Experiments
Overview
In our project, we established a platform for the detection of heavy metal pollution in aquatic environments, where the expression of fluorescent proteins is initiated by nucleic acid aptamer sequences of heavy metals for visualization and detection. We developed a cell-free protein expression system based on stem-loop switch to achieve rapid detection by paper-based microarrays, and at the same time, heavy metal adsorption proteins can be expressed, and heavy metal adsorption can be realized after detection, which is expected to realize the harmless treatment of contaminated water.
Extraction of DNA from polluted water environment
Procedure:
Take 500mL of sewage water and filter it utilizing a 0.45 micron aqueous filter membrane, collect the filter membrane and use Water DNA kit for total DNA extraction. First the filter membrane was added to the Powerbead Tubes, then vortexed and shaken to mix;
Add 60μL of solution C1 and mix upside down several times, then shake vigorously with a vortex shaker for about 5-10 minutes;
In room temperature, centrifuge the solution at 10000g for 30 seconds, transfer the supernatant to another uncleaned collection tube, add 250μL solution C2, vortex and mix well, 4°C for 5 minutes, centrifuge at 10000g for 1 minute at room temperature;
Transfer the supernatant to another collection tube, add 200μL solution C3, 4°C for 5 minutes, centrifuge at 10000g for 1 minute, transfer the supernatant to a new collection tube, add 1200μL solution C4, vortex mix for 5 seconds;
Then gradually transfer the solution (about 675μL each time) to Spin Filter, centrifuge at 10000g for 1 min at room temperature, discard the filtrate, and continue to transfer the supernatant to the filter membrane until all the transfer is completed;
Add 500μL of solution C5 to the Spin Filter, centrifuge at 10000g for 30 seconds at room temperature, discard the supernatant and centrifuge at 10000g for 1 minute;
Carefully transfer the Spin Filter to a two ml collection tube, add 100μL solution C6 for DNA dissolution, centrifuge at 10000g for 30 seconds at room temperature for 2 minutes, the collection solution is the DNA solution, UV spectrophotometer quality control for backup.
Extraction and purification of Escherichia coli plasmid DNA: alkaline cleavage method
PCR amplification of target DNA fragments
Product recovery and purification (QIAGEN)
Purification and recovery of the product according to the steps of the manual
PCR product ligation and transformation
According to the pre-set digestion site for the vector and ligation of PCR products for digestion and ligation. Firstly, Hind III and Nde I were double digested, then CBP was ligated, and the ligand was transformed into E.coli BL21(DE3) receptor cells, and a certain number of transformants were obtained by screening with benzylpenicillin.
Screening and identification of recombinants
Pick the colonies growing on the resistant plate, inoculate them into 4 ml of LB liquid medium respectively (add the corresponding antibiotics to a certain concentration), and incubate them overnight at 37℃ with oscillation.
Plasmids were extracted by alkaline lysis.
Restriction endonuclease digestion was performed for identification.
For related steps of cloning subclones, see Molecular Cloning Laboratory Guide, 2nd edition p55-56
Expression of target protein
Transformation: the recombinant plasmid was transferred into BL21(DE3) E. coli receptor cells, heat-excited at 42°C and then coated on plates containing 50µg/mL ampicillin and cultured at 37°C;
Activation: monoclonal colonies were picked into liquid medium containing 50µg/mL ampicillin and incubated at 37°C;
Induction: when the OD value reaches 0.6, add 0.5 mM inducer IPTG and continue to incubate, respectively, overnight at 20°C and 6 h at 37°C. The one without inducer is the negative control;
Collection of organisms: centrifuge at 4000 rpm for 10 min, discard the supernatant and collect the organisms;
Expression assay: add buffer A suspension to the collected bacilli, and use ultrasonic crusher to make them fully dissolved. Centrifuge to collect the supernatant and precipitate, precipitate using buffer B for dissolution, respectively, the supernatant and precipitated proteins for sampling, ready for gelatinization detection.
Detection of expression products
SDS-PAGE: Protein samples were processed, samples were prepared, 12% separator gel, 5% concentrate gel, gel was run and molecular weight was detected.
