Protocols

• Materials: An artificially synthesized Prunus persica chitinase gene fragment (~5 ng).
• Reagents: Ultra-pure water, 10X Pfu Buffer (with Mg²⁺), dNTP (2.5 mM each), Primer mixture (10 μM each), Pfu DNA Polymerase (5 U/μl)
• Equipment: Pipetters and pipetter tips, PCR thermocycler, mini-centrifuge, Vortex.
• Steps:
1. Setup of PCR Reaction System
2. Thaw and mix well all solutions required for the PCR reaction. Place the Pfu DNA Polymerase on an ice bath or in an ice box.
3. Set up the PCR reaction on an ice bath with reference to the table below. If multiple similar PCR reactions are needed, first prepare a large-volume mixture containing water, buffer, dNTPs, and Pfu enzyme, then aliquot it into each PCR reaction tube.

   Reagent	Final Concentration	Volume
   Ultra-pure water	-	35.75 μl
   10X Pfu Buffer (with Mg²⁺)	1X	5 μl
   dNTP (2.5 mM each)	0.2 mM each	4 μl
   Template DNA	~5 ng	1 μl
   Primer mixture (10 μM each)	0.8 μM	4 μl
   Pfu DNA Polymerase (5 U/μl)	1.25 U/50 μl	0.25 μl
   Total Volume		50 μl

4. Gently pipette up and down to mix, or vortex slightly to mix. Centrifuge at room temperature for a few seconds to collect the liquid at the bottom of the tube.
5. Place each prepared PCR reaction tube in a PCR thermocycler and start the PCR reaction.
6. Setup of PCR Reaction Parameters:
   • STEP 1 (Initial Denaturation): 94ºC for 3 min
   • STEP 2 (Denaturation): 94ºC for 30 sec
   • STEP 3 (Annealing): 55ºC for 30 sec
   • STEP 4 (Extension): 72ºC for 1 min
   • STEP 5 (Cycling): Go to STEP 2 for 30 cycles
   • STEP 6 (Final Extension): 72ºC for 10 min
   • STEP 7 (Temporary Storage): 16ºC indefinitely

The following reaction system was used, and the operation was performed on an ice bath.

1. After adding all liquids sequentially according to the table above, mix gently by pipetting or flicking the tube wall (do not vortex), then centrifuge briefly to settle the liquid at the bottom of the tube.
2. Incubate at 37°C for around 4 hours (as we’ve failed for 30 min) in a water bath.

Reagents: GelRed, Agrose, TAE buffer.

Equipments: Pipetters and pipetter tips, Horizontal Electrophoresis Apparatus, centrifuge, Vortex, water bath, Blue Light Gel Cutting Instrument.

Gel with target band was cut by a knife under Blue Light Gel Cutting Instrument and collected in an 1.5 ml centrifuge tube.

A DNA Gel Extraction kit was used for purification of plasimids.

Method I: pET-28a(+)-CH1 was constructed by using ClonExpress II One Step Cloning Kit (Vazyeme)

Method II: pET-29a(+)-F4 was constructed by using T4 DNA Ligase

1. Vector Enzyme Digestion

Take 1 μg of vector and perform enzyme digestion overnight to ensure complete digestion, which reduces the occurrence of self-ligated clones in subsequent steps.

2. Vector Purification

After the vector enzyme digestion is completed, run agarose gel electrophoresis and perform gel extraction to recover the vector.

3. Reaction System Setup

pET-28a(+): F4 Fragment = 3:1 (molar amount)

4. Ligation Incubation

Incubate overnight at 16°C using a PCR instrument.

5. Transformation

Directly take the ligation product to transform competent bacteria.

Reagents: E.coli Colony Direct PCR kit

Equipments: Pipetters and pipetter tips, centrifuger, PCR thermocycler,

Steps:

1. Thaw the E. coli Colony Direct PCR Mix (Green, 2X) at room temperature. Invert gently to mix well, then centrifuge at low speed for a few seconds.
2. Set up the PCR reaction system on an ice bath.
3. Mark the clones to be picked on the plate. Use a sterile pipette tip to pick the corresponding clone from the plate, add it to the 20 µl pre-prepared PCR reaction system above. Mix well by gentle pipetting or slight vortexing, then centrifuge at room temperature for a few seconds to collect the liquid at the bottom of the tube.
4. Place the PCR reaction tube into a PCR thermocycler.
5. After PCR, directly take 5-10 µl of the reaction product for electrophoresis detection.

Materials: DH5α competent cells, both liquid LB medium and LB broth, Ampicillin, etc.

