Experiment

Experiments

Stage 1: Transformation

Purpose:

  • Transform the pET plasmid with our construct into BL21(DE3) E. coli for later protein expression.

Materials:

  • BL21(DE3) E. coli
  • pET plasmid with contruct (ordered from Twist Bioscience)
  • LB Broth (ThermoFisher, Cat# 12780052)
  • LB agar plates (ThermoFisher, Cat# 22700-025 ) with kanamycin (ThermoFisher, Cat#11815024)

Procedures:

  1. Add 5μL plasmid into a microcentrifuge tube with 100µL of competent BL21(DE3) cells.
  2. Incubate on ice for 20 minutes.
  3. Transfer the tube directly from the ice to the pre-heated 42°C dry bath for exactly 45 seconds.
  4. Immediately return the tubes to ice for 2 minutes.
  5. Add 1ml LB solution in each tube.
  6. Incubate the tube in a 37°C shaker (220 rpm) for 60 minutes.
  7. Centrifuge the tube(15000rpm, 10mins), discharge the supernatant.
  8. Add 100ul LB Broth solution in tube and resuspend the cell pellets
  9. Spread 100 µL of the plasmid transformation culture onto a LB agar plate.
  10. Invert the plates and place them in a 37°C incubator overnight

Stage 2: Verification of Plasmid Construction

Purpose:

  • To isolate the plasmid from transformed E. coli and confirm its identity by releasing a diagnostic DNA fragment via restriction enzyme digestion.

Part 1: Plasmid DNA Extraction

Materials:

  • LB Broth (ThermoFisher, Cat# 12780052)
  • Kanamycin (ThermoFisher, Cat#11815024)
  • GeneJET Plasmid Miniprep Kit(ThermoFisher, Cat# K0502)
  • Transformed E. coli colonies on LB agar plate with Kanamycin

Procedures:

  1. Starter Culture: Using a sterile pipette tip, pick a single, well-isolated colony from the transformation plate. Inoculate it into a culture tube containing 5 mL of LB liquid medium supplemented with the appropriate antibiotic (Kanamycin, 50 µg/mL).
  2. Overnight Incubation: Incubate the tube at 37°C for 24 hours with vigorous shaking (≥ 220 rpm).
  3. Cell Harvesting: Pellet the bacterial cells by centrifuging 5mL of the overnight culture at 12,000 × g for 1 minute. Carefully pour off the supernatant.
  4. Resuspension: Resuspend the pelleted cells completely in 250 µL of Resuspension Solution by pipetting or vertexing. Ensure no cell clumps remain.
  5. Lysis: Add 250 µL of Lysis Solution. Mix gently by inverting the tube 4-6 times until the solution becomes viscous and clear. Do not vortex, as this will shear genomic DNA. Do not allow lysis to proceed for more than 5 minutes.
  6. Neutralization: Add 350 µL of Neutralization Solution. Mix immediately and thoroughly by inverting the tube 4-6 times until a white precipitate forms.
  7. Clarification: Centrifuge at 12,000 × g for 5 minutes. The white precipitate (cell debris and denatured proteins) will form a tight pellet.
  8. Column Binding: Carefully transfer the supernatant (cleared lysate) to the GeneJET Miniprep Column. Centrifuge at 12,000 × g for 1 minute. Discard the flow-through.
  9. Washing: Add 500 µL of Wash Solution (Buffer W1) to the column. Centrifuge at 12,000 × g for 1 minute. Discard the flow-through.
  10. Additional Washing: Add 750 µL of Wash Solution (Buffer W2, ensure ethanol has been added) to the column. Centrifuge at 12,000 × g for 1 minute. Discard the flow-through.
  11. Final Spin: Centrifuge the empty column for an additional 1 minute to remove residual ethanol.
  12. Elution: Transfer the column to a clean 1.5 mL microcentrifuge tube. Add 50 µL of Nuclease-Free Water (pre-warmed to 65-70°C can increase yield) directly to the center of the column membrane. Let it stand for 2 minutes, then centrifuge at 12,000 × g for 2 minutes to elute the purified plasmid DNA.

