This part contains plasmid construction and protein function test. We already have four plasmids including pCadA-RpmG-Feruloyl esterase plasmid, ArsA-amilGFP, ArsD-amilGFP, and ArsR-amilGFP. In addition, we need to construct pVeg-RpmG-Feruloyl esterase plasmid for detecting the cadmium and zinc.
1. plasmid construction
The LB medium was autoclaved to sterilize at 120 °C for 20 mins. In addition, the solid medium needs to add the corresponding antibiotic and mix it until the solution cooled to approximately 55 ℃. The solution was poured into the petri dishes and store the plates in the fridge. The recipe of lipid and solid medium are shown as below.
Lipid LB medium: Yeast extract 5 g/L, Tryptone 10 g/L, NaCl 10 g/L
Solid LB medium: Yeast extract 5 g/L, Tryptone 10 g/L, NaCl 10 g/L, 20 g/L agar
1.2 inoculated the biosensor and pHY300PLK backbone plasmids
In order to prepare the subsequent assay, the bacteria containing biosensor and pHY300PLK backbone plasmids were inoculated into the lipid LB medium, and cultivated in the incubator at 37 ℃ overnight.
1.3 DNA fragment PCR
The biosensor fragment was amplified by PCR using primers Veg-F and Veg-R. And, the vector fragment was obtained by PCR.
Template 1ul
primer F 1µL
primer R 1µL
Pfu polymerase 25µL
ddH2O 22µL
The mixture was gently stirring and put into the thermal cycler. The PCR system set the program in 55℃ for 10 seconds, 72℃ for 20 seconds (promoter) or 6 minutes (Feruloyl esterase), 4℃ for preservation.
1.4 gel electrophoresis
The agarose gel is used to separate the DNA fragments.
1.4.1 weight out 0.5 g Agarose in a conical flask. Add 50 mL 1×TAE buffer to the conical flask.
1.4.2 Melt the conical flask using a microwave oven until the solution become clear.
1.4.3 Add 5 μL nucleic acid staining dye (Gel Red) to Agarose gel when this solution cooled down to ~55℃.
1.4.4 Pour the molten gel to into casting tray. And, insert the comb at the end of the casting tray.
1.4.5 When it completely solidified, remove the comb to form the wells. Put the gel into the electrophoresis
chamber, add 1×TAE until it covered the gel.
1.4.6 The enzyme treated product mix with DNA loading and added into the wells
1.4.7 The gel was exposed to the UV and save the result
1.5 plasmid extraction
Plasmid extraction were performed according to plasmid extraction kit from Sangon Biotech. Briefly, the overnight cultivated bacterial (pHY300PLK) centrifuged to obtain the precipitation. The precipitation containing the plasmids were treated using the solution I, solution II and solution III. The plasmids were extracted in the soluble solution, further purified in the column. The final solution containing the plasmids were collected in the centrifuge tube.
1.6 Restriction enzyme treated biosensor and pHY300PLK backbone plasmids
The pHY300PLK backbone plasmids were digested by the double restriction enzymes to produce cohesive ends. The configuration is shown as below:
10x Buffer 5µL
Plasmid 1-2µg 20µL
EcoRI 1µL
BamHI 1µL
ddH2O 23µL
In total 50µL
The mixture placed in a water bath at 37 ℃ for 30 min.
1.7 gel electrophoresis
For isolate the vector, we performed gel electrophoresis which is same as 1.4.
1.8 gel extraction
The correct bands were cut and weighted the gel. The subsequent steps were performed according to the gel extraction kit.
1.8.1 add buffer B2 to gel
1.8.2 melt the mixture in the water bath until the gel was completely dissolved
1.8.3 add the solution to the column and centrifuge at 8000 xg for 30 sec
1.8.4 add 500 µL wash buffer to clean the solution and repeat this step
1.8.5 add elution buffer to collect the final DNA fragments
1.8.6 measure the concentration of fragments in the Nanodrop
1.9 Recombination plasmid construct
We took two ways to link the biosensor and pHY300PLK vector. One is jointed by the T4 ligase, the other is Gibson assembly. The mixtures were incubated at 4 ℃ overnight.
1.7.1 T4 ligase
10x Buffer 2μl
T4-DNA-ligase 1μl
vector 0.020pmol (4kb)
DNA fragment 0.060pmol (1kb)
ddH2O 20μl
in total 50μl
1.7.2 Gibson assembly
DNA fragment40μg/μl 1μl
Vector 4μl
2x Mix Express 5μl
Total 10μl
1.10 Transformation
The ligation products were transformed into the competent cells DH5α using heat shock method.
