Experiments

Reagent List

  1. 1×TAE
  2. 1% agarose powder
  3. BL21 competent cell
  4. DH5α competent cell
  5. Ampicillin
  6. kanamycin
  7. LB Broth (25 g/L)
  8. LB Broth Agar (40 g/L)
  9. HRV-3C Protease
  10. 1 M & 0.1 M IPTG
  11. Purification Buffer (500 ml): 20 mM Tris-HCl pH 8.0, 300 mM NaCl
  12. Wash Buffer (500 ml): 20 mM Tris-HCl pH 8.0, 300 mM NaCl, 20 mM Imidazole
  13. Balance Liquid BL
  14. Buffer P1: 25 mM Tris-HCl (pH 8.0), 10 mM EDTA, 50 mM Glucose
  15. Buffer P2: 250 mM NaOH, 1% SDS
  16. Buffer P3: 3 M potassium acetate, 5 M acetate acid
  17. Buffer PW (wash buffer): 10 mM Tris-HCl (pH 7.5), 80% ethanol
  18. Buffer EB (elution buffer): 10 mM Tris-HCl (pH 7.5)
  19. Coomassie Brilliant Blue G250
  20. 1×Tris-Glycine-SDS electrophoresis running buffer
  21. DTT
  22. 2×Loading Buffer
  23. 2×Phanta Flash Master Mix
  24. Ensitrelvir
  25. Disulfiram
  26. Dpnl
  27. Color prestrained protein marker (10-180 kDa)
  28. Feto Protein straining buffer
  29. MCA-AVLQS GFRK (DnP) K 9MG 95
  30. ClonExpress II One Step Cloning Kit

Laboratory Protocol

1. Plasmid extraction

  • Column equilibration：place the adsorption column CP3 into the collecting pipe and add 500 μl equilibrium liquid BL. Centrifuge the liquid at 12000 rpm for 1 min. Pour out the liquid in the pipe.
  • Take 1.5 ml bacteria solution which has been cultured overnight and add into centrifuge tube. Centrifuge it at 12000 rpm for 1min then pour out the supernate. Repeat this step until all the bacteria solution is used.
  • Add 250 μl P1 solution and suspend the bacteria in the solution by using the vortex oscillator (The P1 solution mainly consists glucose, EDTA and Tris C. Glucose increases the viscosity of the solution, reduces the mechanical shear force, and prevents plasmid DNA damage;EDTA chelates divalent metal ions, such as Mg²⁺, to prevent the degradation of plasmids by DNA enzymes; Tris CI maintains the optimal pH range of the lysate to ensure the activity of lysozyme.)
  • Add 250 μl P2 solution and turn it upside down 6-8 times soft to make the bacteria fully cleavage. (The P2 solution consists SDS and NaOH.SDS binds to proteins and denatures them, and NaOH (pH>12) breaks hydrogen bonds and denatures the DNA molecule. This step focuses on disrupting the cell wall and releasing the DNA.)
  • Add 350 μl P3 solution and turn it upside down for 6-8 times immediately.Now there is white flocculent precipitate.Then centrifuge it at 12000rpm for 10min. (The P3 solution contains KAc and HAc which form a high-salt solution with a pH of 5.5. It can neutralize the alkalinity of P2 solution and restore chromosomal DNA and plasmid DNA. K⁺ ions form low solubility salts with SDS and form precipitates with proteins for removal. The high-salt environment helps the formation of macromolecular precipitates, Thus isolating plasmid DNA.)
  • Remove the supernate into the adsorption column CP3 by the pipette.(Do not remove the precipitate into the column).Then centrifuge it at 12000 rpm for 60 s.Then pour out the liquid in the collecting tube.
  • Add 600μl PW bleach wash solution which should be added absolute ethyl alcohol in advance.Then centrifuge it at 12000 rpm for 60 s and pour out the liquid in the collecting tube.
  • Repeat the 7th step
  • Put the adsorption pipe into the collecting tube and centrifuge it at 12000 rpm for 2 min in order to wipe off the remaining bleach wash solution.
  • Put the adsorption pipe into clean centrifuge tube and add 50 μl elution buffer.Place at the indoor temperature for 2 min.Centrifuge it at 12000 rpm for 2 min. Now the plasmid solution is in the centrifuge tube.

2. Digestion vectors

The plasmid was digested with a specific endonuclease at a temperature of 37°C and a digestion time of 1 h.

3. PCR

The system of PCR included: nsp-5 template plasmid, 2×Phanta Flash Master Mix, forward primer reverse primer , ddH2O.

PCR procedure：

  1. 98℃-30 s
  2. 98℃-10 s
  3. 60℃-5 s
  4. 72℃-30-180 s（extension rate 30s/kb）
  5. 72℃-10 min
  6. 25℃-5 min

Steps ②-③-④ repeat 30-35 times

4. Agarose Gel Electrophoresis & Gel Extraction

  • Run PCR products in the 1% agarose gel, lets the product undergo 120V electrophoresis for about 30-40 min. After AGE is completed, analyze the position of the product in the agarose gel, should be about 6000 bp. After comfirm the target sequences, the gel recovery of the nsp-5 begin.
  • Place the single target DNA band cut from the agarose gel into a clean centrifuge tube and weigh
  • Add an equal volume of solution PN to the gel in a 50℃ water bath
  • Add the solution to adsorption column CA2 and place it for 2 min at room temperature
  • Centrifuge (12000 rpm, 60 s)
  • Wash with 600 μl PW, centrifuge (12000 rpm, 60 s) , pour the discard solution, put the adsorption column CA2 into the collection tube and repeat this step for once
  • Place the adsorption column CA2 back to the collection tube, centrifuge (12000 rpm, 2 min)
  • Dry the adsorption column CA2 at room temperature
  • Place the adsorption column CA2 in a clean centrifuge tube, add elution buffer EB to the middle of the adsorption membrane, place it for 2min at room temperature
  • Centrifuge (12000 rpm, 2 min)
  • Collect DNA solution

5. Homologous recombination ligation transformation

The PCR fragments and gel-recovered digestion vectors were ligated with the ClonExpress II One Step Cloning Kit, a homologous recombinase from Novozan.

