Methods & Experiments
The aim of the project was to contribute to the development of a microbial wastewater treatment system for the detoxification of pharmaceutical waste, especially targeting the non-steroidal anti-inflammatory drug diclofenac (2-[2-(2,6-dichloroanilino)phenyl]acetic acid). The approach was to overexpress and extract three heterologous laccase enzymes in engineered E. coli. Laccases are enzymes which are capable of catalyzing the conversion of diclofenac into less harmful derivatives. The laccases compared in this study were CotA from B. subtilis, CueO from E. coli and Yak from Y. enterocolitica subsp. palearctica strain 7.
First, we will describe our generally done experiments and then our protein production and purification methods. Finally, we will go through the measurements we performed to measure laccase activities. To get to know more about our project, read our project description. And if you want to know the results we obtained with these methods, please check our results page!
Generally done experiments
Here we describe the general experiments we performed, such as making competent cells, transformation of cells and preparing of gel electrophoresis.
Preparation of plates:
LBA plates were made from LB media which contained LB Broth (Sigma-Aldrich) and BactoAgar (Sigma-Aldrich). Kanamycin and ampicillin were used as antibiotics since pUCIDT-Kan and pET36b plasmids carry the kanamycin resistance gene and pNiv and pDF-lac2 plasmids, in turn, carry the ampicillin resistance gene. The plates were stored at +4°C.
Preparation of 1X BG11 medium for growth and maintenance of Synechocystis spp. PC 6803:
To be able to prepare 1X BG11 medium for cyanobacteria, multiple stock solutions need to be done. In Table 1, you can see all the reagents or solutions that are needed for the preparation of BG11. In the same table, there are also the reagents needed for 1X BG11. All the solutions are made in MQ water.
Making competent cells:
Competent E. coli cells were made according to the following protocol.
- Grow the cells in 5 ml LB until the absorbance at OD600 is between 0.4-0.5. (It will take approximately 1-2 hours, check every 30 minutes).
- Keep on ice for 5 minutes.
- Centrifuge at 5 000 rpm at +4°C for 30 seconds (or 2 min if swing-out). Collect the pellet. Discard the supernatant.
- Keep the tubes on ice from now on.
- Resuspend the pellet in 1 ml of 0.1 M MgCl2 (sterile) gently with a pipette and transfer to 1.5 ml microcentrifuge tubes.
- Centrifuge at Max speed for 30 seconds with a tabletop centrifuge. Collect the pellet. Discard the supernatant.
- Repeat the MgCl2 washing steps (5 and 6) for two times.
- Resuspend the pellet in 100 µl of 0.1 M CaCl2 (sterile) gently with a pipette.
- Keep on ice for 15 minutes and use for transformation.
Transformation of the cells:
To transform our E. coli cells with laccase-containing plasmids, the following protocol was used. After the transformation, plasmids were isolated and DNA digested for further measurements.
Heat-shock transformation protocol for E. coli cells
- Thaw the competent cells on ice for 15 min.
- Add 3 µl DNA to the cells.
- Incubate on ice for 30 min.
- Place the cell + DNA mixture on a water bath (or in a heat block), keep 45 s at 42 °C.
- Keep 2 min on ice.
- Add 500 µl of LB (+ copper) in the laminar.
- Incubate at +37°C for 1 h with 200 rpm shaking.
- Plate 100 μl on a LA plate with appropriate antibiotics. (plate 1)
- Plate the remaining cell culture on another LA plate with appropriate antibiotics. (plate 2)
- Incubate plates at +37°C for 16 h.
- Count the colonies from the plates and take a picture.
To isolate plasmids from cell cultures, QIAGEN QIAprep® Spin Miniprep Kit was used according to the following manufacturer’s instructions:
- Pellet 1-5 ml bacterial overnight culture by centrifugation at 6 800 x g for 3 min at room temperature.
- Resuspend pelleted bacterial cells in 250 μl Buffer P1 and transfer to a microcentrifuge tube.
- Add 250 μl Buffer P2 and mix thoroughly by inverting the tube 4-6 times until the solution becomes clear. Do not allow the lysis reaction to proceed for more than 5 min. If using LyseBlue reagent, the solution will turn blue.
- Add 350 μl Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times. If using LyseBlue reagent, the solution will turn colourless.
- Centrifuge for 10 min at 13 000 rpm in a table-top microcentrifuge.
- Apply 800 μl supernatant from step 5 to the QIAprep 2.0 spin column by pipetting. Centrifuge for 30-60 s and discard the flow-through.
- Wash the QIAprep 2.0 spin column by adding 0.5 ml Buffer PB. Centrifuge for 30-60 s and discard the flow-through.