Western Blot validation: protein samples were processed, samples were prepared. 5% gel concentrate, 12% separator gel. Primary antibody was mouse anti-His tag, secondary antibody was sheep anti-mouse, TMB color development was used, and labeled antibody was used for validation.
Preparation of E. coli BL21 cell lysate
A single colony of the engineered bacteria constructed above was selected and inoculated in 5 ml of LB medium and incubated overnight at 37°C in a shaker at 220 rpm.
On the next day, the above medium was transferred to 300 mL of 2 × YTPG medium and incubated at 37°C on a shaker at 220 rpm.
Bacteria were collected when the 600 OD value of the bacterial concentration increased in the late logarithmic cycle. Centrifuge 10,000 g
Construction of cell-free system
Prepare 11 mixtures in advance: 9 mM magnesium acetate, 90 mM potassium glutamate, 80 mM ammonium acetate, 57 mM HEPES-KOH, 0.171 mg/mL tRNA, 0.034 mg/mL folate, 2 mM dithiothreitol, 1 mM putrescine, 1.5 mM spermidine, 4 mM oxalic acid, 33 mM sodium pyruvate.
Cell-free reaction system (15 μL) was prepared: 6 μL 11 mixture, 1.2 mM ATP, 0.86 mM GTP, CTP and UTP, 5% (V/V) PEG-8000, 0.1 mM phosphoenolpyruvic acid (PEP), 0.27 U/μL RNase inhibitor, 2 mM 20 amino acids, 25% (V/V) E. coli BL2121, 0.1 mM PEP, 0.27 U/μL RNase inhibitor, 2 mM 20 amino acids, 25% (V/V) E. coli PEP. V/V) E. coli BL21 Δ Lac Z cell lysate extract, 5% (V/V) 20 mg/mL X-gal chromogenic substrate.
Add 5 nM pET-22b(+)-CBP-GFP- Neck-loop-switch plasmid
Incubate at 37°C for 1 hour and record the colour change of the solution.
Note: Use coloured proteins as indicators, add the appropriate toe-switch plasmid and do not add the X-gal substrate.
Preparation of paper-based sensors based on cell-free expression system
Strips of Whatmann filter paper (0.6 × 4 cm) were placed in petri dishes, 5% bovine serum protein solution was added to cover the paper completely, and closed at 4°C overnight.
The next day, discard the 5% bovine serum protein solution from the petri dish, add ddH2O to wet the filter paper, place the petri dish in a decolourisation shaker, incubate for 5 min and discard the ddH2O,
Repeat step 2 and wash the filter paper 5 times.
Open the lid of the petri dish and place it in an electric thermostatic drying oven. Wipe the filter paper dry for use.
Spot a dotted volume of 7 μL E. coli BL21 Δ LacZ cell lysate extract on a strip of Whatmann filter paper on the spots pre-marked with a pencil
Placed in an ultra-low temperature refrigerator at -80°C for 6h.
After pre-cooling in a freeze dryer, the paper sheets were sealed with cling film and the cling film was perforated and vented to freeze dry overnight.
Store filter paper induction strips at 4°C until use.
Additional experimental steps:
The PCR products cleanup and fragment recovery from gel using QIAGEN kit according to manual.
Qiagen MinElute® Reaction Cleanup kit
Add ethanol (96–100%) to Buffer PE before use (see bottle label for volume).
All centrifugation steps are carried out at 17,900 x g (13,000 rpm) in a
conventional tabletop microcentrifuge at room temperature.
Add 1:250 volume pH indicator I to Buffer PB (i.e., add 120 μl pH indicator I to
30 ml Buffer PB or add 600 μl pH indicator I to 150 ml Buffer PB). The yellow
color of Buffer PB with pH indicator I indicates a pH of 7.5.
Add pH indicator I to entire buffer contents. Do not add pH indicator I to buffer aliquots.
If the purified PCR product is to be used in sensitive microarray applications, it may be beneficial to use Buffer PB without the addition of pH indicator I.
Procedure
1. Add 5 volumes of Buffer PB to 1 volume of the PCR reaction and mix. It is not necessary to remove mineral oil or kerosene. For example, add 250 μl of Buffer PB to 50 μl PCR reaction (not including oil).
