Overnight culture of bacteria transformed with plasmid
Plasmid Mini Kit
Sterilized distilled water
Equipment
Eppendorf tube
Desktop microcentrifuge
Desktop vortex mixer
Thermostat water bath
Procedures
Grow an overnight culture of bacteria.
Centrifuge the 1~5 mL culture to pellet the bacteria before proceeding with DNA preparation.
Remove the supernatant and resuspend the bacteria in buffer.
Add a denaturing solution from the Plasmid Mini Kit to the resuspended bacteria.
Add a renaturing solution from the Plasmid Mini Kit to the denatured bacteria.
Pellet the proteins and genomic DNA by centrifugation and remove the plasmid-containing supernatant.
Add ethanol to precipitate the plasmid DNA.
Centrifuge at 12000 rpm for 10 min to pellet the DNA.
Wash the pellet with 70% ethanol to remove excess salt.
Resuspend the DNA pellet in sterilized distilled water.
2. Acquisition of Target Gene Fragment
Materials
Template DNA containing the target fragment
Forward Primer
Reverse Primer
2 × Proofast Master Mix (Dye)
Sterilized distilled water
Equipment
Eppendorf tube
Desktop microcentrifuge
Desktop vortex mixer
Ice box
PCR instrument
Procedures
Place thin-wall PCR tubes on ice. Set up a 50 μL reaction:
800 ng template DNA
2.5 μL of 100 μM reverse primer
2.5 μL of 100 μM forward primer
25 μL of 2 × Proofast Master Mix (Dye)
H₂O to a total of 50 μL
Place reaction tubes in PCR machine. Set the standard time and temperature for each step in a PCR cycle:
Initial Denaturation for 2 min at 98°C.
Denature for 10 s at 98°C.
Anneal primers for 15 s at 55°C below.
Extend DNA for 40s at 72°C.
Repeat b-d steps for 30 cycles
Extension for 5 min at 72°C
The products were stored at 4°C.
3. Preparation of Linearized Plasmids
Materials
Purified plasmid DNA
Restriction endonucleases: EcoRI and BamHI
10× Restriction enzyme buffer
Sterilized distilled water
Equipment
Thermostatic water bath
Microcentrifuge tubes
Agarose gel electrophoresis system
Procedures
Prepare a 50 μL digestion reaction containing:
1 μg purified plasmid DNA
1 μL EcoRI enzyme
1 μL BamHI enzyme
Nuclease-free water to 50 μL
Mix gently and incubate at 37°C for 1 hour in a thermostatic water bath.
Verify complete linearization by analyzing the product via agarose gel electrophoresis.
4. Construction of Recombinant Plasmids
Materials
Linearized vector plasmid DNA
Target gene segment
DNA ligase and buffer
Luria-Bertani media
Competent state Escherichia coli DH5α
Equipment
Eppendorf tube
Shaking incubator at 37°C
Stationary incubator at 37°C
Water bath at 42°C
Ice box
Microcentrifuge tubes
Sterile spreading device
Procedures
Combine the following in an Eppendorf tube:
2.5 μL plasmid vector DNA
2 μL insert DNA (for a control group, add H₂O instead of insert DNA)
2 μL ligase Buffer
1 μL DNA Ligase
H₂O to a total of 20 μL
Incubate at 37°C for 30 min before cooling in an ice water bath for 5 min.
Retrieve competent cells from -80°C refrigerator and defrost on ice for approximately 10 min.
Retrieve the agar plates containing the appropriate antibiotic from storage at 4°C, and let them warm up to room temperature.
Mix 10 μL of plasmid DNA into 100 μL of competent cells in a microcentrifuge tube. Gently mix the components. For a control group, add 10 μL of water to verify that the LB agar’s antibiotic functions properly.
Incubate the competent cell/DNA mixture on ice for approximately 30 min.
Heat shocks each transformation tube by placing the bottom half of the tube into a 42°C water bath, leaving it for approximately 45 s.
Put the tubes back on ice for 2 min.
Add 300 μL LB media (without antibiotic) to the bacteria, and grow in 37°C shaking incubator for 30 min.
Plate an appropriate amount of the transformation onto the LB agar plate containing the corresponding antibiotic. Incubate plates at 37°C overnight.
5. Agarose Gel Electrophoresis
Materials
DNA samples
Gel loading dye
10 X TAE buffer
Agarose
4S green plus
Equipment
Well combs
Gel box
Microwave
Electrophoresis chamber
Voltage source
UV light source
Casting tray
Procedures
Mix 0.2 g of agarose powder and 1 μL of 4S green plus with 20 mL 1 X TAE in a microwavable flask.
Microwave for 1-3 min until the agarose is completely dissolved.
Let agarose solution cool down to about 50°C in about 5 min.
Pour the agarose into a gel tray with the well comb in place.
