Experiments

PCR

• 1. Set up assembly reactions in PCR-tubes for a total volume of 50 µL as follows:

• 2. Mix gently and spin down.
• 3. Place PCR-tubes in thermocycler and run the following program:

Transformation

• 1. Thaw chemically competent cells on ice.
• 2. Add DNA (30ng), and pipette gently to mix.
• 3. Incubate the suspension for 25 minutes on ice.
• 4. Heat shock is done by incubating the cells for 45 seconds at 42℃.
• 5. Chill on ice for 2 min.
• 6. Add 700μl of LB medium and incubate for 1 hour at 37℃, 200rpm in order to obtain antibiotic resistance.
• 7. Centrifuge at 5000rpm for 1min, discard the supernatant to 100 μl and spread onto a plate with appropriate antibiotics.
• 8. Incubate overnight at proper temperature.

Plasmid Extraction

• 1. Transfer 1 - 5 ml of overnight (12 - 16 h) culture to a centrifuge tube, and centrifuge at 10,000 rpm (11,500 × g) for 1 min. Discard the culture medium and place the tube inverted on a blotting paper to drain the liquid.
• 2. Add 250 μl of Buffer P1 (make sure that RNase A has been added) to the centrifuge tube containing the precipitated bacterial cells, and mix thoroughly by pipetting or vortexing.
• 3. Add 250 μl of Buffer P2 to the mixture from Step 2. Mix by gently inverting the tube 8 - 10 times to completely lyse the cells.
• 4. Add 350 μl of Buffer P3 to the mixture from Step 3. Immediately invert the tube gently 8 - 10 times to fully neutralize Buffer P2. At this time, white flocculent precipitates should form. Centrifuge at 12,000 rpm (13,400 × g) for 10 min.
• 5. Place FastPure DNA Mini Columns into a Collection Tube 2 ml. Carefully transfer the supernatant from Step 4 to the FastPure DNA Mini Columns with a pipette, taking care not to disturb the precipitates. Centrifuge at 12,000 rpm (13,400 × g) for 30 - 60 sec. Discard the filtrate and place the FastPure DNA Mini Columns back into the Collection Tube.
• 6. Add 600 μl of Buffer PW2 (make sure that absolute ethanol has been added) to the FastPure DNA Mini Columns. Centrifuge at 12,000 rpm (13,400 × g) for 30 - 60 sec. Discard the filtrate and place the FastPure DNA Mini Columns back into the Collection Tube.
• 7. Repeat Step 6.
• 8. Place the FastPure DNA Mini Columns back into the Collection Tube. Centrifuge the empty column at 12,000 rpm (13,400 × g) for 1 min to completely remove the residual wash buffer.
• 9. Place the FastPure DNA Mini Columns in a new sterile 1.5 ml centrifuge tube. Add 30 - 100 μl of Elution Buffer to the center of the spin column membrane. Leave the system at room temperature for 2 min, and centrifuge at 12,000 rpm (13,400 × g) for 1 min to elute the DNA.
• 10. Discard the FastPure DNA Mini Columns and store the extracted DNA at -20°C to prevent degradation.

