(1) Prepare extraction buffer
800 mM guanidine hydrochloride, 50 mM Tris, 0.5% Triton X-100, 1% Tween-20, 40 μg/mL proteinase K.
(2) Prepare wash buffer (10 mM Tris, 0.1% Tween-20)
(3) Prepare circular filter paper
Use the hole punch to punch out the circular filter papers with diameter of 3 mm, 4 mm, 5 mm, 6 mm. Before the experiment, a circular filter paper was placed in each 1.5 mL tube.

(1) Add 300 μL DNA solution (1 ng/mL, 5 ng/mL, 10 ng/mL) to the 1.5 mL tube containing a circular filter paper for approximately 1 minute.
∗If the sample is bacteria culture: Dilute the bacteria culture after shaking (10 times, 100 times, 1000 times). Add 100 μL sample to 200 μL extraction buffer and vortex for 10 seconds for cell lysis, then add this cell lysate to the 1.5 mL tube containing a circular filter paper for approximately 1 minute.
(2) Use the pipette to draw out the fluid, then add 200 μL wash buffer to this tube for 1 minute to remove contaminants including amplification inhibitors.
(3) After 1 minute, the circular filter paper containing nucleic acid was then transferred into an amplification reaction.
(4) After amplification, agarose gel electrophoresis was used to determine the effect.

(1) Add 2.4 μL of each primer at 10 μM concentration to 0.2 mL PCR tubes.
(2) Prepare a pre-master mix (per reaction) in the order below.

Vortex and spin briefly.
(3) To the pre-master mix, add 2.5 μL 20x Core Reaction Mix to tube lid. Spin briefly. Master mix is now complete.
(4) Add 41.7 μL of master mix to primers prepared in tubes (step 1) and evenly mix.
(5) Add 2.5 μL of 280 mM MgOAc and 1 μL template or cellulose-based paper separately to tube lids. Spin briefly.
(6) Incubate at 37°C for 20-40 minutes.
(7) Separate pieces of DNA using gel electrophoresis.

(1) Preheat water bath to 37 °C.
(2) Completely thaw the myTXTL Mix and DNA on ice completely. Keep reagents on ice till use.
(3) Directly before use, vortex the myTXTL Mix for 2-3 seconds and spin down briefly. If any precipitate is visible hereafter, resuspend master mix solution gently about 10 times to ensure homogeneity. Avoid formation of bubbles and foam.
(4) Add 3.1 uL DNA or other reagents into the myTXTL Mix.
(5) Vortex the reaction mixture for 2-3 seconds gently and centrifuge the assembled myTXTL reaction briefly to collect the entire volume at the bottom of the tube.
(6) Incubate the myTXTL positive control reaction(s) for 3~6 h at 37 °C.
(7) Stop the myTXTL positive control reaction by inserting the tube(s) into ice.

Prepare two groups of 50-mL conical flask, breathable sterile sealing film covers the bottleneck. 10 mL of LB liquid medium with corresponding antibiotics was added to each flask.

(1) Inoculate 100 µL bacteria glycerol stock into 10 mL LB liquid medium with corresponding antibiotics, 37 ℃, 200 rpm overnight.
(2) Inoculate 100 µL bacteria culture of experimental and control group into 10mL liquid LB culture medium with corresponding antibiotics. Incubate at 37°C for 4–6 h until the OD₆₀₀=0.6-0.8.
(3) The bacterial culture was induced with 1‰ IPTG and cultured at 37°C for 1 h.
(4) Add 200 µL bacteria culture to the 96-well plate and OD₆₀₀ was measured by a microplate reader.
(5) Add liquid to the 96-well plate in the following order according to the following table:

(6) The relative absorbance unit (RAU) was measured by a microplate reader (490 nm), every 2 minutes for 20 minutes.

The relative absorbance unit (RAU) at different time points for a sample culture was determined by the blank corrected output signal divided by OD₆₀₀, and after subtracting its triplicate-averaged counterpart of the negative control cultures (reporter-free) at the same time. Unless indicated otherwise, each reporter within different sensors was tested with three biological replicates. All the data shown are mean values with standard deviation as error bars.

(1) Prepare two groups of 50 mL conical flask, ventilate sterile sealing film covers the bottleneck. 10 mL of LB liquid medium with corresponding antibiotics was added to each flask.
(2) Nano-Glo® Luciferase Assay Reagent Preparation:
Mix Nano-Glo® Luciferase Assay Substrate with 50-fold Nano-Glo® Luciferase Assay Buffer.