Place the gel at room temperature for 20-30 min, until it has completely solidified.
Place the gel into the gel box of electrophoresis unit. Fill gel box with 1 X TAE until the gel is covered.
Carefully load a molecular weight ladder.
Add loading buffer to each DNA sample. Carefully load samples into the additional wells of the gel.
Run the gel at 120 V for approximately 15 min.
Turn off power, disconnect the electrodes, and remove the gel from the gel box.
Visualize DNA fragments using UV light source.
6. Recombinant Protein Expression
Materials
Competent Pseudomonas kunmingensis HL22-2T
Antibiotic
20 % L-Arabinose
Luria-Bertani media
Equipment
250 mL shaking flask
Shaking incubator at 30°C
Procedures
2 mL of the bacteria solution was added to 100 mL of LB medium, incubated at 30°C with shaking until the OD600 was between 0.60, add 100 µL of 20 % L-Arabinose solution and incubate at 30°C for 12 h.
Centrifuge at 4°C and discard the supernatant to collect the bacteria.
7. Extraction Protein
7.1 Extraction of Protein
Materials
Sterilized distilled water
SDS buffer
5×Protein loading buffer
Equipment
Eppendorf tube
50mL Centrifuge tube
Desktop centrifuge
Metal bath pot
Centrifugal machine
Ultrasonic cell breaker
Procedures
Add 40mL Sterilized distilled water to the cell, resuspend the cells, and ultrasonically break the cells on ice for 1h.
After centrifugation at 11000 rpm for 20 min at 4°C, the supernatant was collected and passed through a 0.22 µm filter membrane.
7.2 Verification of Protein
Materials
SDS-PAGE gel
SDS-PAGE running buffer
Loading buffer
Protein Ladder
Coomassie brilliant blue G-250
Coomassie Blue Staining Destaining Solution
Equipment
SDS-PAGE electrophoresis tank
SDS-PAGE transfer tank
Microwave
Imager System
Procedures
The SDS-PAGE gel was placed in the electrophoresis tank. Connect electrode and load 20 µL of each sample into the gel.
Run the gel at 100 V for approximately 20 minutes, until the dye has migrated into the running gel.
Increase the voltage to 150 V and continue for approximately 30 min, until the dye front has reached the bottom of the gel.
Place the gel into a small tray. Add Coomassie brilliant blue G-250 and heat for 1 min.
Pour off the Coomassie brilliant blue G-250 and add dehydration solution for rinsing.
Put the gel into the imager and select the appropriate exposure time, and save the image.
8. REEs Adsorption
Materials
Sterilized distilled water at pH4.5
Recombinant Pseudomonas kunmingensis HL22-2T
Sterilized rare earth solution
Antibiotic
20% L-Arabinose
Luria-Bertani media
Equipment
50 mL Centrifuge tube
Desktop centrifuge
Centrifugal machine
250 mL shaking flask
100 mL shaking flask
Shaking incubator at 30°C
Procedures
2 mL of the bacteria solution was added to 100 mL of LB medium, incubated at 30°C with shaking until the OD600 was between 0.6, add 100 µL of 20% L-arabinose solution and incubate at 30°C for 12 h.
Centrifuge at 20°C and discard the supernatant to collect the bacteria, weigh after washing with sterilized distilled water once.
The washed and weighed bacterial (wet weight 0.3g) was added to 30 mL PH 4.5 sterilized distilled water, and then add 30 µL 100 µM sterilized rare earth solution.
The bacteria adsorbed rare earth elements for 4 days in a constant temperature Shaking incubator at 30°C, 200 rpm.
After 4 days, Centrifuge at 20°C and discard the supernatant to collect the bacteria, washing with sterilized distilled water once, and the qualitative and quantitative analysis of rare earth elements in the bacterial sediment was performed by ICP-MS.
9. Phosphate Detection
Materials
Malachite Green Phosphate Detection Kit
Sterilized ddH₂O
Rare earth solution after bacterial incubation
Equipment
1.5 mL Centrifuge tube
Microplate reader
Desktop centrifuge
96-well plates
Shaking incubator at 30°C
Procedures
Collect the rare earth solution after bacterial incubation, centrifuge at 14,000 rpm for 5 min, and retain the supernatant.
Add an appropriate volume of the test sample to a 1.5 mL centrifuge tube. If the sample volume is less than 200 µL, dilute with the standard sample diluent to a total volume of 200 µL. Record the dilution factor at this stage.
Pipette 200 µL of each phosphate standard and test sample from the tubes sequentially into the sample wells of the 96-well plate.
Add 70 µL of color developer to each well. Mix thoroughly using a multi-well plate mixer or gently tap the pipette to blend. Incubate at room temperature for 30 minutes.
Measure the absorbance at 630 nm using a microplate reader.
Calculate the phosphate concentration of the sample based on the standard curve and the dilution factor of the test sample.