Molecular Cloning

DNA Agarose Gel Electrophoresis

Gel electrophoresis is the standard lab procedure for separating DNA by size for visualization and purification.

1 Pouring a Standard 1% Agarose Gel

• Purpose: prepare the agarose gel for electrophoresis
• Estimated bench time: 10 mins
• Estimated total time: 30 mins

1.1 Materials

• TAE buffer (Mei5Bio, Beijing, China)
• Agarose (Mei5Bio, Beijing, China)
• GelRed (Mei5Bio, Beijing, China)

1.2 Equipment

• Microwave
• HE-120 Multifunctional Horizontal Gel Electrophoresis Cell (Tanon, Shanghai, China)
• pipettes & tips
• glass media bottles

1.3 Setup & protocol

1. Measure 0.3 g of agarose.
2. Mix agarose powder with 30 mL 1xTAE in a microwavable flask.
3. Microwave for 1-3 min until the agarose is completely dissolved.
4. Let agarose solution cool down to about 50 °C (about when you can comfortably keep your hand on the flask), about 5 mins.
5. Add 3 μL of Gelred to the gel.
6. Pour the agarose into a gel tray with the well comb in place.
7. Place newly poured gel at 4 °C for 10-15 mins OR let sit at room temperature for 20-30 mins, until it has completely solidified.

2 Loading Samples and Running Electrophoresis

• Purpose: run electrophoresis
• Estimated bench time: 10 mins
• Estimated total time: 40 mins

2.1 Materials

• TAE buffer (Mei5Bio, Beijing, China)
• 10× Loading Buffer
• M5 HiClear DL2000 plus DNA marker (Mei5Bio, Beijing, China)
• DNA Marker A (25~500 bp) (Sangon, China)
• pipettes & tips
• agarose gel prepared as above

2.2 Equipment

• PowerPac™ Basic Power Supply (Bio-Rad, USA)
• HE-120 Multifunctional Horizontal Gel Electrophoresis Cell (Tanon, Shanghai, China)
• Gel Image System (Tanon 2500)

2.3 Setup & protocol

1. Add loading buffer to each of your DNA samples.
2. Once solidified, place the agarose gel into the gel box (electrophoresis unit).
3. Fill gel box with 1xTAE until the gel is covered.
4. Carefully load 5 μL molecular weight ladder into the first lane of the gel.
5. Carefully load your samples into the additional wells of the gel.
6. Run the gel at 80-150 V until the dye line is approximately 75-80% of the way down the gel.
7. Turn OFF power, disconnect the electrodes from the power source, and then carefully remove the gel from the gel box.
8. Using any device that has UV light, visualize DNA fragments.

Gel DNA Extraction

To obtain purified DNA fragments from PCR products, performing gel DNA extraction is essential. This process enables the isolation and purification of specific DNA fragments from an agarose gel following electrophoresis.

1 Gel Slice Preparation

• Purpose: Cutting off the gel where the DNA bands is located
• Estimated bench time: 10 mins
• Estimated total time: 10 mins

1.1 Materials

• agarose gel after electrophoresis

1.2 Equipment

• pipettes & tips
• Gel Image System (Tanon 2500)
• 1.5 mL tubes
• scalpels

1.3 Setup & protocol

1. After DNA electrophoresis, place the agarose gel on Gel Image System to visualize the DNA bands.
2. Use a scalpel to carefully excise the desired DNA band from the gel. Minimize the amount of excess agarose around the DNA fragment.
3. Weigh the gel slice in a pre-weighed 1.5 mL microcentrifuge tube to determine its volume (1 mg of gel ≈ 1 μL).

2 DNA Extraction & Collection

• Purpose: extract & collect DNA from gel slices
• Estimated bench time: 30 mins
• Estimated total time: 40 mins

2.1 Materials

• gel slices as above
• FastPure® Gel DNA Extraction Mini Kit (Vazyme)
• ethanol (Bio&Chem Reagent Management Plat, Peking University)

2.2 Equipment

• Pipettes & tips
• Dry Bath Incubator
• 1.5 mL tube centrifuge

2.3 Setup & protocol

2.3.1 DNA Extraction

1. Add 1 volume of Buffer GDP (from the FastPure® Gel DNA Extraction Kit) to the gel slice (e.g., if the gel slice weighs 100 mg, add 100 μL of Buffer GDP).
2. Incubate the tube at 50-55°C for 7-10 minutes, or until the gel slice is completely dissolved. Invert the tube 2-3 times during incubation to facilitate melting.
3. Insert a FastPure DNA Mini Column into a clean 2 mL collection tube.
4. Transfer the dissolved gel solution to the DNA mini column. Centrifuge at 12,000 rpm (~13,400 × g) for 30-60 seconds.
5. Discard the flow-through and place the column back into the collection tube.

2.3.2 Washing

1. Add 500 μL of Buffer GW (diluted with ethanol according to the kit instructions) to the column. Centrifuge at 12,000 rpm for 30-60 seconds.
2. Discard the flow-through and repeat the wash step with another 500 μL of Buffer GW.
3. After the second wash, centrifuge the empty column at 12,000 rpm for an additional 2 minutes to remove any residual wash buffer.

2.3.3 DNA Elution

1. Place the DNA mini column in a clean 1.5 mL microcentrifuge tube.
2. Add 20-30 μL of ddH₂O (pre-warmed to 55-65 °C) directly to the center of the column membrane.
3. Incubate at room temperature for 2 minutes, then centrifuge at 12,000 rpm for 1 minute to elute the DNA.
4. Discard the column and store the eluted DNA at -20 °C.

Gibson Assembly

Gibson Assembly is a method for seamlessly joining multiple DNA fragments into a single, circular DNA molecule in a one-step isothermal reaction. It uses exonucleases, polymerase, and ligase to facilitate efficient and accurate assembly, making it ideal for genetic engineering and cloning.

1 DNA Fragments & Backbone Preparation

• Purpose: prepare fragments & backbone for gibson assembly
• Estimated bench time: 15 mins
• Estimated total time: 1.5 hrs

1.1 Materials

• plasmids contain target fragments/backbones
• primers designed for gibson assembly
• Materials needed for PCR & DNA Agarose Gel Electrophoresis & Gel DNA Extraction

1.2 Equipment

• Equipment needed for PCR & DNA Agarose Gel Electrophoresis & Gel DNA Extraction

1.3 Setup & protocol

• PCR target fragments following PCR protocol.
• obtain pure product following DNA Agarose Gel Electrophoresis & Gel DNA Extraction protocols.

