Protocol

DNA Extraction

• Take 500 mL of wastewater and filter it through a 0.45 μm hydrophilic filter membrane. Collect the membrane and extract total DNA using the Water DNA kit.
• First, add the filter membrane to Powerbead Tubes and vortex to mix thoroughly.
• Add 60 μL of solution C1, invert several times, then vigorously vortex using a shaker for about 5-10 minutes.
• Centrifuge at room temperature for 30 seconds at 10,000g, transfer the supernatant to a clean collection tube. Add 250 μL of solution C2, vortex, and centrifuge at 4°C for 5 minutes at 10,000g, followed by centrifuging at room temperature for 1 minute.
• Transfer the supernatant to another collection tube, add 200 μL of solution C3, incubate at 4°C for 5 minutes, and centrifuge at 10,000g for 1 minute. Transfer the supernatant to a new collection tube.
• Add 1,200 μL of solution C4, vortex for 5 seconds.
• Gradually transfer the solution (approximately 675 μL each time) to a Spin Filter. Centrifuge at room temperature for 1 minute at 10,000g and discard the filtrate. Continue transferring the supernatant to the filter membrane until completed.
• Add 500 μL of Solution C5 to the Spin Filter, centrifuge at room temperature for 30 seconds at 10,000g, and discard the supernatant by centrifuging for 1 minute at 10,000g.
• Carefully transfer the Spin Filter to a 2 mL collection tube, add 100 μL of Solution C6 for DNA dissolution. After 2 minutes at room temperature, centrifuge for 30 seconds at 10,000g. The collected liquid is the DNA solution, ready for quality control using a UV spectrophotometer.

PCR for target DNA
Thaw Taq, dNTP, primers, template DNA on ice.
To a new PCR tube, add:

Mix solution well.
Place tube in PCR thermocycler. Set thermocycler program:
Inititial denaturation: 5 min at 95℃;
Loop (35 cycles), Denaturation: 30s at 95℃,Annealing: 30s at 60℃,Elongation: 1min at 72℃;
Final elongation: 10min at 72℃;
Store: 10℃.
We use 5ul of the PCR product for electrophoresis and 45ul for purification.

3、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

• 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).
• 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.
• Place a MinElute column in a provided 2 ml collection tube in a suitable rack.
• 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.
• Discard flow-through. Place the MinElute column back into the same tube.
• To wash, add 750 μl Buffer PE to the MinElute column and centrifuge for 1 min.
• 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.
• Place the MinElute column in a clean 1.5 ml microcentrifuge tube.
• 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.
• 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.

4、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.

5、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.

6、Transform into Escherichia coli

l 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.

7、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.

• Load 5μL samples, Start electrophoresis under 80V until the bromophenol blue moving into separation gel, then increase the voltage to 100V.
• 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.
• 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. 

8、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.

9、Gel Electrophoresis

• Prepare 100mL 1xTAE buffer with 1g agarose, and boil it three times, shake completely, and waiting for cool.
• Pour the agarose gel into gel tray, assemble gel pouring apparatus by inserting gate into slots.
• Allow agarose to cool, place the gel in the apparatus rig with the wells facing the negative end (black-colored).
• Fill the rig with 1x TAE buffer.
• Load 8μL of DNA maker into lane.
• Mix 1μL of 10x loading buffer with 5μL DNA sample, load them into lane.
• Run at 100V for 30 min.  
• Use the Gel imaging system to check the gel and take a picture.  
• Deal with the gel carefully as medical waste.

10、Preparation of E. coli BL21 cell lysate extract

• Choose the single colonies of the engineered bacteria constructed above and inoculate them in 5 ml LB medium, and culture them overnight at 37°C at 220 rpm in a shaker.
• The next day, the medium above was transferred to 300 mL of 2 × YTPG medium, and cultured at 37°C on a shaker at 220 rpm.
• When the OD600 value of the bacterial concentration grows at the later logarithmic period, the bacteria are collected. Centrifuge 10000g for 1 min and remove the supernatant.
• Suspend the bacteria with the precooled S30 buffer, mix well with a shaker, and mix at 4°C and 8000g for 7 min and remove the supernatant. This step is repeated 3 times, and all traces of the supernatant is finally discarded.
• Add 1 mL precooled S30 buffer to every 1g of bacteria and mix well.
• Cells were crushed with a ultrasonic cell breaker, turn on for 2S, turn off for 2S, the total time is 15m, and the temperature alarm is set at 40°C.
• Centrifuge the tube at 4°C, 12000g for 20 min and take the supernatant into a new tube, freeze it in liquid nitrogen, and store it in the refrigerator at -80°C. Ready for use as the lysate extract.
• Note: 2 × YTPG medium:22 mM potassium dihydrogen phosphate, 40 Mm dipotassium hydrogen phosphate, 100 mM glucose, 16 g/L tryptone, 10 g/L yeast extract, 5 g/L sodium chloride. S30 Buffer: 10 mM Tris-acetate (pH8.2), 14 mM magnesium acetate, 60 mM potassium glutamate, 2 mM DTT.

11、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 folic acid, 2 mM dithiothreitol, 1 mM putrescine, 1.5 mM spermidine, 4 mM oxalic acid, 33 mM sodium pyruvate.
• Prepare cell-free reaction system (15 µL): 6 µL 11 mixtures, 1.2 mM ATP, 0.86 mM GTP, CTP and UTP, 5% (V/V) PEG-8000, 0.1 mM phosphoenolpyruvate （PEP）, 0.27 U/µL RNase inhibitor, 2 mM 20 kinds of amino acids, 25% (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-stemloop switch plasmid.
• Incubate at 37°C for 1 h, and record the color change of the solution.
• Note: use colored protein as indicator, add corresponding toehold switch plasmid, and do not add X-gal substrate.

12、Preparation of Filter-Paper-Based Sensing Strips

• Put the Whatmann filter paper strips (0.6 × 4 cm) into the culture dish, add 5% bovine serum protein solution to completely cover the paper, and block it overnight at 4°C.
• The next day, discard the 5% bovine serum protein solution in the culture dish, add ddH2O to moisten the filter paper, place the culture dish in decoloration shaker, incubate for 5 min, and discard ddH2O.
• Repeat step 2 and wash the filter paper 5 times.
• Open the lid of the culture dish and place it in the electric constant temperature drying oven. Dry the filter paper for use.
• Spot a volume of 7 μL of E. coli BL21 Δ LacZ cell lysate extract on Whatmann filter paper strips at spots pre-marked with a pencil.
• Put it in an ultra-low temperature refrigerator at - 80°C for 6 h.
• After precooling in the freeze dryer, the paper pieces are sealed with cling film, and the cling film is perforated for ventilation to freeze dry overnight.
• Stored the filter paper sensing strips at 4°C until used.