Team:HZAU-China/Protocol

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Protocol

Molecular Cloning

DNA Synthesis

Primer synthesis service is provided by Tsingke Biotechnology Co., Ltd. Gene synthesis service is provided by Genscript.

PCR

Regular PCR, Overlap PCR is carried out with PrimeSTAR® Max DNA Polymerase from TaKaRa and the protocol it provided online. 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 98℃.

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 E.coli DH5α and E. coli BL21 is shown below:
  1. Melt the competent Bacteria on ice for 5 minutes.
  2. Add proper amount of plasmid into the competent Bacteria and place it on ice for 20-30 minutes.
  3. 42℃ heat shock for 45 seconds. Place the tube on ice immediately for 2-3 minutes.
  4. Add 700μL 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℃ overnight.
The protocol we used to transform plasmids into competent cells E.coli Nissle1917 is shown below:
  1. Melt the competent Bacteria on ice for 5 minutes.
  2. Add proper amount of plasmid into the competent Bacteria and place it on ice for 20-30 minutes.
  3. 50℃ heat shock for 60 seconds. Place the tube on ice immediately for 2-3 minutes.
  4. Add 700μL antibiotic free LB medium into the competent Bacteria. Incubate for 1 hour, 37℃, 200 rpm.
  5. Inoculate 200μL of the bacteria from the previous step into 5mL LB medium with proper antibiotic, 37°C, 200 rpm, incubate overnight.
  6. Coat the competent Bacteria on a LB medium agar plate with proper antibiotic.
  7. Incubate at 37℃, 10h.

Competent Cell

Commercial competent cells E.coli DH5α and E.coli BL21 were obtained from Shanghai Weidi Biotechnology Co., Ltd. Competent cells E.coli Nissle1917 were made by ourselves, the protocol we made the Calcium transfer competence is shown below:
  1. Take out the preserved E.coli Nissle1917 from -80℃, and transfer a small amount of bacteria to the appropriate LB liquid medium for expansion.
  2. Coat the Bacteria on a LB medium agar plate with proper antibiotic, incubate at 37℃ overnight.
  3. Pick a moist and smooth single colony and inoculate it in 5mL LB medium without resistance at 37℃, referring to the late logarithmic growth stage.
  4. Inoculate the bacterial suspension in 100mL LB medium at a ratio of 1:100~1:500, and incubate at 37°C and 200rpm for 2~3h until OD600=0.3~0.5.
  5. Transfer the bacterial solution to a 50mL centrifuge tube pre-cooled on ice, and place on ice for 10-30 minutes
    6. .
  6. Centrifuge the cooled bacterial solution at 4°C, 4000 rpm for 10 min.
  7. Use 10mL pre-chilled 0.1mol/L CaCl2 solution to gently suspend the cells evenly, place them on ice for 30min, and centrifuge at 4℃, 4000 rpm for 10min.
  8. Discard the supernatant and invert for 1 min to drain the liquid.
  9. Add 4 mL of pre-chilled 0.1mol/L CaCl2 solution containing 15% glycerol, gently suspend the cells, and place on ice for 10 minutes.
  10. Divide the prepared competent cells into 100μL per 1.5mL EP tube and store at -80℃.

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.

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.

Protein quantitative test

Protein quantitative test were carried out with Bradford Protein Assay Kit from Sangon Biotech (Shanghai) Co., Ltd.

Detection Verification

  1. Methods for molecular cloning and transformation are described above.
  2. Plasmid pSC101-NarXL-ThsSR is transformed to E.coli DH5α. The E. coli strain is cultured in LB medium containing 10 μg/mL kanamycin to OD600=0.4.
  3. Add 196μL bacteria samples into a 96-well plate，and different concentration of sodium thiosulfate(0μM、0.1μM、1μM、10μM、100μM、1000μM) and potassium nitrate(0μM、1μM、10μM、100μM、1000μM) are added into each well to 200μL.
  4. Cultivate the samples in hybrid multi-mode reader (Synergy H1) at 37°C, 282cpm. The OD600 and fluorescence intensity (excitation: 485nm, emission: 528nm) are determined every 15 minutes for 12 hours.
  5. Data taken from the plate reader are exported to Excel.

Report module Verification

Cytotoxicity Protocol for Geosmin

The test is conducted in order to define whether geosmin is toxic to the engineered bacteria.
  1. Dilute geosmin standard solution (10mg/L, purchased from Dr. Ehrenstorfer™, Augsburg, Germany) with LB to 0μg/L, 0.1μg/L, 1μg/L, 10μg/L, 50μg/L, 100μg/L, 1mg/L. The concentrations are arranged referring to theoretical human’s olfactory system to geosmin(4-10ng/L).
  2. Test OD600 of E.coli BL21(DE3)、E.coli DH5α、E.coli Nissle 1917 adding with geosmin diluted above every 15min for 12h at 37℃.

HS-LPME

  1. Add IPTG to induce E.coliBL21(DE3) with pET-28a(+)-ScGS(with His-tag) when OD600 reach 0.6-0.7 in 100 mL TB medium with 5% glycerol. Induce protein expression at 18 °C overnight following by continuing cultivation at 25 °C for 72h.
  2. Add NaCl to the culture into the concentration of saturate, introduce a magnetic stirring bar into the container and seal it with preservative film.
  3. Put the vial on a magnetic agitator(800rpm) and preheat to 55°C.
  4. Use a 10μL syringe(containing 3μL 1-hexanol) to penetrate the film, then fix it. Press the plunger, hold the organic solvent for 6-9 min to let the analytes to be extracted by the micro drop into the syringe.
  5. Remove the micro drop into the syringe and inject it into GC system.