Gel Extraction

• 1. Column equilibration: add 500 µl Buffer BL to the Spin Column CA2 (put Spin Column CA2 into a collection tube). Centrifuge for 1 min at 12,000 rpm in a table-top microcentrifuge. Discard the flowthrough, and put Spin Column CA2 back into the collection tube.
• 2. Cut the DNA fragment from agarose gel with a clean, sharp scalpel. Weigh the gel slice in a clean 1.5 ml EP tube.
• 3. Add equivalent volume of Buffer PN to the gel (If the gel is 0.1 g, it is defaulted to be 100μl, then add 100μl Buffer PN). Incubate at 50°C by inverting up and down the tube until the agarose gel dissolves completely. (If DNA fragment is <300 bp, it is recommended to add isopropanol which is 1/2 volume of Buffer PN to the agarose gel sample after the gel is completely dissolved.)
• 4. When the gel dissolves completely and the solution temperature turns to room temperature, transfer the mixture to the Spin Column CA2 (put Spin Column CA2 into a collection tube). Let the column stand for 2 min at room temperature, then centrifuge for 30-60 sec at 12,000 rpm in a table-top microcentrifuge. Discard the flow-through; place the Spin Column CA2 back into the collection tube again.
• 5. Wash the Spin Column CA2 with 600 µl Buffer PW (ensure that ethanol has been added) and centrifuge for 30-60 sec at 12,000 rpm. Discard the flow-through and place the Spin Column CA2 back into the collection tube.
• 6. Repeat Step 5.
• 7. Place the Spin Column CA2 back to the collection tube and centrifuge at 12,000 rpm for 2 min to remove residual Buffer PW. Discard the flow-through, and place column with the cap open for several minutes to air dry the membrane.
• 8. Transfer the Spin Column CA2 to a clean 1.5ml centrifuge tube. Add appropriate volume of Buffer EB to the center of the membrane, incubate at room temperature for 2 min, then centrifuge at 12,000 rpm for 2 min.

Purification of PCR Products

• 1. Column equilibration: add 500 μl Buffer BL to the Spin Column CB2 (put Spin Column CB2 into a collection tube). Centrifuge for 1 min at 12,000 rpm. Discard the flow-through, and then place Spin Column CB2 back into the collection tube.
• 2. Add 5 volumes of Buffer PB to 1 volume of the PCR reaction or enzymatic reaction and mix.
• 3. Transfer the mixture to the Spin Column CB2, incubate at room temperature for 2 min. Centrifuge for 30-60 sec at 12,000 rpm in a table-top microcentrifuge. Discard the flow-through, and then place Spin Column CB2 back into the same collection tube.
• 4. Add 600 µl Buffer PW (ensure that ethanol (96-100%) has been added) to the Spin Column CB2 and centrifuge for 30-60 sec at 12,000 rpm. Discard the flow-through, and place Spin Column CB2 back in the same collection tube.
• 5. Repeat step 4.
• 6. Centrifuge at 12,000 rpm (~13,400×g) for 2 min to remove residual Buffer PW. Discard the flow-through, and allow the column to air dry with the cap open for several minutes to dry the membrane.
• 7. Place the Spin Column CB2 in a clean 1.5 ml microcentrifuge tube. Add 30-50 μl Buffer EB to the center of membrane, incubate for 2 min, and centrifuge for 2 min at 12,000 rpm.

Restriction Cloning

1.Digestion

• (1) Reaction system:

• (2) Mix components by pipetting the reaction mixture up and down, or by "flicking" the reaction tube.
• (3) Quick ("touch") spin-down in a microcentrifuge. Do not vortex the reaction.
• (4) Incubate for 1 hour at the enzyme-specific appropriate temperature.

2. Ligation

• (1) Reaction system:

• (2) Program: 16℃ for 1-5h

Seamless Cloning

Agarose Gel Preparation

Agarose Gel Electrophoresis

Protein Analysis

Coomassie Blue Staining

GST Affinity Column Protein Purification

Based on experimental results and observations, the protocol from beyondtime kit has been revised and optimized as follows:

1. IPTG-Induced Expression of GST-Tagged Protein in E. coli

The following protocol describes the widely used IPTG-induced expression system. For optimization of induction conditions, please refer to the detailed instructions specific to your expression system. Other induction systems should follow their respective guidelines.

