Bacillus subtilis natto strain screening

Purpose

To get a single colony of Bacillus subtilis natto.

Method

  1. Get commercially-available natto.
  2. Stir natto well.
  3. Add normal saline solution.
  4. Vortex and wait for soybean sediment.
  5. Spread bacteria solution on LB agar plate and pick up the colony.
  6. Using streak-culture to screen the right colony several times.

Materials

  • Commercially available natto
  • LB agar plate
  • Saline solution

Natto making

Purpose

  1. Transformation
  2. Using transformation species to get natto extract.

Method

  1. Culture EP2(B. subtilis natto PQQ) at 37°C for 8-12 hr.
  2. Wash the soybeans and soak overnight.
  3. Cover the beans with water and cook them until they are soft.
  4. Cool down beans and smooth 200g soybeans out into containers.
  5. Each container adds 1mL of bacterial broth and mixes well.
  6. Cover each container with food wrap and keep several whole on it.
  7. Put containers in room temperature for 16-24 hr.

Materials

  • Soybeans 500g
  • Bacillus subtilis natto PQQ
  • A constant temperature space such as an electric cooker or an incubator

DNA electrophoresis

Propose

Examining the length of DNA fragments.


Method

  1. Release the agarose gel from the mold, wipe off the exposed residual glue, put the gel together with the mold into the electrophoresis gel, and slowly add 0.5x TAE buffer until the well is submerged.
  2. Take 1 volume of DNA sample, add 1/5 volume of gel loading buffer, mix well with pipetting, and add to the wells respectively (the total amount here does not exceed 30 µL)
  3. Add 3 µL of DNA marker to the wells at both ends, add the sample to be tested in the middle, and perform electrophoresis at 100V.
  4. Wait for about 30 minutes, until the blue dye is close to the bottom of the gel.
  5. Take out the gel under exposure to UV.

Materials

  • DNA sample
    1. Put 20 mL 0.5x TAE buffer and 0.16 g agarose into a conical flask.
    2. Heat in the microwave for 40s.
    3. View the situation to heat 5s until boiling.
    4. Cool down to about 60° C and add 1 μL safe view mixing evenly.
    5. Pour the gel into the mold,if there are any bubbles should pop it.
    6. Insert comb till cool down to solid.
    7. Remove comb slowly to prevent gel from breaking.
  • 0.5x TAE buffer
  • 6x Gel loading buffer
  • Safe view

16S rDNA PCR

Purpose

Sequencing Bacillus subtilis natto gene.


Method

  1. 200 ~ 500µL broth/per Eppendorf.
  2. Centrifuge at 12000 xg for 10 sec, and remove supernatant(nearly empty).
  3. Add 50µL ddwater/per Eppendorf, and vortex
  4. Add a micro tube holder, and take liquid nitrogen.
  5. Dry bath 92 Celsius degrees for 3~5 mins, put in liquid nitrogen to cool down until there is no sound, put it back into the dry bath, and repeat 3 to 5 times.
  6. Add reagents in following order: ddwater, buffer, dNTPs, MgCl2, template primers, DMSO, phusion polymerase.
  7. Prepare a negative control reaction without template DNA.
  8. Prepare a positive control reaction with a template of known size and appropriate primers.
  9. Program thermal cycler for PCR reaction.
  10. Analyze the results of PCR reaction via gel electrophoresis.

Materials

  • Pure water
  • Liquid nitrogen
  • Buffer
  • dNTPs
  • MgCl2
  • Template primers
  • DMSO
  • Phusion polymerase
  • Negative control
  • Positive control
  • Thermal cycler

Plasmid DNA extraction

Purpose

Extract plasmid for transformation.


Method

    Culturing

  1. Inoculate 5 mL LB broth, supplement with 5 µL of kanamycin. Take an adequate amount of E. coli into the broth, culture it in the incubator for 8~12hr.