- Wash the QIAprep 2.0 spin column by adding 0.75 ml Buffer PE. Centrifuge for 30-60 s and discard the flow-through. Transfer the QIAprep 2.0 spin column to the collection tube.
- Centrifuge for 1 min to remove residual wash buffer.
- Place the QIAprep column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50 μl Buffer EB (10 mM Tris-Cl, pH 8.5) or water to the center of the QIAprep 2.0 spin column, let stand for 1 min, and centrifuge for 1 min.
- If the extracted DNA is to be analyzed on a gel, add 1 volume of loading dye to 5 volumes of purified DNA. Mix the solution by pipetting up and down before loading the gel.
Digestion reaction mixtures are prepared into PCR tubes by calculating and adding following reagents to it.
|DNA||Calculate to equal 1 µg or 2 µg|
|rCutSmart Buffer (NEB)||5 µl|
|Restriction enzyme||Equal to DNA (1 µl if 1 µg, 2 µl if 2 µg)|
|Reaction volume||50 µl|
After preparing reaction mixtures, incubate mixtures at 37°C for 4 h to allow enzymes to work efficiently. Then, inactivate the enzymes by incubating at 80°C for 20 min. Store the products at 4°C.
The restriction enzymes we used over this project were NdeI, XhoI, NsiI, BspHI, SalI, SepI and PvuII. Read more about the specific phases in which they were used from our laboratory notebook.
Agarose gel electrophoresis
To separate and analyze digestion samples, gel electrophoresis was performed.
Agarose gel & Agarose gel electrophoresis
- Weigh the correct mass of agarose into an erlenmeyer flask (0.78 g for 1.2 % gel and 0.98 g for 1.5 % gel).
- Add 65 ml of 1xTAE buffer into the agarose.
- Boil in a microwave until all agarose has melted and slightly simmered.
- Add 6.5 µl SyBR Safe DNA Gel Stain 10 000x to the gel mixture and pour it onto the tray.
- When the gel has solidified, put the tray in the gel electrophoresis apparatus. Add 10 µl of NEB purple loading dye 6x to samples and pipet the samples into wells. Start running the gel, for example, at 50 V for 60-100 min (read more specific times we used from our laboratory notebook.
After running agarose gel electrophoresis, the desired fragments are cut from a gel and the DNA is extracted using the QIAGEN QIAquick® Gel Extraction Kit according to the following manufacturer's instructions:
- Weigh the gel slice in a colorless tube. Add 3 volumes of Buffer QG to 1 volume gel (100 mg gel ~ 100 µl buffer). The maximum amount of gel per spin column is 400 mg.
- Incubate at 50°C for 10 min (or until the gel slice has completely dissolved). Vortex the tube every 2-3 min to help dissolve gel. After the gel slice has dissolved completely, check that the colour of the mixture is yellow. If the color of mixture is orange or violet, add 10 µl 3 M sodium acetate, pH 5.0, and mix. The mixture turns yellow.
- Add 1 gel volume isopropanol to the sample and mix.
- Place a QIAquick spin column in a provided 2 ml collection tube or into a vacuum manifold. To bind DNA, apply the sample to the QIAquick column and centrifuge at 13 000 rpm for 1 min using a table-top microcentrifuge or apply vacuum to the manifold until all the samples have passed through the column. Discard flow-through and place the QIAquick column back into the same tube. For sample volumes >800 µl, load and spin/apply vacuum again.
- To wash, add 750 µl Buffer PE to QIAquick column and centrifuge at 13 000 rpm for 1 min using a table-top microcentrifuge or apply vacuum. Discard flow-through and place the QIAquick column back into the same tube. Centrifuge the QIAquick column in the provided 2 ml collection tube at 13 000 rpm for 1 min using a table-top microcentrifuge to remove residual wash buffer.
- Place the QIAquick column into a clean 1.5 ml microcentrifuge tube.
- To elute DNA, add 50 µl Buffer EB (10 mM tris-Cl, pH 8.5) or water to the center of the QIAquick membrane and centrifuge the column for 1 min. For increased DNA concentration, add 30 µl Buffer EB to the center of the QIAquick membrane, let the column stand for 1 min, and then centrifuge at 13 000 rpm for 1 min using a table-top microcentrifuge. After the addition of Buffer EB to the QIAquick membrane, increasing the incubation time to up to 4 min can increase the yield of purified DNA.
- If purified DNA is to be analyzed on a gel, add 1 volume of loading dye to 5 volumes of purified DNA. Mix the solution by pipetting up and down before loading the gel.
