Experiment protocol

Preparation of gel extraction reaction system

PCR system

material	dosage
Primer F	2µL
Primer R	2µL
Temaplate	1µL
ddH2O	20µL
2× Phanta Mix	20µL

Put the sample into PCR machine, set the parameters:

Reaction condition

Temperature	Time	Number of times
95℃	3min	×1
95℃	15min	×34
65℃	15min
72℃	1Kb/30s
72℃	5min	×1
4℃	infinite	×1

Preparation of gel extraction reaction system

• Cut the gel as thin as possible and put it in 1.5mL tubes, add an 500μL of Buffer GL in kit, and place the gel in a 65 ℃ water bath to dissolve for 4-6 min.
• Transfer the liquid to the Spin column and then centrifugate at 12000rmp for 1 min.
• Discard the solution that is in the collection tube.
• Add 700μL Buffer W2(in kit), and centrifugate at 12000 rpm for 1 min, then discard the solution that is in the collection tube.
• Repeat step 4.
• Discard liquid, centrifugate the empty column at 12000 rpm for 2 min.
• Discard the collection tube and put the column to a new 1.5mL tube, then open the lid for 2~3 min to remove ethanol in adsorption membrane.
• Add 35μL eluent solution(in kit) preheated at 65 ℃ to the center of the membrane, and stand it for 2-3 min.
• Centrifugate at 12000 rpm for 2 min.
• Pipette the liquid back to the column, centrifugate at 12000 rpm for 2 min.
• Discard the column, measure the concentration and purity.

• We use 2% agarose gel so we add 2g agarose per 100mL 1×TAE.
• After adding agarose we need, heat with microwave until the solution become apparently.
• When the temperature of the solution get lower, add 1/10000 nucleic dye.
• Pour into the plate with fixed comb and when the gel coagulate, unplug the comb.
• Mix with the equal volume 2×loading buffer(needn’t loading buffer if use the product of PCR),add the samples to the well.
• Set the voltage to 120V and run the electrophoresis for 25-35 minutes.
• 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.

• Add the 10 µL plasmid into 100 µL competent bacterium.(Note that don't touch the beneath part of the tube by hand)
• Put the bacterium at ice for 30 min.
• Bathe the tube in 42℃ water for 30-60s, and transfer it to the ice immediately to bathe for 2 min.
• Add 500-750µL non-antibiotics liquid LB medium.
• Place the bacteria in a 37℃ shaker for 1h.
• Centrifuge in 7000-8000 rpm for 2-3 min.
• Dump the supernatant and resuspend precipitation with remaining supernate.
• Inoculated the remaining liquid onto solid medium containing certain antibiotics and incubated at 37℃ in an incubator.

• 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 ul Buffer I with addition of RNase A.
• Add 250 ul Buffer I and mix thoroughly by gently inverting the tube 4 - 6 times.
• Add 350 ul Buffer N8, mix gently and thoroughly by inverting the tube until the color of precipitate changes from blue to light yellow completely.
• Centrifuge at full speed 12,000 rpm for 2 min.
• 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.
• Discard the filtrate. Place the spin column back to the collection tube. Add 800 ul Buffer W2 to the spin column. Closed the lid and centrifuge at 12,000 rpm for 30 s.
• Discard the filtrate. Place the spin column back to the collection tube. Centrifuge at full speed 12,000 rpm for 1 min.
• Discard the collection tube and place the spin column in a new 1.5 ml microcentrifuge tube. To elute DNA, add 60~100 ul 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.
• Discard the spin column.

• 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).
• 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.
• Put 0.1M CaCI2 solution on ice for pre-cooling ; The following steps should be performed on clean bench and ice.
• Absorb 1.5ml cultured bacterial solution into 1.5ml centrifuge tube and cool it on ice for 10 minutes.
• Centrifuge for 5 min at 3000rpm at 5.4℃.
• Discard the supernatant, add 100wl of pre-cooled 0.1M CaCl2 solution, gently suction up and down with a pipette gun to beat well, re-suspend the cells, and place them on ice for 20 minutes.
• Centrifuge for 5 min at 3000rpm at 7.4℃.
• Discard the supernatant, add 100wl of pre-cooled 0.1M CaCl2 solution, gently suction up and down with a pipette gun to beat well, re-suspend the cells.
• The cell suspension can be immediately used for conversion experiment or add cryoprotectant (15%-20% glycerin mixed) and then frozen at ultra-low temperature for storage.

