PROTOCOL

The specific procedures of our experiments


Molecular construction experiments

  1. PCR system
    Material dosage
    Primer F 2µL
    Primer R 2µL
    Template 1µL
    ddH2O 20µL
    2× Phanta Mix 25µL
  2. Put the sample into PCR machine, set the parameters:
    Temperature Time Number of times
    95℃ 3min ×1
    95℃ 15min ×n
    67℃ 15min
    72℃ 1Kb/10s
    72℃ 5min ×1
    4℃ infinite ×1
  3.            n=34 for vector-PBE2R and RBS2-F3’H;n=30 for RBS1-FNS1

E.coli

  1. The recipient bacterium (BL21 (DE3)) was inoculated the night before, and a single colony was selected and cultured in LB liquid medium in a shaking bed at 37℃ overnight (about 16 hours).
  2. Transfer 1ml of overnight culture to 100ml LB (1:100) medium, and violently shake culture on a shaking table at 37℃ for about 2.5-3 hours (250-300 RPM) until OD600 is 0.4-0.6.
  3. When the OD600 value of the cultured bacteria reaches 0.4-0.6, collect the bacteria by centrifugation at 2000×g and 4°C for 5 minutes(When the bacterial volume before centrifugation and precipitation is 50 mL, proceed with the subsequent operations as instructed; for other volumes, perform the subsequent operations after proportional conversion).
  4. Resuspend the bacterial pellet with 5 mL of pre-chilled LB medium, add 5 mL of competent cell preparation reagent that has been thawed in an ice bath or ice-water bath, mix well, and incubate on ice for 5 minutes.
  5. Mix gently, then aliquot appropriately into 1.5 mL or 0.5 mL centrifuge tubes as needed for the experiment, and store at -70°C.

Preparation of E. coli Electrocompetent Cells

  1. Pick a single colony of Escherichia coli and culture it overnight in SOC recovery medium.
  2. Inoculate the overnight culture into LB medium at a ratio of 1:200 and incubate with shaking until the OD₆₀₀ reaches approximately 0.6.
  3. Incubate on ice for 10 minutes, then centrifuge at 4000 rpm at 4 °C for 10 minutes and discard the supernatant.
  4. Resuspend the pellet in 10% glycerol, centrifuge at 4000 rpm at 4 °C for 10 minutes and discard the supernatant.
  5. Repeat step 4 (the resuspension volume can be reduced).
  6. Resuspend the pellet in 10% glycerol at a ratio of 1:400, aliquot 80 μL per tube, and store at −80 °C.

Preparation of Super Competent Bacteria

  1. When the OD₆₀₀ of the cultured bacteria reaches 0.6–0.7, place the bacterial culture in an ice bath and cool for 15 minutes. Note: All subsequent operations must be performed at 4 °C or in an ice bath.
  2. Centrifuge at 4000 g at 4 °C for 5 minutes to collect the bacteria, and discard the supernatant. Note: The centrifuge must be pre-cooled.
  3. If the bacterial volume before centrifugal precipitation is 50 mL, proceed with the subsequent operations. If it is another volume, convert it proportionally before proceeding with the subsequent operations.
  4. Gently resuspend the bacterial pellet with 20 mL of pre-cooled Super Competent Cell Preparation Reagent A. Be sure to resuspend very gently and slowly; otherwise, the effect will be affected.
  5. Ice bath for 15 minutes.
  6. Centrifuge at 4000 g at 4 °C for 5 minutes to collect the bacteria, and discard the supernatant. Note: The centrifuge must be pre-cooled.
  7. Gently resuspend the bacterial pellet with 2 mL of pre-cooled Super Competent Cell Preparation Reagent B. Be sure to resuspend very gently and slowly; otherwise, the effect will be affected.
  8. Ice bath for 15 minutes.
  9. Aliquot on ice, and you can appropriately aliquot into 50–200 μL per tube as needed.
  10. Use immediately or quick-freeze in liquid nitrogen or an ethanol–dry ice bath and store at −70 °C. Usually, the efficiency will not change significantly within 6 months.

