Team:HZAU-China/Protocol
Molecular Cloning
DNA Synthesis
Primer synthesis service is provided by Sangon Biotech (Shanghai) Co., Ltd.Gene synthesis service is provided by Genscript.
PCR
Regular PCR, OE-PCR is carried out with PrimeSTAR®Max DNA Polymerase from TaKaRa and the protocol it provided online. PCR products with similar length to its template are digested with QuickCut Dpn I from TaKaRa and the protocol it provided.
Colony PCR is carried out with 2×Taq PCR MasterMix from Aidlab Biotechnologies Co.,Ltd and the protocol they provided. The solution system we used is 10 μL. The initialization and denaturation temperatures are set at 95℃.
Homologous Recombination
Two-fragment recombination is carried out with ClonExpression® II One Step Cloning Kit from Vazyme biotech co., ltd and the protocol it provided.
Three-fragment recombination is carried out with ClonExpression ® MultiS One Step Cloning Kit from Vazyme biotech co., ltd and the protocol it provided.
Plasmid Extraction
Plasmid Extraction is carried out with Plasmid Mini Kit I from Omega Bio-Tek biotech co., ltd and the protocol it provided.
Gel Extraction
Gel Extraction is carried out with Gel Extraction Kit from Omega Bio-Tek biotech co., ltd and the protocol it provided.
Transformation
The protocol we used to transform plasmids into competent cells are shown below:
1. Melt the competent Bacteria on ice for 5 minutes.
2. Add proper amount of plasmid into the competent Bacteriaand place it on ice for 25 minutes.
3. 42℃heat shock for 45 seconds. Place the tube on ice immediately for 2-3 minutes.
4. Add antibiotic free LB medium into the competent Bacteria. Incubate for 1 hour, 37℃, 200 rpm.
5. Coat the competent Bacteria on a LB medium agar plate with proper antibiotic.
6. Incubate at 37℃ overnights.
Competent Cell
Commercial competent cells E.coli DH5α E. coli BL21 were obtained from Shanghai Weidi Biotechnology Co., Ltd.
Protein purification
protein purification is carried out with 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.
Protein concentration
Protein concentration is carried out with Amicon® Ultra-4 Centrifugal Filter Units from Millipore co.,ltd and the protocol it provided.
Preparation of M9 solid medium
1. A packet of 5×M9 salt is mixed with 3.2 g agarose, dissolve in 200 mL sterilized water, and autoclave for 15 minutes.
2. The sterilized M9 salt solution is left to cool until the temperature drop to 50°C, the solution is mixed with 2 ml 1 mol/L MgSO4 and 0.1 ml 1 mol/L CaCl2, (both filter by a 0.22 μm bacterial filter) with 20 ml 20% dextroglucose solution and shake gently.
3. Then quickly pour 15 ml the mix solution into the plastic petri dish and gently shake the solution evenly.
Freeze-drying bacteria
1. 0.01% of 1.0 mL LB medium was inoculated overnight and cultured at 26℃ until OD600= 0.15 (about 1.0x 107 cells/mL), corresponding to the early exponential growth stage.
2. Cells were taken and centrifuged (4000 rpm, 10 mins) , and reduce the supernatant to a final concentration of 2.0x 107 Cells/ml.
3. The final concentration of trehalose in the solution was 15% by adding trehalose to the re-suspended bacterial solution.
4. Transfer 50 uL of the liquid into a glass vial and transfer to a freeze-dryer (Advantage, VirTis, USA). The freeze-drying operation lasted for 40 h (freezing for 2 h at 200 mTorr to 40 C, then drying for 38 h at 40 C and 25 mTorr).
5. The vials are sealed under vacuum and stored at temperature of 4℃ or -20℃.
Identification of proteins
Determination of protein molecular weight of normal size protein were carried out with SDS-PAGE Preparation kit from Sangon Biotech (Shanghai) Co., Ltd and the protocol it provided.
Determination of protein molecular weight of proteins below 10 kd were carried out with Tris-Tricine-SDS-PAGE gel preparation kit and the corresponding reagents 2×tris Tricine SDS-PAGE loading buffer(including DTT), 1×tris Tricine SDS PAGE electrophoresis buffer (cathode -), 1×tris Tricine SDS PAGE electrophoresis buffer (anode +) from Beijing solarbio science﹠technology co.,ltd. The specific experimental steps are the protocols they provided.
Protein quantitative test
Protein quantitative test were carried out with Bradford Protein Assay Kit from Sangon Biotech (Shanghai) Co., Ltd.
Suicide module verification
1. Methods of molecular cloning and transformation are described above. Transform this plasmid into E. coli DH5α. Then spread it onto a LB medium plates with 60 μg/mL ampicillin and incubate overnight at 37℃ in a incubator.
2. Pick three clonies from the same plate as parallel repeats. With the 5 ml LB medium containing 60μg/mL ampicillin, the seed culture media are grow at 37℃ while shaking at 200 rpm overnight.
