. Protocols .
1.Preparation of Media
LB liquid (solid) medium: For the preparation of 1000 mL of LB medium, add 10 g tryptone, 5 g yeast extract, 10 g sodium chloride, and 15 g agar (for solid medium). Dissolve these components in approximately 800 mL of RO water. Adjust the pH to 7.0–7.2 using 1 M sodium hydroxide (NaOH) or 1 M hydrochloric acid (HCl). Add RO water to bring the total volume to 1000 mL. Sterilize by autoclaving at 121°C for 20 minutes. Once cooled to 50–55°C, antibiotics (kanamycin/ampicillin) can be added in a sterile environment to a final working concentration of 50 µg/mL.
2.PCR Amplification
(1)Take an ice box, put template,primer,dNTPs,polymerase,reaction buffertemplate plasmid, double distilled water, into the ice box and wait for melting. Take the corresponding number of microtubules and put them into the ice box.
(2)Configure the reaction system. Note the amount of template used (concentration determines volume).
| Digestion reaction system | |
|---|---|
| Component | Volumes |
| Template | 1 μL |
| Primer_F | 1 μL |
| Primer_R | 1 μL |
| dNTPs (10mM) | 1 μL |
| Pfu DNA polymerase | 2 μL |
| 10X KOD DNA polymerase | 5 μL |
| ddH2O | 39 μL |
| Total | 50 μL |
(3)Set the program on the PCR instrument for amplification.
3.Enzymatic Reaction for Vector-Fragment Ligation
(1)Molar Ratio: Maintain a vector-to-insert molar ratio of 1:3. Calculate the required volumes based on fragment concentration and length.
(2)Prepare the following reaction system on ice.
| Digestion reaction system | |
|---|---|
| Component | Volumes |
| Plasmid digestion product | a |
| PCR recovery product | 3a |
| Total | 50 μL |
(3)Briefly centrifuge the reaction mixture and incubate at 16°C for 2 hours, or overnight at 4°C.
4.Nucleic Acid Gel Electrophoresis
(1)Gel Preparation: For a 1% agarose gel, dissolve 1.0 g agarose in 100 mL of 1×TAE buffer. Heat until completely dissolved, cool to ~50°C, pour into a casting tray, and insert a comb.
(2)Sample Preparation: Mix 4 µL of PCR product with 3 µL of loading dye.
(3)Run Gel: Place the gel in the electrophoresis tank, cover with 1×TAE, and load the samples. Run at 150 V until separation is complete.
(4)Staining: Stain the gel for 15 minutes and image using a gel documentation system.
5.Western Blot
5.1 Protein Sample Preparation
(1)Culture bacteria.
(2)Recover and wash the bacterial cells using PBS buffer.
(3)Add 80 μL of Urea (8M) to fully lyse the cells and release proteins.
(4)Centrifuge at 12,000 rpm for 15 minutes at 4°C and remove the supernatant.
5.2SDS-PAGE electrophoresis
(1)Gel Casting: Prepare and pour 10% resolving gel and a 5% stacking gel. Allow the gel to polymerize fully.
(2)Run Gel: Load the samples and run at 80 V initially, then increase to 120 V once the dye reaches the resolving gel.
5.3Membrane Transfer
(1)Immerse the gel in the transfer buffer and equilibrate for 10 minutes.
(2)Cut the PVDF membrane and filter paper into 6 pieces according to the size of the gel, and equilibrate in the transfer buffer for 10 minutes. (The PVDF membrane needs to be soaked in pure methanol for 1 minute first).
(3)Assemble the transfer sandwich: Place them in the order of filter paper, gel, membrane, filter paper successively, remove air bubbles to ensure close contact between each layer.
(4)Transferring the membrane: Place it in the membrane - transfer device, connect to the power supply, and select appropriate membrane - transfer conditions (such as voltage, time, current, etc.) according to the protein size and the type of membrane to transfer the proteins in the gel onto the membrane.
(5)After transferring the membrane, cut off the power supply and take out the hybridization membrane.
5.4 Blocking and Hybridization
(1)Block the membrane in 5% non-fat dry milk in TBST for 2 hours.
(2)Incubate with primary antibody at 37°C for 2 hours or overnight at 4°C.
(3)Wash and incubate with secondary antibody at 37°C for 1 hour.
(4)Develop the membrane and visualize protein bands.
6.Plasmid Transformation
(1)Take out the cloned competent cells from the - 80°C freezer and thaw them on ice.
(2)Take 1 μl of the recombinant product and add it to 100 μl of recipient cells, gently pipette up and down, and ice - bath for 30 minutes.
(3)Heat in a 42°C water bath for 45 seconds.
(4)Immediately cool on ice for 3 - 5 minutes.
(5)Add 200 μl of NZY medium and shake at 37°C, 180 rpm for 1 hour.
(6)Take 100 μl of bacteria and spread on the plate. Invert in a 37°C incubator for 12 - 16 hours.
7.Protein Expression with pCold Plasmid
(1)Transform the pCold plasmid containing the target gene into an Escherichia coli host and culture with shaking at 37°C in a medium containing antibiotics.
(2)Induce protein expression with IPTG (0.1–1.0 mM final concentration) when the culture reaches OD600 = 0.8–1.0, and incubate at 18°C for 16 hours.
8.Plasmid extraction (Plasmid extraction kit)
(1)Column equilibration step: Add 500 μl of equilibration buffer BL to the adsorption column CP3 (the adsorption column is placed in the collection tube), centrifuge at 12,000 rpm (≈13,400 ×g) for 1 min. Discard the waste liquid in the collection tube and put the adsorption column back into the collection tube.
