Materials:

Procedures:

1. Gently Pipette DNA (Chr-T7-amilCP, ChrR, YieF) 2 μL, 25 μL 2xPCR Master Mix, 2 μL, 19 μL ddH₂O into the 6 PCR tubes, all placed on ice.

2. Place tubes in the PCR machine.

3. Initial Denaturation: 95 ℃, 5 min.

4. Further Denaturation: 95 °C, 30 sec.

5. Annealing: 55 ℃, 30 sec.

6. Extension: 72 ℃, 1 min.

7. Final Extension: 72 ℃, 10 min.

8. Hold: ∞.

Materials:

Procedures:

1. Measure about 50 mL of 1xTAE.

2. Measure 0.5g of Agarose.

3. Put into the microwave, and heat for about 4 minutes until everything has melted.

4. Pipette 3 μL of Gel red into the solution.

5. Make sure everything is distributed evenly.

6. Clean the mold and the mold piece thoroughly.

7. Insert the mold piece, and pour the solution into the mold.

Materials (Liquid\Solid):

Procedures:

Liquid\Solid:

1. Weigh 2.5g of yeast extract powder with weighing paper and scale, and add it to the beaker.

2. Weigh 5g of NaCl (s) with weighing paper and scale, and add to the beaker.

3. Weigh 5g of tryptone with weighing paper and scale, and add it to the beaker.

4. Measure 500 mL of ddH₂O with a graduated cylinder, and add it to the beaker.

5. Weigh 5 portions of 1.5g agar powder, add to 5 beakers separately, and stir until all has dissolved(solid).

Innoculation

Procedures:

1. Disinfect both hands and supplies.

2. Pipette 5x5mL of LB liquid into each test tube.

3. Pipette 50 μL of E. coli solution (2 different types) into each test tube.

4. Pipette 5 μL of antibiotic into each test tube.

5. Cap test tubes and seal firmly.

6. Put in the machine, shake at about 37℃.

Materials:

Procedures:

1. Use pipette to get 2mL of pET28a (same for pSC101) bacterial suspension liquid, add into 2mL collection tube.
2. Put in centrifuge at 10000 rpm, for 1 min.
3. Bacteria form a pellet at the bottom of the tube, and dump the supernatant.
4. Add in 250 μL Buffer P1 (make sure Buffer P1 has RNase A), use pipette at smaller volume (eg, 200 μL) to resuspend bacteria back into the liquid (blow the pipette repeatedly towards the mixture to disperse the bacteria).
5. Add in 250 μL Buffer P2 (softly tilt the tube repeatedly upside down to mix).
   • Lyses bacterial cells, releasing the plasmid DNA, chromosomal DNA, proteins, etc
6. Add 350 μL Buffer P3 and tilt to mix; a white, flocculent precipitate should appear. P3 simultaneously rids the mixture of bubbles.
• Chromosomal DNA + proteins become the precipitate
• Plasmid DNA is soluble in the mixture
7. Put in centrifuge at 12000 rpm, for 10 min.
   • Precipitate pellets down while the plasmid DNA is still in the liquid mixture
8. Put FastPure DNA Mini Columns in a 2 mL collection tube. Add in the mixtures from Step 7 using a pipette (250 μL, smaller volume) into the FastPure DNA Mini Columns, avoiding aspirating the precipitate into the mixture. In the end, we combined 2 columns of liquid into one FastPure DNA Mini Column.
   • Plasmid binds in the silica membrane (filter) in the middle; everything else flows through
9. Put the FastPure DNA Mini Column in a centrifuge at 12000 rpm for 1 min. Some liquid should’ve dropped in the collection column below the FastPure DNA Mini Column; dump that liquid out and continue using the same collection tube.
10. Add in 500 μL of Buffer PW1, and centrifuge the mixture at 12000 rpm for 1 min. Dump the filtered liquid out and continue using the same collection tube.
    • PW1 denatures nucleases that could be in the mixture (that could destroy plasmid DNA)
11. Add in 600 μL of Buffer PW2 (ensure that 80mL of ethanol is added into the bottle of PW2 before usage), and centrifuge the mixture at 12000 rpm for 1 min. Dump the filtered liquid out and continue using the same collection tube.
    • Ethanol makes nucleic acids less soluble, so they bind to silica better; PW2 washes away other residues.
12. Repeat step 11.
13. Do not add any liquid into the FastPure DNA Mini Columns and use an empty collection tube. Centrifuge this at 12000 rpm for 1 min, to get some remaining liquid to the bottom of the collection tube. Dispose of this liquid and keep the FastPure DNA Mini Column.
    • Get rid of the remaining ethanol, since ethanol could disrupt reactions performed later on
14. Get another tube of 1.5mL volume. Put the FastPure DNA Mini Columns on top of this empty tube, and use a pipette to transfer 40 μL of ddH₂O into the top of the FastPure DNA Mini Column. Make sure the ddH₂O is transferred on top of the silica membrane portion of the FastPure DNA Mini Column.
    • To make sure pure plasmid DNA comes off into the column and into the 1.5 tube
15. Wait 2 min for it to settle.
16. Put the mixture in a centrifuge at 12000 rpm for 2 min.
17. Check the DNA concentration in the remaining solution by putting it in a Ulatramicro-volume spectrophotometer. Pipette onto the small hole, record the concentration (ng/ μL) and purity (A260 ratio).