2 Gibson Assembly

• Purpose: perform gibson assembly
• Estimated bench time: 30 mins
• Estimated total time: 1 hrs

2.1 Materials

• DNA Fragments & Backbone as above
• ddH₂O
• ClonExpress® II One Step Cloning Kit

2.2 Equipment

• pipettes & tips
• 1.5 mL Tubes
• T100 Thermal Cycler (Bio-Rad)

2.3 Setup & protocol

2.3.1 Reaction Setup

• Calculate the required amounts of vector and insert using the following formula:

$${\rm The\ optimal\ mass\ of\ vector\ required = [0.02 \times number\ of\ base\ pairs]\ ng\ (0.03\ pmol) }$$

$${\rm The\ optimal\ mass\ of\ insert\ required = [0.04 \times number\ of\ base\ pairs]\ ng\ (0.06\ pmol) }$$

• On ice, prepare the Gibson Assembly reaction mixture as follows (for a 20 μL total reaction volume):

• Gently mix the reaction components by pipetting up and down. Avoid introducing air bubbles. Briefly spin down the reaction mixture.

2.3.2 Incubation

• Incubate the reaction mixture at 37 °C for 30 minutes using a thermal cycler or heat block.
• After incubation, immediately place the reaction on ice or store it at -20 °C until further use.
• Transform the assembled DNA into competent E. coli cells using plasmids transformation protocol.

Plasmid Extraction

To isolate and purify plasmid DNA from bacterial cultures for downstream applications such as cloning, sequencing, or transformation.

1 Plasmid Extraction

• Purpose: purify plasmid DNA from bacterial cultures
• Estimated bench time: 30 mins
• Estimated total time: 30 mins

1.1 Materials

• bacterial cultures containing target plasmids
• TIANprep Mini Plasmid Kit (TianGen, Beijing , China)
• ethanol (Bio&Chem Reagent Management Plat, Peking University)

1.2 Equipment

• 1.5 mL tubes
• Pipettes & tips
• Dry Bath Incubator
• 1.5 mL tube centrifuge

1.3 Setup & protocol

1.3.1 Cell Harvesting

• Transfer 1-5 mL of overnight bacterial culture into a 1.5 mL microcentrifuge tube.
• Centrifuge at 12,000 rpm (~13,400 × g) for 1 minute to pellet the cells.
• Carefully remove and discard the supernatant, ensuring not to disturb the cell pellet.

1.3.2 Cell Lysis

• Resuspend the cell pellet thoroughly in 250 μL of Buffer P1 (ensure RNase A has been added).
• Add 250 μL of Buffer P2 and gently invert the tube 6-8 times to mix. The solution should become clear and viscous.
• Add 350 μL of Buffer P3 and immediately invert the tube 6-8 times to mix. A white precipitate should form.
• Centrifuge the lysate at 12,000 rpm for 10 minutes to pellet the cell debris.

1.3.3 DNA Binding

• Carefully transfer the supernatant to a TIANprep spin column (placed in a collection tube).

• Centrifuge at 12,000 rpm for 1 minute to bind the plasmid DNA to the column. Discard the flow-through.

1.3.4 Washing

• (Optional) Add 500 μL of Buffer PD to the column, centrifuge at 12,000 rpm for 1 minute, and discard the flow-through. This step is recommended for strains with high nuclease activity (e.g., endA+ strains).
• Add 600 μL of Buffer PW (diluted with ethanol) to the column. Centrifuge at 12,000 rpm for 1 minute and discard the flow-through.
• Repeat the wash step with another 600 μL of Buffer PW.
• Centrifuge the empty column at 12,000 rpm for 2 minutes to remove any residual wash buffer.

1.3.5 DNA Elution

• Place the spin column in a clean 1.5 mL microcentrifuge tube.
• Add 50-100 μL of pre-warmed Elution Buffer EB (or ddH2O) directly to the center of the column membrane.
• Incubate at room temperature for 2 minutes, then centrifuge at 12,000 rpm for 2 minutes to elute the plasmid DNA.
• Store the eluted plasmid DNA at -20 °C.

Polymerase Chain Reaction (PCR)

The following PCR enzymes were used in wet lab. In brief, the high- fidelity enzymes KOD and phanta were used in DNA amplification or colony PCR.

1 2 × Phanta® Max Master Mix (Vazyme)

• Estimated bench time: 10 mins

• Estimated total time: 1.5 hrs

1.1 Materials

• 2 × Phanta® Max Master Mix (Vazyme)
• plasmids (10 pg - 30 ng) OR cDNA ( 1 - 5 μL)
• primers
• ddH₂O

1.2 Equipment

• T100 Thermal Cycler (Bio-Rad)
• pipettes & tips
• PCR tubes (0.2 mL)

1.3 Setup & protocol

1.3.1 Prepare 2 × Phanta® Max Master Mix System

• Prepare 50 μL system as following:

1.3.2 Perform 2 × Phanta® Max Master Mix PCR Program

1. 98 ℃ pre‐heating for 30sec/3 min (30sec for plasmid DNA, 3min for genome DNA)

2. 98 ℃ unwinding DNA for 15 sec

3. Annealing DNA for 15 sec (The annealing temperature is the same as primer Tm)

4. 72 ℃ for 30-60 sec/kb

5. Go to Step 2 for 31 cycles

6. 72 ℃ elongation and complement for 5 min

7. Cool down to 12 ℃

8. End

2 2 × KOD One™ PCR Master Mix (Toyobo)

• Estimated bench time: 10 mins

• Estimated total time: 1.5 hrs

2.1 Materials

• 2 × KOD One™ PCR Master Mix (Toyobo)
• plasmids ( < 50 ng) OR cDNA (< 750 ng)
• primers
• ddH₂O

2.2 Equipment

• T100 Thermal Cycler (Bio-Rad)
• pipettes & tips
• PCR tubes (0.2 mL)

2.3 Setup & protocol

2.3.1 Prepare 2 × KOD One™ PCR Master Mix System

• Prepare 50 μL system as following:

2.3.2 Perform 2 × KOD One™ PCR Master Mix PCR Program

1. 98 ℃ pre‐heating for 2 min

2. 98 ℃ unwinding DNA for 10 sec

3. 60 ℃ annealing DNA for 5 sec

4. 68 ℃ for 4-5 sec/kb

5. Go to Step 2 for 31 cycles

6. 68 ℃ elongation and complement for 1 min

7. Cool down to 12 ℃

8. End

Aptamer Affinity Measurement

Electrophoretic Mobility Shift Assay (EMSA)

The Electrophoretic Mobility Shift Assay (EMSA), also known as the Gel Shift Assay or Gel Retardation Assay, is a commonly used technique in molecular biology and biochemistry to study protein-DNA or protein-RNA interactions. We use the EMSA to verify the binding ability between aptamers and the target protein.