Steps:

Cell culture: E. coli containing the target plasmid are inoculated into a liquid LB medium, where they are vigorously shaken at 37 °C overnight so that the plasmids replicate in large numbers.

Collection of bacteria : Transfer the cultured bacteria to centrifuge tubes, and then centrifuge to collect the bacteria.

Cell lysis: A lysate, usually a solution containing stain remover and enzymes, is added to the collected cell to lyse the cell and release plasmid DNA.

Plasmid DNA separation: By centrifugation, plasmid DNA are separated from cell debris and other impurities, and the supernatant contains plasmid DNA. Precipitation and washing: Add alcohol and other precipitating agents to precipitate the plasmid DNA, and then wash with washing buffer twice to remove waste.

Plasmid DNA elution: Finally, the plasmid DNA is eluted with an appropriate buffer and the plasmid DNA is collected.

Plasmid DNA Quantification by Qubit:

1. On the 'Home' screen, touch 'dsDNA HS', then select 'dsDNA High Sensitivity' as the assay type. Touch 'Read standards' to proceed.

2. Insert the tube containing Standard #1 into the sample chamber, close the lid, then touch 'Read standard'. When the reading is complete (~3 seconds), remove Standard #1.

3. Insert the tube containing Standard #2 into the sample chamber, close the lid, then touch 'Read standard'. When the reading is complete, remove Standard #2.

4. Touch 'Run samples'.

5. On the assay screen, select the Sample volume and units.

6. Insert a sample tube into the sample chamber, close the lid, then touch 'Read test tube'. When the reading is complete (-3 seconds), remove the sample tube. The top value ( in large font) is the concentration of the original sample and the bottom value is the dilution concentration.

7. Repeat step 6 until all samples have been read.

• Materials: E. Coli collected 0 h, 2 h, 4.5 h after IPTG treatment.

• Reagents: Universal Total Protein Extraction Kit with Mild Lysis Buffer and Spin Column. *We didn't use PMSF.

• Equipments:Pipetters and pipetter tips, centrifuge, Vortex, water bath.

• Steps:
  1. Take 1 ml of bacterial culture.
  2. Centrifuge at max speed for 2 minutes.
  3. After thoroughly removing the liquid, gently vortex or flick the bottom of the tube to disperse the bacteria or yeast as much as possible.
  4. Add 100-200 μl of lysis buffer, gently vortex or flick the bottom of the tube to mix well, and lyse on ice for 2-10 minutes.

  *We tried to wash once with PBS and lysozyme, but residues of lysozyme interfered with the observation of experimental results. So we just added 100-200 μl of lysis buffer for lysis.

  5. After the sample is fully lysed, transfer it to a spin column, nest the column into a collection tube, centrifuge at room temperature*, 12,000 x g for 30-60s.

  *As we didn't have a refrigerated centrifuge.

  6. Discard the spin column and the liquid in the collection tube is the total protein sample.
  7. The isolated protein sample can be used for downstream experiments or stored at -20°C for later use.

• Materials: Total protein extracted from E. Coli collected 0 h, 2 h, 4.5 h after IPTG treatment.
• Reagents: Precast PAGE Gel for Tris-Gly System (8-20%), Coomassie Blue Super Fast Staining Solution (Low Background), Protein Marker, SDS-PAGE Electrophoresis Buffer with Tris-Gly (Powder), Ultra-pure water.
• Equipments: Vertical electrophoresis apparatus, pipetters and pipetter tips, centrifuge, water bath.
• Steps:
1. Take the precast PAGE gel out of the packaging bag and peel off the blue sealing tape at the bottom*.

*Don't forget to peel off the blue sealing tape!

2. Fix the precast gel in the electrophoresis tank, then pull out the comb smoothly and slowly.
3. Prepare the SDS-PAGE electrophoresis buffer (Tris-Glycine buffer).
4. Fill the inner tank completely with the electrophoresis buffer, and add the buffer to the outer tank to a level of at least 1/3 of the tank height; do not let the buffer overflow the gel plate.
5. Sample denature and loading: Denature the proteins by heating in boiling water for 5 min. Gently insert the tip of a 10 μL pipette tip vertically into the sample well for loading. Ensure the pipette tip does not pierce the gel, and never deform the gel plate—deformation may cause sample leakage, leading to band tailing and excessive signal.
6. Cover the electrophoresis tank, and plug the power cord into the power jack of the electrophoresis apparatus (match red to red, black to black). Generally, run electrophoresis at 180 V for approximately 60 minutes.
7. Remove the gel plate, use a knife to separate the two plates along the gap between them, and gently take out the gel with a scraper.
8. Place (the gel) into Coomassie Brilliant Blue rapid staining solution and shake for 10 minutes. Take the photo of the gel.