• a. Pick a single colony expressing the GST-tagged protein and inoculate it into 3-10 ml of LB medium containing the appropriate antibiotic. Incubate overnight at 37°C with shaking.
• b. Dilute the overnight culture 1:20 into fresh, pre-warmed (37°C) LB medium containing the appropriate antibiotic. For example, inoculate 5 ml of overnight culture into 100 ml of pre-warmed, antibiotic-supplemented LB. The culture volume depends on the required protein yield: 3-10 ml for initial small-scale verification; 100-200 ml for routine expression and purification; and 1 L or more for preparative-scale purification. For improved expression outcomes, a 1:100 dilution of the overnight culture is recommended, although this will require a longer incubation time to reach the target OD.
• c. Incubate the culture at 37°C with shaking for approximately 30-60 minutes, or until the OD₆₀₀ reaches 0.6-1.0.
• d. Add IPTG to a final concentration of 1 mM and continue incubation at 37°C for 2-4 hours to induce protein expression.

Note: Prior to IPTG addition, remove a small aliquot of the culture to serve as an uninduced control. This control can be incubated separately for the same duration or sampled directly. Optimal IPTG concentration, induction temperature, and induction time should be determined experimentally for each specific protein.

• e. Transfer the culture to centrifuge tubes. Harvest the cells by centrifugation at 4,000 × g for 20 minutes or 15,000 × g for 1 minute at 4°C. Discard the supernatant. The cell pellet can be processed immediately for lysis or stored at -20°C or -80°C for future use. Frozen pellets should be thawed on ice for 15 minutes before proceeding.

2. Reagent Preparation

• a. 10X GSH Solution: Dissolve the 184 mg of GSH provided in the kit in 6 ml of the supplied Elution Buffer (requires GSH addition). Mix thoroughly to obtain the 10X GSH solution. The prepared solution can be stored at -20°C and remains stable for at least one year.
• b. Elution Buffer: Mix the provided Elution Buffer (requires GSH addition) with the 10X GSH solution at a 9:1 ratio (e.g., combine 9 ml of Elution Buffer with 1 ml of 10X GSH solution). The resulting mixture is the working Elution Buffer. Due to the susceptibility of GSH to oxidation, the Elution Buffer should be prepared fresh and can be stored at 4°C for up to two weeks. The final composition of the Elution Buffer is 50 mM Tris, 150 mM NaCl, 10 mM GSH, pH 8.0.

3. Large-Scale Purification of GST-Tagged Protein

This method is suitable for purifying larger quantities of protein (e.g., from a starting culture volume of 150 ml or more).

• a. Following step 1(e), for fresh or thawed bacterial pellets, add Lysis Buffer at a ratio of 4 ml (a range of 2-5 ml is acceptable) per gram of wet cell pellet. Resuspend the cells thoroughly. If necessary, add an appropriate protease inhibitor cocktail (e.g., Beyotime's P1025/P1026 for bacterial extracts or P1005/P1006 general-use protease inhibitor cocktail) to the lysis buffer before cell disruption.
• b. Add lysozyme to a final concentration of 1 mg/ml and mix thoroughly. Incubate the suspension on ice or in an ice-water bath for 30 minutes.

Note: Lysozyme can be prepared as a 100 mg/ml stock solution in Lysis Buffer, aliquoted, and stored at -20°C. It should be added to the sample just before use.

• c. Lyse the cells using sonication on ice. Apply sonication at a power of 200-300 W, using pulses of 10 seconds followed by 10-second intervals, for a total of 6 cycles.

Note: The specific sonication protocol should be optimized for the particular ultrasonic instrument used.

• d.(Optional) If the lysate remains highly viscous after sonication, add RNase A to a final concentration of 10 µg/ml and DNase I to 5 µg/ml, and incubate on ice for 10-15 minutes. Alternatively, shear the genomic DNA by repeatedly passing the lysate several times through a syringe fitted with a narrow-gauge needle.
• e. Centrifuge the lysate at 10,000 × g for 20-30 minutes at 4°C. Collect the clarified supernatant and keep it on ice or in an ice-water bath. Save 20 µl of the supernatant for subsequent analysis.