1 Samples & Buffers Preparation

• Purpose: prepare samples and buffers used in EMSA
• Estimated bench time: 15 mins
• Estimated total time: 20 mins

1.1 Materials

• Aptamers (Customized from RuiBiotech, Beijing, China)
• Thrombin from human plasma (Merck KGaA, Darmstadt, Germany)
• ddH₂O
• Tris-HCl (1 mol/L, pH8.0) (Shanghai yuanye Bio-Technology, Shanghai, China)
• KCl (Innochem, Beijing, China)
• MgCl₂ (Tgreag, Beijing, China)
• CaCl₂ (Tgreag, Beijing, China)
• NaCl (Macklin, China)
• M5 Gelred Plus (Mei5bio, Beijing, China)

1.2 Equipment

• T100 Thermal Cycler (Bio-Rad)
• pH Meter (Mettler Toledo)
• UV light source (Tanon, Shanghai, China)
• pipettes & tips
• glass media bottles
• 1.5 mL Tubes

1.3 Setup & protocol

1. prepare bingding buffer as Mears et al.¹:

• 20 mL 1 mol/L Tris-HCl (20 mM);
• 372 mg KCl (5 mM);
• 95 mg MgCl₂ (1 mM);
• 111 mg CaCl₂ (1 mM);
• 2.925 g NaCl (50 mM);
• dissolve all solutes with 1 L ddH₂O, adjust the pH to 7.4 with Concentrated hydrochloric acid.

2. folded aptamer solution should be prepared as following steps:

• dissolve aptamer in powder form with binding buffer to final concentration 10 ng/μL;
• incubate aptamer solution in Thermal Cycler for 5 mins and cool to room temperature slowly to form correct folded conformation.

3. thrombin solution (10 μM) should be dissolved with binding buffer.

2 EMSA Experiment

• Purpose: prepare and perform EMSA binding test
• Estimated bench time: 1 hr
• Estimated total time: 2.5 hrs

2.1 Materials

• thrombin/aptamer solution and EMSA buffer as described above
• DNA PAGE electrophoresis Kit (Coolaber, Beijing, China)

2.2 Equipment

• Vertical Electrophoresis Tank
• Pipettes & tips
• 1.5 mL Tubes & PCR Tubes

2.3 Setup & protocol

1. Incubation of different concentrations of aptamer and thrombin:

• Add the following reagents in the table to a PCR tube.

• Incubation at 37℃ for 30min in PCR amplifier.

2. Electrophoresis

• Use DNA PAGE electrophoresis Kit to prepare a 15% non-denaturing PAGE gel.
• Add 2ul 6xDNA loading buffer (from the DNA PAGE electrophoresis Kit) to each sample.
• Load samples and run the gel at 160V for 30min.
• Stain the gel with GelRed for 30 minutes.
• Wash the stained gel with ddH₂O softly.
• Use UV light source to see the DNA bands.

References

Surface Plasmon Resonance (SPR)

Surface plasmon resonance (SPR) spectroscopy is an advanced technique that enables real-time, quantitative measurement of biomolecular binding interactions. By monitoring changes in the resonance angle as molecules bind to or dissociate from a metal surface, SPR provides valuable insights into the dynamics of these interactions, making it a powerful tool in biosensing and molecular research. In our protocol, SPR experiment specifically examines the interactions between aptamers and their targets.

1 Samples & Buffers Preparation

• Purpose: prepare samples and buffers used in SPR
• Estimated bench time: 15 mins
• Estimated total time: 20 mins

1.1 Materials

• 5’-end biotinylated aptamers (Customized from RuiBiotech, Beijing, China)
• Thrombin from human plasma (Merck KGaA, Darmstadt, Germany)
• ddH₂O
• Tris-HCl (1 mol/L, pH8.0) (Shanghai yuanye Bio-Technology, Shanghai, China)
• KCl (Innochem, Beijing, China)
• MgCl₂ (Tgreag, Beijing, China)
• CaCl₂ (Tgreag, Beijing, China)
• NaCl (Macklin, China)

1.2 Equipment

• T100 Thermal Cycler (Bio-Rad)
• pH Meter (Mettler Toledo)
• pipettes & tips
• glass media bottles
• 1.5 mL Tubes
• HBS-EP+ Buffer 10× (GE HealthCare, Gift from Prof. Qian Wang)

1.3 Setup & protocol

1.3.1 prepare SPR buffer as Mears et al.²:

• 20 mL 1 mol/L Tris-HCl (20 mM);
• 372 mg KCl (5 mM);
• 95 mg MgCl₂ (1 mM);
• 111 mg CaCl₂ (1 mM);
• 2.925 g NaCl (50 mM);
• dissolve all solutes with 1 L ddH₂O, adjust the pH to 7.4 with Concentrated hydrochloric acid.

1.3.2 dilute 100 mL HBS-EP+ Buffer 10× into 1 L with ddH₂O to obtain HBS-EP+ Buffer.

1.3.3 folded aptamer solution should be prepared as following steps:

• dissolve aptamer in powder form with SPR buffer to final concentration 10 ng/μL, with volume at least 200 μL;
• incubate aptamer solution in Thermal Cycler for 5 mins and cool to room temperature slowly to form correct folded conformation.

1.3.4 thrombin solution (10 μM) should be dissolved with SPR buffer.

2 SPR Experiment

• Purpose: prepare and perform SPR binding test
• Estimated bench time: 1 hrs
• Estimated total time: 6 hrs

2.1 Materials

• thrombin/aptamer solution and SPR/ HBS-EP+ buffer as described above
• Sensor Chip CM5 (Cytiva, Gift from Prof. Qian Wang)
• Bovine Serum Albumin (BSA) (Gift from Prof. Qian Wang)
• Streptavidin (SA) (Gift from Prof. Qian Wang)

2.2 Equipment

• Biacore™ 8K SPR system (Cytiva)
• Pipettes & tips
• 1.5 mL Tubes

2.3 Setup & protocol

2.3.1 SA immobilization (use HBS-EP+ buffer):

• wash the chip with HBS-EP+ buffer;
• chip surface activation with EDC/NHS;
• amine couple SA with chip using ligand contact process;
• use BSA to block unbound sites in order to prevent non-specific binding of thrombin;
• chip surface blocking with ethanolamine.

2.3.2 immobilize biotinylated aptamer with SPR buffer in one flow cell, leave the other flow cell as reference channel.

2.3.3 thrombin kinetic analysis performed as following:

• select gradient diluted thrombin concentrations to perform (50 nM - 0.39 nM for aptamer 29, 2000 nM - 7.8 nM for aptamer 40), dilute thrombin to appropriate concentrations;
• perform multi-cycle kinetics process;
• partial experimental results should be fitted with 1:1 binding kinetic model in order to calculate dissociation constant (K_d).