3. Take 2 mL seed culture medium into 2 mL EP tube and centrifuge for 2 minutes at 5000 rpm. Then wash the centrifugated deposit with PBS buffer to remove ferrous ions for three times. After that resuspend the deposit in the M9 medium (60 μg/ml ampicillin) and make the final volume 5 mL.
4. Measure the OD600 value of the resuspending culture media in an automatic microplate reader (SynergyH1 hybrid multimodal reader). The dilute the resuspending culture medium to OD600 = 0.02 and add FeSO4 solution (40 mM) to the final concentration of 0 μM, 10 μM , 20 μM, 40 μM. The blank control groups are the same volume of M9 medium containing 60 μg/mL ampicillin which are added the corresponding concentration gradient FeSO4 solution respectively.
5. Incubate the cultures for 9 h at 37℃ while shaking at 200 rpm.
6. Samples are taken from each group once an hour and measured by UV spectrophotometer. And then convert the raw data into OD600.
7.Take 50 μL from the seed culture medium and add it into 5 ml LB medium containing 60 μg/mL ampicillin. Incubate for 8-9 hours at 37℃ while shaking at 200 rpm.
8. Dilute the incubated bacterial fluid to 10-2 and 10-3 times respectively.
9. Take 10 μL bacterial fluid from the two diluted groups, spread it onto M9 medium containing 60 μg/mL ampicillin respectively. Then two filter papers with a drop of 10 μL of FeSO4 (40 mM) and 10 μL of double distilled water respectively are put onto each plate. Blank controls are fresh M9 medium containing ampicillin (Amp, 60 μg/mL) with the engineered bacteria. Incubate them overnight at 37℃ in a incubator.
10. All experiments above are carried out in three biological repeats.
Sterilization module Validation
Expression and purification of ClyR
1. Methods of molecular cloning and transformation are described above.
2. Plasmid pET-28a(+)-ClyR (with His-tag) is transformed to E. coli BL21(DE3). The E. coli strain is cultured in LB medium containing 10 μg/mL kanamycin.
3. The E. coli strain is cultured to OD600 = 0.6, induced with 0.2 mM IPTG, and grow overnight at 16℃.
4. The harvested bacteria are resuspended with a binding/washing buffer (Sangon Biotech, Shanghai, China), and then the bacteria are lysedby ultrasonication. Purification is performed following the instructions of Ni-NTA SefinoseTM Resin (Sangon Biotech, Shanghai, China). The protocol are described above.
5. Purified ClyR is desalted and concentrated by Amicon ® Ultra-4 Centrifugal Filter Units (Millipore). After quantitation by Bradford assay (Sangon Biotech, Shanghai, China), the ClyR solution is stored at -20℃. The protocol are described above.
6.The samples are electrophoresed by SDS-PAGE Preparation kit (Sangon Biotech, Shanghai, China), observing of Coomassie's brilliant blue staining. The protocol are described above.
Activity tests of ClyR
1. S. mutans UA159 is static cultured in BHI medium at 37℃.
2. Resuspend the overnight cultured S. mutans UA159 by PBS. All activity tests are implemented in PBS resuspended S. mutans.
3. Culture the PBS resuspended S. mutans UA159 to OD600 = 0.8. Measure the drop of OD600 at 37℃ for 1 h by automatic microplate reader (SynergyH1 hybrid multimodal reader) . In the 96-well plates, 20 μl ClyR solution is added to the final concentration of 0 μg/mL, 0.1 μg/mL , 0.5 μg/mL, 1.0 μg/mL, 2.5 μg/mL, 5.0 μg/mL, 10 μg/mL, 15 μg/mL, 20 μg/mL, 39 μg/mL with 140 μl cell suspension in each well.
4. Implement the S. mutans UA159 treated by dilution plating of ClyR on BHI agar. Culture the plates overnight to count CFU.
5. All experiments above are carried out in three biological repeats.
Detection report module Verification
1. Methods of molecular cloning and transformation are described above.
2. Transform this plasmid into E. coli DH5α. Then spread it onto a LB medium plates with 20 μg/mL ampicillin and incubate overnight at 37℃ in a incubator.
3. Pick three clonies from the same plate as parallel repeats. With the 5 ml LB medium containing 20 μg/mL ampicillin, the seed culture media are grown at 37℃ while shaking at 200 rpm overnight.
4. Dilute the seed culture medium and culture it to OD600 = 0.8 with shaking at 200 rpm. Each seed culture medium is divided into five 2 ml EP tubes, each EP tube is filled with 1 ml medium, and sCSP is added to the final concentration of 0 mg/ml, 0.125 mg/ml, 0.25 mg/ml, 0.5 mg/ml, 1 mg/ml for gradient induction.
5. Pipette 160 μl of seed culture medium from each EP tube into 96-well plate, and add LB medium to 3 wells as blank control. Place the 96-well plate in an automatic microplate reader (SynergyH1 hybrid multimodal reader). Incubate at 37°C for 24 hours, then measure the fluorescence value (excitation: 584 nm, emission: 610 nm) and OD600 of each well every 20 minutes.
