for 1h to let the medium cool down

4. The Culture will be moved into different tubes for 3ml in each and add IPTG for final concentration 0 0.2 0.4 0.6 0.8 1.0mM (Day2)

5. For each tube, 200ul of bacteria culture was added into each well of Corning(®)96well plate(CLS3610-48EA) (3repeated wells), blank using sterilized LB medium.

Well	Sample
A1 A2 A3	sterilized LB medium
B1 B2 B3	Bacteria Culture with 0mM IPTG
C1 C2 C3	Bacteria Culture with 0.2mM IPTG
D1 D2 D3	Bacteria Culture with 0.4mM IPTG
E1 E2 E3	Bacteria Culture with 0.6mM IPTG
F1 F2 F3	Bacteria Culture with 0.8mM IPTG
G1 G2 G3	Bacteria Culture with 1.0mM IPTG

6. Using the plate reader, measure the emission of fluorescent at 520nm with excitation 485nm, and also absorbance density at 600nm(A600).

13. Measuring the Efficiency of UV promoter SulAp

1. E. Coil was cultured in 50ml M9 medium* till OD ~0.6 . The container was coated with aluminum foil to prevent interference from the environment.

2. The culture was equally transferred into different petri dishes and exposed to 254nm light source with 15mW/cm2 energy density(around 10cm from our device)

3. The culture was transfer into new tubes and coated again and incubate at 37℃ 210rpm

4. At different time stages, using the plate reader, measure the emission of fluorescent at 520nm with excitation 485nm same as protocol 12.

*We found the LB medium has a very high fluorescent background when measuring, M9 medium could reduce the noise.

14. Measure the efficiency of self-lysis device

1. 10ul of glycerol stock bacteria was added into 5ml of LB medium containing kanamycin and culture overnight (Day1)

2. 0.5ml of the start up culture was added into 50ml LB medium containing Ampicillin and culture at 37℃ 210rpm for 3-4h until the OD600 reach 0.6-0.8.

3. Final concentration of 0.2％ arabinose* was added into the culture, the culture was put back into the incubator.

4. At different time stages, samples were collected to test absorbance density at 600nm(A600) and colony-forming unit(CFU).

* According to how the arabinose regulator works, the concentration of arabinose usually will not affect the strength(Siegele and Hu, 1997) . Our experiment also proves this concept.