References

Protein Acquisition and Activity Verification

Double Plasmids Co-transformation³

Double plasmids co-transformation is a sophisticated genetic engineering technique whereby two distinct plasmids are introduced simultaneously into a host cell, here the Escherichia coli BL21(DE3) strain. This method enables the co-expression of multiple genes, thereby facilitating complex biochemical pathways within a single organism. In this study, we detail the co-transformation of plasmids pEvol-pAzFRS.2.t1 and pET28a_cPPVp_mut, with the goal of inducing subsequent protein expression.

1 Plasmids Transformation & Bacteria Cultivation

• Purpose: obtain bacteria containing both plasmids

• Estimated bench time: 1 hr

• Estimated total time: 2.5 days

1.1 Materials

• E. coli BL21(DE3) competent cell 100 μL (EarthOx, USA)

• pEvol-pAzFRS.2.t1 plasmid (200-300 ng) (MiaoLing Plasmid Platform, China)

• pET28a_cPPVp_mut plasmid (200-300 ng) (prepared as molecular cloning section)

• kanamycin sulfate solution 10 mg/mL (Shanghai yuanye Bio-Technology, Shanghai, China)

• chloramphenicol (Macklin, China)

• tryptone (Aobox, Beijing, China)

• yeast extract (Oxoid, USA)

• NaCl (Macklin, China)

• NaOH (Bio&Chem Reagent Management Plat, Peking University)

• agar

• ddH₂O

1.2 Equipment

• high pressure sterilizer
• water bath
• ice machine
• oscillation incubator
• constant temperature incubator
• ultra-clean bench
• pipettes & tips
• conical flasks
• glass media bottles
• 90mm culture dish
• spreaders
• scales (weigh things)

1.3 Setup & protocol

1.3.1 Prepare LB liquid & solid medium:

• Prepare LB liquid medium, for every 800 mL glass media bottle, add:

  • ddH₂O 700 mL

  • tryptone 8 g

  • yeast extract 4 g

  • NaCl 8 g

• Shake the bottle until the solutes dissolve, adjust the pH to 7.0 with 5M NaOH;

• For LB solid medium, add 1.5-2g agar powder per 100ml of liquid medium;

• Autoclave all the mediums at 121 °C for 20 mins, take out when the temperature is reduced to 50-60 °C;

• (following operations should be performed in an ultra-clean bench) Add kanamycin (final concentration 40 μg/mL) and chloramphenicol (final concentration 34 μg/mL) to all solid medium and a portion of liquid medium, leave at room temperature;

• Pour the solid medium into culture dishes, leave until solidification,place them upside down;

• Storage all medium in 4 °C freezer.

1.3.2 Plasmids Transformation:

• Thaw E. coli BL21(DE3) competent cells on ice (30-40 mins);

• Add plasmids into competent cells and mix well. For efficient co-transformation, use 200-300 ng of each plasmid.

• Leave the cells on ice for 30 mins.

• Water bath at 42℃ for exactly 45 secs, slowly and gently but rapidly cooled on ice for 2-3 mins.

• Add 900 μL of LB liquid medium without antibiotics, shake at 180rpm for 45 minutes;

• Centrifuge at 6000 rpm for 5 minutes, leaving only 100 μL of supernatant to mix the bacterial cells;

• Pipette the cells (100 μL) on the plate, spread the cells on the plate using the spreaders;

• Transfer the agar plate to the 37°C incubator, cultivated for 12-16 hours.

1.3.3 Bacteria Cultivation:

• The monoclonal clones on the plate can be considered to contain both plasmids, colony PCR and plasmid sequencing can be performed to double check.

• Select single colonies and inoculate into 100 mL of LB medium containing the aforementioned antibiotics and then grow overnight at 37 °C in a shaking incubator, until OD₆₀₀ reaches 0.75.

2 Protein Induced Expression

• Purpose: Add inducers to enable expression of target proteins

• Estimated bench time: 20 mins

• Estimated total time: 1 day

2.1 Materials

• p-Azidophenylalanine (Cenmed)
• isopropyl β-d-1-thiogalactopyranoside (IPTG) (Innochem, Beijing, China)
• L(+)-Arabinose (Aladdin)

2.2 Equipment

• oscillation incubator
• ultra-clean bench
• pipettes & tips
• bacterial cultures in conical flasks as mentioned above
• scales (weigh things)

2.3 Setup & protocol

2.3.1 Protein Induced Expression

• Add 25 mg of the p-Azidophenylalanine and induce expression with final concentration of 1mM IPTG and 0.02% L-arabinose, shake overnight at 30 °C;
• Follow protein extraction & purification protocols to obtain target proteins.

Reference

Gravity column purification of proteins

This experiment is used to purify proteins of interest from a complex mixture, such as a cell lysate or culture supernatant. Here we use Nickel Affinity Chromatography through Gravity column is used to purify and at the same time doing on-column refolding of target proteins that are in the solution of inclusion bodies in a denaturation condition.

1 Washing the nickel column

• Purpose: Prepare and wash the gravity column packed with nickel beads.
• Estimated bench time: 15 mins
• Estimated total time: 20 mins

1.1 Materials

• Wash and binding buffer: 20mM Tris-HCl(pH = 8.0), 0.5M NaCl, 20mM imidazole and 8M urea (or other levels of concentration if needed)
• Ni Sepharose 6 Fast Flow (cytiva, Sweden)

1.2 Equipment

• Vacuum pump
• Gravity column

1.3 Setup & protocol

1. Assemble the gravity column with vacuum pump.
2. Pack the gravity column with a suitable volume of nickel beads(2-3ml beads for 100ml bacterial culture).
3. Wash the beads with the wash buffer.

2 Load Samples and Incubate

• Purpose: Bind the target protein to beads.
• Estimated bench time: 5 mins
• Estimated total time: 60 mins

2.1 Materials

• Washed beads in step 1.
• Protein samples

2.2 Equipment

• Gravity column
• Rotary shaker

2.3 Setup & protocol

1. Add protein samples to washed beads.
2. Use rotary shaker to gently mix the beads and samples in 4℃ for 1hr.

3 Refolding and Elution

• Purpose: Refold and purify the target protein.
• Estimated bench time: 60 mins
• Estimated total time: 60 mins

3.1 Materials

• Protein-binded beads in step2.
• Refolding buffer: 20mM Tris-HCl (pH = 8.0), 0.5M NaCl and 20mM imidazole ( or other concentration if needed)
• Elution buffer: 20mM Tirs-HCl (pH = 8.0), 0.5M NaCl and 500mM imidazole (or other levels of concentration if needed)

3.2 Equipment

• Gravity column

3.3 Setup & protocol

1. Prepare buffers with different urea concentrations(8M, 7M, 6M, 4M, 3M, 2M, 1M, 0M) using binding buffer and refolding buffer.
2. Wash the beads with these buffers one by one from high urea concentration to low urea concentration.
3. Add suitable volume of elution buffer to gravity column and collect the eluted protein.