After the gel extraction, the ligation reaction was performed. It was done with a 3:1 ratio, i.e. the amount of DNA was three times bigger than the amount of a plasmid. The reagents needed for the reaction are as follows:
|DNA||Calculate to equal 150 ng|
|plasmid||Calculate to equal 50 ng|
|T4 Ligase Reaction Buffer||2 µl|
|T4 DNA Ligase||1 µl|
|Reaction volume||20 µl|
After preparing ligation reaction mixtures, incubate the samples at 19°C for 16 h to enable the enzymes to work efficiently. After that, incubate them at 65°C for 10 min to inactivate the enzymes. Store the ligation products at 4°C.
Protein production and purification
The laccase production of E. coli BL21(DE3) cells was induced by the IPTG system and the His-tagged laccases were extracted with Ni2+ affinity chromatography.
- Incubate transformed cells in LB media at 37°C at 220 rpm for 16 h.
- Add 400 µl kanamycin and 5 ml of cell culture in 400 ml LB media. Incubate at 37°C at 220 rpm for an hour. Measure OD600 values every 30 min until they are over 0.6A.
- Add 400 µl of 1M IPTG to cells and incubate with the same settings for 3 h.
- Measure OD600 values.
- Centrifuge at 4°C at 20 000 rpm for 10 min. Discard the supernatant and add 15 ml of TrisA buffer to the pellets.
- Sonicate in 4 x 30 s cycles to break the cell membranes. Amplitude should be between 8 and 12.
- Centrifuge at 4°C at 20 000 rpm for 30 min.
- Measure the volume of the supernatant and discard the pellet.
Two buffers, Tris A buffer and B buffer, needed in the affinity chromatography were prepared according the following amounts of reagents:
Tris A buffer, pH 7.5:
- 20 mM Tris-Cl
- 150 mM NaCl
- 20 mM imidazole
- 20 mM Tris-Cl, pH 7.5
- 150 mM NaCl
- 300 mM imidazole
- Add 1 ml Ni2+ -affinity resin and a filter to the bottom of the column.
- Add 3 CV (column volume) of MQ to remove the ethanol and wash the hartz.
- Balance column by adding 3 CV of Tris A buffer.
- Add produced proteins to the column.
- Wash the column with 3 CV of Tris A buffer.
- Add 7 CV of B buffer to the column.
- Collect the run-through.
- Wash column with 10 ml MQ.
SDS-PAGE was performed to verify the sizes of purified laccase proteins. Bio-Rad Ready Gel® Precast Polyacrylamide Gel was used and the gel was run using Bio-Rad Mini Protean 3. The SDS buffer was prepared as follows:
10x SDS stock buffer, pH 8,3
- 0.25 M Tris
- 1.92 M glycine
- 1 % SDS
Samples for SDS-PAGE were made from both raw extracts (1-2 mg/ml) and purified proteins (0.1-0.5 mg/ml). The total sample size for both was 40 µl and they were made as follows:
- Add 10µl of 4 x SDS buffer and 30µl of protein
- Boil samples with SDS for 5 minutes.
- Centrifuge at 12 000 x g for 1 minute.
- Add 10 µl of samples to gel.
Visualization of samples:
To visualize the proteins separated in the SDS-PAGE run, the gel was dyed with Coomassie blue stain. It was done as follows:
- Mix Coomassie Brilliant Blue G-250 stain (70 mg) with 950 ml of MQ for 2-3 hours.
- Add 3 ml of HCl and adjust the volume to 1 liter with MQ.
An ABTS assay was conducted to measure the laccase activities. ABTS was dissolved in water according to the manufacturer’s recommendations. To find out if the laccases were active at all, the following filter paper test was performed. A colour change would indicate the laccase activity.
Filter paper test
- First, soak the filter paper in ABTS solution (2 mM ABTS, 100 mM phosphate-citrate buffer, pH 4).
- Dry the paper at 60°C and add 10 µl of enzymes glycerol preps.
- Incubate at 30°C for 10 min.
In addition, a 96-well plate assay was used to test the activity of the laccases. The experiments were done in two different pHs, pH 5 and pH 7, of phosphate-citrate buffer (100 mM). Absorbances were measured at OD530 in one minute intervals.
Due to a failure of the ABTS assay, the syringaldazine assay was performed to measure laccase activities. In the assay, the substrate was syringaldazine and a commercial SAE0050-laccase from Aspergillus SP. was used as a control. Syringaldazine was dissolved in methanol and the Potassium-phosphate buffer (0.1 mM, pH 6.5) was used. The absorbances were measured at OD530 in one minute intervals.
- Qiagen. (2015). Quick-Start Protocol for QIAprep® Spin Miniprep Kit.
- Qiagen. (2018). Quick-Start Protocol for QIAquick® Gel Extraction Kit.