1. DNA Synthesis
Primer synthesis service is provided by Sangon Biotech (Shanghai) Co., Ltd. Gene synthesis service is provided by Genscript.
2. PCR
Preparation: The simple fragment amplification system is generally 50 μl, and the components of the non-50 μl system can be increased or decreased in proportion. Using different DNA polymerases, the optimal amount of template and primer may vary. 2X Phanta Max Master Mix is commonly used in our laboratory. The following is the general reaction system for PCR using this enzyme.
While overlapping-extension, the concentration of bridge primer is 1/20 of the normal primer's usage.
3. Homologous Recombination
Two-fragment recombination is performed using ClonExpression® II One Step Cloning Kit from Vazyme biotech co., ltd and following the provided protocol.
4. Plasmid Extraction
Plasmid Extraction is performed using Plasmid Mini Kit I from Omega Bio-Tek biotech co., ltd and following the provided protocol.
5. Gel Extraction
Gel Extraction is performed using SanPrep Column DNA Gel Extraction Kit from Sangon Biotech (Shanghai) Co., Ltd and following the provided protocol.
1. Remove the competent cells from -80℃ and immediately melt them in an ice water bath for 5min.
2. Add the transposition DNA into 100μL competent cells, gently stir well, and incubate on ice for 30min.
3. Place the centrifuge tube in the 42℃ water bath without shaking. After the heat shock of 90s, immediately place it in the ice water bath for 2-3 min.
4. Add 900μl LB medium preheated at room temperature or 37℃ to the centrifuge tube, and then put it in a 37℃ shaker for 45 min.
5. Take different volumes of transformation products, spread them on the correct resistant plates with sterile coating sticks, and incubate them overnight at 37℃ in an incubator.
1. PCR by 2X Phanta Max Master Mix.
2. Gel Extraction by SanPrep Column DNA Gel Extraction Kit.
3. Digestion by DpnI enzyme from Takara Biomedical Technology (Beijing) Co., Ltd. and the protocol it provided.
4. Transformation.
1. Cell lysis
The bacteria were centrifuged at 8000rpm at 4℃ for 10 minutes. After washing with PBS buffer (Sangon Biotech, Shanghai, China) and resuspension with 1x Cell lysis buffer (Sangon Biotech, Shanghai, China), add 40 μl (Sangon Biotech, Shanghai, China) and perform ultrasonic broken for 10 minutes, breaking 1s every 2s. The broken cell fluid was centrifuged at 5000rpm at 4℃ for 30 min, and the supernatant was collected.
2. purification
Protein purification is performed using Ni-NTA 1 ml (Pre-Packed Gravity Column), and the corresponding reagents Binding/Wash Buffer (pH 7.9 = 8.1) for Ni-NTA or Ni-IDA Sefinose (TM) Resin, Elution Buffer (pH 7.9 = 8.1) for Ni-NTA or Ni-IDA Sefinose (TM) Resin from Sangon Biotech (Shanghai) Co., Ltd. The specific experimental steps are the protocols they provided.
3. Identification of proteins
Determination of protein molecular weight of normal size protein is performed using SDS-PAGE Preparation kit from Sangon Biotech (Shanghai) Co., Ltd and following the provided protocol.
4. Protein quantitative test
Protein quantitative test is performed using Bradford Protein Assay Kit from Sangon Biotech (Shanghai) Co., Ltd.
5. Take different volumes of transformation products, spread them on the correct resistant plates with sterile coating sticks, and incubate them overnight at 37℃ in an incubator.
1. Flag-tag purification
Flag-tag purification is performed using Anti-Flag Affinity gel purified kit (Acid elution) from Sangon Biotech (Shanghai) Co., Ltd and the protocol it provided.
2. Sandwich-ELISA
1) Microtiter plates were coated overnight at 4 ℃ with FelD.
2) Plates were blocked with 1% BSA (Sangon Biotech, Shanghai, China) for 1 h at RT.
3) Add 200 μl 1x ELISA Washing Buffer (Sangon Biotech, Shanghai, China) to wash.
4) Incubate 100 μl Standard IgE sample (25 ng/ul) (Sangon Biotech, Shanghai, China) or 100 μl sera from allergic donors which was diluted 5-fold at 37℃ for 90 min.
5) Incubate 100 μl biotin-labeled IgE antibody (Sangon Biotech, Shanghai, China) at 37℃ for 1 h.
6) Add 350 μl 1x ELISA Washing Buffer to wash 4 time.
7) Add 100 μl HRP-conjugated Streptavidin (Sangon Biotech, Shanghai, China) at 37℃ for 30 min.