Inclusion Body Protein Extraction

After Protein Solubility Analysis, if the results indicate that the target protein we aim to extract is present in inclusion bodies, this method will be required to extract the protein from the inclusion bodies using a chemical extraction kit. Names of components during the inclusion body protein extraction process are shown below.

<img src="https://static.igem.wiki/teams/5321/protocol/750ee8bb6c3915df0f53c5816aa311b.jpg" alt="Names of components during the inclusion body protein extraction process" style="max-width: 100%; height: auto;">  

1 Reagent Preparation

• Purpose: prepare buffers used in protein extraction
• Estimated bench time: 15 mins
• Estimated total time: 15 mins

1.1 Materials

• Tris-HCl (1 mol/L, pH8.0) (Shanghai yuanye Bio-Technology, Shanghai, China)
• Ethylenediaminetetraacetic acid (EDTA) (Innochem, Beijing, China)
• Urea (Innochem, Beijing, China)
• Triton X-100 (Harveybio, Beijing, China)
• NaCl (Macklin, China)

1.2 Equipment

• scales (weigh things)
• pipettes & tips
• glass media bottles

1.3 Setup & protocol

• Some reagents are needed to be prepared in advance. Notice that these reagents are preferably prepared fresh and used on the same day, as high-concentration urea solution decomposes over time.

1.3.1 Cell Lysis Buffer:

• The amount of cell lysis buffer is fixed and relatively small, where 10 mL is sufficient for each strain.
• Every 50mL contains:

1.3.2 Inclusion Body Solubilization Buffer:

• The amount of inclusion body solubilization buffer is variable, depending on the initial biomass weight. It is best to reserve at least 40 mL for each strain.
• Every 50mL contains:

2 Inclusion Body Washing

• Purpose: wash away unwanted proteins and lipids
• Estimated bench time: 10 mins
• Estimated total time: 40 mins

2.1 Materials

• bacterial solution after ultrasonication (AU)
• cell lysis buffer as prepared above

2.2 Equipment

• pipettes & tips
• 1.5/50 mL Tubes
• 50 mL tube Centrifuge

2.3 Setup & protocol

1. After ultrasonication centrifugation, first take 100 μl of the sample from the 50ml tube into a 1.5 ml EP tube as reserved sample. (labeled as: strain number + AU + date).

2. Centrifugate the 50ml tube at 12000xg for 20 min at 4°C. Transfer the supernatant into another 50 ml tube for storage (labeled as: number + SU + date).

3. Resuspend the precipitate (PU) thoroughly in 10 ml of cell lysis buffer, then centrifuge at 12000 g, 4°C for 15 min. Pour the supernatant into another 50 ml tube for storage (labeled as: number + SW + date).

4. Store SU, AU, PU, SW in the -20°C freezer if needed.

3 Inclusion Body Dissolution

• Purpose: Dissolve inclusion body proteins into supernatant
• Estimated bench time: 20 mins
• Estimated total time: 1.5 hrs

3.1 Materials

• inclusion body solubilization buffer as prepared above
• precipitate after wash (PW)
• NaOH (Bio&Chem Reagent Management Plat, Peking University)
• PBS buffer

3.2 Equipment

• pipettes & tips
• 1.5/50 mL Tubes
• 50 mL tube Centrifuge
• pH test strips
• 50 mL plastic beakers
• magnetic stirrers

3.3 Setup & protocol

1. Based on the weight of the cells obtained at the time of harvesting, 𝑚 (g), add 20×𝑚 (ml) of inclusion body solubilization buffer to the precipitate (PW) and resuspend thoroughly.

2. Pour in the resuspended solution obtained in step 1. into a 50ml plastic beaker and stir at room temperature for 1 hour using a magnetic stirrer. Note that the stirring speed of the magnetic stirrer should be adjusted beforehand, or it may splash out during stirring.

3. Use pH test strips to measure the pH of the resuspended solution. Add 1 M NaOH to adjust the pH to 8.0 if the pH measured is lower.

4. Transfer the solubilized inclusion body solution at room temperature to a 50 ml centrifuge tube and centrifuge at 12000g, 4°C for 15 minutes.

5. Transfer the supernatant to a 50 ml centrifuge tube(label it as: ID + FS + Date). Here we have obtained the solution containing the target protein .

6. Resuspend the precipitate with 20 ml of PBS (label it as: ID + PD + Date).

7. The dissolved inclusion solution (FS) can be used for downstream on-column refolding and ultrafiltration purification. Note that it is highly recommended to perform on-column renaturation immediately, and do not store dissolved inclusion bodies in urea for a long time.

Nickel Affinity Chromatography through ÄKTA pure™ chromatography system

Nickel affinity chromatography using the ÄKTA pure™ system is employed to purify target proteins while simultaneously performing on-column refolding of proteins from inclusion bodies in denaturing conditions. The ÄKTA pure™ chromatography system is highly efficient and user-friendly. However, a limitation is that the sample volume cannot be too large.

1 Buffers & Samples Preparation

• Purpose: Prepare all the buffers and samples needed.
• Estimated bench time: 20 mins
• Estimated total time: 20 mins

1.1 Materials

• ddH₂O
• dissolved inclusion solution (FS)
• Tris-HCl (1 mol/L, pH8.0) (Shanghai yuanye Bio-Technology, Shanghai, China)
• NaCl (Macklin, China)
• Imidazole (Macklin, China)
• Urea (Innochem, Beijing, China)
• 0.45 μm filter membrane for vacuum pump
• 0.45 μm syringe-driven filter

1.2 Equipment

• Vacuum pump
• syringes
• scales (weigh things)
• pipettes & tips
• glass media bottles

1.3 Setup & protocol

1.3.1 Prepare the buffers:

• Wash and binding buffer NiA should be prepared for sample washing. Every 50mL contains:

• Refolding buffer NiA will be used to perform on-column refolding. Every 50mL contains:

• Elution buffer NiB will be used to elute the target protein. Every 50mL contains:

1.3.2 Filter the buffers and samples:

• Assemble the filter with vacuum pump and filter the buffers with 0.45 μm filter membrane.
• Assemble the syringe-driven filter and filter the samples with 0.45 μm syringe-driven filter.