(1) Inoculate 100 µL bacteria glycerol stock into 10 mL LB liquid medium with corresponding antibiotics, 37°C, 200 rpm overnight.
(2) Inoculate 100 µL bacteria culture of experimental and control group into 10 mL liquid LB culture medium with corresponding antibiotics. Incubate at 37°C for 4–6 hours until the OD₆₀₀ = 0.6-0.8.
(3) The bacterial culture was induced with 1‰ IPTG and cultured at 37°C for 1 h.
(4) Add 200 µL bacteria culture to the 96-well plate and OD₆₀₀ was measured by a microplate reader.
(5) Add liquid to the 96-well plate according to the following table:

Vortex briefly.
(6) The relative luminescence unit (RLU) was measured by microplate reader (460 nm), every 5 minutes for 30 minutes.

The relative luminescence unit (RLU) at different time points for a sample culture was determined by the blank corrected output signal divided by OD₆₀₀. Unless indicated otherwise, each reporter within different sensors was tested with three biological replicates. All the data shown are mean values with standard deviation as error bars.

Use sterile water to prepare the resuspension buffer: 50 mM HEPES-0.85% NaCl buffer, pH = 7.4. Then filter it through 0.22-μm filter.

(1) Inoculate 200 µL bacteria glycerol stock into 10 mL LB liquid medium with 34 μg/mL chloramphenicol, 37°C and 200 rpm overnight.
(2) Inoculate 1 mL bacteria culture into flasks contained 50 mL LB liquid medium with 34 μg/mL chloramphenicol, incubate the culture at 37°C and 200 rpm until the OD₆₀₀ reaches 0.6-0.8.
(3) If necessary, add appropriate amount of inducer into bacteria culture, and then incubate the culture at 28°C and 200 rpm for 16 hours. Or directly incubate the culture at 37°C and 200 rpm for 12 hours.
(4) After incubation, 50 mL of the culture is centrifuged at 10000 ×g for 15 minutes at 4°C to remove cells.
(5) Then, the supernatant was filtered through the 0.45-μm filter once then 0.22-μm filter for a second time.
(6) The cell-free supernatant is used to extract the OMV via ultracentrifugation at 140,000 ×g for 2 hours at 4°C.
(7) The OMV pellets (may not be easily visible) are suspended in 100 μL resuspension buffer, then stored at -20°C.
(8) The diluted OMV samples were spotted onto LB agar plate with 34 μg/mL chloramphenicol to verify that there is no bacteria component.
(9) The OMV samples are negatively stained for TEM observation.

When the culture supernatant is filtered, be very careful to prevent bacterial contamination!

(1) OMVs are extracted according to the OMV purification protocols.
(2) The OMVs are lysed by 1% (w/v) SDS (final concentration) at room temperature for 10 minutes.
(3) Take 4 μL lysate, measure protein concentration of each group’s OMV according to the user’s manual of BCA Protein Assay Kit.
(4) According to the standard curve, calculate the protein concentration and normalize it to OD₆₀₀.

Measure the OD₆₀₀ of each group when they are taken out from the shaking incubator as soon as possible. Subsequently, centrifuge at 10,000 ×g for 15 minutes at 4°C to separate the bacterial precipitation and supernatant in order to obtain a more convincing data.

(1) Treat the extracted vesicles with DNase I at 37°C for 30 minutes.
(2) Deactivate DNase I at 75°C for 15 minutes.
(3) Denature in 100℃ water bath for 10 minutes.
(4) Pipet 3 μL of treated solution for PCR amplification.
(5) Apply the PCR products to agarose gel electrophoresis.
(6) Check whether there are correct strips on the gel illuminated by transilluminator.

(1) Incubate the culture of the experimental group and the control group respectively with the OMVs in different proportions at 37 °C.
(2) Dilute each group to 10^-2 and 10^-4 concentration respectively.
(3) Apply each diluted culture to agar spot test.
(4) Select single colonies for colony PCR and then apply the PCR products to agarose gel electrophoresis to verify whether the plasmid is transferred during incubation.
(5) Select the colonies with the correct band for culture.
(6) Each culture is applied to agar point test on the medium with arabinose induction and the medium without arabinose induction respectively to verify the killing effect of the plasmid.