• Configure lysis buffer, wash buffer and elution buffer.



• Filter the supernatant obtained from 2 through a filter membrane.
• Connect the affinity chromatography system: inlet tube - constant flow pump - nickel column upper inlet - nickel column lower outlet - waste tank. Flush with Lysis buffer at constant-flow pump -30 rpm for half an hour, and observe whether the liquid flows out evenly from the mouth of the tube.
• Suspend the constant-flow pump, connect the nickel column outlet to the protein detector, turn on the constant-flow pump, wait for the protein detector to stabilize the number of signals, zero the protein detector.
• Into the liquid for cell crushing fluid, flow rate is adjusted to -10 rpm, the protein detector began to rise when the number of collected flow-through liquid.
• After the sample is added, rinse the inlet port with a wash bottle, and change the injection to Lysis buffer until the protein detector is stable.
• Change the sample to Wash buffer and collect the stray proteins in a 50mL centrifuge tube when the protein tester starts to show a rise until the number stabilizes.
• Change the sample to Elution buffer, use a 5mL centrifuge tube to collect PETase when the protein tester number rises, and change the centrifuge tube every 4mL.
• Put the collected proteins into dialysis bag, prepare 2L of 20mmol/L Tris-HCl buffer as dialysis solution, and dialyze three times to get the purified proteins.

• 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.
• 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 glue: 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.
• 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.
• Protein Sampling
• Place the protein sample on an ice box, take 30µL of protein sample and 7µL of 6×Loading buffer and add it to a 200µL centrifuge tube.
• Centrifuge the sample to the bottom of the tube.
• Place the tube in a metal bath heater and heat at 100°C for 10 min.
• Take 20µL of sample and 10µL of marker and add to the top sample well.
• Turn on the electrophoresis machine, the mode is constant voltage, the first stage is 80V for 20min, the second stage is 140V for 80min, the current is 1200mA.
• After electrophoresis is completed. Place the gel in a box containing Coomassie Blue Staining Solution, shake in the shaker and stain for 30min.
• The stained gel was rinsed several times with tap water until the bottom color of the gel became transparent.

• Cut out the desired length of the dialysis bag.
• Put it into a beaker, add pure water to cover the dialysis bag.
• Microwave for 20 minutes on medium heat until the bag softens.
• Seal one end of the dialysis bag with a sealing clip, add the protein to be purified and then seal the other end with a sealing clip.
• After confirming that there is no leakage, spill p olyethylene glycol on the dialysis bag and place it in the refrigerator at 4℃. When the liquid in the dialysis bag is less than 4mL, collect it into a 5mL centrifuge tube and add purified water to volume to 4mL.

• Make SDS-PAGE gel for detected protein.
• Membrane transfer
• Prepare membrane transfer: 10×Transfer Buffer 100mL + 200mL Methanol + 700mL 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 PVDF 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 360mA, PETase transfer membrane for 30min.
• Detect the efficiency of membrane transfer with Ponceau S: pour Ponceau S into the box, put the membrane in and shuffle it, observe it with naked eyes, add TBS buffer and shake for a few minutes to decolorize it, and then soak the containment solution.
• Closure
• Prepare closure solution: 1g skim milk powder + 20mL TBS shake well.
• After electrophoresis, take out the membrane and soak it in the sealing solution overnight at 4℃.
• Primary antibody incubation
• Discard the containment solution and rinse the membrane and box with 1×TBS.
• Add 1×TBS to wash the membrane and shake for 10min.
• Discard the TBS, add the primary antibody. 4 ℃ shaker incubation overnight.
• Secondary antibody incubation
• Recover primary antibody. Wash with 1×TBS, shake for 10min and wash three times.
• Wrap the box with tin foil.
• Add the secondary antibody and shake for 2h away from light.
• Recover secondary antibody.
• Wash with 1×TBS, shake for 10min, and wash three times, taking care to avoid light.
• Developing
• Scan with a near infrared developer. First spray alcohol to wipe the instrument, use tweezers to clip the NC film, placed above the zero scale line, avoiding the fault area, pay attention not to produce bubbles.
• Open the software draw new, select the range. Select 700nm channel, adjust the contrast, save.