8) Add 300 μl 1x ELISA Washing Buffer to wash 4 time.
9) Add 90 μl color developer (Sangon Biotech, Shanghai, China) (dark) at 37℃ for 15 min.
10) Add 50 μl termination solution (Sangon Biotech, Shanghai, China) and measuring OD value immediately with microplate reader at 450 nm wavelength.
3. Blocking ELISA
1) Microtiter plates were coated overnight at 4 ℃ with FelD.
2) Plates were blocked with 1% BSA for 1 h at RT.
3) Add 200 μl ELISA Washing Buffer to wash.
4) Different combinations of proteins(NeuA, NeuB, NeuA-NeuB, ClyR) were mixed on the microtiter plates for 90 min at 37℃.
5) Add 200 μl ELISA Washing Buffer to wash 2 time.
6) Incubate 100 μl sera from allergic donors which was diluted 5-fold at 37℃ for 90 min.
7) Incubate 100 μl biotin-labeled IgE antibody at 37℃ for 1 h.
8) Add 350 μl ELISA Washing Buffer to wash 4 time.
9) Add 100 μl HRP-conjugated Streptavidin at 37℃ for 30 min.
10) Add 300 μl ELISA Washing Buffer to wash 4 time.
11) Add 90 μl color developer (dark) at 37℃ for 15 min.
12) Add 50 μl termination solution and measuring OD value immediately with microplate reader at 450 nm wavelength.
1. A method for measuring the dose-dependent and time-dependent lytic activity of ClyR
1) Streptococcus mutans UA159 was adjusted to OD600~0.8 by PBS buffer.
2) In a 96-well plate, 35 μl of ClyR solution at different concentrations and 165 μl of bacterial suspension was added.
3) The OD600 was continuously measured using a Synergy H1 microplate reader under constant shaking at 37℃ for 1 hour.
1. Adhesion tests based on detection of chemiluminescence
1) The preserved E. coli BL21(DE3) strain imported into the target plasmid was cultured in the liquid medium of LB+25 ㎍/mL Kan, until OD600 was within the range of 0.6-0.8. At this time, IPTG was added to the culture medium to produce gradient induced concentrations of 0.1 mM, 1 mM, 2 mM and 5 mM. Induction at 16℃ and 160 rpm for 16 h.
2) The induced bacterial solution was removed from the culture bottle, centrifuged at 8000 rpm at 4℃ for 10 min, and the supernatant was discarded. 2 ml of 1x cell lysis buffer (Sangon Biotech ,Shanghai,China) was added to each tube and the body weight of the bacteria was suspended. PMSF was added to inhibit possible protease degradation. The procedure of ultrasonic cell crushing was 1 s, the interval was 1 s, and the cycle was 10 min. The breaking time of each tube is 20 min. The crushing solution was centrifuged at 5000 rpm at 4℃ for 30 min, the supernatant was taken, the total protein content was controlled at 3 mg/mL with PBS buffer dilution solution, and the protein sample was stored at 4℃.
3) The cat tooth presented by the Pet-King Veterinary Hospital were cut into 5 pieces of similar size by mechanical method, and the 5 tooth fragments were incubated in the same tube of cat saliva (presented by the same veterinary hospital) at 37℃ and 200 rpm for 14 h.
4) The cat tooth incubated with saliva were removed, and each tooth fragment was placed on a petri dish with a diameter of 10 cm and washed twice with 2 mL PBS buffer for 2-3 min each time.
5) The cleaned tooth fragments were placed in the previous bacterial crushing supernatant and incubated at 37℃ and 200 rpm for 20 h to bind Hsa to sialic acid.
6) The tooth fragments in the cell crushing solution were removed and placed in a petri dish with the same specifications as before. The unbound proteins were washed with PBS buffer, and washed with 2mL PBS buffer for 2-3 mins each time, washing twice.
7) Use 5% skim milk with 1: 5000 Dilute Anti-6X His rabbit polyclonal antibody (Sangon Biotech ,Shanghai,China), add 2 mL diluent of antibody to each plate, and shake in a shaker for 2 h at room temperature. Then the uncombined primary antibody was cleaned with 1x TBST buffer, and the washing dosage of each plate was 3 mL, the washing time was 10 min, and washing three times. After washing, 2 mL HRP-conjugated Goat Anti-Rabbit IgG (Sangon Biotech ,Shanghai,China) diluted 1:5000 with 5% nonfat milk was added to each plate and shaken for 2 h at room temperature on a shaker. After incubation, 1x TBST buffer was added to rinse the unbound goat anti-rabbit antibody. Wash each plate with 3 mL TBST buffer for 10 min each time, washing three times.