2. If pH indicator I has been added to Buffer PB, check that the color of the mixture is yellow. If the color of the mixture is orange or violet, add 10 μl of 3 M sodium acetate, pH 5.0, and mix. The color of the mixture will turn to yellow.
3. Place a MinElute column in a provided 2 ml collection tube in a suitable rack.
4. To bind DNA, apply the sample to the MinElute column and centrifuge for 1 min. For maximum recovery, transfer all traces of sample to the column.
5. Discard flow-through. Place the MinElute column back into the same tube.
6. To wash, add 750 μl Buffer PE to the MinElute column and centrifuge for 1 min.
7. Discard flow-through and place the MinElute column back in the same tube. Centrifuge the column for an additional 1 min at maximum speed.
IMPORTANT: Residual ethanol from Buffer PE will not be completely removed unless the flow-through is discarded before this additional centrifugation.
8. Place the MinElute column in a clean 1.5 ml microcentrifuge tube.
9. To elute DNA, add 10 μl Buffer EB (10 mM Tris·Cl, pH 8.5) or water to the center of the membrane, let the column stand for 1 min, and then centrifuge for 1 min.
10. If the 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.
QIAGEN MinElute® Gel Extraction Kit
This protocol is designed to extract and purify DNA of 70 bp to 4 kb from standard or low-melt agarose gels in TAE or TBE buffer resulting in high end-concentrations of DNA. Up to 400 mg agarose can be processed per MinElute column.Important points before starting
The yellow color of Buffer QG indicates a pH ≤7.5.
Add ethanol (96–100%) to Buffer PE before use (see bottle label for volume).
All centrifugation steps are carried out at ≥10,000 x g in a conventional table-top microcentrifuge at room temperature.
Procedure
1. Excise the DNA fragment from the agarose gel with a clean, sharp scalpel. Minimize the size of the gel slice by removing extra agarose.
2. Weigh the gel slice in a colorless tube. Add 3 volumes of Buffer QG to 1 volume of gel (100 mg or approximately 100 μl). For example, add 300 μl of Buffer QG to each 100 mg of gel. For >2% agarose gels, add 6 volumes of Buffer QG. The maximum amount of gel slice per spin column is 400 mg; for gel slices >400 mg use more than one MinElute column.
3. Incubate at 50°C for 10 min (or until the gel slice has completely dissolved). To help dissolve gel, mix by vortexing the tube every 2–3 min during the incubation. IMPORTANT: Solubilize agarose completely. For >2% gels, increase incubation time.
4. After the gel slice has dissolved completely, check that the color of the mixture is yellow (similar to Buffer QG without dissolved agarose). Note: If the color of the mixture is orange or violet, add 10 μl of 3 M sodium acetate, pH 5.0, and mix. The color of the mixture will turn to yellow. The adsorption of DNA to the membrane is efficient only at pH ≤7.5. Buffer QG contains a pH indicator which is yellow at pH ≤7.5 and orange or violet at higher pH, allowing easy determination of the optimal pH for DNA binding.
5. Add 1 gel volume of isopropanol to the sample and mix by inverting the tube several times. For example, if the agarose gel slice is 100 mg, add 100 μl isopropanol. Do not centrifuge the sample at this stage.
6. Place a MinElute column in a provided 2 ml collection tube in a suitable rack.
7. To bind DNA, apply the sample to the MinElute column, and centrifuge for 1 min. For maximum recovery, transfer all traces of sample to the column. The maximum volume of the column reservoir is 800 μl. For sample volumes of more than 800 μl, simply load and spin again.
8. Discard the flow-through and place the MinElute column back in the same collection tube.
9. Add 500 μl of Buffer QG to the spin column and centrifuge for 1 min.
10. Discard the flow-through and place the MinElute column back in the same collection tube.
11. To wash, add 750 μl of Buffer PE to the MinElute column and centrifuge for 1 min.
12. Discard the flow-through and centrifuge the MinElute column for an additional 1 min at ≥10,000 x g.
13. Place the MinElute column into a clean 1.5 ml microcentrifuge tube.
14. To elute DNA, add 10 μl of Buffer EB (10 mM Tris·Cl, pH 8.5) or water to the center of the membrane, let the column stand for 1 min, and then centrifuge for 1 min.