Note: The supernatant must be clear and free of insoluble material before proceeding to the purification step. The presence of insoluble impurities will significantly compromise the purity of the final protein product.

• f. Take 1 ml of well-resuspended BeyoGold™ GST-tag Purification Resin. Centrifuge at 1,000 × g for 10 seconds at 4°C and discard the storage solution. Add 0.5 ml of Lysis Buffer to resuspend and equilibrate the resin. Centrifuge again and discard the liquid. Repeat this equilibration step 1-2 more times. After the final equilibration, add approximately 4 ml of the clarified bacterial lysate supernatant to the resin. Incubate at 4°C with gentle shaking on a orbital shaker for 60 minutes.
• g. Load the mixture of lysate and BeyoGold™ GST-tag Purification Resin into the provided affinity chromatography empty column (3 ml).

Note: An alternative method is to first prepare a column with 1 ml of the 50% BeyoGold™ GST-tag Purification Resin slurry, equilibrate it with 0.5 ml of Lysis Buffer (2-3 times), then load the ~4 ml of clarified lysate supernatant. The flow-through can be collected and reloaded onto the column 3-5 times to enhance binding efficiency. While the batch binding method (mixing before loading) is slightly more cumbersome, it often promotes better binding of the GST-tagged protein to the resin.

• h. Open the bottom cap of the purification column and allow the liquid to drain out by gravity. Collect approximately 20 µl of the flow-through for subsequent analysis.
• i. Wash the column 5 times, each time with 0.5-1 ml of Lysis Buffer. Collect about 20 µl of the wash flow-through after each wash for subsequent analysis. During the washing and subsequent elution steps, the protein content in the washes and eluates can be monitored quickly using the Bradford method (P0006) to determine if the number of washes or elutions should be adjusted.

Note: If the purity of the final protein is insufficient, the number of washes can be increased by 2-3 times. If required, the GST tag can be cleaved from the fusion protein using enzymes such as PreScission Protease (P2302/P2303), TEV Protease (P2307/P2308), or Thrombin to release the target protein. Refer to step 5 for detailed methods on GST tag cleavage and removal.

• j. Elute the target protein 6-10 times, each time with 0.5 ml of Elution Buffer. Collect each elution fraction into separate centrifuge tubes. The collected eluates contain the purified GST-tagged protein samples.

Functional Verification Assays

Liquid Culture Characterization

• 1. The optical density of overnight cultures is measured at 600 nm and then diluted at a specific ratio.
• 2. The diluted culture is incubated at 37 °C with shaking for 3 h. (If induction of AHL receptor protein expression is required, 0.5 mM IPTG is added at a final concentration when the OD600 reaches 0.6, followed by a 1-hour induction period.)
• 3. Bacterial culture and the appropriate signal molecule solution are mixed in the wells. The total volume in each well is adjusted to 200 µL using medium.
• 4. The plate is placed in the plate reader and incubated at 37 °C with continuous shaking for 3 h. Absorbance (600 nm) and fluorescence (excitation 484 nm, emission 510 nm) are measured every 6 min.

Agar Plate Colony Experiments

This protocol is adapted from Fedorec et al. (2024).

• 1. The plates, which are filled with about 12 mL of solid medium containing the appropriate antibiotic, are allowed to dry at 55 °C for ~2.5 min in a laboratory oven to prevent interference from condensed water.
• 2. The optical density of overnight cultures is measured at 600 nm, and the cultures are diluted to an OD600 of 0.6.
• 3. 0.3 μL of the diluted culture and 1.0 μL of the appropriate signal molecule solution are dispensed onto the surface of the agar at the positions shown in the corresponding spotting patterns (placed beneath the culture dish).
• 4. The plates are left to dry until the droplets are no longer visible (approximately 15 min).
• 5. The plates are then placed in an incubator at 37 °C and imaged with a fluorescence microscope at 12 h, 15 h, 18 h, 21 h, and 24 h to monitor growth and fluorescence.