Bacillus subtilis

  1. Targeted training overnight.
  2. Pick monoclonal colonies from the plate with gun muzzle and drive it in 3mL LB cultural medium. And then culture it in the environment of 37℃、200rpm overnight.
  3. Prepare cultural medium one:
    Reagent Volume
    Competent cell preparation reagent A 5mL
    Competent cell preparation reagent B 0.625mL
    Competent cell preparation reagent C 0.5mL
    Competent cell preparation reagent D 0.5mL
    Sterile ultrapure water 43.375mL
    Prepare cultural medium two:
    Reagent Volume
    Cultural medium one 39.6mL
    Competent cell preparation reagent E 0.4mL
    1. Get 80uL bacterial solution and put it into 4mL cultural medium one and culture it in the environment of 37℃、220rpm till the number of OD600 reaches 0.6 and is lower than 0.8.(about 4 to 5h)
    2. Put the above solution into 36mL cultural medium two and culture it in the environment of 37℃、100rpm for 90 min.
    3. Add 8uL Competent cell preparation reagent F and accurately culture it in the environment of 37℃、100rpm for 10min.
    4. Centrifuge under the condition 4℃、4000g for 3min and reserve 4mL the above solution. And then re-suspend.
    5. Package at 200uL/tube.
  1. We use 1% agarose gel so we add 2g agarose per 100mL 1×TAE.
  2. After adding agarose we need, heat with microwave until the solution become apparently.
  3. When the temperature of the solution get lower, add 1/10000 nucleic dye.
  4. Pour into the plate with fixed comb and when the gel coagulate, unplug the comb.
  5. Add the samples to the well.
  6. Set the voltage to 120V and run the electrophoresis for 25-35 minutes.
  7. To checking the results, the location of the stripe is determined by shining a UV torch(don’t shine for long time) on the gel.
  1. Add the 10 µL plasmid into 50-100 µL competent bacterium.(Note that don't touch the beneath part of the tube by hand)
  2. Put the bacterium at ice for 30 min.
  3. Bathe the tube in 42℃ water for 90s(for reconstituted product is 60s), and transfer it to the ice immediately to bathe for 2 min(for reconstituted product is 5 min) .
  4. Add 500-750µL non-antibiotics liquid LB medium.
  5. Place the bacteria in a 37℃ shaker for 45-50 min.
  6. Centrifuge in 4000 rpm for 1-2 min.
  7. Pour the supernatant liquid into the remaining 50-100uL and resuspend precipitation with remaining supernate.
  8. Inoculated the remaining liquid onto solid medium containing certain antibiotics and incubated at 37℃ in an incubator.
  1. Put the electrospinning cup on the ice and SOC cultural medium at the environment of 37℃ before the electrical transformation starts.
  2. Add 10ng plasmids into 80µL electrically competent cells. Pay attention not to make any bubble.
  3. Open the electrical transformation equipment, and set the parameters for 1.8kv, 200Ω, 0.5ms, 1mm electroporation cuvette.
  4. If there does not occur any electric spark, then add the transformed cells to SOC cultural medium for 1h for revival.
  1. Harvest 1 - 5 ml overnight cultures of bacteria cells in LB (Luria-Bertani) medium by centrifuging at 12,000 rpm for 30s, discard the medium. Fully suspend pelleted bacterial cells in 250 µL Buffer I with addition of RNase A.
  2. Add 250 µL Buffer I and mix thoroughly by gently inverting the tube 4 - 6 times.
  3. Add 350 µL Buffer N8, mix gently and thoroughly by inverting the tube until the color of precipitate changes from blue to light yellow completely.
  4. Centrifuge at full speed 12,000 rpm for 2 min.
  5. Place a spin column in a 2 ml collection tube (provided), transfer the supernatant into the spin column, close the lid and centrifuge at 12,000 rpm for 30 s.
  6. Discard the filtrate. Place the spin column back to the collection tube. Add 800 µL Buffer W2 to the spin column. Close the lid and centrifuge at 12,000 rpm for 30 s.
  7. Discard the filtrate. Place the spin column back to the collection tube. Centrifuge at full speed 12,000 rpm for 1 min.
  8. Discard the collection tube and place the spin column in a new 1.5 ml microcentrifuge tube. To elute DNA, add 60~100 µL Buffer E to the center of the column, close the lid, let stand for 1 min, and centrifuge at 12,000 rpm for 30 s.
  9. Discard the spin column.
  1. Prepare the following reaction system in an ice-water bath:
    Component Reaction system Negative control c Positive control d (if necessary)
    DNA Assembly Mix 5 μl 5 μl 5 μl
    Linearized carrier a 50~200 ng 50~100 ng pUC19 Control Plasmid, Linearized, 1 µl
    Inserted fragment b 10~200 ng 500 bp Control, Fragment, 1 µl
    ddH2O To 10 µl
  2. Place the reaction system at 50℃ and incubate for 5–15 minutes. 
  3. Place the centrifuge tube containing the reaction solution on ice to cool it down, followed by either performing transformation or storing it at -20℃
  1. Cut the gel as thin as possible and put it in 1.5mL tubes, add 500 µL of Buffer GL in kit, and place the gel in a 65 ℃ water bath to dissolve for 4-6 min.
  2. Transfer the liquid to the Spin column and then centrifuge at 12,000 rpm for 1 min.
  3. Discard the solution that is in the collection tube.
  4. Add 700 µL Buffer W2 (in kit), and centrifuge at 12,000 rpm for 1 min, then discard the solution that is in the collection tube.
  5. Repeat step 4.
  6. Discard liquid, centrifuge the empty column at 12,000 rpm for 2 min.
  7. Discard the collection tube and put the column into a new 1.5 mL tube, then open the lid for 2-3 min to remove ethanol in the adsorption membrane.
  8. Add 35 µL eluent solution (in kit) preheated at 65 ℃ to the center of the membrane, and stand it for 2-3 min.
  9. Centrifuge at 12,000 rpm for 2 min.
  10. Pipette the liquid back to the column, centrifuge at 12,000 rpm for 2 min.
  11. Discard the column, measure the concentration and purity.
  1. Briefly centrifuge the PCR product, enzymatic reaction solution, or crude DNA product (including genomic DNA). Measure its volume with a pipette and transfer it to a sterile 1.5 ml or 2 ml centrifuge tube. If the sample volume is less than 100 µL, make it up to 100 µL with sterile water. For high-concentration genomic DNA, it is better to dilute it to 300 µL with sterile water to improve the recovery efficiency.
  2. Add 5 times the volume of Buffer GDP, invert or vortex to mix well. If it is necessary to recover DNA fragments less than 100 bp, add an additional 1.5 times the volume (of the sample + Buffer GDP volume) of anhydrous ethanol.
  3. Place the adsorption column into the collection tube. Transfer ≤700 µL of the solution to the adsorption column. Centrifuge at 12,000 rpm (13,800 × g) for 30 - 60 seconds. If the volume of the mixture is >700 µL, place the adsorption column back into the collection tube, transfer the remaining solution to the adsorption column, and centrifuge at 12,000 rpm (13,800 × g) for 30 - 60 seconds.
  4. Discard the filtrate and place the adsorption column into the collection tube. Add 700 µL of Buffer GW (to which anhydrous ethanol has been added) to the adsorption column. Centrifuge at 12,000 rpm (13,800 × g) for 30 - 60 seconds.
  5. Please add Buffer GW along the periphery of the adsorption column wall, or cap the tube and invert it 2 - 3 times after adding Buffer GW to help completely wash off the salts adhering to the tube wall.
  6. Repeat step 5.
  7. Washing with Buffer GW twice ensures that salts are completely removed, eliminating their impact on subsequent experiments.
  8. Discard the filtrate and place the adsorption column into the recovery tube. Centrifuge at 12,000 rpm (13,800 × g) for 2 minutes.
  9. Place the adsorption column into a sterile 1.5 ml centrifuge tube, add 20 - 30 µL of Elution Buffer to the center of the adsorption column, and let it stand for 2 minutes. Centrifuge at 12,000 rpm (13,800 × g) for 1 minute. Discard the adsorption column and store the DNA at -20℃.