(2)Take 1-5 ml of the overnight-cultured bacterial liquid and add it to a centrifuge tube. Use a conventional benchtop centrifuge to centrifuge at 12,000 rpm (≈13,400 ×g) for 1 min. Aspirate the supernatant as much as possible (when there is a large amount of bacterial liquid, the bacterial pellets can be collected into one centrifuge tube through multiple centrifugations).
(3)Add 250 μl of solution P1 to the centrifuge tube with the bacterial pellet left (please first check whether RNase A has been added). Thoroughly resuspend the bacterial pellet using a pipettor or a vortex mixer.
(4)Add 250 μl of solution P2 to the centrifuge tube and gently invert the tube 6-8 times to fully lyse the bacteria. Note: Mix gently without vigorous shaking to avoid breaking genomic DNA, which may result in genomic DNA fragments mixed in the extracted plasmid. At this time, the bacterial liquid should become clear and viscous, and the process should not take more than 5 min to prevent plasmid damage. If it does not become clear, it may be due to excessive bacteria and incomplete lysis, so the amount of bacteria should be reduced.
(5)Add 350 μl of solution P3 to the centrifuge tube, immediately and gently invert the tube 6-8 times to fully mix, and a white flocculent precipitate will appear at this time. Centrifuge at 12,000 rpm (≈13,400 ×g) for 10 min.
(6)Transfer the supernatant collected in the previous step to the adsorption column CP3 (the adsorption column is placed in the collection tube) using a pipettor. Be careful not to aspirate the precipitate as much as possible. Centrifuge at 12,000 rpm (≈13,400 ×g) for 30-60 sec. Discard the waste liquid in the collection tube and put the adsorption column CP3 into the collection tube.
(7)Add 600 μl of wash buffer PW to the adsorption column CP3 (please first check whether anhydrous ethanol has been added), centrifuge at 12,000 rpm (≈13,400 ×g) for 30-60 sec. Discard the waste liquid in the collection tube and put the adsorption column CP3 into the collection tube.
(8)Repeat step 7.
(9)Put the adsorption column CP3 into the collection tube and centrifuge at 12,000 rpm (≈13,400 ×g) for 2 min to remove the remaining wash buffer in the adsorption column.
(10)Place the adsorption column CP3 in a clean centrifuge tube, add 50 - 100 μl of elution buffer EB to the middle part of the adsorption membrane, leave it at room temperature for 2 min, and centrifuge at 12,000 rpm (≈13,400 ×g) for 2 min to collect the plasmid solution into the centrifuge tube.
9.Genomic modification of EcN (Transformation of λDE3 lysogenization kit)
10.Protoplast preparation
(1)Collect and wash the bacterial cells. Wash them twice with hypotonic Tris-HCl (pH = 7.0, 0.01M), centrifuge at 4000 rpm for 15 minutes, and discard the supernatant.
(2)Measure the wet weight of the bacterial cells.
(3)Take a certain amount of hypotonic Tris-HCl and dilute it until the OD600 is between 0.8 - 1.0.
(4)Suspend the bacterial cells in hypertonic Tris-HCl, add lysozyme to a concentration of 3 mg/ml, and incubate in a 37°C water bath for 50 minutes.
(5)Add EDTA to a final concentration of 10 mM and incubate at 37°C for 20 minutes.
(6)Centrifuge at 4000g for 20 minutes at 4°C and wash three times with hypotonic Tris-HCl.
11.Extrusion by a liposome extruder
Prepare EcN into protoplasts, dilute to OD = 0.8 (CFU = 8×10⁷/mol), wash three times with PBS (pH 7.4), and perform physical extrusion using a liposome extruder from Avanti Company, passing through Whatman polycarbonate membranes with pore sizes of 10 μm, 5 μm, 2 μm, 1 μm, 0.8 μm, 0.6 μm, 0.4 μm, 0.2 μm, and 0.1 μm in sequence.
12.Purification of CL7-sfGFP by Nickel Column
(1)Resuspension: Resuspend the bacterial cells with 30 ml of lysis buffer and add 1/100 PMSF.
(2)High - pressure mechanical cell disruption: At 4°C, wash the instrument with RO water three times, rinse with cell lysis buffer three times, slowly increase the pressure above 800, add the sample, collect the lysate, start cell disruption, repeat 3-4 times until the bacterial lysate is clear, and increase the pressure to 1100-1200.
(3)Sampling: Take the NI-NTA column, wash it three times with 5-fold volume of RO water, then wash it three times with 5 - fold volume of buffer, and finally wash it once with 10 mM imidazole and bind for 3 minutes.
(4)Purify: Pour the supernatant in (2) into the purification column, add imidazole to a final concentration of 10 mM, seal the purification column, and incubate with rotation in a 4°C refrigerator for 1.5 hours.Prepare imidazole gradient solutions of 20 mM, 50 mM, 80 mM, 100 mM, 150 mM, 200 mM, 300 mM, and 500 mM in cell lysis buffer for elution. Take the incubated column for elution in sequence, and take 10 μL of the flow - through liquid and use 150 μL of G250 for color development until the color does not change.
(5)Gel running detection: Prepare a protein gel to detect the purification result.
13.Co - Stain Vesicles with CL7 - sfGFP and Dil
(1)Fix glass slides with 0.01% poly-L-lysine.
(2)Stain vesicle samples with Dil and sfGFP for 30 minutes. Fix with 4% paraformaldehyde, wash with PBS, and mount the slides for imaging.