(1) Bacteria culture was inoculated into LB liquid mediums at 2% (v/v) and kept at 37 °C, 200 rpm and last 12-16 h.
(2) Bacteria culture from step 1 was inoculated into LB liquid mediums at 2% (v/v) and kept at 37 °C, 200 rpm and last 2-4 h.
(3) When the OD₆₀₀ of bacteria culture from step (1) is 0.6-0.8, add 0.2% (v/v) arabinose and keep at 37 °C, 200 rpm and last 2-4 h.
(4) Pellet 1 mL bacteria culture from step (3) in a clean 1.5-mL microcentrifuge tube. Centrifuge at 6500 rpm for 3 minutes at room temperature. Decant or aspirate medium and discard.
(5) Add 1 mL PBS buffer, evenly mix the sample. And centrifuge at 6500 rpm for 3 minutes at room temperature. Decant or aspirate medium and discard.
(6) Repeat step (5) twice.
(7) Add 500 μL - 1 mL 5% defatted milk, evenly mix the sample, incubated at 37°C for 0.5-1 h.
(8) Centrifuge at 6500 rpm for 3 minutes at room temperature. Decant or aspirate medium and discard.
(9) Repeat step (5) three times.
(The following operations should be performed in darkness)
(10) Add 490 uL PBS buffer, evenly mix the sample. Add FITC anti-6xHis Antibody（1: 1000）(Abcam), incubate at 37°C for 1 h.
(11) Repeat step (8) once, step (5) three times.
(12) Inoculate 200 μL bacteria culture from step (11) into 96-well microtiter plates, use microplate reader reads the fluorescence intensity (Absorption wavelength: 492 nm, Emission wavelength: 518 nm) and OD₆₀₀.

(1) Bacteria culture was inoculated into LB liquid mediums at 2% (v/v) and incubated at 37 °C, 200 rpm, 12-16 h.
(2) Bacteria culture from step 1 was inoculated into LB liquid mediums at 2% (v/v) and incubated at 37 °C, 200 rpm, last 2-4 h.
(3) When the OD₆₀₀ of bacteria culture from step 1 is 0.6-0.8, add 0.2% (v/v) arabinose and keep at 37 °C, 200 rpm and last 2-4 h.
(4) Pellet 1mL bacteria culture from step 3 in a clean 1.5-mL microcentrifuge tube. Centrifuge at 6500 rpm for 3 minutes at room temperature. Decant or aspirate medium and discard.
(5) Add 1mL TBST buffer, evenly mix the sample. And centrifuge at 6500 rpm for 3 minutes at room temperature. Decant or aspirate medium and discard.
(6) Repeat step (5) twice.
(7) Add 500μL - 1mL 5% defatted milk, evenly mix the sample, incubated at 37°C for 0.5-1 h.
(8) Centrifuge at 6500 rpm for 3 minutes at room temperature. Decant or aspirate medium and discard.
(9) Repeat step (5) three times.
(10) Add 500 μL-1 mL TBST, evenly mix the sample. Add appropriate concentration of protein culture to positive control while negative control not. Incubated at 4°C for 10 h.
(11) Repeat step (8) once, repeat step (5) three times.
(The following operations should be performed in darkness)
(12) Add 490 uL TBST buffer, evenly mix the sample. Add FITC anti-6xHis Antibody（1: 2500）(Abcam), incubate at 37°C for 1 h.
(13) Repeat step (8) once, repeat step (5) three times.
(14) Inoculate 200 μL bacteria culture from step (13) into 96-well microtiter plates, use microplate reader reads the fluorescence intensity (Absorption wavelength: 492 nm, Emission wavelength: 518 nm) and OD₆₀₀.

(1) Wipe clean with tweezers and scissors.
(2) Prepare soluton:
TBST(1L) : Tris-HCl(1M) 50 ml, NaCl 8 g, KCl 0.2 g, Ttwain 0.5 ml
5% milk, 1x TBST : Dissolve 5 g non-fat dry milk in 100 mL 1x TBST and filter.