Materials:

Procedures:

1. Add 1x TAE until the water level in the gel electrophoresis machine.
2. In each individual tube of amilCP/ChrR/YieF, add 8μL of Loading Buffer to each.
3. Prepare 10 μL of 100~2000 bp Marker and 10μL of 500~15000 bp Marker.
4. Insert fluids (10μL) into the holes in the gel, run the gel electrophoresis at 180 volts for 20 minutes.
5. Bring the gel to the Gel Imaging System, under UV light, to see the results.

Materials:

Procedures:

Once the mixture is complete, place it in the centrifuge for a bit (a couple of seconds), then into the PCR machine, where it can incubate at 37℃ for 20 min. It will be cutting the plasmids and making them linear.

Materials:

Procedures:

1. Place fluids into holes in the gel for gel electrophoresis. Wait 10 min for gel electrophoresis (see second experiment steps 5 & 6 after inserting liquid). Check the nature of the gel after electrophoresis underneath the Gel Imaging System.
2. Take out a knife and carefully slice through the bands that are kept for purification (all bands except for the markers); the same slices (eg, A & A) are put in the same 2 mL collection tube.
3. Measure the mass of the gels after subtracting the tube + gel mass from the tube’s mass.
4. Add in equal quantities (500 μL) of Buffer GDP in all gel tubes.
5. Place gels in a water bath, wait for 7 min, though ensure that throughout the process, the gels are occasionally taken out to tilt upside down and mix slightly. A mix was done after the first 5 min and after the total 7 min had passed. Ensure that the gel is completely melted.
6. Attach FastPure DNA Mini Columns to 2 mL collection tubes, transfer 700 μL gel liquid into the FastPure DNA Mini Columns using a pipette.
7. Place in centrifuge at 12000 rpm for 60s. Discard the liquid in the collection tube after.
8. Transfer 300 μL Buffer GDP to FastPure DNA Mini Columns. Wait for 1 min. Then place in the centrifuge at 12000 rpm for 60s once more. Discard the remaining liquid in the collection tube after.
9. Transfer 700 μL Buffer GW to FastPure DNA Mini Columns. Place in centrifuge at 12000 rpm for 60s. Then, discard the remaining liquid in the collection tube. Repeat this step twice.
10. Place the empty collection tube back below the FastPure DNA Mini Columns and add it to the centrifuge at 12000 rpm for 2 min. Discard the remaining liquid.
11. Place the FastPure DNA Mini Columns on a 1.5mL Column. Add 20 μL of ddH₂O to the center of the silicon membrane. Wait for 2 min. Then place in a centrifuge at 12000 rpm for 1 min.
12. Discard the FastPure DNA Mini Column, and place the resulting liquid on the Ultramicro-volume spectrophotometer to test the concentration of the linear pET28a/pSC101, as well as that of the ChrR, YieF, and amilCP.

Materials:

Procedures 1 :

pET28a - Chr-T7-amilCP

pSC101 - ChrR

pSC101 - YieF

pSC101 - ChrR-YieF

Once all are ready, place in a water bath at 50℃ for 20 min, then put in 4℃.

