This experimental protocol details the various molecular biology techniques used in the construction and characterization of arsenic biosensors, including medium preparation, DNA manipulation, plasmid construction, and sensor performance testing.
To prepare 1000 mL of LB medium, add 10 g tryptone, 5 g yeast extract, and 10 g NaCl. For solid medium, additionally add 15 g agar. Dissolve these components in approximately 800 mL of Reverse Osmosis (RO) water. Adjust the pH to 7.0-7.2 using 1 mol/L Sodium Hydroxide (NaOH) or 1 mol/L Hydrochloric Acid (HCl). Add RO water to bring the total volume to 1000 mL. Autoclave at 121°C for 20 minutes. After the medium cools to 50-55°C, antibiotics (Kanamycin / Carbenicillin) can be added under sterile conditions to a final working concentration of 50 μg/mL.
Prepare an ice box. Place the template, primers, 2×Phanata Unifi Master Mix, template plasmid, and double-distilled water (ddH₂O) in the ice box to thaw. Place an appropriate number of microcentrifuge tubes in the ice box as well.
Prepare the reaction mixture. Note the amount of template used (template concentration determines the volume to be added).
| Component | Volume |
|---|---|
| Template | 2 μL |
| Forward Primer (Primer_F) | 2 μL |
| Reverse Primer (Primer_R) | 2 μL |
| 2×Phanata Unifi Master Mix | 25 μL |
| ddH₂O | 19 μL |
| Total Volume | 50 μL |
Molar Ratio: Maintain a molar ratio of vector to insert of 1:5. Calculate the required volumes based on fragment concentration and length.
| Component | Volume |
|---|---|
| Fragment I | x μL |
| Fragment II | y μL |
| 2×Unicclone Seamless Cloning Mix | 5 μL |
| Total Volume | 10 μL |
PCR Reaction: 50°C for 15 min.
| Component | Volume |
|---|---|
| DPNⅠ | 0.5 μL |
| DNA | 5 μL |
| ddH₂O | 19 μL |
| rCutSmart Buffer | 5 μL |
| Total Volume | 25 μL |
PCR Reaction: 37°C for 90 min + 80°C for 20 min.
Gel Preparation: For a 1% agarose gel, dissolve 1.0 g agarose in 100 mL of 1×TAE buffer. Heat until completely dissolved, cool to ~50°C, pour into a casting tray, and insert a comb.
Sample Preparation: Mix 4 μL of PCR product with 3 μL of loading dye.
Running the Gel: Place the gel in the electrophoresis tank, add 1×TAE buffer, then load the samples. Run at 150 V, 300 mA until separation is complete.
Take competent cloning cells from the -80°C freezer and thaw them on ice.
Take 1 μL of the recombinant product and add it to 100 μL of recipient cells. Gently pipette up and down, then incubate on ice for 30 minutes.
Heat-shock in a 42°C water bath for 45 seconds.
Immediately cool on ice for 3 - 5 minutes.
Add 200 μL of LB medium and shake at 37°C, 180 rpm for 1 hour.
Take 100 μL of the bacterial culture and spread evenly onto a plate. Incubate inverted at 37°C for 12-16 hours.
Column Equilibrium: Add 500 μL of Balance Buffer BL to the CP3 adsorption column (placed in a collection tube). Centrifuge at 12,000 rpm (≈13,400 ×g) for 1 minute. Discard the flow-through from the collection tube and place the CP3 column back into the collection tube.
Take 1-5 mL of an overnight bacterial culture and transfer it to a centrifuge tube. Centrifuge at 12,000 rpm (≈13,400 ×g) for 1 minute using a standard benchtop centrifuge. Carefully aspirate and discard the supernatant completely. (For large culture volumes, collect the bacterial pellet into one tube through multiple centrifugation steps).
Add 250 μL of Solution P1 to the centrifuge tube containing the bacterial pellet. Resuspend the pellet thoroughly using a pipette or vortex mixer. (Ensure RNase A has been added to P1 beforehand).
Add 250 μL of Solution P2 to the centrifuge tube. Gently invert the tube 6-8 times to lyse the bacteria completely. Note: Mix gently; avoid vigorous shaking to prevent shearing genomic DNA, which could contaminate the plasmid prep. The solution should become clear and viscous. Do not exceed 5 minutes for this step to avoid plasmid damage. If the solution does not clear, it may indicate excessive bacteria and incomplete lysis; use less bacterial culture next time.
Add 350 μL of Solution P3 to the centrifuge tube. Immediately invert the tube gently 6-8 times to mix thoroughly. A white flocculent precipitate will form. Centrifuge at 12,000 rpm (≈13,400 ×g) for 10 minutes.
Carefully transfer the supernatant from the previous step to the CP3 adsorption column (placed in a collection tube) using a pipette. Avoid transferring the precipitate. Centrifuge at 12,000 rpm (≈13,400 ×g) for 30-60 seconds. Discard the flow-through and place the CP3 column back into the collection tube.
Add 600 μL of Wash Buffer PW to the CP3 column. Centrifuge at 12,000 rpm (≈13,400 ×g) for 30-60 seconds. Discard the flow-through and place the CP3 column back into the collection tube. (Ensure ethanol has been added to PW beforehand).
Repeat Step 7.
Place the CP3 column in the collection tube and centrifuge at 12,000 rpm (≈13,400 ×g) for 2 minutes to remove residual wash buffer.
Place the CP3 column into a clean centrifuge tube. Add 50-100 μL of Elution Buffer EB to the center of the adsorption membrane. Let it stand at room temperature for 2 minutes. Centrifuge at 12,000 rpm (≈13,400 ×g) for 2 minutes to collect the plasmid solution in the centrifuge tube.
Sample Treatment: Inoculate E. coli harboring the sensor plasmid into LB liquid medium (containing antibiotic) and culture at 37°C until OD₆₀₀ reaches 0.6-0.8. Add NaAsO₂ gradiently and continue culturing at 37°C for 6 hours.
Fluorescence Detection: Measure GFP fluorescence intensity using a microplate reader (excitation wavelength 488 nm, emission wavelength 520 nm). Use the uninduced group as a control. Calculate the relative fluorescence intensity (a.u.), fit the dose-response curve, and obtain key parameters.