Experiments | SJTU-BioX-Shanghai

1. Add 500 μl of equilibrium solution BL to the adsorption column CP3 (adsorption column placed in the collection tube), and centrifuge for 1 min at 12,000 rpm. Centrifuge for 1min, pour out the waste liquid in the collection tube and put the column back into the collection tube.

2. Take 5 ml of the overnight culture, add it to the centrifuge tube and centrifuge it at 12,000 rpm for 1 min using a conventional table-top centrifuge to remove as much of the supernatant as possible.

3. Add 250 μl of solution P1 (with RNase A added) to the centrifuge tube with the bacterial precipitate, and use a pipette or vortex shaker to thoroughly suspend the bacterial precipitate.

4. Add 250 μl of Solution P2 to the centrifuge tube and gently turn the tube up and down 6-8 times to fully lyse the bacteria.

5. Add 350 μl of Solution P3 to the centrifuge tube and immediately gently turn it up and down 6-8 times to mix thoroughly and produce a flocculent precipitate. centrifuge at 12,000 rpm for 10 min.

6. Transfer the supernatant collected in the previous step to the adsorbent column CP3 with a pipette (adsorbent column in a collection tube), taking care not to aspirate the precipitate as much as possible. centrifuge at 12,000 rpm for 30-60 sec, pour off the waste liquid in the collection tube, and place the adsorbent column CP3 in a collection tube.

7. Add 600 μl of rinse solution PW (with anhydrous ethanol added) to the adsorbent column CP3, centrifuge at 12,000 rpm for 30-60 sec, pour out the waste liquid from the collection tube, and place the adsorbent column CP3 into the collection tube. 8. Repeat step 8.

9. Place the adsorbent column CP3 into the collection tube and centrifuge at 12,000 rpm for 2 min.

10. Place the adsorbent column CP3 in a clean centrifuge tube, add 50-100 μl of 37°C ddH2O dropwise to the middle part of the adsorbent column, leave it at room temperature for 2 min, and centrifuge at 12,000 rpm for 2 min to collect the plasmid solution into the centrifuge tube.

1. Bacteria on the successfully transformed plate were picked into 10ml shaking tube with the tip of a gun, and 5ml of LB liquid medium and 5μl of corresponding antibiotics were added.

2. Constant temperature shaker amplification: 37 ℃, 200r, 4h

3. Amplify the target fragment, configure 10μl system: 1μl template + 0.5μl upstream primer + 0.5μl downstream primer + 5μl 2xTaq Master Mix + 3μl ddH2O

4. Set up the PCR program:

5. Ice bath for 5 min

6. Add 700ul of LB into the EP tube on the ultra-clean bench.

7. Shake the recovery at constant temperature: 37℃, 200r, 1h.

8. Take out the EP tube and aspirate 100ul to coat the plate.

1. Prepare 1% agarose gel with TAE and agarose, heat it in microwave oven until completely dissolved.

2. When the gel cools down to about 60 ℃ Celsius, add 20xGel Red dye and pour it into the electrophoresis tank.

3. Mix the sample into 6xLoading Buffer (this step can be ignored if Taq enzyme or other staining enzymes are used).

4. Add samples after the gel has solidified, and add DNA maker of appropriate size (5μl for small wells; 10μl for large wells).

5. Electrophoresis: 120v-150v, 15-30min

1. Cut off the target band after agarose gel electrophoresis.

3. Add an equal volume of Buffer GDP, and use a 50 ℃ water bath until the gel completely melts.

4. After brief centrifugation, place the adsorbent column in a 2ml collection tube, transfer the gel solution to the adsorbent column, and centrifuge at 12000rpm for 60sec. If there are many gel solutions, multiple transfers can be made.

5. Discard the filtrate, place the column in a collection tube, add 300μl Buffer GDP to the column, let it stand for 1min, and centrifuge at 12000rpm for 60sec. 6. Discard the filtrate, place the column in a collection tube, add 700μl Buffer GW (anhydrous ethanol has been added) to the column, and centrifuge at 12000rpm for 60sec.

6. Discard the filtrate, place the column in a collection tube, add 700μl Buffer GW (anhydrous ethanol has been added) to the column, and centrifuge at 12000rpm for 60sec.

8. Discard the filtrate, put the column back into the collection tube and centrifuge at 12000rpm for 2min.

9. Place the column in a 1.5 ml centrifuge tube, add 20-30 μl of 37 ℃ ddH2O, leave for 2 min, centrifuge at 12000 rpm for 1 min, discard the column and store the DNA at -20 ℃ Celsius.

1. Centrifuge the bacterial culture media for 15 min at 7,000 x g at 4 C to pellet intact bacteria

2. Decant, and save, the media of the top of the cell pellet post centrifugation.

3. Add an equal volume of Buffer GDP, and use a 50 ℃ water bath until the gel completely melts.3. Further purify the bacterial culture media using a 0.45 um syringe filter to remove residual bacteria and undesired large cellular material

4. Add the filtered culture media into centrifugation tubes and balance opposing tubes in the centrifuge carefully

5. Pellet at -150,000 x g for 3 hr at 4 in an ultracentrifuge using the corresponding rotor matching both the instrument and tubes being utilized.