    2. Cell harvest and lysis

  2. Transfer the cultured medium into a 1.5 mL Eppendorf (Tube1), centrifuge at maximum speed in RT for 10 seconds.
  3. Completely remove the supernatant, resuspend in 100 µL of Solution I (containing lysozyme), incubated at RT for 30~45 minutes.

  4. Add 200 µL of solution II, gently mix by inverting for approximately 10 times, and incubate for up to 5 minutes at RT.
  5. Add 150 µL of solution III, mixed by inverting.
  6. Centrifuge for 5 min at RT and maximum speed.
  7. Carefully transfer supernatant to a fresh tube(Tube 2), with as little contamination of pallet as possible. (450 µL)

    8. If necessary, redo step 6 again.


    Phenol/Chloroform extraction

  8. Add equal volume of phenol/chloroform solution into the centrifuge tube(Tube 2).
  9. Invert for approximately 10 times.
  10. Centrifuge for 5 min at RT and maximum speed.
  11. Transfer the top layer into new tube(Tube 3) (320 µL)

    12. Ethanol precipitation

  12. Add 2.5 volumes of 100% ethanol.
  13. Centrifuge the sample for 5 minutes at 4°C and max speed. Carefully remove the supernatant without discarding the DNA pallet.
  14. Wash the pallet by adding 900 µL of 75% ethanol ( no need to resuspend ). Centrifuge sample for 5 minutes at RT using max speed.
  15. Redo 17. for 2-3 times.
  16. Dry off the pallet in a dry-bath at 65°C for 10 min.
  17. Resuspend pellet in 33 µL pure water or buffer of choice (e.g. TE buffer).
  18. Add 1 µL RNase to remove RNA.
  19. Using a nanodrop spectrophotometer to analyze the A260/A280 and A260/A230 ratio, and DNA concentration.
  20. Using gel electrophoresis to check the final product of plasmid DNA.

Materials

  • Escherichia coli JM109
    • (Storage in -80° C freezer)
  • LB broth
  • Kanamycin (50 mg/mL)
    • (Storage in -20° C freezer)
  • Phenol / chloroform
    • (Storage in 4° C freezer)
  • 75 % ethanol
  • 100 % ethanol
  • RNase
  • Solution I
  • Solution II
  • Solution III
  • Germ-free water or TE buffer

Instrument

  • Ultra high centrifugation
  • Dry bath
  • Nanodrop spectrophotometer

Transforming plasmid DNA into electrocompetent cells

Purpose

Transformating PQQ plasmid from E.coli to Bacillus subtilis natto.


Tool preparation:

All the tools used in the experiment need to cool in 4 degree Celsius(usually ice water) at any stage in the protocol. To make sure the bacteria will not grow.

Cell preparation:

  1. Inoculate cell in a flask containing 25mL of LB medium. Incubate the culture overnight 37 degree Celsius with vigorous aeration(usually 250rpm shaker).
  2. Expansion: Inoculate 250mL of prewarmed LB medium in a 2-L flask with 12.5mL of the overnight bacterial culture. Inoculate the flask at 37 degree Celsius with agitation(300rpm shaker).
  3. Measure the OD600 of the growing bacterial culture every 20 mins.(usually 2.5h incubation; best to OD600=0.4, so begin to harvest when the OD600=0.35)
  4. As the OD600=0.4, rapidly transfer the flask to an iced water bath for 15-30 min. Swirl to make cooling evenly.
  5. Transfer culture to the iced-cold centrifuge bottle. Centrifuge at 1000g for 15min at 4 degree Celsius. Remove the supernatant and resuspend the pellet in 500mL of iced-cold pure water by gently pipetting.
  6. Centrifuge at 1000xg for 20min at 4 degree Celsius. Remove the supernatant and resuspend the pellet in 250mL of iced-cold pure water and iced-cold 10% glycerol by gently pipetting.
  7. Centrifuge at 1000xg for 20min at 4 degree Celsius. Remove the supernatant and resuspend the pellet in iced-cold 10% glycerol by gently pipetting.
  8. Centrifuge at 1000xg for 20min at 4 degree Celsius. Remove any of the supernatant and resuspend the pellet in 1mL of iced-cold GYT medium.
  9. Measure the OD(cells/mL, blank by GYT medium) of a 1:100 dilution of the cell suspension.
  10. Dispense 40 μL aliquots of the cell suspension into sterile, iced-cold 0.5 mL microcentrifuge tubes, drop them into a bath of LN before transfer into -80 degree Celsius freezer.