• Add 0.606g of Tris to 60m of pure water to dissolve it completely.
• Clean the electrode of pH meter with pure water.
• Put the electrode into the standard buffer pH = 6.86, the pH meter display to 6.86, remove the electrode, clean the electrode of the pH meter with pure water and then wipe with filter paper.
• If you want to configure alkaline buffer, put the electrode into the standard buffer pH = 9.18, press “linear”, the pH meter display will be adjusted to 9.18; if you want to configure acidic buffer, put the electrode into the standard buffer pH = 4.01, press “linear”, the pH meter display will be adjusted to 9.18; if you want to configure acidic buffer, put the electrode into the standard buffer pH = 4.01, press “linear”, the pH meter display will be adjusted to 6.86, remove the electrode, wash the pH meter electrode with pure water and wipe it with filter paper. linear” and set the pH meter to 4.01.
• Remove the electrode, wash the pH meter electrode with pure water and wipe it with filter paper.
• Deepen the electrode into the Tris solution and add 1 mol/L HCl solution while stirring until the pH reaches a specific value.
• Take out the electrode, clean the electrode of pH meter with pure water, wipe it with filter paper and put it into the electrode protection liquid.
• Pour the solution into a 100mL volumetric flask, and then set the volume with purified water and store it.

• Prepare a substrate solution of PNPB at a concentration of 8 µmol/mL in anhydrous ethanol as a solvent, and wrap it in tin foil to protect it from light.
• Prepare 54 5mL centrifuge tubes, divided into experimental group and control group, set the pH of the buffer as a pH gradient of 2-10.
• Every three centrifuge tubes in the experimental group were added with 1.8mL of Tris-HCl buffer + 100µL PNPB of the same pH value.In the control group, every three centrifuge tubes were filled with 1.8mL of Tris-HCl buffer + 100µL PNPB + 100µL ddH2O at the same pH, and preheated at 65℃ for 5min.
• Add 100µL of PETase to the experimental group and react for 5min.
• Add 500µL TCA to terminate the reaction.
• Take 100µL of liquid and add it into 96-well plate.
• Detect the absorbance at 410nm with a full-wavelength microplate reader.

• Weigh 0.04g of BHET using one over ten thousand of electronic balance.
• Prepare 9 15mL centrifuge tubes, add 10mL of Tris-HCl buffer (pH=2-10) and 0.04g of BHET, and sonicate in an ultrasonic cleaner until BHET is dissolved.
• Prepare 36 2mL centrifuge tubes, of which 27 were for the experimental group and 9 were for the control group, set the pH of the buffer for the pH gradient of 2-10, every three centrifuge tubes in the experimental group were added with 0.9mL of the same pH value of BHET solution, and nine centrifuge tubes in the control group were added with 0.9mL of the different pH value of the BHET solution and 100µL of water, and put in a 65℃ water bath to preheat. Preheat at 65℃ for 5min.
• 100µL of PETase was added to the experimental group, and the reaction was carried out for 1h.
• Add 1mL of methanol to terminate the reaction and place in the refrigerator at 4℃ overnight.
• The next day, the sample was filtered through a membrane and added to the liquid phase vial.
• Place the vial into HPLC or UPLC.

• 1. The protein concentration of two batches of purified PETase samples was determined
• (1) Gradient concentration PBS protein standard product configuration, 96-well plate in order to add standard product, ultra-pure water, diluent, ultra-pure water, configure a series of gradient protein standard products;
• (2) Sample loading: two batches of purified protein samples (the first batch on 7/14 and the second batch on 8/12) were added into the 96-well plate successively;
• (3) The absorbance of A595 was measured with a 96-well plate enzyme-labeled instrument in the instrument platform.
• (4) Standard curve was constructed by analyzing standard protein data, linear/polynomial function relationship of A595 and protein concentration was established, and sample protein concentration was calculated by using calculation tools. The fitting relation R value was compared, and the fitting relation of fourth power polynomial function was better.
• 2. Construction of reaction component standard curve:
• (1) Configure series gradient concentration BHET, TPA standard, gradient series is 0.05, 0.1, 0.2, 0.4, 0.8, 2 mM;
• (2) HPLC liquid phase analysis of the standard product was carried out to collect peak data, GC/LC analysis was used to calculate peak area and collect data;
• (3) Linear fitting of the relationship between the concentration of the standard substance and its characteristic peak-to-peak area to establish a standard curve.
• 3. Configure reaction systems to characterize enzyme activity at different temperatures:

From 20 to 70 degrees, each 10 degrees is a gradient, a total of 6 gradients, 3 groups of each gradient experimental group and 2 groups of blank control group parallel,two experimental groups were treated with a batch of newly purified enzyme at 60 ° C and 40 ° C to observe the enzyme activity.

• (1) Configure the substrate liquid: 30ml BHET solution (0.04g/10ml) for each 0.04g BHET, use 10ml of 50mM Tris-HCl with PH=8 for constant volume, and use a vortex oscillator to suspend the suspension after ultrasonic dissolution.
• (2) Reaction solution: 0.9ml substrate solution + 0.1ml PETase purification enzyme solution
  Blank solution: 0.9ml substrate solution + 0.1ml ddH2O
• (3) Control the time for reaction: preheat each temperature gradient of the constant temperature water bath, place the substrate liquid in the water bath, add one part of purified enzyme solution (or ddH2O) every 15 seconds between groups, and add one part of purified enzyme solution (or ddH2O) every 40 seconds between groups.The purification enzyme solution was added to the first sample to start the timing, and the reaction was performed in a constant temperature water bath for 0.5 h
• (4) Stop the reaction: 1ml methanol was added every 15 seconds between groups, and 1ml methanol was added every 40 seconds between groups. Sample reaction solution was collected
• (5) HPLC analysis: Set the column temperature to 30, sample 20 micro, 240nm mobile phase: 80% formic acid water 20% acetonitrile.
  GC/LC was used to analyze the liquid phase data to calculate the peak area, and the data was analyzed according to the standard curve.
  BHET and temperature decreased linearly, TPA increased linearly, and MHET peak area data were basically consistent under different temperatures.

• Prepare the target gene from PCR.
• Do Agarose gel electrophoresis to separate genes.
• Get the DNA from gel through gel extration.
• Homologous Recombination: Homologous recombination

  Meterial	Dosage
  Gene of interest	2µL
  Vector	1.5µL
  Basic mix	3.5µL
  Incubation in 50℃for 18 min.

  Note:In case of homologous recombination of multiple fragments, it is necessary to adjust the amount of target genes and vectors (no more than 10µL in the total reaction system) and prolong the incubation time.
• Transform the plasmid constructed in step 4 into E. coli Trans1-T1 strain

• 1 Sample preparation:
• add methanol to the material in the ratio of 1:1, to dissolve the material to be detected. Let it sit overnight. Centrifuge at 8000 rpm for 5 minutes, use a 1 ml syringe to take the supernatant, and filter with 0.22 μm organic solution filtration membrane, and inject the sample in time.



• 2 Mobile phase preparation:
• Phase A: chromatography grade formic acid reagent and up water to configure 0.1% formic acid water, using 0.22 μm organic system filtration membrane filtration, in the ultrasonic instrument open lid sonication for 30min to remove the gas in the solution.
• Phase B: Using chromatography grade acetonitrile reagent, sonicate in a sonicator with the lid open for 30 min to remove gases from the solution.



• 3 Sample analysis:
• HPLC method
• Instrument: Aglient 1260 Infinity II, C18 column, 240nm UV detector
• Mobile phase:
  Phase A: 0.1% formic acid in water
  PhaseB: 20% acetonitrile
• Method：
• Column temperature 30，Injection 20μ
• Phase B=20% constant
• Flow rate 0.8 ml/min. Detection time 20min per sample



• UPLC method
• Instrument: SHIMA Nexera LC-40 （Ultra High Performance Liquid Chromatograph）, C18 column, 240nm UV detector
• Mobile phase:
  Phase A: 0.1% formic acid in water
  B: acetonitrile
• Method：>
• Column temperature 30，Injection 20μ
• Phase B slowly rises from 10% acetonitrile to 40% in 10 min.
• Flow rate 0.2 ml/min. Detection time 10min per sample