Molecular characterization experiment

  1. Installation of the glue maker and template tank
    • Take one long glass plate and one short glass plate, align the left, right and bottom ends, and insert the short glass plate into the template groove with the short glass plate facing inward.
    • Insert a slanting plate on the outside of the long glass plate to fix it.
    • Place the above template slot on the gel maker and clamp it tightly, check the leakage with water.
  2. Preparation of the gel
    • Prepare the lower layer of gel: 4mL of lower layer of gel solution + 4mL of lower layer of gel buffer + 80µL of improved coagulant promoter, mix well, take appropriate amount of the lower layer of gel and inject it into the slit of the glass plate, with the liquid level in the vicinity of the white crossbar.
    • Prepare upper layer gel: 1mL of upper layer glue solution + 1mL of upper layer glue buffer + 20µL of improved coagulant promoter, mix well, take the appropriate amount of upper layer glue and inject it into the slit of the glass plate, with the liquid level equal to the upper part of the short glass plate.
    • Insert a comb, try to avoid air bubbles at the upper sample hole, remove the comb after the gel is solidified, remove the template tank from the glue maker and put it into the electrophoresis tank.
  3. Mix 400mL UP water with 100mL 5× SDS-PAGE solution to get the electrode buffer, add the electrode buffer to the template tank, and the liquid level inside and outside the template tank should be equal.
  4. Protein Sampling
    • Centrifuge the sample at 8,000 rpm for 5 minutes.
    • Discard the supernatant.
    • Add 500 μL of PBS to the centrifuge tube and resuspend the sample.
    • Pipette 10 μL of the sample into an EP tube, and place the tube on an ice box.
    • Add 40 μL of instant protein loading buffer (5×).
    • Place the tube in a metal bath heater and heat at 100°C for 10 min.
    • Centrifuge the heated sample at 12,000 rpm for 2 minutes.
  5. Turn on the electrophoresis machine, the mode is constant voltage, 120V for 65min, the current is 1200mA.
  6. After electrophoresis is completed, place the gel in a box containing Coomassie Blue Staining Solution, shake in the shaker and stain for 30min.
  7. The stained gel was rinsed several times with tap water until the bottom color of the gel became transparent.
  1. Make SDS-PAGE gel for detected protein.
  2. Membrane transfer
    • Prepare membrane transfer: 5× Transfer Buffer 200 mL + 200 mL Methanol + 600 mL UP water.
    • Pour the membrane transfer buffer onto a white porcelain plate and a flat dish, and prepare filter paper and cotton in the same size as the size of the clamp. Prepare three pieces of filter paper on each side of the clamp and soak them in the buffer together.
    • Cotton is placed on the black clamp surface, above the three layers of filter paper in turn, there can be no air bubbles between the filter paper, use the roller to roll a few circles to discharge the air bubbles.
    • After electrophoresis is completed, remove the template tank, pry up the glass plate with a spatula, and cut away the upper layer of glue. Put the glue into the buffer and transfer it to the filter paper.
    • Take the NC membrane and soak it in methanol for 30 seconds.
    • Carefully put the membrane into UPH2O and soak for 2 minutes.
    • Carefully put the membrane into transfer buffer and equilibrate for at least 5 minutes.
    • Put the membrane on the adhesive (if the membrane is smaller than the adhesive, just put the membrane on the target strip position), the membrane is near the white clip part, the adhesive is against the black color.
    • Put three layers of filter paper on the membrane to drive out air bubbles.
    • Put another layer of filter paper, clip in buffer, put into the electrophoresis tank (pay attention to the direction of the electrode, black to black).
    • Put in the WB exclusive ice pack, pour in the membrane transfer buffer.
    • Let the electrophoresis tank let that as in the ice box, surrounded by ice packs, and fill the remaining area with water, but do not go over the electrophoresis tank.
    • Electrophoresis instrument set constant current 300mA, transfer membrane for 100min.
  3. Closure
    • Prepare closure solution: 0.1 g BSA + 20 mL TBST, shake well.
    • After electrophoresis, take out the membrane and soak it in the sealing solution, shake for 1 hour.
  4. Primary antibody incubation
    • Discard the containment solution and rinse the membrane and box with 1× TBST.
    • Add 1× TBST to wash the membrane and shake for 10 minutes.
    • Discard the TBST, add the primary antibody. Incubate at 4°C on a shaker overnight.
