E. coli competence preparation

1) Select the single colonies of Escherichia coli, inoculate in 20mL LB medium, and culture at 37℃ for one night.

2) Inoculate in 20mL LB medium at 1% of the inoculum, and shake the culture at 37℃ and 200 rpm for 3h.

3) Take 1mL culture, ice bath for 30 min, then centrifugate at 4℃ 4,000 rpm for 3 min to remove the supernatant.

4) Add 500ul of ice-cold 0.1mol/L CaCl₂ solution, resuspend the bacteria pellet, centrifugate at 4,000 rpm at 4°C for 3 min, and remove the supernatant.

5) Add 100μL of ice-cold 0.1 mol/L CaCl₂ solutions, resuspend the bacterial pellet, centrifugate at 4,000 r/min at 4°C for 3 min, and remove the supernatant.

6) Add 50μL of ice-cold 0.1 mol/L CaCl₂ solutions, resuspend the bacterial pellet, and ice bath for 3h-24h to get the E. coli competent cells.

E. coli heat shock transformation

1) Take 50μL of the competent Escherichia that melted on ice to the precooled 1.5 mL EP tube.

2) Add 2 μL plasmid to the tube and avoid touching the bottom of the EP tube. Then gently blow and stir and place the tube on the ice for 30 min.

3) Place the tube at 42 ℃ for 90 s and transfer it to the ice immediately to bathe for 2-3min.

4) Add 700 μL non-antibiotics liquid LB to the tube and incubate it in a shaker at 37 ℃ for 1 h.

5) Take solid LB medium, add antibiotics and pour the plate.

6) Centrifugate at 5000 r/min for 2 min, pour out part of the supernatant, re-suspend it, spread it on a plate, and incubate it at 37 ℃.

Escherichia coli conservation

Add 800ul 50% glycerol aqueous solution to the seed preservation tube for high-pressure steam sterilization, mix with 800ul bacteria solution, and store at -80℃.

Plasmid extraction

1) Take 2 mL bacteria solution to centrifugate at 12000 rpm for 1 min, and pour out the supernatant. (If the plaque is small, repeat until getting a large one.)

2) Add 250ul solution I. Blow and stir repeatedly to ensure that there is no bacterial block to cleave the inside of the bacterial block adequately.

3) Add 250 ul solution II, slowly invert, and turn the test tube several times to mix gently. Stand for two minutes to get a transparent cleavage solution. In order not to break the chain, the operation must be very gentle. The standing time can not be too long since DNA will also break under alkaline conditions, otherwise, the genomic DNA will be broken. It is difficult to separate broken DNA from the plasmid.

4) Add 350 ul solution III, immediately invert and mix.

5) Centrifugate at 12000 rpm for 15 min.

6) Take 100 ul of 3M NaOH in the purification column for column equilibration, and centrifugate at 12,000 rpm for 1 min to discard excess sodium hydroxide. After column equilibration, the silica matrix membrane can be activated to the maximum extent and the plasmid yield can be improved.

7) Carefully pipette the supernatant into the column. Stand it for 10 minutes to make sure that DNA is fully adsorbed to the membrane. Centrifugate at 12000 rpm for 1 min. Repeat once without standing.

8) Add 500 ul HBC washing Buffer, centrifugate at 12000 rpm for 1min and discard the filtrate.

9) Add 700 ul DNA washing Buffer (80% ethanol) to clean the purification column. Centrifugate at 12000 rpm for 1 min and discard the filtrate. Repeat the wash operation twice. Use the cleaning solutions to wash away impurities such as small molecular proteins and inorganic ions.

10) Centrifugate empty column at 12000 rpm for 2min.

11) Dry in an oven until there is no ethanol (15 min), and remove ethanol in these two steps, otherwise, the subsequent elution efficiency will be affected.

12) Put the purification column into a clean 1.5 ml EP tube and add 30~100ul of the elution buffer preheated at 60℃ to the column, and stand it for 2 min. Preheat can improve the elution efficiency, and add it to the center of the silicone gel membrane to ensure that the eluent will completely cover the surface of the silicone membrane to achieve maximum elution efficiency. Centrifugate at 12000 rpm for 1 min.

13) Repeat the elution with the above eluent without standing. The first standing is to let the eluent cover the silicone membrane and let the DNA fall off the column.

Gel extraction(purification)

1) Cut the gel as thin as possible, add an equal volume of Bind Buffer, and place the gel in a 65°C water bath to dissolve.

2) Transfer the liquid to the purification column and let it stand for 5 min.

3) Centrifugate at 12000 rpm for 1 min, pipette the liquid back to the suction column, and centrifugate at 12000 rpm for 1 min.

4) Discard the solution, add 300 μL Bind Buffer, and centrifugate at 12000 rpm for 1 min. (purification doesn’t include this step)

5) Add 700 μL SPW wash Buffer, stand it for 5 min, centrifugate at 12000 rpm for 1 min. (only add 400 μL for purification)

6) Repeat step 5.