1.10.1 The competent cells were thrown on ice until the ice crystals disappear
1.10.2 pipette the ligation products into the competent cells on ice for 30 minutes
1.10.3 Heat shock at 42 ℃ for exact 90 seconds
1.10.4 place the tube on ice for 3 minutes
1.10.5 pipette 500 μl LB medium and cultivated in the shaker for 30 minutes
1.10.6 centrifuge the solution and discard 500 μl supernatant
1.10.7 mix the precipitant with solution and spread it onto a selection plate
1.10.8 incubate at 37 ℃ overnight
1.11 double enzyme digestion and colony PCR
The double enzyme digestion and colony PCR were performed in order to test whether the colonies contain recombinant plasmids. Thus, the colonies selected from plates and inoculated them into LB medium for plasmid extraction. The bacteria were cultivated in shaker at 37 ℃ overnight. The plasmid extraction assay is same as 1.3 and the plasmid further verified by double enzyme digestion which is same as 1.4 and colony PCR. Gel electrophoresis was used to analyze the digested products and PCR products.
1.12 colony PCR using primers pVeg-verf-up and pVeg-verf-dn
Colonies 1µl
primer F 1µL
primer R 1µL
Taq polymerase 10µL
ddH2O 7µL
The positive colonies were picked to DNA sequencing to confirm the recombinant plasmid.
2.1 The correct sequenced recombinant plasmid transformed B. subtilis using the electroporation.
2.1.1 the competent cells mixed recombinant plasmid in the electroporation cuvette (1mm electrode gap)
2.1.2 the cells exposed to electrical pulse which set at 25F and 200Ω, lasting time for 4-5ms
2.1.3 quickly add 1 ml RM solution (0.5M sorbitol, 0.38M mannitol).
2.1.4 the competent cells incubated at 37 ℃, 100rpm for 3 hours in order to recovery the cells
2.1.5 following, the cells spread on the LB agar plate with tetracycline
2.1.6 the plate incubated at 37 ℃ overnight
2.2 plasmid extraction of B. subtilis
The plasmid from gram-positive bacteria using the Yeast kit. The procedure is similar to E.coli plasmid extraction, different lie supplement with lytic enzyme solution to open up the cells. Then, the mixture incubated 30 min at 37 ℃.The subsequent steps following the protocol.
2.3 verified positive colony using double enzymes digestion (1.11)
The bacteria containing the ArsA-amilGFP, ArsD-amilGFP, and ArsR-amilGFP plasmids enlarged cultivation in 50 ml LB medium at 37 ℃. The GFP intensity was detected in the different cultivation time such as 0h, 1h, 2h, and 3h.
23.1 inoculate the single colonies of the engineered strains in a flask containing 10mL of fresh LB medium with antibiotics, and incubated at 37°C, 220 rpm overnight.
23.2 1 mL of bacterial cultured medium was incubated to 100mL of fresh LB medium with antibiotics and incubated at 37°C, 220 rpm until the 0D600 was around 0.6.
23.3 the medium was divided into 6 conical flasks which 50ml of cultured medium with arsenic ions to a final concentration of 10 µg/L, 20 µg/L, 50 µg/L, 100 µg/L, 150 µg/L, and 200 µg/L at 22°C, 220 rpm for 7 hours. Samples were monitor the GFP intensity at 0h, 1h, 2h, 3h.
23.4 fluorescence intensity was detected by using 200 μL bacterial solution samples to a 96-well black microplate plate
The pHY300PLK-PcadA-biosensor and pHY300PLK-Pveg-biosensor expressed in B. subtilis. The reporter feruloyl esterase is used to test cadmium and zinc concentration. Specifically, feruloyl esterase catalyzes 4-Nitrophenyl Butyrate into 4-Nitrophenol, which is easy to measure at 410 nm wavelength.
24.1 preparing of solution:
0.1 M potassium phosphate buffer, pH 6.5
10.5 mM 4-Nitrophenyl Butyrate dissolved in DMSO, prepared freshly
substrate solution is mixing potassium phosphate buffer with 4-Nitrophenyl Butyrate in the ratio of 9:1
supplement with 2.5% Triton X-100
24.2 inoculate the single colonies of the engineered pHY300PLK-Pveg-biosensor strains in a flask containing 10mL of fresh LB medium with antibiotics, and incubated at 37°C, 220 rpm overnight.
25.2 1 mL of bacterial cultured medium was incubated to 100mL of fresh LB medium with antibiotics and incubated at 37°C, 220 rpm overnight
24.3 reaction mixture contains 0.1 ml crude enzyme and 2 ml substrate solution in 10-mmcuvettes
Various concentration of cadmium nitrate and zinc sulfate solutions were used to induce reporter feruloyl esterase expression. Thus, we test the reporter’s enzymatic activities to analyze the effect on protein expression under heavy metals induction.
25.1 inoculate the single colonies of the engineered pHY300PLK-PcadA-biosensor strains in a flask containing 10mL of fresh LB medium with antibiotics, and incubated at 37°C, 220 rpm overnight.
25.2 1 mL of bacterial cultured medium was incubated to 100mL of fresh LB medium with antibiotics and incubated at 37°C, 220 rpm until the 0D600 was around 0.6.
25.3 the medium was divided into 6 conical flasks which 50ml of cultured medium with arsenic ions to a final concentration of 10 µg/L, 20 µg/L, 50 µg/L, 100 µg/L, 150 µg/L, and 200 µg/L at 22°C, 220 rpm for 7 hours. Samples were monitor the GFP intensity at 0h, 1h, 2h, 3h.
25.4 the enzymatic activities test is same as 24