2 Nickel Affinity Chromatography through AKTA

• Purpose: Doing on-column refolding and purification of proteins.
• Estimated bench time: 2 hrs
• Estimated total time: 2 hrs

2.1 Materials

• Filtrated buffers and samples
• ddH₂O
• 20% Ethanol

2.2 Equipment

• ÄKTA pure™ chromatography system

2.3 Setup & protocol

1. Connect the laptop with AKTA and start the UNICORN program.
2. Wash the flow path of A, B and sample valve, respectively, with ultra-pure water for 5min at a flow rate of 1mL/min.
3. When the ultra-pure water flows through the path, install the nickel column (Make sure that there’s no bubbles in the column).
4. Wash the flow path of B valve with refolding buffer for 5min at a flow rate of 1mL/min, wash the flow path of A valve with wash and binding buffer for 5min at a flow rate of 1mL/min, and wash the sample valve with the same buffer as A for 5min at a flow rate of 1mL/min.
5. Before fractionation process start, set the fraction volume as 10mL to collect the transmission peak. And before sample Loading (approximately 1min), start fractionation.
6. Load samples through sample valve at a flow rate of 1mL/min before the tube of sample valve is exposed to air (usually 1mL sample left).
7. Pause the program and exchange the buffer of sample valve to wash and binding buffer to push the remaining sample onto the column.
8. When the UV curve starts to decline, exchange the valve to A to balance the flow path.
9. When the UV curve reaches baseline, keep the A valve balancing the flow path for 3min.
10. On-column refolding: set the program to exchange the buffer from 100% wash and binding buffer to 100% refolding buffer at a gradient of 15mL. When 100% refolding buffer is balancing the flow path, keep the state for 5 min.
11. Start elution peak fractionation. Set the fraction volume as 1mL.
12. Start elution process. Set the program to exchange the buffer from 100% refolding buffer to 100% elution buffer at a gradient of 15mL. When 100% refolding buffer is balancing the flow path, keep the state for 5 min. When the UV curve drops to baseline for some time, stop elution peak fractionation.
13. Exchange the buffer of A, B, and sample valve to ultra-pure water and wash the flow path for 3min respectively. Do the same with 20% ethanol.
14. When the flow path is balanced by 20% ethanol, uninstall the nickel column (Make sure that there’s no bubbles in the column).
15. End the program, select and collect the samples needed, and export the data of this experiment.

Protease Activity Verification⁴

After extracting the target protein, including both split and intact proteases along with their corresponding substrates, from the inclusion body, it is crucial to verify the enzyme activity. This verification aims to determine whether the extracted protease can effectively react with its respective substrates. The following protocol outlines the method for confirming the enzymatic activity.

1 Reagents Preparation

• Purpose: prepare reagents used in protease activity verification
• Estimated bench time: 20 mins
• Estimated total time: 20 mins

1.1 Materials

• Tris-HCl (1 mol/L, pH8.0) (Shanghai yuanye Bio-Technology, Shanghai, China)
• KCl (Innochem, Beijing, China)
• NaCl (Macklin, China)
• Dithiothreitol (DTT) (Innochem, Beijing, China)
• rapamycin (Aladdin)
• ethanol (Bio&Chem Reagent Management Plat, Peking University)

1.2 Equipment

• Pipettes & tips
• glass media bottles
• 50 mL Tubes
• scales (weigh things)

1.3 Setup & protocol

1.3.1 Prepare Enzyme Reaction Buffer:

• This Enzyme Reaction Buffer as been tested to promote the activity of proteases.
• Every 100mL contains:

1.3.2 Prepare 10^-2 g/L rapamycin solution

• The 10^-2 g/L rapamycin solution is used to facilitate the dimerization of split proteases.
• Due to the hydrolytic instability of rapamycin in water, we have pre-prepared a 0.1 g/L rapamycin solution in ethanol.
• For each use, the required amount is withdrawn and subsequently diluted with water to a final concentration of 0.01 g/L for application.

2 Configuring Enzyme-Substrate Reaction System

• Purpose: prepare enzyme-substrate reaction system
• Estimated bench time: 10 mins
• Estimated total time: 10 mins

2.1 Materials

• Protease (intact or split) and its corresponding substrates extracted from the inclusion body
• Enzyme Reaction Buffer as mentioned above
• 10^-2 g/L rapamycin solution as mentioned above

2.2 Equipment

• Pipettes & tips
• 1.5ml tubes

2.3 Setup & protocol

1. The total reaction volume for the enzyme activity assay is 110 µL, including 2 µg of protease (or 2 µg of each part if using split proteases) and 100 µg of the corresponding substrate. The required volumes of enzyme and substrate should be calculated based on their respective concentrations in the extracted protein samples.

2. After determining the required volumes of enzyme and substrate, aliquot the calculated volumes into a 1.5 mL EP tube.

3. If using split proteases, add rapamycin; in our experiment, three concentrations of rapamycin were used for a gradient experiment (350nM, 700nM, 1400nM).

4. Subsequently, adjust the volume of the reaction mixture to a final volume of 110 μL with enzyme reaction buffer.

3 Reaction Termination and Sample Processing

• Purpose: Termination of the reaction at different times and prepare electrophoretic samples
• Estimated bench time: 30 mins
• Estimated total time: 150 mins

3.1 Materials

• 1.5ml tubes
• SDS loading buffer
• Enzyme-Substrate Reaction System as above

3.2 Equipment

• Dry Bath Incubator

• constant temperature incubator

3.3 Setup & protocol

1. After preparing the reaction mixture, place the 1.5 mL EP tube containing the mixture in a 30°C incubator.
2. At 10, 30, and 120 minutes into the reaction, withdraw 20 μL of the reaction mixture.
3. Immediately after each sampling, add 5 μL of SDS loading buffer to terminate the reaction, and heat the sample in a 95°C metal bath for 5 to 10 minutes.
4. The processed samples can be stored at -20 °C for subsequent electrophoresis or used directly for electrophoretic analysis.

Protein Electrophoresis (SDS-PAGE)

Protein electrophoresis is performed through out our Protein Acquisition and Activity Verification part to validate the expression, extraction and cleavage result of protein samples. We use SDS-PAGE because we only need to separate proteins based on their molecular weight, and we mainly focus on purity than the natural conformation of protein sample. Go to Inclusion Body Protein Extraction, Protein Purification and Protease Activity Verification to see when SDS-PAGE is needed in each part.