6. All experiments above are carried out in three biological repeats.
Repair module verification
LRAP protein purification
1. Methods of molecular cloning and transformation are described above.
2. Transform this plasmid into E. coli BL21(DE3) and pick three clonies from the same plate as parallel repeats. With the 5 ml LB medium containing 50 μg/mL ampicillin, the seed culture media are grown at 37℃ while shaking at 200 rpm overnight.
3. Expand the seed culture medium to OD600 = 0.6, then add IPTG to a final concentration of 0.3 nM, and induce it for 36 h at 16℃ while shaking at 200 rpm.
4. Bacteria are gathered at 4℃, 5000 rpm, and resuspended in binding/washing buffer (Sangon Biotech, Shanghai, China), and then ultrasonically lysed. Purification is performed following the instructions of Ni-NTA SefinoseTM Resin (Sangon Biotech, Shanghai, China). The protocol are described above.
5. The samples are electrophoresed on Tricine-SDS-PAGE gels (50% glycerol (w/v), Solarbio), observing of Coomassie's brilliant blue staining. The protocol are described above.
Combined proficiency testing
1. The plasmid is transformed into E. coli BL21(DE3) and induced as in LRAP protein purification above. The E. coli BL21(DE3) with plasmid transformation but without induction and the E. coli BL21(DE3) without plasmid transformation and induction are as control groups.
2. The bacteria with plasmid transformation are cultured in LB medium containing 50 μg/mL ampicillin, and those without plasmid are cultured in LB free medium. Lyse the bacteria by ultrasonication and centrifuge at 10000 rpm, 4℃. Take the supernatant for characterization.
3. Broke the teeth of domestic pigs into fragments with a side length of about 5 mm, and the fragments with more exposed sections are selected and stored in 75% alcohol at room temperature.
4. Take three pieces of pig teeth, and wash with PBS buffer to remove the alcohol on the surface. Place them in three 2 mL centrifuge tubes respectively, and then the three supernatants with uniform protein concentration are added respectively, and labeled on the centrifuge tubes.Incubate at 37℃ for 20 h. Centrifuge the tubes upside down for several times every 5 h to eliminate the influence of protein deposition processes as much as possible. Wash the incubated pig tooth fragments with PBS buffer solution by pipet-gun for 2-3 mins to remove the protein that is not bound to the teeth.
5. Incubate the rinsed pig teeth in plastic petri dishes with diameter of 10c m, and dilute with 50 mL 5% skimmed milk at 1:10000 and hybridize with His-Tag, Rabbit pAb (YEASEN Biotech,Shanghai, China) at room temperature for 2 h. Then wash with TBST buffer 3 times for 10 mins each. After 50 mL 5% skim milk is diluted with 1∶10000 peroxidase-conjugated Goat anti-rabbit IgG (H+L) (YEASEN Biotech,Shanghai, China), it is cross-bred at room temperature for 2 h and then washed with TBST buffer solution for 3 times, 10 mins each.
6. Use A Super ECL Detection Reagent ECL (YEASEN Biotech,Shanghai, China)for development operations.
Nucleation proficiency verification
The above three kinds of broken supernatants are added into artificial saliva (8.7 mM KCl, 0.6 mM MgCl2, 10 mM CaCl2, 4.6 mM K2HPO4, 2.7 mM KH2PO4) respectively until the final protein concentration was 5 mg/ml. The samples are incubated at 37 ℃ for 24 h to observe the crystallization precipitation.
Control loop verification
1. Methods of molecular cloning and transformation are described above.
2. Transform this plasmid into E. coli DH5α and pick three clonies from the same plate as parallel repeats. With the 1 ml LB medium containing 15 μg/mL Kanamycin Sulfate, the seed culture media are grown at 37℃ while shaking at 200 rpm for 3 hours.
3. Add 10 μl seed culture media and 1 ml LB medium containing 15 μg/mL Kanamycin Sulfate to 24 well plate, and add each sample to four wells. And add IPTG to the final concentration of 1 mM, 100 μM, 50 μM, 0 μM. Place the 24-well plate in an automatic microplate reader (SynergyH1 hybrid multimodal reader). Incubate at 37°C for 10 hours, and measure the fluorescence value (excitation: 570 nm, emission: 610 nm) and OD600 of each well every 30 minutes.
4. All experiments above are carried out in three biological repeats.
Parts Improvement
1. Methods of molecular cloning and transformation are described above.
2. Plasmid pUC57-PfhuA1-mCherry and pUC57-PfhuA-mCherry is transformed to E. coli DH5α, cultured in LB medium containing 60 μg/mL ampicillinum overnight.
3. Inoculated Seed culture into 3 types of medium, as M9 medium，LB medium，LB medium containing 500 μM FeSO4.
4. Take 1 ml of each sample and add it into the wells. Place the 24-well plate in an automatic microplate reader (SynergyH1 hybrid multimodal reader). Incubate at 37°C for 14 hours, and measure the fluorescence value (excitation: 584 nm, emission: 610 nm) and OD600 of each well every hour.
5. All experiments above are carried out in three biological repeats.
Part characterization
Part of MazF characterization
This part is the same as the suicide module verification.
Part of nlmAB promoter characterization
This part is the same as the Detection report module Verification