  5. Secondary antibody incubation
    • Recover primary antibody. Wash with 1× TBST, shake for 10 minutes and wash three times.
    • Wrap the box with tin foil.
    • Add the secondary antibody and shake for 2 hours away from light.
    • Recover secondary antibody. Wash with 1× TBST, shake for 10 minutes, and wash three times, taking care to avoid light.
  6. Developing
    • Use the PerkinElmer EnVision 2103 Multilabel Reader for scanning. First, spray alcohol and wipe the instrument. Then, hold the NC membrane with tweezers and place it on the microplate.
    • Mix the ELISA Femto Luminol and ELISA Femto Stabilizer at a ratio of 1:1 to prepare 400 μL of developing solution. Then, add the developing solution dropwise onto the NC membrane to make it completely cover the membrane.
    • Observe and capture images via the software.
HPLC Conditions
Component Condition
Equipment Agilent 1200LC
HPLC Column TC-C18(2), 4.6 x 250 mm, 5 µm, 400 bar
Mobile Phase A 100% Methanol
Mobile Phase B 0.1% Formic Acid in Water
Mobile Phase Composition A: 60%, B: 40%
Flow Rate 1 mL/min
Detection Wavelength (Naringenin) 285 nm
Detection Wavelength (Luteolin) 350 nm
  1. Culture & Harvest: The engineered BS168 and wild-type BS168 strains were cultured overnight, and the cells were harvested by centrifugation.
  2. Lysate Preparation: A portion of the cell pellet was resuspended and disrupted (e.g., by sonication or bead-beating) to create a crude lysate.
  3. Clarification: The lysate was centrifuged (12,000 × g, 10 min) to remove cell debris, and the resulting supernatant was collected as the enzyme source.
  4. Reaction Setup: The reaction mixture was assembled by combining the supernatant with the substrate and co-factors as detailed in Table X. The final volume was adjusted with an appropriate buffer.
  5. Incubation & Termination: The reaction was incubated at 37°C for 9 hours and then terminated by heat inactivation at 98°C for 10 minutes.
  6. Sample Workup: The terminated reaction was centrifuged (12,000 × g, 10 min) to precipitate denatured proteins.
  7. Analysis: The clarified supernatant was analyzed by HPLC to quantify the reaction products.
  • Preparation of host bacteria
    1. All frozen bacterial stocks were stored at −80 °C in 25% v/v glycerol.
    2. Inoculate 3 mL of bacterial media with glycerol stock in LB media, the inoculated media was incubated at 37 °C and 180 rpm for 16–18 h to promote bacterial growth.
    3. Overnight cultures were subsequently diluted 1:100 in 50 mL of fresh bacterial cell media and incubated for 2–3 h to allow bacterial cells to reach the mid-exponential growth phase.
  • Preparation of phage stocks solution
    PB solution:
    Component Concentration
    NaCl 68 mM
    MgSO4 10 mM
    Tris HCl 10 mM
    pH 7.5
    1. Take 20 mL soil and water samples in 50 mL centrifuge tubes. For the soil sample, add 20 mL phage buffer PB, mix well, and let it stand for a certain period of time.
    2. Centrifuge both the soil and water samples at 5000g for 10 minutes.
    3. Filter through a 0.22μm filter to obtain the phage stocks solution.
  • Preparation of phage concentrated solution
    LB powder: Mix well in advance for rapid preparation of culture medium containing phage, with the ratio of peptone: yeast extract: NaCl = 2:1:2.
    1. Pour 20 mL of the phage stocks solution into a conical flask (usually a 50 mL conical flask), add 0.5 g of LB powder and 20 μl of 2M CaCl2 solution. Filter through a 0.22 μm filter. Inoculate the host bacteria solution at a ratio of 1:200.
    2. Cultivate at 37℃ and 220 rpm for overnight.
    3. Centrifuge at 5000g for 10 minutes to obtain the phage concentrated solution.
  1. Spread 5 ml of solid medium with a 1.5% agar concentration onto the plate.
  2. Mix 5, 50, and 500 μL of phage concentrated solution with 500 μL of host bacteria that are in the logarithmic growth phase, and then combine it with 4.5 mL of solid medium with a 0.7% agar, immediately pour it onto the top layer of the agar plate.
  3. Place the plate in a 37°C incubator for overnight cultivation, and then count the number of plaques at the end.
  1. Mix 500 μL of the host bacteria that are in the logarithmic growth phase with 4.5 mL of 0.7% agar medium, and then pour it onto the 1.5% agar plate.
  2. Centrifuge the phage concentrated solution at 5000g for 10 minutes.
  3. Take the supernatant and perform a 10-fold gradient dilution.
  4. Take 5 μl of the diluted solution and drop it onto the double-layer plate, marking it accordingly.
  5. When the droplets have completely dried, incubate overnight at 37 ℃ in room air, and count the plaques.