7) Discard liquid, centrifugate the empty column at 12000 rpm for 2 min.

8) Discard the collection tube and dry the column at 37°C incubators for 15~20 min.

9) Place the purification column on a 1.5 mL EP tube, add 20 μL ddH2O preheated at 60 °C to the center of the membrane, and stand it for 5 min.

10) Centrifugate at 12000 rpm for 2min.

11) Pipette the liquid back to the suction column, centrifugate at 12000 rpm for 2min.

12) Discard the column, measure the concentration and purity.

Construction of plasmid

1) Amplify fragments of 1-1~2-2

Correspondence between template and primer

PCR system

2) Purify the correct linear fragment by running electrophoresis.

3) Extract the gel.

4) Recombinate the fragments with homologous recombinase.

5) Transform E. coli.

Protein purification

Sample preparation

1) Centrifugate at 6500rpm and 4°C for 5min.

2) Add the supernatant to an μLtrafiltration tube, centrifugate at 4000 rpm for 15 min at 4°C, and take the supernatant.

Nickel column preparation

3) Prepare the Ni-NTA affinity chromatography column.

4) Elute the column with deionized water slowly to avoid introducing air bubbles into the column bed.

5) Pre-equilibrate the column with 10 times the volumes’ binding buffer.

Gradient buffer preparation

6) Buffer 1: 50 mM PBS buffer, pH 7.4.

Preparation: 0.5M NaH₂PO₄ 19mL, 0.5M Na₂HPO₄ 81mL, NaCl 29.3g, add appropriate amount of water to dissolve and dilute to 1000mL.

7) Buffer 2: 50 mM phosphate buffer, PBS buffer pH 7.4.

Preparation: 0.5M NaH₂PO₄ 19mL, 0.5M Na₂HPO₄ 81mL, NaCl 29.3g, and imidazole 34g, add the appropriate amount of water to dissolve and dilute to 1000mL.

8) Buffer 3: Buffer B with different concentrations of imidazole:

Sample purification

9) Pipette the concentrated supernatant into a Ni-NTA affinity chromatography column. Shake it slowly for 60min at 4ºC on a side or horizontal shaker.

10) Wash with 10 times the column’s volume of binding buffer and collect the filtrate.

11) Elute with gradient elution respectively ( buffer 3 containing 10, 20, 50, 100, 200, 300, and 400 mM imidazole) and collect the elution peaks at each stage.

12) Column recovery: Wash with 5 times the column’s volume of pure water, and 3 times the volume of 20% ethanol.

SDS-PAGE validated analysis

Gel preparation

1) Set up the device, align the lower ends of the two glass plates on the template groove, clamp the template groove on the in-situ glue maker to the 1.0 scale and detect leakage.

2) Separating gel preparation (in order): 6.9 mL redistilled water + 4.0 mL gel stock solution + 3.8 mL separating gel buffer (pH 8.8) + 0.15 mL 10% SDS + 0.15 mL 10% AP + 0.009 ml TEMED.

3) Perfuse the separation glue, add water to seal, and wait for 10-30 minutes to solidify the gel.

4) Pipette part of the water out, and gently suck out the remaining water with filter paper.

5) Stacking gel preparation (in order): 4.1 mL redistilled water + 1.0 mL gel stock solution + 0.75 mL separating gel buffer (pH 8.8) + 0.06 mL 10% SDS + 0.06 mL 10% AP + 0.006 mL TEMED.

6) Perfuse the stacking gel and insert the comb in time. The gel will solidify in about 5 minutes, and wait for the glue to polymerize for 30 minutes.

For precast gels, start from here:

7) Mix the protein solution with Loading buffer in a 1.5mL EP tube in the proportion of 4:1. Seal and heat in a metal bath at 95°C for 5 min to fully stretch the protein space structure.

8) Perfuse the diluted electrophoresis buffer and fill it up to about 0.5cm above the glass plate in the groove, and take out the comb.

9) Pipette 30 μL of the treated sample to pass through the buffer, and add the sample vertically.

10) Add electrode buffer to the electrophoresis tank to make the liquid levels equal on both sides. Connect the positive and negative electrophoresis tanks of the electrophoresis apparatus at 80V for 30min.

11) After entering the separation gel, 120V was maintained, and the electrophoresis was continued for 1h at a constant voltage.

12) Peel the board, and dye with 0.1% Coomassie brilliant blue R250 dyeing solution for about 20min (baking dyeing: heating and boiling in microwave oven 6-7 times).

13) Soak and rinse with rinsing solution twice for 10min each time, change ddH2O and continue rinsing until the band is clear.

14) Observe the bands and analyze the results.

Membrane protein expression validation

1) Culture the engineered bacteria in an LB medium containing kanamycin at 37 °C and 200 rpm overnight.

2) Inoculate the culture into fresh LB medium (pH 6.0, 50 µg/mL kanamycin) for cultivation at 37 °C and 200 rpm, and add 0.5 mM IPTG and 50 µM Tb³⁺ to the culture medium when the OD₆₀₀ value reached 0.6, and incubate overnight to induce the expression of the target protein.