Part 2: Diagnostic Restriction Digest

Materials:

  • SphI (FD2164) & NcoI (FD0574) (Thermo Scientific, Cat#B49)
  • Kanamycin (ThermoFisher, Cat#11815024)
  • Purified plasmid DNA from Part 1

Procedures:

  1. Prepare the following reaction mix on ice:
  2. Mix the components gently by pipetting and collect the liquid at the bottom of the tube by brief centrifugation.
  3. Incubate the reaction mixture at 37°C for 30 minutes in a thermal cycler or water bath.

Part 3: Agarose Gel Electrophoresis

Materials:

  • 1X TAE Buffer (Thermo Scientific, Cat#B49)
  • Agarose powder (ThermoFisher, Cat# R0491)
  • DNA ladder (ThermoFisher, Cat# 10787026)
  • SYBR safe DNA stain (ThermoFisher, Cat# S33102)

Procedures:

  1. Prepare the Gel: Prepare a 1% (w/v) agarose solution by dissolving 0.5 g of agarose in 50 mL of 1X TAE buffer. Microwave to dissolve completely, let it cool slightly, add DNA stain, and pour into a gel casting tray with a well comb.
  2. Load the Samples: Once the gel has solidified, place it in the electrophoresis chamber filled with 1X TAE buffer. Carefully load the samples.
  3. Run the Gel: Run the gel at 100 V for 30 minutes, or until the dye front has migrated sufficiently through the gel.

Stage 3: Optimization of IPTG Induction and Expression Duration

Purpose:

  • To determine the suitable concentration of IPTG for inducing protein expression.
  • To establish the ideal duration of induction for maximum signal and sensitivity.

Materials:

  • BL21(DE3) E. coli
  • pET plasmid- glyphosate-sensitive aptamer-EGFP (ordered from Twist Bioscience)
  • Glyphosate
  • IPTG (ThermoFisher, Cat#34060)
  • LB Broth (ThermoFisher, Cat# 12780052)
  • LB agar plates (ThermoFisher, Cat# 22700-025) with kanamycin (ThermoFisher, Cat#11815024)

Procedures:

  1. Following the protocol from Stage 1, transform the pET plasmids (glyphosate-sensitive aptamer-EGFP) into BL21(DE3) competent cells respectively. For each transformed BL21(DE3), proceed with the following steps separately.
  2. Inoculate a single colony of each transformed BL21(DE3) into 5 mL of LB medium containing kanamycin.
  3. Incubate the starter cultures overnight at 37°C with shaking at 220 RPM.
  4. Dilute 0.6 mL of each overnight starter culture into 60 mL of fresh, kanamycin-supplemented LB medium.
  5. Incubate the cultures at 37°C with shaking at 220 RPM.
  6. Monitor the OD600 every 30 minutes. When the OD600 reaches 0.5–0.6, proceed to the next step.
  7. Aseptically split each culture into 11 separate tubes, each containing 5 mL culture, using 15 mL conical centrifuge tubes.
  8. Label the tubes from 1 to 11. Add IPTG and glyphosate to each tube according to the concentrations specified in Table 1 below.

    9. Table 1: IPTG and glyphosate concentration in different set-up in step 3 experiment.

  9. Induce protein expression by incubating all tubes at 30°C with shaking at 220 RPM for 30 hours.
  10. Transfer 1mL from each tube to a microcentrifuge tube at different timeslot (12hrs, 24hrs and 36hrs after the expression). Analyze the results using the designated hardware (Smartbox) and software (PestiGuard biosensor platform).

Stage 4a: Glyphosate-binding Aptamer Specificity Testing

Purpose:

  • Validate the specificity of each pesticide-specific aptamers.

Materials:

  • pET plasmid with glyphosate-binding aptamers with EGFP gene (ordered from Twist Bioscience)
  • LB Broth (ThermoFisher, Cat# 12780052)
  • LB agar plates (ThermoFisher, Cat# 22700-025) with kanamycin (ThermoFisher, Cat#11815024)
  • Glyphosate
  • IPTG (ThermoFisher, Cat#34060)

Procedures:

  1. Following the protocol from Stage 1, transform the pET plasmids (glyphosate-binding aptamer) into BL21(DE3) competent cells respectively. For each transformed BL21(DE3), proceed with the following steps separately.
  2. Inoculate a single colony of each transformed BL21(DE3) into 5 mL of LB medium containing kanamycin.
  3. Incubate the starter cultures overnight at 37°C with shaking at 220 RPM.
  4. Dilute 0.5 mL of each overnight starter culture into 50 mL of fresh, kanamycin-supplemented LB medium.
  5. Incubate the cultures at 37°C with shaking at 220 RPM.
  6. Monitor the OD600 every 30 minutes. When the OD600 reaches 0.5–0.6, add IPTG to reach suitable concentration (based on the results from stage 3) and proceed to the next step.
  7. Aseptically split each culture into 9 separate tubes, each containing 5 mL culture, using 15 mL conical centrifuge tubes.
  8. Label the tubes from 1 to 9. Add corresponding pesticide to each tube according to the concentrations specified in Table 2 below.