Materials

  • LB medium(5 mL, 25mL, 250mL)
  • Bacillus subtilis natto
  • Pure water(iced-cold)
  • 10% glycerol(iced-cold)

Reagent preparation

  1. Withdraw two of the microcentrifuge tubes and measure the transformation efficiency of the preparation using 10 and 50 pg of plasmid DNA. If everything is to work to plan, the efficiency of transformation of the preparation should be 2.1×104 transformants per μg DNA(best) and the number of transformants should be proportional to DNA concentration.

Materials

  • SOC medium
  • Water bath
  • LB + antibiotics plate/medium(for selecting)
  • 17 mm x 100 mm round-bottom culture tubes
  • Competence cell
  • SOB
  • OS buffer
  • OS-LB buffer

Method

Electroporation(coefficient & parameter takes 1mm cuvette for instance)

  1. Prepare 17 mm x 100 mm round-bottom culture tubes at room temperature.
  2. Place SOC recovery medium in a 37°C water bath. Pre-warm selective plates at 37°C for 1 hour.
  3. Place electroporation cuvettes (1 mm) and microcentrifuge tubes on ice.
  4. Thaw electrocompetent cells(exponential stage, OD600 value of about 0.35-0.4) on ice and mix cells by flicking gently. (Freshly prepared electrocompetent cells may be used immediately.)
  5. Transfer 25 μL of the cells to a chilled microcentrifuge tube. And add 1 μL of the DNA solution.
  6. Carefully transfer the cells and plasmid DNA mix into a chilled cuvette without introducing bubbles and make sure that the cells deposit across the bottom of the cuvette.
  7. Wipe off excess moisture from outside of the cuvette using a kimwipe.
  8. Place the cuvette in an electroporator. Close the lid.
  9. Have 975 μL of SOC ready to add to your cells.
  10. Electroporate conditions:
    • 1kV(2mm cuvette uses 4.2kV)
    • 25 μF
    • 100 Ω
    • The typical time constant is ~2.6 milliseconds
  11. Press the “Pulse” button on the electroporator to shock cells.
  12. Immediately add 975 µl of 37°C SOC to the cuvette in RT, gently inverse twice, then transfer to the 17 mm x 100 mm round-bottom culture tube.
  13. Transfer SOC-cell mixture to an eppendorf tube.
  14. Incubate tube in 37°C shaker for at least 1 hr (gently)to permit expression of antibiotic resistance gene.
  15. Place selective agar plate(s) supplemented with appropriate antibiotics in a 37 °C incubator to warm.
  16. Plate 200μL of cells onto pre-warmed selective plate(s). (The appropriate volume depends on the efficiency of the transformant.)
  17. Incubate plate overnight at 37°C.
  18. Leave remaining SOC-cell mixture on the benchtop overnight.

Medium

GYT Medium

Sterilize by a pre-rinsed 0.22 μm filter, store at 4 degree Celsius.


SOC Medium

Add 20 ml of sterile 1 M glucose per liter of SOB medium immediately before use. Or make frozen aliquots and store at -20°C.
Sterilize by a pre-rinsed 0.22 μm filter, store at 4 degree Celsius.

SOB Medium

  1. Adjust pH to 7.0 with 10N NaOH.
  2. Autoclave to sterilize on LIQUID cycle 15 minutes.
  3. Sterilize by a pre-rinsed 0.22 μm filter, store at 4 degree Celsius.