Phage concentration (number of plaque forming units per milliliter, PFU/mL) was determined using this technique. Phage stock was serially diluted in PBS and plated on bacteriallawns to enumerate the number of infectious virions present inthe stock.

  1. Dilute the overnight culture of the host bacteria at a ratio of 1:40, add it to the LB medium, and shake for 2 hours to achieve an OD600 value of 0.6 in the bacterial solution.
  2. Take 4 ml of the bacteria, centrifuge at 3000g for 2 minutes, discard the supernatant, and resuspend the bacteria in 1 ml of ddH2O.
  3. Repeat Step 2 three times.
  4. Centrifuge the phage concentrated solution at 5000g for 10 minutes.
  5. Add phage (10^11 pfu/ml) to the bacteria solution, incubate for 30 minutes, and then aspirate 20 μl and drop it onto the copper mesh (dark side up) for Transmission electron microscopy observation.
  1. Phage purification
    • Pick a clear phage spot and draw lines on the 1.5% agar plate.
    • Mix 500 μL of the host bacteria that are in the logarithmic growth phase with 4.5 mL of 0.7% agar medium, and then pour it onto the streaked plate.
    • Place the plate in a 37°C incubator for overnight cultivation, pick out clear plaques and repeat the process once more to isolate a single phage.
  2. Phage enlarged cultivation
    • Pick out clear plaque and elute it in the host bacteria, place the plate in a 37°C incubator for overnight cultivation.
    • Centrifuge at 5000g for 10 minutes to obtain phage stock solution, 4℃ for preservation.
  3. Planktonic activity of phage against host bacteria
    • Centrifuge the phage concentrated solution at 5000g for 10 minutes.
    • Based on the concentration of virus particles in the phage enrichment solution obtained from Spot titer assay, an appropriate amount of supernatant was mixed with the host bacteria with an OD600 value of 1.0, so that the concentration of virus particles in the mixture was 10 times that of the bacteria.
    • Every two hours, the bacterial cell concentration in the mixture was determined using spectrophotometry, with the detection wavelength set at 600 nm.


Caenorhabditis elegans (C. elegans) experiment

1. Amplification of Target Gene - PCR

  1. PCR Reaction System
    Component Volume Added
    Template DNA (Cry5Ba, Cry6Aa) 2µL
    F Primer 2µL
    R Primer 2µL
    ddH2O 19µL
    Mix 25µL
    Total 50µL
  2. PCR Program Settings
    Fig. 1 PCR Parameters (for Cry5Ba)
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2. Electrophoresis

  1. Gel Preparation
    Component Amount
    agarose 0.25g
    10X TAE buffer 2.5ml
    ddH2O Top up to 25mL
  2. Heat the mixture in a microwave, add 2.5μL of nucleic acid dye, stir, pour the hot mixture into a mold, and wait for solidification.
  3. Load the sample into the well and perform gel electrophoresis.
  4. After electrophoresis, observe under UV light, then cut and recover the target band.