3) Centrifugate 10 mL of culture medium at 6000 rpm for 5 min, and resuspend the cell pellet with 5 mL of PBS buffer for ultrasonic decomposition.

4) Centrifugate at 5000 rpm for 10 min to remove the uncracked cells in the pellet, and centrifugate the supernatant at 50,000 rpm (12,000 rpm can be used instead) for 1 h, and membrane proteins will exist in the pellet.

5) Place the membrane protein pellets on ice and wash them with 400 µl TDSET buffer for 30 min.

Components of TDSET buffer: 1% TritonX-100, 0.2% sodium deoxycholate, 0.1% SDS, 10 mM tetrasodium EDTA, and 10 mM Tris-HCl

6) Add 40 µl of sample to 10 µl of SDS-PAGE loading buffer, heat at 99 °C for 10 min, and then analyze membrane proteins by running a precast SDS-PAGE gel. At the same time, excise the candidate target protein and identify it by mass spectrometry.

Immunofluorescence Validation

1) Centrifugate 1 mL of the culture medium induced by IPTG and Tb³⁺ at 12,000 rpm for 1 min.

2) Wash the cell pellet twice with PBS buffer.

3) Resuspend the cell pellet in 50 µl of PBS containing 1 mg/mL BSA, add 1 µl of His-tag primary antibody, and incubated at 28 °C for 3 h.

4) Centrifugate and wash the pellet twice with PBS buffer.

5) Resuspend the cell pellet in 50 µl PBS containing 1 mg/mL BSA, add 1 µl FITC-labeled affinity-purified goat anti-mouse IgG (H+L) secondary antibody, and incubate at 28 °C for 3 h in the dark.

6) Centrifugate and wash the pellet twice with PBS buffer, then resuspend the cell pellet in 100 µl PBS buffer.

7) Take 10 µl drops on a glass slide to make samples, and observe the fluorescence under an upright fluorescence microscope.

Rare earth element adsorption test

1) Culture the bacteria overnight in LB medium with kanamycin at 37 °C and 200 rpm.

2) Inoculate 1 mL of the overnight culture into a fresh 100 mL LB medium (pH 6.0, 50 µg/mL kanamycin) and cultivate at 37 °C and 200 rpm. Add 0.5 mM IPTG and 50 µM Ca2+ to the culture medium when the OD₆₀₀ value reaches 0.6 to induce the expression of the target protein.

3) Culture for 11 h, centrifugate at 6000 rpm for 5 min, wash the cells twice with 10 mM MES buffer (pH 6.0) and resuspend the cell pellet in 100 mL of 10 mM MES buffer (pH 6.0) to obtain Rare earth metal ion adsorption experimental system.

4) To determine the optimal addition concentration of Tb³⁺, we tried several initial addition concentrations. Incubate the adsorption system at 100 rpm and 30 °C for two hours, then centrifugate at 12,000 rpm for 10 min, pipette the supernatant and measure the concentration of Tb³⁺ in the supernatant by ICP-OES.

5) Add 150 µM Tb³⁺ to the adsorption system, and take samples at different time points to determine the concentration of Tb³⁺ in the supernatant to analyze the adsorption kinetics.

6) To determine the selective adsorption capacity of engineered bacteria for different rare earth elements, add 150 µM of Sm³⁺, Y³⁺, La³⁺, Gd³⁺, Eu³⁺, Sc³⁺, Nd³⁺ or Ce³⁺ to the adsorption system respectively, and determine the rare earth metal ions in the supernatant of the system after adsorption, and calculate the rare earth adsorption capacity of engineered bacteria.

Recycling Rare Earth Elements Using Silicon Columns

1) Pack a 30 mL column with silica particles (2-7 mm) and pretreat the column with 150 mL Tris buffer.

2) Inoculate 1 mL of the overnight culture of engineered bacteria into fresh 100 mL LB medium (pH 6.0, 50 µg/mL kanamycin) and cultivate at 37 °C and 200 rpm/min, and add the culture 0.5 mM IPTG and 150 µM Tb3+ to the medium when the OD600 value reaches 0.6.

3) Take 2 mL samples every two hours for OD₆₀₀ and Tb³⁺ concentration determination. during the incubation.

4) Pour the culture medium into the pretreated silica column after 13 h of induction and incubate for 5 h, during which take 1 mL of the effluent every half hour to measure OD₆₀₀.

5) Take some Tb3+-adsorbed silicon particles, gently rinse with 1 M NaCl, then freeze-dry and observe them under field emission scanning electron microscopy to characterize the adhesion of cells to the surface of silicon particles.

6) Incubate for 5 h, open the cover under the silica column to let the liquid in the silica column flow out, and then wash and desorb the Tb element on the silica particles with 5 times the volume of sodium citrate on the silica column respectively to recover the Tb element in the silica column. Measure the Tb element concentration in the desorption solution five times.