Procedures 2 :

1. Take 10 μL of the combined mixture out, then transfer it into 100 μL E. coli Competent Cells. Place the mixture in ice for 20 min.
2. Transfer to a 42℃ water bath for 45s, immediately take it out and put it in ice for 2 min.
3. Add 800 μL of LB without antibiotics to the mixture.
4. Place the combined mixture into a centrifuge at 220 rpm, 37℃, for 1 hour, in order to let the bacteria revive.
5. Prepare another set of LB that contains 100μL of kanamycin(50mg/mL)→ LB is heated slightly because it gets conjugated and hardens into a jello-like solid form after a day. When cooled down to a temperature around 37℃, add kanamycin.
6. After an hour is up, place the bacteria mixture in a centrifuge at 5000 rpm for 2 min.
7. Take 100 μL of supernatant through a pipette (resuspend), extract 50 μL each of pET28a-chr-T7-amilCP (4 times), pSC101-ChrR (2 times), pSC101-YieF (2 times), and pSC101-ChrR-YieF (2 times), and spread the fluid (the strands of E. coli) throughout the entirety of the agar plate.
8. Place in an incubator at 37℃ for the whole night to culture E. coli.

Materials:

Procedures:

1. Split groups of cultures by the target genes in the plasmids they contain. Prepare for PCR separately.

2. Prepare a PCR test solution with the above materials in one tube, and repeat for each culture. Add DNA, use a pipette to gently pick up one distinct cluster of cells from a culture, and place them inside the tube.

Materials:

Procedures:

1. Measure 0.5g Agarose and place it into a 100 mL conical flask.

2. Add 50mL 1xTAE buffer into the flask.

3. Stir thoroughly so no powdered Agarose is visible.

4. Heat in microwave for around 3minutes until Agarose is fully dissolved.

5. Add 3 μL of GelRed and mix until the solution is uniform in color.

6. Pour the solution into the tray with the ladder, and leave to cool.

7. Add the previously prepared PCR DNA into the wells, wait for 20 min.

8. View results under UV light.

Make the solution for the test: each concentration group for 0mg/L, 10mg/L, 30mg/L, 50mg/L, 80mg/L, 100mg/L of chromium makes three big test tubes of solution, respectively, sampling them after each hour for a total of 6 hours.

Making the Test Tube Solution: add 30 mL of LB and 30 μL of Kana to every tube. Then, add 300 μL of pSC101-ChrR bacterium, pSC101-YieF bacterium, and pSC101-ChrR-YieF bacterium in each tube, respectively. Label each tube with the bacteria gene and the concentration of chromium in the solution.

Lastly, add the corresponding concentration of previously diluted chromium in each solution:

0mg/L: no chromium added to the prior solution.

10mg/L: add 300 μL of chromium to the prior solution.

30mg/L: add 900 μL of chromium to the prior solution.

50mg/L: add 1.5mL of chromium to the prior solution.

80mg/L: add 2.4mL of chromium to the prior solution.

100mg/L: add 3mL of chromium to the prior solution.

After each hour (0/1/2/3/4/5/6), participants of each group sample the solution by extracting 2ml from the test tube solution from each of the 3 test solutions, and placing it into three 2ml centrifuge tubes, respectively.

Materials:

Procedure:

1. Evenly distribute sample of pSC101-ChrR, pSC101-YieF, and pSC101-ChrR-YieF using a vibrator (Labgic L-VM-C) for a few seconds.
   • Samples were made of LB + kan + Cr(VI) at 0, 30, 50, 80, 100 mg/L concentrations collected every hour from 0-6.
2. Using three 24-well plates (3 rows x8 columns), collect 3 rounds of 20 μL from the vial into the well for each hour of bacterial growth. This was repeated for every single concentration of Cr(VI).
   • The three microwell plates corresponded to different bacterium strains (pSC101-ChrR, pSC101-YieF, and pSC101-ChrR-YieF). Each row corresponded to an hour (0-6), and 3 samples were taken for each hour.
   • Rows 1-3, 5-7, and 9-11 represent pSC101-ChrR, pSC101-YieF, and pSC101-ChrR-YieF, respectively.
   • Columns represented hours from the top to the bottom (0-6)
3. OD600 was tested via SpectraMax’s i3x machine at 600 nm, to measure the optical density of samples, for 100-0 Cr(VI) mg/L density. The entire plate was selected for reading.
4. A growth curve was graphed for each concentration and bacteria.
5. OD600 values for each Hour6 sample was graphed.

Procedure:

1. Collect the Cr(VI) treated bacterium (which has been incubated for 20 hours), and collect 20 μL to test if it can grow.