  • Mix the digested vector, PCR fragments, and a certain amount of enzyme.
  • The mixture reacts at 50°C for 30 min.
  • Mix chemically competent cells with ligated plasmid DNA.
  • Incubate the cells with DNA on ice for 30 min.
  • Perform heat shock at 42°C for 45 s as appropriate for the bacterial strain and DNA used.
  • Return the heat-shocked cells to the ice for more than 2 min.
  • Prepare the medium with appropriate antibiotics.
  • Prepare the cell suspension that is required to be cultured or screened.
  • Spread cell suspension with a cell spreader.
  • Incubate at 37℃ in an incubator for 12-16 h.
  • The colonies are sent for sequencing.

6. Protein expression purification

  • Extract Amplified Bacterial Culture: After confirming the completion of amplification, add 500 ul of antibiotics (Amp) to 500ml culture LB liquid, remove the bacterial culture from the incubator, mix Amp with LB liquid together.
  • Temperature Reduction Treatment: Place the bacterial culture in a 4°C refrigerator and set a timer（20 min） to ensure accurate cooling time. Simultaneously, adjust the shaker temperature to 16°C and begin preheating.
  • Sterile Operation to Remove Seal Film: Within the laminar flow hood, ignite the alcohol burner and use sterile tweezers to swiftly and smoothly remove the seal film from the culture bottle beside the flame.
  • Addition of IPTG to Medium: Precisely measure out 100μL of IPTG solution and carefully add it to the 500 ml liquid medium, thoroughly mixing to ensure uniformity.
  • Inoculation of Bacterial Culture into Medium: Inoculate the cooled bacterial culture into the IPTG-containing medium at an appropriate ratio, gently mixing to combine.
  • Low-temperature Cultivation: Transfer the inoculated medium to the preheated 16°C shaker, set the speed to 180rpm, and conduct continuous cultivation for 18 hours.
  • Establishment of Control Group:Prepare a group of BL21 strains without plasmids, perform a 10 ml shaking operation similarly, serving as the experimental control group.
  • Cultivation of Bacterial Cells : Centrifuge the bacterial solution at 16℃ for 4000 g for 10 min, discard the supernatant and collect the bacteria until 1 l of bacterial solution is used up.
  • Add 30 ml of purified buffer into the centrifuge tube to resuspend the cells.
  • Put the suspended bacterial solution on the ice and set the ultrasonic crushing program 300 W to work for 10 s and stop for 10 s for 15 min.
  • The solution was then centrifuged at 13000 g at 4℃ for 40 min, and the supernatant was collected.
  • 2 ml nickel medium was added to the gravity column, 2 volumes of digestion buffer was added for balance, the supernatant passed through the column and the miscellaneous laundry operation will be performed.
  • Plug the lower end of gravity column and add 3 ml digestion buffer.
  • Add 5 μl Ppase and digest the enzyme for 12-16 h at 4℃ .
  • Protein Elution and Collection: Wash the protein with 15 ml of purification buffer, collect eluted proteins into a tube, and store 40 μl of enzymatically cleaved nickel resin into two separate tubes for later use; and add 50 μl of G250 to 20 μl of bacterial culture (nsp5/nsp5_native) to perform the dye test and measure protein concentration.
  • Centrifugation: Centrifuge at 3600 g, 4°C for 10 min. Perform dye tests for each tube: nsp5 and nsp5_native. Add 50 μl of coomassie brilliant blue G250 dye. Add 20 μl of supernatant into one test tube and 20μl of collected liquid in the column into another tube
  • Store the remaining purified protein at -80°C

7. SDS-PAGE

  • Add melted 2×SDS (10 μL) loading into containers and heat around 100℃ in a water bath for 6 min
  • Add SDS loading buffer to the buffer chamber
  • Inject samples into the precast gel
    • Gel specifications: Tris-Glycine, 12%, 15 holes, 1.5 mm
  • Conduct electrophoresis at 120 V for 60 min
    • Increased voltage above reccomended value to ensure maximal Marker performance
  • Stain the gel with FETO SDS-PAGE staining buffer for 15 min, horizontal shaker 70 rpm
  • Rinse with ddH2O twice

8. Co-transformation and enzyme activity

Verify FlipGFP Cleavage and Fluorescence Relief Co-transformation allowed the simultaneous expression of both split fluorescent protein fragments and nsp5 within the same host cell. This setup enabled nsp5 to cleave the linker between the GFP fragments, leading to their correct assembly and the generation of a detectable fluorescence signal. Without co-transformation, the fragments would remain unassembled, and fluorescence would not occur. The presence of active nsp5 facilitated the proper assembly of GFP, confirming its crucial role in enabling fluorescence.

9. Inhibitors impact

By testing the co-transformation function, adding effective inhibitors and ineffective inhibitors to measure the enzyme activity of the nsp5. Using ELISA to show the fluorescent signal to determine the substrate's concentration. A decrease or loss of fluorescence indicated successful inhibition of the protease, preventing cleavage of the linker and assembly of the fluorescent protein.