• 4. Rinse the column
• HPLC：
• 0min 80% Phase A, 20% Phase B Flow rate 0.8 ml/min
• 0-40 min, Phase B was increased from 20% to 100%, and the flow rate was increased to 1.0 ml/min
• 40-60min, 100% Phase B, flow rate maintained at 1.0ml/min
• 60-100 min, the Phase B is reduced to 10%,
• 100-120min, 10% Phase B
• 120-123 min, Phase B up to 20%
• 123-140min, 20% Phase B
• 140-141min, 20% Phase B, flow rate reduced to 0.8 ml/min



• UPLC：
• 0-6min 90% Phase B for washing
• 6-15min 10% Phase B for balancing



• 5 Liquid phase data processing and analysis
• HPLC Data processing: Instrument supporting analysis software, using GC/LC quantitative external standard method to analyze and calculate the peak area, record data
• UPLC Data analysis: LabSolutions instrument supporting analysis software, calculate peak area and peak height, record data.

Preparation of S-medium:

Materials: NaCl, K₂HPO₄, KH₂PO₄, EDTA, 200×Trace metal solution, 5mg/ml cholesterol, 1mol/L CaCl₂, 1mol/L MgSO₄

Preparation of nano PET, TPA solution:

Materials: S-medium, nano-size PET, TPA.

C. elegans liquid culture protocol:

1. Transfer 20 day 1 adult worms to normal nematode growth medium (NGM) plates.
2. Remove parental worms after 3-4 hours of egg laying.
3. 24 hours after removal of parent worms, transfer worms to NGM plates with no OP50 seeded for 2 minutes to remove bacteria.
4. After bacteria removal, 15 worms per well are added to 12 wells culture plates with 1.5 ml 20 mg/mL bacterial solution with certain concentration of nano PET/ TPA, always keep rotating on a slow shaker at 20 degree, 120 rpm.
5. After 48 hours of cultivation, the worms grow to day 1 adult stage and are ready for fluorescence detection.

C. elegans synchronization:

1. Transfer 20 day 1 adult worms to fresh 60-mm plates with different concentration of nano PET/ TPA.
2. Remove parental worms after 3-4 hours of egg laying.
3. After 72 hours of cultivation at 20ºC, Day 1 adult worms were ready for fluorescence detection.

Microscopy:

1. Prepare an agarose pad by dropping a 2% agarose solution onto a slide and place a second slide on top. Wait for the agarose solution to solidify (~1 min) and carefully remove the top slide.
2. Pick 7 Day 1 adult worms onto an unseeded NGM plate in order to remove the remaining bacteria sticky to the worms.
3. Add a drop (3–15 μL) of 5 mM levamisole solution onto the agarose pad.
4. Transfer 7 worms into the drop of 5 mM levamisole solution and wait for its effect (~30 s). Once the worms do not move anymore, they can be mounted carefully with a pick or eyelash tool. Try to mount the worms as close to each other as possible.
5. Acquire images with IX73 invert microscope (Olympus) under 10X objectives with MetaMorph (Molecular Devices Inc.). Each treatment had three replicates.
6. Analyze fluorescence images with ImageJ (NIH).

Experiment 1: transform tdTomato plasmid into competent cells of Escherichia coli strain BL21 or OP50

• Take out 50µL of Escherichia coli BL21/OP50 competent cells from a -80° C refrigerator and put the cells on an ice bath, waiting for cells thawing out.
• Dip the plasmid with tdTomato gene with appropriate amount, and add to BL21/OP50 competent cells, then immediately bath the cells in ice for 30min.
• Perform a 90s heat shock in a 42° C water bath.
• Ice bath for 3min.
• Add BL21/OP50 competent cells on solid medium containing ampicillin antibiotic.
• Culture at 37 ° C for 14 to 16h.

Experiment 2: expanded cultures of transformed E. coli

• Pick monoclonal colonies and add it to 50mL LB liquid medium, and at the same time add 50µL ampicillin antibiotic to the medium, then incubate for 12h at 37° C with shaking table.
• The two bacteria were collected separately in a 50mL centrifuge tube and stored in a refrigerator at 4 ° C.