2. Pipette 20 μL of varying density Cr (VI) treated bacteria.

3. Pippete into the middle of the LB Plate, spread evenly.

4. Incubate at 37 ℃ overnight.

Materials:

Procedure:

1. Preparation: Properly sterilize and take out 15 tubes, 5 different inducer concentrations for each bacterial strain. Label each according to plasmid and inducer concentration.
2. Prepare LB culture: Add 30 mL LB broth to all tubes.
3. Inoculation: For each tube, add 1.5 mL of the corresponding bacterial strain (5% inoculum). For all tubes, add 30 µL of Kanamycin solution.
4. Growth to logarithmic phase: Seal the tubes, bundle together using elastic bands, then incubate at 37 °C, shaking at 220 rpm, until OD₆₀₀ ≈ 0.6 (~4 hours).\
5. Prepare the five groups from each of the three bacterial strains. Add L-rhamnose (50 mM stock) to reach final concentrations in 30 mL cultures as follows (C₁V₁ = C₂V₂):

0.10 mM: add 60 µL stock

0.25 mM: add 150 µL stock

0.50 mM: add 300 µL stock

0.75 mM: add 450 µL stock

1.00 mM: add 600 µL stock

6. Induction phase: After adding the inducer, first seal using the sealing film, bundle together using elastic bands, then shift the incubator to 16 °C, continue shaking at 220 rpm for 20 hours.

3.2.1 Lysis and Purification

Procedure:

1. Centrifuge bacterial suspension at 5400 rpm for 15 minutes at 4℃. (Eppendorf centrifuge 5810 R, make sure to balance the centrifuge.)
2. Decant the supernatant and pipette out residual liquid until only the pellet is left. (Make sure not to disrupt the cell pellet, and use the pipette volume according to the residual liquid.)
3. Add 5 mL of Beyotime Denaturing Lysis Buffer to the pellet. Mix evenly to avoid clumping or condensate formation. Use the Labgic L-VM-C vibrator in order to mix samples.
4. Add 50 μL of lysosome (enzyme stock,60 mg stored at 4℃) to the sample.
5. Incubate samples in ice bath or on ice for 20 minutes, or until sonicator is ready.
6. Use the BioSafer Sonicator to disrupt the cell and complete cell lysis. Keep samples on ice to prevent heat denaturation. Set settings to 50 W, 10 minutes, 3 seconds off, 3 seconds on, 50 percent yield. (Ensure that when undergoing sonification, the sample remains submerged in the ice bucket without too much ice melting, and the sample is in the center, with the probe not touching the sides of the tube. Remove samples if it starts bubbling or a muffled sound emerges instead of a sharp one.)
7. Bring samples to centrifuge for 10 minutes, 4000 rpm. After centrifugation, pipette 2 mL into a 2 mL tube (crude protein)
8. Pipette remaining liquid into 15 mL tubes for purification. Add 1mL Ni-NTA (His-tag purification resin) into the tubes. ( 6x His-tag on the end of ChrR and YieF to mark the protein, and Ni-NTA combines with 6x His-tag and keeps the product to ensure purification.)
9. Take a 15 mL tube and put it in a vortex mixer at 4℃ for 2 hours.

3.2.2 Extraction

Procedure:

1. Label Affinity columns with protein name (ChrR, YieF, ChrR-YieF) and its concentration of rhamnose (0.25, 0.5, 0.75, 1 mM), and remove the bottom cap of the column. Slowly pipette all the protein-Ni-NTA solution into the Affinity column. Add 1 mL of non-denaturing Washing Buffer to the column dropwise. Apply 5 times to remove the unwanted proteins
2. Label microcentrifuge tubes with the protein’s name and rhamnose concentration, preparing two tubes for each protein-concentration pair. Add 0.5 mL of non-denaturing elution buffer 6 times to each of the microcentrifuge tubes. Collect the eluate into the labeled tube.
3. Put into the Microvolume Spectrophotometer (Dade Chuangtong (Shanghai) Technology Co., Ltd.) and read results.