(1) Label the nitrocellulose blotting membrane for the ease to locate your individual sample after blotting.
(2) Place the labeled membrane in an incubation box.
(3) Pipette 1.5 uL of each sample onto separate pre-determined locations on the blot, four dots for each sample. The samples should be absorbed to the membrane immediately without any beading on the surface.
(4) Block the membrane in 5% milk, 1x TBST for 1 hour on a rotating shaker.
(5) While blocking, dilute the protein in 5% milk, 1x TBST.
(6) Decant blocking solution, rinse the membrane with 1x TBST, wash the membrane 3 times with 1x TBST buffer, each time 5 minutes with shaking on a rotating shaker.
(7) Incubate the membrane with the diluted protein for 2 hours on a rotating shaker.
(8) Discard the diluted protein and wash with 5% milk, 1x TBST for 5 minutes on a shaking incubator for 3 times.
(9) While washing, dilute the primary antibody in 5% milk, 1x TBST.
(10) After washing, incubate the membrane with the diluted primary antibody. Incubate for 1 hour on a shaking incubator.
(11) Discard the diluted primary antibody and wash with 5% milk, 1x TBST for 5 minutes on a shaking incubator for 3 times.
(12) While washing, dilute the second antibody in 5% milk, 1x TBST.
(13) After washing, incubate the membrane with the diluted second antibody. Incubate for 1 hour on a shaking incubator.
(14) Discard the diluted primary antibody and wash with 1x TBST for 5 minutes on a shaking incubator for 3 times.
(15) Proceed with incubation using EasySee® Western Blot Kit for 1 min (using freshly prepared working solution) . Wrap the membrane in a plastic wrap and remove bubbles and wrinkles. Place the membrane in a chemilunescence instrument to capture the image.

Expression and purification of Lysqdvp001 gene (his-edl060)
(1) Incubate E. coli BL21(DE3) with I0500-B0034-his-edl060-B0015_ pUC57-Simple plasmid in LB medium with 1 μL/mL ampicillin at 37°C until the OD₆₀₀= 0.6.
(2) Pipet 4 mL of the culture and incubate in 400 mL medium with ampicillin for 2-4 hours at 37 °C.
(3) Add arabinose with the final concentration of 0.2%. Then add the same amount of arabinose after 1 hour, and then repeat inducing every 2 hours until the induction time overall reaches 12 hours.
(4) Pour the culture into 50 mL centrifuge tubes.
(5) Centrifugate at 6500 rpm for 15 minutes, then resuspend the precipitate in 1% PBS solution.
(6) Repeat step (5) twice.
(7) Froze the precipitate overnight in the refrigerator at -80 °C.
(8) Suspend the frozen precipitate with 1% PBS solution.
(9) Apply the solution to ultrasonic disruption.
(10) Filtrate the lysate supernatant with 0.45-micron pore size filter membrane.
(11) Use AKTA to purify edl060 protein.
(12) Measure the protein concentration with Bradford method.
(13) Dialyze the protein.
(14) Freeze the dialyzed protein in a -80°C refrigerator.
(15) Freeze dry the solution to concentrate.
(16) Measure the concentration of edl060 protein with Bradford method.
Verification of the killing effect of endolysin
(1) Incubate E. coli BL21(DE3) and Vibrio alginolyticus ATCC33787 in LB medium, Vibrio natriegens strain VnDX in LBv2 medium.
(2) Pipette 1 mL of each culture. Incubate with 1 mL 150 mM EDTA for 30 minutes.
(3) Centrifuge the solution at 6500 rpm for 10 minutes to remove EDTA, and remove the supernatant. Then add 1mL corresponding medium and resuspend the precipitation.
(4) Repeat step (3) twice.
(5) Dilute each solution, and the dilution concentration is 10^-3, 10^-4, 10^-5, 10^-6.
(6) The solution of each dilution concentration shall be treated as follows:
a. each with different diluted concentration shall be treated with isochoric sterile water and then be applied to agar spot test;
b. each with different dilution concentration gradient shall be treated with 150 mM EDTA for 30 minutes and then be applied to agar spot test;
c. each with dilution concentration of 10^-5 shall be treated with endolysin edl060 for 5 minutes, 10 minutes, 15 minutes and 20 minutes, and then apply each group to agar spot test;
d. each with different dilution concentration shall be treated with 10% NaOH for 20 minutes and be applied to agar spot test.
(7) Incubate overnight at 37 °C.
(8) Observe the results of agar spot test.

(1) Inoculate 200 µL bacteria glycerol stock into 10 mL LB liquid medium with 40 μg/mL kanamycin (final concentration), 37°C and 200 rpm overnight.
(2) Inoculate 200 µL bacteria culture into flasks contained 10 mL LB liquid medium with 40 μg/mL kanamycin, then the culture was incubated at 37°C and 200 rpm until the OD₆₀₀ reaches 0.6-0.8.
(3) Add the L-rhamnose and L-mannose stock solution to different groups respectively to reach different final concentrations 0, 0.1, 0.4, and 0.7 mg/mL.
(4) After cultured at 37 °C, 200 rpm for 12 hours, take 200 µL of culture for detecting OD₆₀₀ and fluorescence intensity.