3. Gel Extraction and Purification

  1. Add an equal volume of Buffer GDP, and heat in a metal bath until the gel melts.
  2. Place the adsorption column into a collection tube, transfer the solution to the adsorption column, and centrifuge at 12,000 rpm for 1 minute.
  3. Discard the lower solution, place the adsorption column back into the collection tube, add 300μL of Buffer GDP to the adsorption column, and centrifuge.
  4. Discard the lower solution, place the adsorption column back into the collection tube, add 700μL of Buffer GW (with anhydrous ethanol added) to the adsorption column, and centrifuge.
  5. Repeat Step 4 (add 700μL of Buffer GW with anhydrous ethanol again and centrifuge).
  6. Dry the adsorption column and centrifuge to remove residual ethanol.
  7. Add 30μL of ddH₂O for elution, centrifuge, discard the upper solution, and the collected DNA is in the lower collection tube.

4. Homologous Recombination

  1. Measure the concentration of the gene recovered in the previous step, calculate the volumes of the linearized vector and target gene to be added (total volume: 5μL).
  2. Add 5μL of 2× Seamless Cloning Mix.
  3. Incubate in a PCR instrument at 50°C for 30 minutes, then at 12°C for 5 minutes. (Note: There are two tubes of Cry5B gene: one has a concentration of over 50 ng/μL (1.6μL of gene + 3.4μL of vector), and the other has a concentration of 41.65 ng/μL. However, the linearized vector used for the first experiment is suspected to be exhausted, so the ratio must be recalculated in subsequent experiments.)

5. Transformation

  1. Add 10μL of the reaction sample (all contents in the PCR tube) to a tube of competent cells (DH5α or BL21), mix gently, and stand on ice for 30 minutes (in a clean bench).
  2. Perform heat shock in a 42°C water bath for 90 seconds, quickly transfer back to the ice bath, and stand for 3 minutes (to restore the cell membrane).
  3. Add 500μL of LB medium without antibiotics, mix well, and incubate with shaking at 37°C for 45 minutes.
  4. Centrifuge at 4,000 rpm for 1 minute to precipitate bacterial cells (skip centrifugation if the concentration is too high). Aspirate the medium, leaving 100μL of medium, and mix well. Take 30μL (both undiluted and diluted as controls) and spread evenly on LB plates containing kanamycin (kana), then incubate at 37°C.

1. Induction of Protein Expression

  1. Inoculation of Culture Medium: Inoculate 1 mL of overnight engineered bacterial culture (1% inoculum) into 100 mL LB liquid medium. Add 1‰ kanamycin (50 mg/mL stock solution).
  2. Initial Culture: Incubate conical flasks in a 37°C constant-temperature shaking incubator at 220 rpm for approximately 2 hours until the culture OD600 reaches 0.3–0.7 (mid-log phase).
  3. Expression induction: Add 0.5‰ IPTG (1M stock solution) to the conical flask, mix thoroughly, and place in a 16°C constant-temperature shaking incubator. Incubate at 160 rpm for 16–24 hours.

2. Bacterial Lysis and Soluble Expression Validation

  1. Bacterial Density Measurement: Transfer 100 μL of induced bacterial culture into a 1.5 mL EP tube. Dilute 10-fold with 900 μL of ultrapure water and vortex to mix. Use ultrapure water as a blank control. Measure the OD600 of the diluted bacterial suspension using a spectrophotometer and record the actual OD value. Assuming a target OD value of 30, calculate the required volume of Buffer A for resuspension using the following formula.
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  2. Cell Centrifugation and Resuspension: Transfer bacterial suspensions to pre-chilled centrifuge tubes. Top up with ultrapure water to achieve equal weights (weight tolerance ≤0.1 g). Centrifuge at 4°C (6000 rpm for 10 min). Discard the supernatant and retain the bacterial pellet. Resuspend each tube of bacterial pellet with pre-chilled Buffer A at the calculated resuspension volume. Vortex gently until bacteria are uniformly distributed throughout the solution.
  3. Ultrasonic disruption: Perform ultrasonic disruption in an ice bath (parameters: 5 seconds on, 3 seconds off, for 30 minutes at 4°C). Transfer the disrupted sample to a pre-cooled centrifuge tube and store at 4°C for later use.