Materials:

Procedure:

1. Assemble mold: Wash the mold with water and securely (with no leakage) set up the gel casting mold with clean 1.00 mm glass plates.
2. Prepare resolving (lower) gel: Mix 2.7 mL 2x lower gel solution with 2.7 mL 2x lower gel buffer in a cup. Add 55 µL modified polymerization initiator, mix gently, avoiding bubbles.
3. Pour resolving gel: Fill the space between plates until 1.5 cm from the top edge. Overlay gently with isopropanol to create a flat surface and prevent oxygen inhibition.
4. Leave at 25 °C for 6–10 minutes until a boundary forms, indicating solidification.
5. Prepare stacking (upper) gel: Remove the overlay carefully. In a new mixing cup, mix 0.75 mL 2X upper gel solution with 0.75 mL 2X coloured upper gel buffer. Add 15 µL modified polymerization initiator, mix gently, and avoid bubbles.
6. Pour stacking gel: Add the upper gel solution on top of the polymerized resolving gel until flush with the top of the glass plates.
7. Insert comb: Slowly insert the comb to form wells, taking care to avoid trapping bubbles. Leave at room temperature for 10-15 minutes.
8. Prepare wells: After gel solidification, carefully remove the comb. Wash wells with electrophoresis buffer using a pipette tip. The gel is now ready for protein loading and electrophoresis.

Procedure:

1. Label each 50 mL centrifuge tube by chromium concentration (10, 30, 50, 80, 100mg/L); hours of sampling (0, 3, 6, 16); and Protein name(ChrR, YieF, and ChrR-YieF).

• [For one concentration ex:100mg/L Cr(VI), there is one tube of 0hr, 3hr, 6hr, 16hr respectively]

2.Add 10mL of Sangon Biotech 1x PBS(with 0.05% Tween-20) Solution. Then, add 1mL of protein (ChrR, YieF, and ChrR-YieF) in each tube, respectively.

3. Add the corresponding concentration of previously diluted chromium in each solution:

10mg/L: add 10 μL of chromium to the prior solution.

30mg/L: add 30 μL of chromium to the prior solution.

50mg/L: add 50ml of chromium to the prior solution.

80mg/L: add 80ml of chromium to the prior solution.

100mg/L: add 100 μL of chromium to the prior solution.

Materials:

Procedure:

1. Use 0 h, 5 h, and 12 h as our target times and gather 17 1.5mL-centrifuge tubes per protein (ChrR, YieF, and ChrR & YieF)for the experiment (5 different concentrations of Cr VI, one standard tube or calibrator (Sangon), and one reagent blank containing ddH₂O).
2. Take out 200 μL from each protein solution under different concentrations of Cr(VI) and transfer it into the 1.5mL centrifuge tube.
3. Take out 200 μL of the calibrator solution (Sangon Biotech) and place it into the calibrator tube, and transfer 200 μL of ddH₂O into the reagent blank tube.
4. Add 10 μL reagents 1 and 2 (Sangon Biotech) to all tubes, place all 17 tubes onto the rack, and put the rack inside the incubator at 37 ℃ for 10 minutes.
5. Transfer 100 μL of the incubated protein solution into the microplate/ELISA plate twice per solution.
6. Transfer 100 μL of calibrator solution (Sangon Biotech) and ddH₂O to the last row of the plate.
7. Place the plate into the microplate reader (Molecular Devices).

Materials:

Procedure:

1. Preparation: Properly sterilize and take out 5 tubes, labelling each with the corresponding plasmid and chromium concentration.
2. Prepare cultures: Add (pipette) 5 mL LB broth into all tubes.
3. Add Antibiotic: Add (pipette) 5 µL per tube.
4. Inoculate bacteria: Add 50 µL of pET-Chr-T7-amilCP culture to each tube.
5. Add chromium stock: Using C₁V₁ = C₂V₂, add the volumes of 1000 mg/L chromium stock to achieve final concentrations of 0, 10, 30, 50, 80, and 100 mg/L.
6. Incubate: Seal the tubes using sealing film, bundle using elastic bands, then incubate overnight at 37 °C, 220 rpm.

Materials:

Procedure:

1. Prepare 12 agar plates (two each for 0, 10, 30, 50, 80, and 100mg/L), label the backside of the agar plates with different Chromium concentrations.
2. Place 6 of the tubes for spreading the bacteria in agar plates, and the remaining ones are used for centrifuging purposes later.
3. Take out 10 μL of the bacterial solution and eject 10 μL at the center of the agar plate with a pipette.
4. Use a L-shaped spreader to spread the solution evenly throughout the agar plate
5. Close the lid and seal the agar plate with parafilm. Close the cap for the 6 test tubes containing pET-Chr-T7-amilcp under different Cr(VI) concentrations.
6. Place the 12 plates into the constant temperature incubator at 37 ℃.