15. If the 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.
Enzyme digestion reaction and reaction of ligation between target gene and plasmid
1) Enzyme digestion
Incubate the tube at 37℃ for about 2hours, then cleanup according to the previous approaches.
2) ligation
Mixed thoroughly according to above table, and incubate the tube at 16℃ over night. The ligation products were also cleaned according to the previous approaches.
Preparation competent cell of Escherichia coli
After culture the E.coli BL21(DE3) on LB medium, collect the cells by centrifuge. Resuspend the cell in 0.1M CaCl2, mixed thoroughly, centrifuge again, remove the culture medium, resuspend the cell 0.1M CaCl2 at 4℃, prepared for the next step. Unusually, the cells from 50mL LB medium could be dissolved in 2mL CaCl2 solution.
Transform into Escherichia coli
Thaw competent cells rapidly by immersing frozen tubes in a 37℃ water bath after remove from 70℃ refrigerator. Draw about 50uL of the competent cells in a clean tube and add 5uL recombined plasmid, throw on ice for about 30 min. then put them in 42℃ for 90 second, immediately turn on ice for 2min. add 900uL LB medium and incubate in a roller drum at 37℃ fro 1hours. Took 100ul LB medium contain ampicillin, upside down when dried, put in 37℃ incubator over night. Validation by agarose electrophoresis after the plasmid DNA was extracted from transforming Escherichia coli.
Sample preparation for SDS-PAGE
5mL of induced products was centrifuged with 10000rpm for 10 min under 4℃, collecting the cells.
Resuspend the precipitate with 2mL 0.5M Tris-HCl (pH 6.8), then centrifuged with 10000rpm for 5 min, repeat this step twice.
Resuspend the precipitate with 5mL buffer for ultrasonication under 200W, 1second, with 2 second interval, 30 cycles, total times with 6 min. then centrifuged with 12000rpm for 25 min, collected the supernatant and precipitation respectively.
Resuspend the precipitate with 80µL ddw, add 20µL 5×loading buffer and 5µL DTT, mixed thoroughly. Prepare another tube with the supernatant with the same procedure. Then boiling in 100℃ for 6min for SDS-PAGE detection.
Procedure of PAGE
Preparation of the separation and concentration gels
AP and TEMED should be the last to added. When it was added, mixed thoroughly immediately, and after the gel polymerization, pull out the comb and wash the sample hole with double distilled water.
1)Load 5μL samples, Start electrophoresis under 80V until the bromophenol blue moving into separation gel, then increase the voltage to 100V.
2)After electrophoresis, strip the gel and immerge in the fixed solution until the bromophenol blue change to faint yellow; replace the fixed solution with staining solution, then dyeing and decolorizing for 4 hours.
3)Removing the staining solution, following another decolonization step, it could be watch and took photo after the band of protein was complete clear. The gel could be stored in distilled water.
Western Blotting
Apparatus: Apparatus of SDS-PAGE, Electroblotting Apparatus, Power supply, PVDF membrane（Millipore Immobion-P #IPVH 000 10）, Whatman 3MM paper, Additional Tools: Forceps, sponge pad, scissor, gloves, small plastic or glass container, Shallow tray.
Cell extracts were prepared by homogenizing cells or tissues in the lysis buffer (50 mM Tris–HCl, pH 7.5, 5 mM EDTA, 150 mM NaCl, 0.5% NP-40) for 45 min. The soluble protein concentration was measured using Bio-Rad Protein Assay (Bio-Rad). The lysates (50 lg) were separated by 12% sodium dodecyl sulfate (SDS)-polyacrylamide gel (SDS–PAGE) and transferred to PVDF membranes for immunoblotting assay. The membrane was blocked in 5% fat-free dry milk, and probed with antibodies against the interest proteins. The blots were visualized using the enhanced ECL reagent. The levels of protein expression were semi-quantified by optical densitometry using Image J software.