Experiment 3: preparation of Nematode Growth Medium(NGM) plates

• For each strain (BL21/OP50), 25 NGM plates were prepared and 200µL of bacterial solution was added to the plates and stored at 20 ° C in the nematode room.

Experiment 4: Passage of nematode

• Fifteen wild-type N2 nematodes (stage L4) were picked by platinum picker onto the previously-prepared NGM plates of E. coli strains with fluorescence, and passage every three days, two plates per group at a time.

Experiment 5: Fluorescence photography of N2 nematodes at L4 & day1 stage

• Prepare an agarose pad by dropping a 2% agarose solution onto a slide and place a second slide on top. Wait for the agarose solution to solidify (~1 min) and carefully remove one of the slides.
• Pick 7 L4/Day1 stage worms onto an unseeded NGM plate to free worms of any remaining bacteria.
• Add a drop (3–15 μL) of 5mM levamisole solution onto the agarose pad.
• Transfer 7 worms into the drop of 5mM levamisole solution and wait for its effect (~30 s). Once the worms do not move anymore, they can be arranged carefully with a pick or eyelash tool. Try to arrange the worms as close to each other as possible.
• Image worms using a fluorescence dissecting microscope with a filter for RFP excitation (excitation maximum at 555 nm), Each treatment had three replicates.
• Quantify fluorescence images using ImageJ.
• Compare the fluorescence intensity in the intestinal tract of nematodes under different strain feeding conditions.

Experiment 6: C. elegans intestine lysis spreading assay

1. Preparation of Worms:
• Culture N2 young adult worms on OP50 or BL21 (tdTomato) NGM plates for several generations.
• For each E. coli strain, select one hundred L4 stage worms from previous NGM plates, place them on new NGM plates with fluorescent strains, and culture overnight until the worms reached day1 stage.
• Using 1mL M9 buffer, gently wash worms off the culture plate and transfer to a sterile microcentrifuge tube.
• Allow the worms to settle for 1-2 minutes, then carefully aspirate the supernatant.

2. Washing Worms:
• Resuspend the worms in 1 mL of sterile M9 buffer.
• Repeat the washing step four more times: allow worms to settle, aspirate the supernatant, and replace with fresh M9 buffer each time.
• After the fifth wash, resuspend worms in 1 mL of M9 + 25 mM levamisole to block pharyngeal pumping.

3. Surface Sterilization:
• Incubate the worms in 1% bleach solution (in M9 + 25 mM levamisole) for 5 minutes at room temperature.
• After incubation, wash the worms five times with sterile M9 buffer to remove residual bleach.

4. Wash Control:
• To assess the effectiveness of surface sterilization, take 100 µL of the final supernatant (after the last wash) and plate it on LB agar. This will serve as a wash control to confirm that external bacteria have been removed.

5. Post-Sterilization Handling:
• Transfer the washed worms to a fresh NGM plate.

6. Collection and Homogenization of Worms:
• Pick 10 young adult worms per condition and transfer them to individual microcentrifuge tubes containing 100 µL of sterile M9 buffer.
• Using a sterile pestle, carefully crush the worms to release their internal contents, ensuring the complete disruption of each worm.

7. Serial Dilution and Plating:
• Serially dilute the homogenized worm samples using sterile M9 buffer.
• Plate each dilution onto LB agar to allow for the growth of bacterial colonies.

8. Colony-Forming Unit (CFU) Quantification:
• Incubate the LB plates at an appropriate temperature to allow colony formation.
• Count the CFU (colony-forming units) from the plated dilutions.
• Normalize the CFU per 10 worms by subtracting the number of colonies observed in the wash control plates from the number of colonies counted on the sample plates.

1. Primer Design

Linearize Vector:
For preparing a linearized vector by reverse PCR amplification, it is recommended to use a high-fidelity polymerase to minimize the introduction of amplification mutations. In a 50 µL PCR system, use 0.1–1 ng of circular plasmid template, or alternatively, a pre-linearized plasmid as the template to reduce the influence of residual circular plasmid template on the cloning efficiency.

Insert Fragment:
When designing primers for amplifying the insert fragment, add homologous sequences corresponding to both ends of the linearized vector at the 5' ends of both forward and reverse primers. After amplification, the 5' and 3' ends of the insert fragment should have homologous sequences (15–20 bp, excluding restriction sites) matching the respective ends of the linearized cloning vector.