GC-MS

GC conditions:

Analytical columns: DB-5 MS(30m×0.25mm×0.25μm) Injector temperature: 250℃ Injection mode: splitless mode Inlet temperature: 250℃ Flow mode: constant Oven temperature: 60℃ for 2min Carrier gas: N2 at 1.0mL/min Transfer line temperature: 270℃

MS conditions:

Scan mode: TIC mode with electron impact (EI) ionization resource (electron energy 70eV) Ion source temperature: 250℃ Quadrupole: 150℃

Health-care module verification

Expression and purification of azurin

  1. Methods of molecular cloning and transformation are described above.
  2. Plasmid pET-28a(+)-azurin (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 strain is cultured to OD600 = 0.6, induced with 0.5 mM IPTG, and grow overnight at 18℃.
  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 is described above.
  5. Purified azurin is desalted and concentrated by Amicon® Ultra-4 Centrifugal Filter Units (Millipore). After quantitation by Bradford assay (Sangon Biotech, Shanghai, China), the azurin solution is stored at -20℃. The protocol is 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 is described above.

Combination of azurin and p53

  1. Plasmids pET-28a(+)-p53(with His-tag) and pET-28a(+)-azurin were transformed into E. coli BL21 (DE3). The p53 protein is fused with a 6×His tag in order to be able to bind to the Ni-NTA column, while azurin is not fused with a 6×His tag.
  2. Cultivate the engineered bacteria in 10mL LB medium containing 50μg/mL Kan at 37°C and 200rpm to OD600=0.6, induced with 0.4 mM IPTG.
  3. These cultures are grown overnight at 18 °C to induce protein expression.
  4. To process bacterial samples for protein purification, the protocol is described above.
  5. Use Binding/Wash Buffer (Sangon Biotech, Shanghai, China) to bind the processed p53+6×His tag protein on a Ni-NTA column (Sangon Biotech, Shanghai, China), and then add azurin protein sample to the column for 2h, So that it can combine with p53.
  6. After combining for 2h, the two proteins are purified together, which is performed following the instructions of Ni-NTA SefinoseTM Resin (Sangon Biotech, Shanghai, China). The protocol is described above. 5、The samples are electrophoresed by SDS-PAGE Preparation kit (Sangon Biotech, Shanghai, China), observing of Coomassie's brilliant blue staining. The protocol is described above.

Anti-inflammatory module verification

Visualization of protein

In order to determination the protein whose molecular weight are under 10 kDa were conducted by Tris-Tricine-SDS-PAGE gel preparation kit and the corresponding reagents 2× Tricine Sample Buffer (including DTT), Discontinuous Buffer System (Tris-Tricine-SDS) (including 1× Tricine-SDS-PAGE Electrophoresis Buffer (Cathode -), 1× Tricine-SDS-PAGE Electrophoresis Buffer (Anode +)) purchased from Sangon Biotech (Shanghai, China). The specific experimental steps are followed by the protocols the company provided.

Expression and purification of LL-37/LTA/HyLα

  1. Plasmid pET-28a (+)-LL-37/ pET-28a(+)-LTA/ pET-28a(+)-HyLα (with His-tag) is transformed to Escherichia. coli BL21(DE3). The E. coli strain is cultured in LB medium containing 10 μg/mL kanamycin.
  2. When the optical density of the cultured bacteria reached approximately OD600 = 0.6, IPTG was added to the final concentration 50mM. And the bacteria were induced at 18ºC overnight.
  3. The harvested bacteria are resuspended with a binding buffer (Sangon Biotech, Shanghai, China), and then the bacteria are lysed by ultrasonication.
  4. Purification is performed following the instructions of Ni-NTA SefinoseTM Resin (Sangon Biotech, Shanghai, China). The protocol is described above.
  5. The samples are electrophoresed by Tricine-SDS-PAGE Preparation kit (Sangon Biotech, Shanghai, China), observing of Coomassie’s brilliant blue staining. The protocols are described above.

Activity Test assay of LL-37/LTA/HyLα

  1. Salmonella Typhimurium SL1344 is cultured in LB medium at 37ºC overnight.
  2. Resuspend the cultured S. Typhimurium by PBS. All activity tests are implemented in PBS resuspended S. Typhimurium.
  3. Culture the PBS resuspended S. Typhimurium to OD600 = 0.35.
  4. In the 96-well plates, 20 μl LL-37/LTA/HyLα solution is added to the final concentration of 8 μg/mL with 180 μl bacteria suspension in each well. Together with 20 μl Elution buffer (Sangon Biotech, Shanghai, China) added with 180 μl bacteria suspension serve as control groups. Each with three biological repeats.
  5. Use the Microplate Reader to count OD600 of the 96-well-plate for 2.5 hours.

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 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 two temperatures(37 ℃ and 28 ℃) respectively while shaking at 200 rpm overnight. Treated the same with the experimental groups, control bacteria are also cultured at the two temperatures.
  3. Measure the OD600 value of the resuspending culture media in an automatic microplate reader (SynergyH1 hybrid multimodal reader) and compare the data of experimental groups with the data of control group.
  4. Put all the media into the 37 ℃ orbital shaker and culture them at 200 rpm for 12 hours.
  5. Plot the OD600 value curves of the resuspending culture media over time in an automatic microplate reader (SynergyH1 hybrid multimodal reader). Incubate the cultures for 12 hours at 28℃ while shaking at 200 rpm. Samples are taken from each group once an hour and measured by UV spectrophotometer. 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.
  6. All experiments above are carried out in three biological repeats.