We constructed three vectors: pET28a-P14-tyrR-Ptyr-6×His -PPO, pET28a -P14-tyrR-Ptyr-GFP and pET28a -PT7-6×His -PPO. The first one is used to test the para-cresol consumption, the second one is use to detect the tyrosine or phenylalanine induction system, and the third one is used to express PPO protein. In order to complete the project, we did experiments below.
Electrophoresis and Make Agarose Gel
To performance electrophoresis, we prepared 100 ml of agarose mixture by adding 1g agarose powder into 100 ml 1x TAE buffer in a beaker and placed it into a microwave oven. After the mixture became homogeneous and transparent, we poured it into a casting tray and inserted the comb, left it cool down under room temperature.
We put the gel into running buffer in an electrophoresis chamber, added the nucleic acid dye and DNA loading buffer into each PCR tube and carefully transferred DNA marker and the dyed solution in each tube. After centrifugation, the samples were pipetted into the holes on the gel then we put the cover back and turned on the power supply.
We turn off the power supply, put the gel into the gel imaging machine and documented the pictures.
We cut off the gel that contained our DNA fragments and collected them into an EP tube. Then we did experiments as below:
- Added buffer DE-A three times volume of the gel into the EP tubes, and placed them into a 72℃ water bath kettle until the gel was completely dissolved.
- We transferred the liquid into the adsorption column, which was later put into an EP tube, and added buffer DE-B half the value of buffer DE-A into the column.
- Centrifuge at 12000rpm for 1min, discard the effluent.
- Add 500 uL buffer W1, centrifuge at 12000rpm for 1min, discard the effluent.
- Add 700 uL buffer W2 into the column, centrifuge at 12000 rpm for 1min, discard the effluent.
- Repeat the last step.
- Add 30 uL Eluent or distilled water into the column and centrifuge at 12000rpm for 1 min.
- The solution left in the EP tube was our recovered DNA.
LB Kanamycin Culture(liquid)
LB medium was used to culture bacteria, we put the medium in an autoclave to sterilize at 120℃.
LB Kanamycin Culture (liquid)
200 ml double distilled H2O
1g Yeast Extract
0.2ml Kanamycin 50mg/ml
LB Kanamycin Culture (solid)
1g Yeast Extract
0.2ml Kanamycin 50mg/ml
- 3 ml E. coli bacteria solution were pipetted into a 1.5ml EP tube, centrifuged at 12000 rpm for 1min.
- The supernatant was discarded and we added 250 uL buffer P1, which contained RNase A that can degrade the RNA.
- Resuspend the precipitate and ensure the bacteria sufficiently soak in the solution.
- Add 250 uL buffer P2 into EP tube and gently flipped it 6-8 times for NaOH to completely lyse the membrane.
- Add 350 uL buffer P3, gently flipped 6-8 times and centrifuged at 12000 rpm for 10 min.
- Transfer the supernatant into CP3 tubes and centrifuge at 12000 rpm for 1min.
- Added 600 uL washing buffer PW, centrifuge at 12000 rpm for 1min to further wash off the impurities. Centrifuge at 12000 rpm for 2 min, put the adsorption column into a new clean 1.5 ml tube.
- Add 80 uL of Eluent into CP3 tubes until the ethanol absolute was completely vaporized.
- Centrifuged at 12000 rpm for 1min after placing under room temperature for 2 min. The plasmid extract was eventually washed off into recovery tubes.
The one-step method is used to construct the recombination carrier. The recovered DNA fragments and the carrier fragments were mixed at a molar ratio of about 2:1, and the homologous recombinant enzyme was added and incubated at 37℃ for 2h.
1. Unfreeze the E. coli competent cells on ice then add plasmid into the competent cells, keep the complex on ice for 30 min;
2. Heat shock the E. coli competent at 42℃ water bath kettle for 1min;
3. Cool the E. coli competent immediately for 2min in ice;
4. Add 500ul LB liquid medium with no antibiotics, keep E. coli competent regrowth in 37℃ shake cultivation for 45 min.
5.Centrifuge at 12000 rpm for 1 min, discard the liquid then spread the deposit on LB solid medium plate with antibiotics.
6.Put the plate in 37℃ constant temperature incubator for growth over night.
E. coli competent cell BL21(DE3) is transformed when PPO protein is expressed and purified.
To test whether the construction is right, we did colony PCR.
The colony PCR is done using Taq Mix instead of high-fidelity enzyme.
We select Petri dishes that had E. coli colonies cultured, then carefully transferred a small portion of the colony into PCR components mixture with pipette tips and put the mixture into a PCR amplifier to do reaction.
Colonies with special band were pipetted into tubes with 3ml LB liquid medium, culture them at 37℃ constant temperature shaker overnight. This medium is added with related Kan or Chloramphenicol.
Plasmid is extracted and sent to company for sequencing by using the T7 promoter and T7 ter Primer.
The overnight cultured bacteria solution is diluted in a scale of 1:100 to 100 ml new LB medium. Then cultured at 37 ℃ constant temperature shaker, and the OD600 is measured using a spectrophotometer. We also collected 2 ml of the fluid as a control. IPTG store solution was added into the 100 ml medium when the OD600 reached the range of 0.6-0.8 and the working concentration was 1mM.The 100ml bacteria solution was cultured at 16℃ constant temperature shaker overnight.