1 Gel Preparation

• Purpose: prepare gels used in Protein electrophoresis
• Estimated bench time: 20 mins
• Estimated total time: 20 mins

1.1 Materials

For precast gels: - precast gels (SurePAGE, GenScript) - running buffer (MOPS)

For self-made gels: - 15% SDS-PAGE Gel Quick Preparation Kit (Beyotime, P0012AC) - running buffer (Tris-Gly)

1.2 Equipment

For precast gels: - gel cassette & clamp - sample well - plastic Buffer Dam

For self-made gels: - pipettes & tips - gel cassette & clamp - sample well - plastic Buffer Dam - glass media bottles - 1.5ml, 15ml tubes

1.3 Setup & Protocol

For precast gels: 1. Take the precast gel from the 4°C freezer. Remove the Gel from the pouch. 2. Rinse the gel cassette with deionized water. Peel off the tape from the bottom of the cassette. 3. In one smooth motion, gently pull the comb out of the cassette. 4. Rinse the sample wells with the appropriate 1X SDS Running Buffer (MOPS running buffer, no Tris-Gly!). Invert the gel and shake the gel to remove the buffer. Repeat two more times. 5. Orient the two gels in the Mini-Cell such that the notched “well” side of the cassette faces inwards toward the Buffer Core. 6. Seat the gels on the bottom of the mini cell and lock into place with the Gel Tension Wedge. Note: If you are using only one gel, the plastic Buffer Dam replaces the second gel cassette. 7. Fill the upper buffer chamber with a small amount of the running buffer to check for tightness of seal. If you detect a leak from Upper to the Lower Buffer Chamber, discard the buffer, reseal the chamber, and refill. 8. Once the seal is tight, fill the Upper Buffer Chamber (inner) with the appropriate 1X running buffer (MOPS running buffer). The buffer level must exceed the level of the wells.

For self-made gels: (Different Gel Preparation Kit could have different instructions. Follow instructions from your Gel Preparation Kit to make gels)

1. Select an appropriate concentration of the SDS-PAGE lower gel (i.e., separating gel) based on the molecular weight of the target protein. Please refer to relevant data for the optimal separation range of SDS-PAGE separating gels of different concentrations.
2. Weigh an appropriate amount of gel polymerization catalyst and prepare a 10% gel polymerization catalyst solution using double-distilled water or other high-purity water.
3. In the pre-mixed solution of 15% SDS-PAGE lower gel, add the corresponding amount of 10% gel polymerization catalyst solution at a ratio of 1%, and add the corresponding amount of TEMED at a ratio of 0.04%. For example, in 10 ml of the 15% SDS-PAGE lower gel pre-mixed solution, add 100 μl of the 10% gel polymerization catalyst solution and 4 μl of TEMED.
4. Mix well and pour it into the gel mold, covering the liquid surface with isopropanol, 0.1% SDS, or distilled water until the lower gel is fully solidified. Typically, the gel will solidify within 10-30 minutes.
5. After the lower gel has solidified, in the pre-mixed solution of SDS-PAGE upper gel, add the corresponding amount of 10% gel polymerization catalyst solution at a ratio of 1%, and add the corresponding amount of TEMED at a ratio of 0.1%, then mix well.
6. Remove the liquid covering the top of the lower gel as thoroughly as possible, then pour the SDS-PAGE upper gel pre-mixed solution containing the 10% gel polymerization catalyst solution and TEMED, and insert the comb to await solidification. Once the upper gel has solidified, it indicates that the gel preparation step is complete, and you can prepare for subsequent electrophoresis.
7. If the prepared gel cannot be used on the same day, it can be stored at 4°C for 1-2 days before use.

Assemble the gel to the gel running device: 1. Seat the gels on the bottom of the mini cell and lock into place with the gel clamp. Note: If you are using only one gel, the plastic Buffer Dam replaces the second gel cassette. 7. Fill the upper buffer chamber with a small amount of the running buffer (Tris-Gly) to check for tightness of seal. If you detect a leak from Upper to the Lower Buffer Chamber, discard the buffer, reseal the chamber, and refill. 4. Add Tris-Gly running buffer to the inner and outer tanks. The buffer level must exceed the level of the wells.

2 Sample Preparation

• Purpose: prepare samples used in Protein electrophoresis
• Estimated bench time: 15 mins
• Estimated total time: 15 mins

2.1 Materials

• samples for electrophoresis
• 5× SDS loading buffer

2.2 Equipment

• pipettes & tips
• 1.5ml or PCR tubes
• hydrothermal bath or metal bath equipment
• centrifuge machine

2.3 Setup & Protocol

1. Thaw in advance the 5×SDS loading buffer.
2. Prepare 1.5ml tube for each sample. Add 5×SDS loading buffer into the samples follow the table below:

3. Heat samples at 95° C for 5-10 minutes.
4. For precipitated/uncentrifuged components (AU, PD), centrifuged at 15,000×g, room temperature for 5min after heating. Keep the supernatant for loading. This is to prevent the lysed cell precipitation from affecting the loading.

3 Sample Loading & Running

• Purpose: load prepared samples for electrophoresis
• Estimated bench time: 15 mins
• Estimated total time: 70 mins

3.1 Materials

• samples (prepared as above)
• protein molecular weight markers (M5 10-180kDa prestained)

3.2 Equipment

• pipettes & tips
• HT-Mini01 Vertical Electrophoresis Cell (HTJY, Beijing, China)
• PowerPac™ Basic Power Supply (Bio-Rad, USA)

3.3 Setup & Protocol

1. Thaw in advance the protein molecular weight markers.

2. Load 15μl of sample onto the gel.

3. Load 5μl protein molecular weight markers. Note: If the space is sufficient, do not to sample the two paths on both edges of the gel to avoid margin effect.

4. Fill the Lower Buffer Chamber with the appropriate 1X running buffer.

5. Place the lid on the Buffer Core. With the power on the power supply turned off, connect the electrode cords to the power supply [red to (+) jack, black to (-) jack].

6. Run your gels according to the following conditions:

   Gel Type	Voltage	Expected Current	Run Time
   10% Precast Gel	~160V constant	?	About 40min. Run the gel until the tracking dye reaches 3/4 ~ 4/5 of the bottom of the gel.
   Self-made Gel	<200V constant	?	About 60min. Run the gel until the tracking dye reaches 3/4 ~ 4/5 of the bottom of the gel.