    9. Table 2: The concentration of pesticide in each set-up

  9. Induce protein expression by incubating all tubes at 30°C with shaking at 220 RPM for a suitable duration (based on the results from stage 2).
  10. Transfer 1 mL from each tube to a microcentrifuge tube and measure OD600 of each tube.
  11. Analyze the results using the designated hardware (Smartbox) and software (PestiGuard biosensor platform).
  12. Repeat step 1 – step 10 two times to get total three replicates for each pesticide.

Stage 4b: Other Pesticide-binding Aptamers Specificity Testing

Purpose:

  • Validate the specificity of each pesticide-specific aptamers.

Materials:

  • Four different pET plasmid with acephate, malathion, chlorpyrifos and acetamiprid aptamers with EGFP gene respectively (ordered from Twist Bioscience)
  • LB Broth (ThermoFisher, Cat# 12780052)
  • LB agar plates (ThermoFisher, Cat# 22700-025) with kanamycin (ThermoFisher, Cat#11815024)
  • Four different pesticides: acephate, malathion, chlorpyrifos and acetamiprid
  • IPTG (ThermoFisher, Cat#34060)

Procedures:

  1. Following the protocol from Stage 1, transform the four pET plasmids (acephate, malathion, chlorpyrifos and acetamiprid) into BL21(DE3) competent cells respectively. For each transformed BL21(DE3), proceed with the following steps separately.
  2. Inoculate a single colony of each transformed BL21(DE3) into 5 mL of LB medium containing kanamycin.
  3. Incubate the starter cultures overnight at 37°C with shaking at 220 RPM.
  4. Dilute 0.5 mL of each overnight starter culture into 50 mL of fresh, kanamycin-supplemented LB medium.
  5. Incubate the cultures at 37°C with shaking at 220 RPM.
  6. Monitor the OD600 every 30 minutes. When the OD600 reaches 0.5–0.6, add IPTG to reach suitable concentration (based on the results from stage 3) and proceed to the next step.
  7. Aseptically split each culture into 9 separate tubes, each containing 5 mL culture, using 15 mL conical centrifuge tubes.
  8. Label the tubes from 1 to 9. Add corresponding pesticide to each tube according to the concentrations specified in Table 2 below.

    9. Table 2: The concentration of pesticide in each set-up

  9. Induce protein expression by incubating all tubes at 30°C with shaking at 220 RPM for a suitable duration (based on the results from stage 2).
  10. Transfer 1 mL from each tube to a microcentrifuge tube and measure OD600 of each tube.
  11. Analyze the results using the designated hardware (Smartbox) and software (PestiGuard biosensor platform).
  12. Repeat step 1 – step 10 two times to get total three replicates for each pesticide.

Stage 5: Control Experiment – Validating the Aptamer Mechanism

Purpose:

  • Rule out false positives.

Materials:

  • pET plasmid with reporter gene but no aptamer (ordered from Twist Bioscience)
  • LB Broth (ThermoFisher, Cat# 12780052)
  • LB agar plates (ThermoFisher, Cat# 22700-025 ) with kanamycin (ThermoFisher, Cat#11815024)
  • Pesticides with ideal results from stage 4 experiment
  • IPTG (ThermoFisher, Cat#34060)

Procedures:

  1. Following the protocol from Stage 1, transform the pET plasmid with reporter gene but no aptamer into BL21(DE3) competent cells.
  2. Inoculate a single colony of each transformed BL21(DE3) into 5 mL of LB medium containing kanamycin.
  3. Incubate the starter cultures overnight at 37°C with shaking at 220 RPM.
  4. Dilute 1mL of each overnight starter culture into 100mL of fresh, kanamycin-supplemented LB medium.
  5. Incubate the cultures at 37°C with shaking at 220 RPM.
  6. Monitor the OD600 every 30 minutes. When the OD600 reaches 0.5–0.6, add IPTG to reach suitable concentration (based on the results from stage 2) and proceed to the next step.
  7. Aseptically distribute 5 mL of each culture into separate 15 mL conical centrifuge tubes. The number of tubes required is determined by the experimental results from Stage 4.
  8. Label the tubes with correct number. Add corresponding pessticide to each tube according to the concentrations specified in Table 3 below.