We prepared three buffers - lysis buffer, binding buffer, and washing buffer -before the protein was purified. These three buffers are consisted of the same components but were used differently in procedure.
Formula of Buffers
Each 500 ml consists of
Tris HCl (pH 8.0) - 25 ml
NaCl - 50 ml
Glycerol - 25 ml
DTT - 0.5 ml
ddH2O - 400 ml
The following steps were done on ice or refrigerated centrifuge.
- The induced E. coli bacteria solution was evenly distributed into two 50 ml centrifuge tubes and centrifuged at 6500 rpm for 10 min.
- We removed supernatant and added 15 ml lysis buffer into the 50 ml centrifuge tubes incubated for 1 hour.
- We dealt the E. coli cells using ultrasonic cell disruptor, 4 sec on, 6 sec off, 20% output power, 30 min. This progress should be done on ice.
- The mixture was transfer into 1.5 ml EP tubes and centrifuge at 10000 rpm for 40min in a precooled centrifuge.
- Transfer the supernatant in a 15 ml centrifuge tube, add 50 𝜇l Ni beads then incubate at 4 ℃ for 2 hours. (The Ni beads were washed use buffer twice, 500 ml buffer each time, centrifuge at 3000 rpm, 3 min each time.)
- Centrifuge at 3000 rpm for 3min then discard the liquid.
- Add 5ml WB buffer into the 15 ml centrifuge tube to wash the beads that adhered with protein, repeat 3 times.
- Transfer the beads to 1.5 ml EP tube from the 15ml tube using WB. Then centrifuge at 3000 rpm for 3min in the precooled centrifuge.
- Discard the liquid, add 200 𝜇l EB and mix gently then centrifuge at 3000 rpm for 3min. Do not discard the liquid which contains protein.
SDS-PAGE Gel Electrophoresis.
We did SDS-PAGE gel electrophoresis to identify our target protein. SDS-PAGE gel is consistent of separating gel and stacking gel, the protocol is showed below.
Separating Gel Protocol
Buffer - 5 ml
Gel solution - 5 ml
Improved APS - 100 ml
Stacking Gel Protocol
Buffer - 1.5 ml
Gel solution - 1.5 ml
Improved APS - 30 ml
- We carefully poured the separating gel mix solution into a casting tray until it reached a scale line on the glass.
- Double distilled water was added to even out the surface and the gel was placed on table until the gel solidified.
- The double distilled water was poured and stacking gel solution was added into the glass using pipette. We carefully inserted the comb and placed the gel into the thermotank under 37 ℃ until it was solidified.
When the stacking gel solidified, we removed the comb carefully and placed the gel into the electrophoresis chamber. Then, we poured the running buffer into the gel and electrophoresis chamber. After pipetting 3-5ul sample into a new EP tube, we added the protein loading buffer and boiled the mixture in 100℃ metal bath for 5 minutes. Then carefully transferred the protein in the EP tubes into holes on gel. After that, we connected the electrodes and then turned on the power supply.
At the same time, we also prepared the Coomassie bright blue dye. Dyeing and decoloration assays were also done. The formula is showed below and the dye is stored at room temperature for use.
95% ethanol 526 mL,
glacial acetic acid 100 mL,
Coomassie bright blue G250 2g,
Add ddH2O constant volume to 1000 mL
Polyphenol oxidase (PPO) quantification
PPO activity was determined using catechol as a substrate. The total reaction mixture volume (2 mL) containing 1 mL of 0.2 M phosphate buffer (pH 7.0), 0.5 mL of culture supernatant and 0.5 mL of 10 mM catechol in 0.2 M phosphate buffer at pH 7.0 was incubated at 50 °C for 3 min and the change in absorbance at 420 nm was measured spectrophotometrically. One unit of enzyme (PPO) activity was defined as the amount of enzyme that increased absorbance of 0.001 per minute.
Degradation of p-cresol by PPO
The effects of initial concentration of p-cresol (10–20 mM) on PPO were examined at the optimum pH. For p-cresol degradation, mushroom PPO (2 mL, 1220 units) was added to 50 mL p-cresol solution (10 mM in 50 mM phosphate buffer, pH 7.0) and stirred, the reaction mixture gradually turned to brown due to degradation of p-cresol.
Measurements of fluorescence intensity
All sensor strains were grown in LB broth at 37°C overnight, and were diluted (1:50; v%) in M9 medium (supplemented with 0.4% glucose, 0.24 mg/mL MgSO4, 11.1 μg/mL CaCl2, 0.3 μM thiamine hydrochloride, and 12.5 μg/mL chloramphenicol) supplemented with Tyr at various concentrations and incubated at 37°C for different hours. The cell densities and fluorescence intensities were measured on a microplate reader (BioTek Synergy HT, Winooski, VT) equipped with an excitation/emission filter 485 nm/528 nm for green fluorescence protein (GFP)). Photograph of fluorescence images were obtained using (GE Amersham Imager 600). For the urine analysis, 10% artificial urine solution was mixed with M9 medium spiked with various concentrations of Tyr.
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