3. Soluble Expression Validation

  1. SDS-PAGE Electrophoresis: Assemble the vertical electrophoresis chamber, insert the prepared gel plates, and pour 300 mL of Tris electrophoresis buffer into the middle compartment of the chamber. Remove the comb and load samples into the loading wells (loading buffer – marker – each sample – loading buffer). Cover the top lid and connect the leads. Start at 80V. Once samples enter the separating gel, increase the voltage to 120V. Stop electrophoresis when the bromophenol blue band reaches the bottom of the gel.
  2. Staining and Decolorization: Remove the gel blocks after electrophoresis and immerse them in Coomassie Brilliant Blue staining solution. Seal and place in a 60°C oven for staining for 40 minutes. Rinse the gel blocks under running water, then immerse them in decolorizing solution and shake overnight until the gel background becomes transparent. Observe the band distribution under a white light transilluminator and analyze the results.

1. Preparation of LB Solid Medium (100mL)

Component Volume Added
LB Broth 2.5g
Agar 1.5g
ddH2O Top up to 100mL
  1. Dissolve the components by sonication, and wrap the container with parafilm.
  2. Sterilize in an autoclave at 121°C for 15-20 minutes.

2. Plasmid Extraction

  1. Column Equilibration: Add 500μL of Equilibration Buffer BL to Adsorption Column CP4 (placed in a collection tube), centrifuge at 12,000 rpm (~13,400×g) for 1 minute. Discard the waste liquid in the collection tube and put the adsorption column back.
  2. Add 5-15mL of overnight-cultured bacterial solution to a centrifuge tube, centrifuge at 12,000 rpm for 1 minute, and aspirate the supernatant as much as possible.
  3. Add 500μL of Solution P1 to the tube with bacterial pellets, and thoroughly resuspend the pellets using a pipette or vortex oscillator.
  4. Add 500μL of Solution P2, gently invert the tube up and down 6-8 times to fully lyse the bacteria. Do not use a vortex oscillator; lysis time should not exceed 5 minutes.
  5. Add 700μL of Solution P3, immediately invert up and down gently 6-8 times (mix quickly to avoid local precipitation) – white flocculent precipitate will appear. Centrifuge at 12,000 rpm for 10 minutes, and a precipitate will form at the bottom of the tube. (Do not use a vortex oscillator.)
  6. Add the supernatant from Step 5 to Filter Column CS (placed in a collection tube) in batches, centrifuge at 12,000 rpm for 2 minutes. Carefully transfer the filtrate (from the collection tube) to Adsorption Column CP4 (placed in a new collection tube) in batches. If there is residual liquid in the filter column, extend the centrifugation time of Step 5; if there is a small amount of precipitate at the bottom of the collection tube, avoid aspirating it.
  7. Centrifuge Adsorption Column CP4 at 12,000 rpm for 1 minute, discard the waste liquid, and put the column back into the collection tube.
  8. Add 600μL of Washing Buffer PW to the column, centrifuge at 12,000 rpm for 1 minute, discard the waste liquid, and put the column back.
  9. Repeat Step 9 (add 600μL of Washing Buffer PW again and centrifuge), then let the column stand to volatilize residual ethanol.
  10. Put Adsorption Column CP4 back into the collection tube and centrifuge at 12,000 rpm for 2 minutes to remove residual washing buffer.
  11. Transfer the column to a clean centrifuge tube, add 100-300μL of Elution Buffer TB to the center of the adsorption membrane (avoid touching the membrane with the pipette tip), stand at room temperature for 2 minutes, and centrifuge at 12,000 rpm (~13,400×g) for 1 minute to collect the plasmid solution.