8) The tooth were transferred to a 96-well plate and 100 μL Ultrasensitive ECL Chemiluminescence Kit (Sangon Biotech ,Shanghai,China) was added to each well to react for 10 min, The luminescence intensity of each well was measured using a microplate reader (SynergyTMH1) as a reference for the amount of adhered Hsa.
2. Crystal violet staining method in 96 well plates
1) 100 μL LB liquid medium with 50 ug/ml kanamycin was added to each well of the 96 well culture plate, and E. coli BL21(DE3) culture medium induced overnight with 0.1 mM IPTG was inoculated and incubate at 37 ℃ for 36 hours. Set the experimental group with 0.1 mM IPTG added, the negative control group without IPTG added, and the pure LB liquid culture medium as the blank control group, with 9 replicates set for each group.
2) We extracted the culture medium and added 200 μL to each well Sterile PBS buffer (Sangon Biotech ,Shanghai,China), washing the flat hole 3 times.
3) To each well, 100 μL of methanol (Sinophasia Chemical Reagent, Shanghai, China) was added for 15 min, and then methanol was extracted from the culture Wells and air-dried naturally.
4) 100 μL of 1% crystal violet solution (Sinopmedicine Chemical Reagent, Shanghai, China) was added to each well and stained for 5 min at room temperature.
5) The crystal violet staining solution was aspirated from the medium and the excess dye was washed with running water.
6) The well plates were inverted to remove residual moisture and dried at room temperature.
7) After complete drying, 100 μL of 33% glacial acetic acid solution (Sinopmedicine Chemical Reagent, Shanghai, China) was added to each well and incubated at 37 ° C for 30 min to dissolve the crystal violet.
8) Measure the OD600 values of the solution in the culture well using an enzyme-linked immunosorbent assay at 600 nm.
3. Observation of Bacterial Membrane Formation by Fluorescence Microscopy
1) E. coli was inoculated overnight (20 h) in LB plates containing 50 ug/ml kanamycin, and individual colonies were selected and shaken for approximately 20 h in 10 ml of LB medium containing 50 ug/ml kanamycin;
2) Six sheets of cover paper soaked in 2% hydrochloric acid (Sinophasia Chemical Reagent, Shanghai, China) overnight and then soaked in 75% ethanol (Wuhan Xingheda Trading, Wuhan, China) for 1 hour were used to extract saliva. Then, clamp the cover on the bottom of the 6-well plate;
3) Six ml of LB medium was added to each well. 200 ul of bacterial solution was added to each well and a 6-well plate was placed in a 37°C incubator;
4) During the cultivation process, we detect the OD600 values and add IPTG when the appropriate OD600 values is reached.
5) IPTG was induced overnight at 16°C, and the slides were removed tomorrow and rinsed repeatedly with PBS buffer to wash away nonadherent bacteria, excess water was absorbed with absorbent paper, and fixed with 2.5% glutaraldehyde (Sinopharmide Chemical Reagent, Shanghai, China) for 1.5 hours;
6) After developing the PBS buffer film, the film was stained with PI(Shanghai Yuanye Biotechnology, Shanghai, China) for 15 minutes at 4°C with closed light. Excess water was rinsed and absorbed with PBS buffer and sealed with 40% glycerol on clean glass slides;
7) At last, we observed and collect images using a fluorescence microscope.
1. Toxin Protein HepT ,Antitoxin Protein MntA and RelE
1) Methods of molecular cloning and transformation are described above. Transform this plasmid into E. coli BL21(DE3). Then spread it onto an LB medium plates with 50 μg/mL kanamycin and incubate overnight at 37℃ in an incubator.
2) Pick four colonies from the same plate as parallel repeats. Each colony is inoculated on two identical media with 5 mL LB medium containing 50 μg/mL kanamycin and cultured at temperature (37℃) respectively while shaking at 200 rpm .
3) Measur the OD₆₀₀ of the resuspending culture media in an automatic microplate reader (SynergyH1 hybrid multimodal reader) until the OD₆₀₀ is in the range of 0.4 and 0.6.
4) Add IPTG to induce the expression of the toxin protein in the bacterial fluid of the experimental group.
5) Plot the OD₆₀₀ curves of the resuspending culture media over time in an automatic microplate reader (SynergyH1 hybrid multimodal reader). Incubate the cultures for 4 hours at 37℃ while shaking at 200 rpm. Use UV spectrophotometer to measure the OD₆₀₀ of bacterial suspension at intervals or continuously, and then convert the raw data into OD₆₀₀. Compare the data of experimental groups and control group and curves in two schemes with each other.