    9. Table 3: Concentration of pesticides in different control set-ups

  9. Induce protein expression by incubating all tubes at 30°C with shaking at 220 RPM for a suitable duration (based on the results from stage 2).
  10. Transfer 1 mL from each tube to a microcentrifuge tube. Analyze the results using the designated hardware (Smartbox) and software (PestiGuard biosensor platform).

Stage 6: Food Sample Validation

Purpose:

  • Validate the biosensor’s performance by testing it on real food samples with known pesticide concentrations.

Materials:

  • Vegetables (from supermarket)
  • Transformed BL21(DE3) with pET plasmid- pesticide sensitive aptamer
  • LB Broth (ThermoFisher, Cat# 12780052)
  • LB agar plates (ThermoFisher, Cat# 22700-025 ) with kanamycin (ThermoFisher, Cat#11815024)
  • Pesticides
  • IPTG (ThermoFisher, Cat#34060)

Procedures:

  1. Obtain various vegetable samples from local markets.
  2. Separate each vegetable type into two groups: an experimental group and a control group.
  3. Spray the experimental group vegetables with a 3mg/L and 10 mg/L pesticide solution four times respectively. Do not apply any pesticide to the control group.

    4. Figure 1: Spray pesticide on samples

  4. Weigh 2 g of sample from both the experimental and control groups.
  5. Mince the samples and immerse each in 20 mL of distilled water in a separate beaker for 10 minutes.

    6. Figure 2: The flowchart of vegetable sample preparation

  6. Transfer 1 mL of the water extract from each beaker into labelled 1.5 mL microcentrifuge tubes.
  7. Repeat steps 1-6 from stage 3 experiment to prepare BL21(DE3) culture with 0.5 OD600.
  8. Aseptically aliquot 5 mL of the BL21(DE3) culture into separate 15 mL conical tubes. Prepare one tube for each water extract sample.
  9. Add 1 mL of a water extract (from either the experimental or control group) to each corresponding BL21(DE3) culture tube.
  10. Induce protein expression by incubating the tubes at 30°C with shaking at 220 RPM for the optimal duration, as determined in Stage 2.
  11. Transfer 1 mL from each reaction tube to a microcentrifuge tube. Analyze the samples using the designated hardware (Smartbox) and software (PestiGuard biosensor platform).

Stage 7: Soil Sample Screening

Purpose:

  • Validate the biosensor's performance by testing it on soil and environmental samples.

Materials:

  • Soil sample (from organic farm)
  • Transformed BL21(DE3) with pET plasmid- pesticide sensitive aptamer
  • LB Broth (ThermoFisher, Cat# 12780052)
  • LB agar plates (ThermoFisher, Cat# 22700-025 ) with kanamycin (ThermoFisher, Cat#11815024)
  • Pesticides
  • IPTG (ThermoFisher, Cat#34060)

Procedures:

  1. Weigh 2g of soil sample
  2. Transfer soil sample to a beaker containing 20ml of distilled water and stir for 10 min
  3. Filter the mixture and obtain the filtrate
  4. Transfer 1ml of water sample into 1.5ml tubes for further treatment

Stage 8: Cell-free Protein Synthesis Kit application

Purpose:

  • Apply our gene construct in a cell-free protein synthesis kit.

Materials:

  • Cell-free Protein Synthesis Kit (BioSharp Life Science)
  • Plasmid construct (Ordered from Twist Bioscience)

Procedures:

  1. Incorporate the insert gene into the PJL1-sfGFP plasmid from the kit by digestion, gel extraction and ligation.
  2. Add different reagents based on the menu from the kit. Table 4 below shows the proportion of different reagents.

    3. Table 4: The proportion of different reagents added in cell-free protein synthesis kit

  3. Induce protein expression by incubating the tubes at 30°C for 8hours.
  4. Analyze the samples using the designated hardware (Smartbox) and software (PestiGuard biosensor platform).