2. Polymerase Chain Reaction (PCR) amplification

• Reaction Setup (50 µL per tube, 2 tubes total):
• - 2x PCR Mix (I5): 25 µL
• - Forward Primer: 2 µL
• - Reverse Primer: 2 µL
• - Template DNA (25-50 ng): 1 µL
• - H₂O (nuclease-free): 20 µL
• Total Volume per Reaction: 50 µL

• PCR Program
• - Initial Denaturation: 98°C for 2 minutes
• - Cycling (35 cycles):
• Denaturation: 98°C for 15 seconds
• Extension: 72°C for 2 minutes
• - Final Extension: 72°C for 10 minutes
• - Hold: 4°C indefinitely

3. Agarose gel electrophoresis

• - Add 2g agarose per 100mL 1×TAE buffer to get 2% agarose gel.
• - Heat with microwave until the solution become transparent.
• - Add 1/10000 nucleic dye after the solution cool down.
• - Pour the solution into the plate with fixed comb and unplug the comb when the gel coagulates.
• - Add equal volume of 2×loading buffer (no loading buffer if PCR product is used) into samples and add the samples to the wells.
• - Set the voltage to 120V and run the electrophoresis for 25-35 minutes.
• - Check the results using UV torch or computer software.

4. Homologous Recombination

• - Calculate the Required Amount of Linearized Vector and Insertion DNA.
• - Dilute the vector and insertion DNA to ensure the volume of each component added to the reaction is not less than 1 µL.
• - Prepare the recombination reaction system on ice.
• - Gently pipette the mixture up and down to mix. Do not vortex.
• - Briefly centrifuge to collect the reaction mixture at the bottom of the tube.
• - Incubate the reaction mixture at 37°C for 30 minutes in a thermocycler.
• - After the reaction, either cool down to 4°C and hold or immediately place on ice to cool.
• - Store the recombination product at -20°C for up to one week.
• - Thaw when ready for transformation.
• - Gently pipette the mixture up and down to mix. Do not vortex.
• - Briefly centrifuge to collect the reaction mixture at the bottom of the tube.
• - Incubate the reaction mixture at 37°C for 30 minutes in a thermocycler.
• - After the reaction, either cool down to 4°C and hold or immediately place on ice to cool.
• - Store the recombination product at -20°C for up to one week.
• - Thaw when ready for transformation.

5. Transformation

• - Place competent cells on ice and allow them to thaw.
• - Add the ligation product to the thawed competent cells.
• - Gently pipette up and down to mix.
• - Incubate the mixture on ice for 30 minutes (work quickly and avoid leaving competent cells at room temperature for too long).
• - Heat shock the cells at 42°C for 90 seconds.
• - Immediately transfer the tube to ice and cool for 3 minutes.
• - Add 800 µL of antibiotic-free LB liquid culture medium to the cells and incubate at 37°C with shaking table for 40 minutes to allow the recovery of plasmid.
• - Centrifuge the recovered cells at 3000 rpm for 3 minutes.
• - In a sterile environment, carefully remove the excess supernatant, leaving about 100 µL of the bacterial pellet.
• - Resuspend the remaining cells by pipetting up and down.
• - Spread the resuspended cells evenly on an LB agar plate containing the appropriate antibiotic using a sterile, cooled spreader.
• - Invert the plate and incubate at 37°C for 12-16 hours.

6. Bacterial Culture

• - Prepare LB Medium: in a sterile environment, add the appropriate antibiotic to liquid LB medium at a 1:1000 dilution.
• - Dispense 5 mL of the LB-antibiotic liquid medium into 15 mL sterile centrifuge tubes for each sample.
• - Using sterile tweezers, pick up a sterile pipette tip.
• - Let the pipette tip attach a single bacterial colony on the LB agar plate.
• - Drop the tip with the colony into one of the tubes containing 5 mL of LB medium.
• - Place the tubes in a shaking table at 37°C (around 200-220 rpm) and incubate for 12-16 hours to allow the bacteria to grow.

7. Plasmid extraction

• - Follow kit instructions.

8. PCR and agarose gel electrophoresis again

9. Gene sequencing

• - Send the samples to the company to get the sequencing results.