4 Staining and Photograph

• Purpose: Stain and take photograph of the gel with sample for further analysis.
• Estimated bench time: 10 mins
• Estimated total time: half-day

4.1 Materials

• gel after electrophoresis
• CBB (Coomassie brilliant blue), stored in 4°C freezer
• deionized water

4.2 Equipment

• a lid (to put the gel)
• shaker
• 50 mL centrifuge tube wrapped in aluminum foil
• Gel Image System (Tanon 2500)

4.3 Setup & Protocol

1. Move the gel from the plate into a lid for staining.
2. Trim a little (you can cut off the gel in front of the indicator frontline since there’s no protein there; the sample wells can also be cut off).
3. Stain with Coomassie Brilliant Blue. The staining solution is kept in the refrigerator at 4°C ; Add enough to cover the gel surface.
4. Stain on a shaker for 30 minutes.
5. After staining, recover the Coomassie Brilliant Blue into a 50 mL centrifuge tube wrapped in aluminum foil (if the dye shows significant sedimentation or clumping, discard it and use fresh dye next time).
6. Change to deionized water for destaining. You can use excess water and let it destain overnight on the shaker.
7. Once destaining is complete, transfer the gel to the gel imaging equipment for photography and recording.
8. After taking the photo, throw the gel into the designated waste bin for gels.

Protein Solubility Analysis

According to former research, TEVp is prone to form inclusion bodies in E.coli. Our attempt of purifying PPVp (which is homologous to TEVp) in the supernatant after ultrasonication ended in failure. Thus, we analyzed the solubility of PPVp and TEVp using the following protocol:

1 Protein expression

• Purpose: To express the protein for solubility analysis.
• Estimated bench time: 2 days
• Estimated total time: 2 days

See Inclusion Body Protein Extraction protocol for details.

2 Solubility analysis

2.1 Materials

• SDS running buffer
• SurePAGE precast gel
• Coomasie bright blue

2.2 Equipment

• Ultrasonic crusher
• Centrifuge
• Protein electrophoresis tank

2.3 Setup & Protocol

1. Firstly, take 100uL of the component after ultrasonication(called AU)

2. Secondly, precipitant after ultrasonication(PU) and supernatant after ultrasonication(SU) are gained by 12000g 4°C 10min

3. AU, PU, SU are subjected to SDS-PAGE. Also, AU, PU, SU from EGFP expression strain are used as positive control(EGFP exists in soluble form). If protein forms inclusion bodies in the cell, its major presence will be in the PU and AU part. In comparison, EGFP mainly appears in the SU and AU part.

Single Plasmid Transformation and Induced Expression

To express the protein of interest, CDS of the protein is cloned into pET28a vector and transformed into BL21(DE3) strain, and expression is induced by IPTG.

1 Transformation of plasmid

• Purpose: To transform the plasmid of interest into BL21(DE3)
• Estimated bench time: 1 day
• Estimated total time: 1 day

1.1 Materials

• yeast extract (Oxoid, USA)
• tryptone (Aobox, Beijing, China)
• agar
• kanamycin sulfate solution 10 mg/mL (Shanghai yuanye Bio-Technology, Shanghai, China)
• NaOH (Bio&Chem Reagent Management Plat, Peking University)
• NaCl (Macklin, China)
• BL21(DE3) competent cells
• pET28a (with target gene inserted)

1.2 Equipment

• conical flasks
• autoclaves
• water baths
• culture dishes
• cryovials
• spectrophotometers

1.3 Setup & protocol

1.3.1 Preparation of LB culture medium

1. Add deionized water 950 mL, tryptone 10 g, yeast extract 5 g, NaCl 10 g
2. Shake the container until the solute is dissolved, and adjust the pH to 7.0 with 5M NaOH. Make up to 1000mL with deionized water.
3. Preparation of solid LB medium: add 1.7g agar powder per 100ml LB liquid medium
4. The above liquid and solid culture media are sterilized by high pressure steam
5. When the temperature of LB solid culture medium drops to around 55℃, add kanamycin to each bottle to make the final concentration 40ug/mL
6. Pour 15-20ml into one culture dish.
7. Store upside down at 4℃ and use within 3 weeks.

1.3.2 Transformation

1. Use nanodrop to measure the plasmid concentration and calculate the transformation volume based on the transfer of 40ng plasmid

2. Thaw the competent cells on ice

3. Add the plasmid, gently rotate the tube to mix the contents, and place it in ice bath for 30 minutes.

4. Place the competent cells in a 42℃ water bath for 75 seconds and then in ice bath for 2 minutes.

5. Add 500ul of antibiotic-free LB medium to the competent cells and culture at 37℃ 180rpm for 1 hour.

6. Take 200uL of the transformed competent cells, apply LB plates containing the corresponding antibiotics, and culture them upside down at 37℃ for 12-16 hours.

2 Induction of protein expression

• Purpose: To induce expression of target protein
• Estimated bench time: 1 day
• Estimated total time: 1 day

2.1 Materials

• isopropyl β-d-1-thiogalactopyranoside (IPTG) (Innochem, Beijing, China)

2.2 Equipment

• spectrophotometers
• large centrifuge

2.3 Setup & protocol

1. Add 10mg/mL kanamycin to the liquid LB medium to a final concentration of 40ug/mL (add 1.6mL per 400mL medium)
2. Take a 1L conical flask, add 400mL of liquid medium containing antibiotics to each, and pick the corresponding single colony.
3. Shake at 37°C 220rpm for 5-7 h
4. Measure the OD600 absorbance with a spectrophotometer (blank LB liquid medium) When OD600 > 0.3, add IPTG to induce protein expression (to a final concentration of 0.4mM)
5. Induce overnight at 20°C, 220rpm for 16h

Others

DBCO-p-AzF Ligation Reaction⁵⁶

Combining the strong binding properties of aptamers with the activity of split proteases relies on covalent cross-linking of the two components. Here, we present a method for achieving this linkage using click chemistry, mediated by dibenzocyclooctyne (DBCO) and 4-Azido-L-phenylalanine (p-AzF). The fundamental principles outlined here are theoretically transferable to other types of biomolecules.

1 Ligation Reaction

• Purpose: obtain aptamer-split protease molecular machine

• Estimated bench time: 1 hr

• Estimated total time: 1 day

1.1 Materials

• DBCO-modified aptamers
• p-AzF incorporated split proteases (concentrated to 50 μM)
• PBS buffer (pH = 7.2)
• materials needed in protein denaturation electrophoresis

1.2 Equipment

• Vortex Mixer
• pipettes & tips
• constant temperature incubator
• equipment needed in protein denaturation electrophoresis

1.3 Setup & protocol

1. Sample Preparation

• Dissolve the aptamers with PBS buffer to 10-20 mM;

2. Ligation Reaction

• Proteins were mixed with aptamer at a molecular ratio of 1:10-100, Vertex-mixed, and reacted at 37°C for 5-20 h;

3. Product Verification

• Perform SDS-PAGE electrophoresis and stain with Coomassie Brilliant Blue as protein electrophoresis protocol.

Reference