3. Toxicity Testing of Obtained Cry5B, Cry6A, and a Series of Fusion Proteins

  1. Survival Analysis
    • Day 1 (D1)
      1. Prepare L4-stage synchronized nematodes and NGM plates (including medium-sized and small-sized plates).
      2. Inoculate a single colony of OP50 E. coli into antibiotic-free LB medium. Culture with shaking at 200 rpm and 37°C for 16 hours. Prepare a fresh culture for each use.
    • Day 2 (D2)
      1. Prepare FUDR-containing OP50 bacterial solution and seed NGM plates:
        1. (1) Dispense 50 μL of 50 mg/mL FUDR into PCR tubes for storage (ensure sterility).
        2. (2) Take 3 mL of freshly cultured OP50 bacterial solution and mix it with 48 μL of 50 mg/mL FUDR (calculate the volume proportion to avoid concentration errors).
        3. (3) Seed 100 μL of the FUDR-OP50 mixture onto each medium-sized NGM plate.
        4. (4) Use a sterilized and cooled spreader to spread the bacterial solution evenly across the plate surface.
        5. (5) Seal the prepared plates with parafilm (to prevent contamination and moisture loss) and incubate at 37°C for 24 hours.
      2. Synchronize nematodes for L4-stage preparation:
        1. (1) For each nematode strain (labeled D1–D2), select 15–25 adult worms (adjust the number based on their egg-laying capacity).
        2. (2) Perform synchronization for 3–4 hours to collect eggs, and remove the parent worms after synchronization (ensure no adult worms remain in the egg culture).
        3. Recommendation: The development from nematode egg to L4 stage takes approximately 56 hours. To avoid delayed picking of L4-stage worms, it is recommended to start synchronization between 10:00 and 14:00 (local time).
    • Day 4 (D4)
      1. Pre-treat L4-stage nematodes with FUDR:
        1. (1) Transfer more than 100 L4-stage synchronized nematodes to the FUDR-seeded NGM plates (prepared on D2).
        2. (2) Incubate the plates at 20°C for 24 hours (this step inhibits nematode egg hatching and ensures only adult worms are used in subsequent toxicity tests).
        3. (3) Adjust the number of nematodes per plate as needed: For small-sized NGM plates, use 30 worms per plate, with 3 replicate plates per protein treatment group (total of n protein solution groups, depending on the number of fusion proteins tested).
      2. Prepare Cry protein-containing OP50 bacterial solution and seed small NGM plates:
        1. (1) Prepare a mixed solution of Cry protein (Cry5B, Cry6A, or fusion protein) and OP50, ensuring the final concentration of the Cry protein is 15 mg/L (verify the concentration with a spectrophotometer if necessary).
        2. (2) Seed 100 μL of the Cry-OP50 mixture onto each small-sized NGM plate.
        3. (3) Spread the solution evenly with a sterilized and cooled spreader.
        4. (4) Seal the plates and incubate at 25°C for 12 hours (to allow the bacterial lawn to form).
        5.   Note: Prepare control plates (seeded with OP50 alone, without Cry protein) simultaneously to eliminate the influence of OP50 on nematode survival.
    • Day 5 (D5)
    • Conduct toxicity treatment and survival counting:
      1. (1) Transfer the FUDR-pretreated nematodes (from D4) to the small NGM plates containing Cry protein (prepared on D4): 30 worms per plate, 3 replicate plates per Cry protein solution.
      2. (2) Incubate all plates at 25°C.
      3. (3) Perform survival counting every 12 hours: Record the number of live nematodes per plate. Continue counting until the survival rate of any group drops below 10% or all nematodes in the group are dead.
      4.   Operation Note: Remove dead nematodes from the plates immediately after counting and incinerate them (to prevent cross-contamination between plates).
    • Example of Survival Counting Table
      strain date strain date
      Date C/D 1 2 3 Date C/D 1 2 3
      / / / / / / / /
      / / / / / / / /
      / / / / / / / /
      / / / / / / / /
      / / / / / / / /
      strain date strain date
      Date C/D 1 2 3 Date C/D 1 2 3
      / / / / / / / /
      / / / / / / / /
      / / / / / / / /
      / / / / / / / /
      / / / / / / / /
      strain date strain date
      Date C/D 1 2 3 Date C/D 1 2 3
      / / / / / / / /
      / / / / / / / /
      / / / / / / / /
      / / / / / / / /
      / / / / / / / /
    •   Note: "C/D" refers to "Censor/Death"; fill in the number of live nematodes in the "/ " blanks.
  2. Median Lethal Concentration (LC₅₀) Determination
  3.   Note: The preparation method for nematode synchronization is consistent with that described in "1) Survival Analysis" (ensure L4-stage synchronization to maintain experimental consistency).
    1. Nematode Culture: Prepare L4-stage larvae of the Caenorhabditis elegans N2 wild-type strain.
    2. The Cry protein was diluted in HEPES buffer to create a concentration gradient (e.g., 10 µM, 5 µM, 2.5 µM, 1.25 µM). For each concentration, a minimum of three replicates were prepared.
    3. Prepare 48-well microtiter plates for toxicity testing: Seed each well of a 48-well microtiter plate with 140 µL of S medium and 40 µL of an E. coli OP50 suspension (OD600 = 0.3), prepared in S medium.
    4. Add FUDR to inhibit nematode reproduction: Add 5 μL of 8×10⁻³ mol/L FUDR to each well
    5. Each well was treated with 10 µL of either the toxin solution or HEPES buffer (which served as the control).
    6. Add 5 µL of L4-stage larvae (approximately 20 larvae resuspended in S medium) to each well.
    7. Wrap the culture plate with moist paper towels, place it in a sealed container to maintain humidity, and incubate at 25°C for 5 days.
    8. Score nematode survival in each well under a microscope. Motile nematodes are scored as alive. For non-motile nematodes, gently prod with a pick; if no response is observed, score them as dead. Record the survival status of at least 100 nematodes per concentration group.
    9. LC50 calculation: Plot the survival rate against the toxin concentration. Determine the LC50 (the toxin concentration that kills 50% of the nematodes) through curve fitting.