6. Decant the OMV culture media from the OMV pellet, carefully, immediately at thecompletion of centrifugation.

7. Save the centrifuged supernatant for later Nano Particle Tracing Analysis (NTA) testing to verify that the OMV partficles were fully deplete from the media

8. Add 0.5-1 ml of sterile filtered PBS to the OMV pellet allowing it to incubate for 30 min at RT.

9. Carefuly pipet the purified OMV solution from the centrifuge tube, mixing gently to ensure the entire pellet has been solubilized

1. Pour the SDS-PAGE gel, either pre-made or manually prepared, consisting of a separating gel and a stacking gel. Insert a comb or create sample wells in the stacking gel. Allow the gel to solidify.

2. Load Samples:Mix protein samples with Loading buffer and denature the proteins by boiling for 5 minutes at 100°C.

3. Cool the samples to room temperature.

4. Carefully load the samples into the sample wells in the separating gel, along with maker.

5. Connect the electrophoresis chamber to the power supply. Start the power supply and set the current and voltage, running at 100-200V until the dye migrates to an appropriate position.

6. Immerse the gel in Coomassie Brilliant Blue staining solution and gently shake or rock it for 1-2 hours or until protein bands become visible.

7. Destain the gel by immersing it in a destaining solution until the background becomes clear, changing the destaining solution as needed.

1. Sample preparation: Determine the protein concentration and ensure equal loading.

2. SDS-PAGE electrophoresis: Prepare 12% SDS-PAGE gel and prepare protein samples with 6× SDS-Page Protein Loading Buffer (YEASEN, Shanghai, China).

3. Electrophoresis was run at a constant voltage (80V for 20 minutes followed by 120V for 30 minutes) to separate proteins.

4. Proteins were then transferred onto PVDF membranes (GE Healthcare, Solingen, Germany).

5. Carrier membrane pretreatment: Wash the PVDF membrane with TBST and block the membrane with 5% skim milk to avoid non-specific binding.

6. Immunoblotting: incubate the carrier membrane in primary antibody overnight at 4°C; wash the carrier membrane with TBST to remove unbound primary antibody; incubate the carrier membrane in HRP-conjugated secondary antibodies at room temperature for 1 h; wash the carrier membrane to remove unbound secondary antibody.

7. Color development and imaging: Blots were formed using the ECL reagent (Share-bio, Shanghai, China).

1.Prepare the reaction mixture at room temperature in the order indicated

2.Mix gently and spin down.

3.Incubate at 37°C in a heat block or water thermostat for 5 min.

4.lnactivate the enzyme (optional).

1. In microcentrifuge tubes, the following reaction systems were prepared.

2. 16 ℃ overnight reaction

3. Conversion of ligation products

3.1. Thaw the chemoreceptor cells for cloning on ice.
3.2. Take 10μl of ligand product and add it to 100μl of chemoreceptor cells, flick the wall of the tube to mix well (do not oscillate to mix well), and leave it on ice for 30 min.
3.3.After heat-excited at 42℃ for 45 sec, immediately put on ice to cool for 2-3 min.
3.4. Add 900 ul SOC or LB medium (without antibiotics) and shake the bacteria at 37℃ for 1 h (200-250 rpm).
3.5. Pre-warm the LB plate solid medium with the corresponding resistance in a 37℃incubator.
3.6. Centrifuge at 5000 rpm for 5 min and discard 900μl of supernatant. Resuspend the bacteria with the remaining medium and spread gently on the plate containing the correct resistance using a sterile applicator stick.
3.7. Incubate in an inverted incubator at 37 ℃ for 12 - 16 h. Overlap PCR

Three gradient experiments were designed to verify the binding ability and conditions of OMV to lead ions

1. Pb2+ concentration gradient

1.1. The OMV concentrate was fixed to 40.4 ml 1.2. Prepare Pb2+ concentration of 250 mg / L of lead ion solution 100 mL, take 10 mL diluted to 25 mg / L solution 100 mL 1.3. Mix and react for 2h.

2.OMV tested after 10x concentration

2.1. OMV concentrated solution was fixed to 12.1 ml 2.2. Prepare 20 mL of lead ion solution with Pb2+ concentration of 500 mg/L. 2.3. Mix and react for 2h.

3.Reaction time gradient

3.1. the OMV concentrate was fixed to 40.4 ml 3.2. Prepare 400 mL of lead ion solution with a Pb2+ concentration of 250 mg/L. 3.3. Mix and react for 2h

1. At the end of the reaction, dilute according to the corresponding multiplicity, ultracentrifugation to separate OMV and supernatant, supernatant 2-fold dilution

2. OMV was presuspended with a small amount of PBS and then resuspended and mixed in equal volume in a thick-walled tube, 1 mL of 10% SDS solution was added, and the reaction was performed at RT for 30 min, 2-fold dilution