2. RelE CFU detection of toxin protein
1) Methods of molecular cloning and transformation are described above. Transform this plasmid into E. coli BL21(DE3), spread it onto an LB medium plates with 50 μg/mL kanamycin, then Incubate overnight at 37℃ in an incubator.
2) Pick four colonies from the same plate as parallel repeats. Each colony is inoculated on two identical media with 5 mL LB medium containing 50 μg/mL kanamycin and cultured at temperature (37℃) respectively while shaking at 200 rpm .
3) Measure the OD₆₀₀ of the resuspending culture media in an automatic microplate reader (SynergyH1 hybrid multimodal reader) until the OD₆₀₀ is in the range of 0.4 and 0.6.
4) Take samples every half an hour and dilute the bacterial solution by 1 * 10⁶ times, then spread it onto an LB medium plates with 50 μg/mL kanamycin and incubate overnight at 37 ℃ in an incubator.
5) On the second day, count the colonies of the plates cultured for the same time, and we draw the image after processing the statistical data.
3. RNA Thermosensor
To validate the function of the RNA thermosensor, we used two methods to experimentally verify it.
Method 1:
1) Methods of molecular cloning and transformation are described above. Transform this plasmid into E. coli DH5α. Then spread it onto an LB medium plates with 170 μg/mL chloramphenicol and incubate it overnight at 37℃ in an incubator.
2) Pick four colonies from the same plate as parallel repeats. Each colony is inoculated on two identical media with 5 mL LB medium containing 170 μg/mL chloramphenicol and cultured at temperatures (36℃) respectively while shaking at 200 rpm .
3) measure the OD₆₀₀ value of the resuspending culture media in an automatic microplate reader (SynergyH1 hybrid multimodal reader) until the OD₆₀₀ is in the range of 0.4 and 0.6.
4) Samples were grouped and incubated at 36°C, 26°C, and 16°C while shaking at 200 rpm for four hours.
5) Fluorescence intensity (Exλ: 453 nm, Emλ.: 486 nm) and OD₆₀₀ were measured continuously using a an automatic microplate reader (Synergy H1 hybrid multimodal reader). At the end of the detection, the measured fluorescence intensity is divided by OD₆₀₀ to obtain the relative fluorescence intensity, and the results were analyzed by plotting.
Method 2:
1) Methods of molecular cloning and transformation are described above. Transform this plasmid into E. coli DH5α. Then spread it onto an LB medium plates with 170 μg/mL chloramphenicol and incubate overnight at 37℃ in an incubator.
2) Pick four colonies from the same plate as parallel repeats. Each colony is inoculated on two identical media with 5 mL LB medium containing 170 μg/mL chloramphenicol and cultured at temperatures (37℃) respectively while shaking at 200 rpm overnight.
3) Measure the OD₆₀₀ value of the resuspending culture media in an automatic microplate reader (SynergyH1 hybrid multimodal reader)until the OD₆₀₀ is in the range of 0.4 and 0.6.
4) Samples were grouped and incubated at 36°C and 26°C while shaking at 200 rpm for four hours.
5) Continuously measure OD₆₀₀ with an automatic microplate. And then convert the raw data into OD₆₀₀. compare the data of experimental groups and control group and compare curves in two schemes with each other.
4. The verification of Asr promoter (Pasr)
1) Methods of molecular cloning and transformation are described above. Transform this plasmid into E. coli BL21(DE3). Then spread it onto an LB medium plates with 50 μg/mL kanamycin and incubate overnight at 37℃ in an incubator.
2) Pick four colonies from the same plate as parallel repeats. Each colony is inoculated on two identical media with 5 mL LB medium containing 50 μg/mL kanamycin and cultured at temperatures (36℃) respectively while shaking at 200 rpm .
3) Measure the OD₆₀₀ value of the resuspending culture media in an automatic microplate reader (SynergyH1 hybrid multimodal reader) until the OD₆₀₀ is in the range of 0.4 and 0.6.
4) Use HCl aqueous solution and NaOH aqueous solution to adjust the pH of the medium, and construct a gradient from pH8 to pH3.
5) Samples were grouped and incubated continuously at 37°C while shaking at 200 rpm for four hours.
6) Fluorescence intensity (Exλ: 453 nm, Emλ: 486 nm) and OD₆₀₀ were measured continuously using a an automatic microplate reader (Synergy H1 hybrid multimodal reader). At the end of the detection, the measured fluorescence intensity was divided by OD₆₀₀ to obtain the relative fluorescence intensity, and the results were analyzed by plotting.