Reference

  1. http://mcb.berkeley.edu/labs/krantz/protocols/electroporation.pdf
  2. https://www.addgene.org/protocols/bacterial-transformation/
  3. https://www.sciencedirect.com/science/article/pii/S0167701298000876?casa_token=1nf_hCAUVFgAAAAA:jRFlA-FCmjVx6fGSMC1xho_Uu4vbimICNMIoM3G_yuH6c1uJA0Wqdrlu3Yy7FkbS_HYCffE78a8
  4. https://international.neb.com/protocols/0001/01/01/electroporation-protocol-c2986

Growth curve

Purpose

Comparing the growth curve of transformation species with wild type.

Method

  1. Activation of culture: Culture bacteria at 37°C for 8-12hr.
  2. Streak culture bacteria on the plates.
  3. Pick up the single colony into 5ml LB.
  4. measure the OD600(benchmark:LB)in 5ml LB then add to the 200ml LB. Let the starting OD600 to be 0.001.
  5. Use spectrophotometer to measure OD600. The dilution ratio: first and second time:1 third time:1/5 other time: 1/10. The first and second time measurements are taken for every two hours, others are every hour.

Materials

  • LB agar plate
  • LB
  • kanamycin

Bacteria

  • Bacillus subtilis natto PQQ
  • Bacillus subtilis natto wild type
  • Bacillus subtilis RM125
  • Bacillus subtilis RM125 PQQ

Cultivation situation

37°C 200rpm light wave: 600 nm


PQQ gene PCR

Purpose

Sequencing PQQ gene in plasmid.

Method

The final volume should be 20 µL.

  1. Thaw all reagents on ice.
  2. Assemble reaction mix into 20 µL volume in a thin walled 0.2 mL PCR tubes.
  3. Add reagents in following order: water, buffer, dNTPs, MgCl2, template primers, Taq polymerase.
  4. Gently mix by tapping the tube. Briefly centrifuge to settle tube contents.
  5. Prepare negative control reactions without template DNA.
  6. Prepare a positive control reaction with a template of known size and appropriate primers.
  7. Program thermal cycler for PCR reaction.
  8. Analyze the results of PCR reaction via gel electrophoresis.

Materials

  • Sample
  • water
  • buffer
  • dNTPs
  • MgCl2
  • template primers
  • Taq polymerase
  • negative control
  • positive control
  • thermal cycler

Reference



PQQ separation and purification

Purpose

Extract pure PQQ from fermentation broth.

Method

  1. Fermentation broth purification
    • Add methanol into the fermentation broth and undergo ultrasonication for 30 min. 0.22 filter removing bacteria and leaving the supernatant. Concentrating 3 times to get purified broth.
  2. Supramolecular solvent (SUPRAS) preparation
    • Add surfactant and alkanol into alkane.
      Stir it. If the solution is clear and transparent, SUPRAS product successfully.
  3. The procedure of PQQ extraction
    • Add SUPRAS into a solution containing PQQ and stir for 10 min.
      Pour it into a separatory funnel and stand for a layer.
      Upper layer is the SUPRAS phase(containing PQQ); the lower layer is the water phase.
  4. Back-extraction
    • Add NaCl solution into SUPRAS.
      Upper layer is the recovered SUPRAS phase; the lower layer is the aqueous phase containing PQQ.

Materials-SUPRAS

BC:n-hexane:n-pentanol(w/v/v)=0.5:9.5:1

  • methanol
  • fermentation broth
  • surfactant
  • alkanol
  • alkane
  • NaCl solution
  • ultrasonicator
  • 0.22 filter

Natto extract

Purpose

Getting natto side products, like γ-PGA,nattokinase,and vitamin K2.


Method

  1. Add 15g natto and 15g water into the centrifuge tube mashing into muddy.
  2. undergo ultrasonication for 10 min.
  3. Centrifuge for 5000 xg for 15 min.
  4. Move supernatant into centrifuge tube and put into ultra-centrifuge for 5000 xg 15 min.
  5. Move supernatant into a serum bottle and put into the autoclave for 121° C 20 min.
  6. Cool down to room temperature and store into 4° C.

Materials

  • Natto
  • Centrifuge tube
  • Ultracentrifuge

Diabetic-alike cell making

Purpose

Producing diabetic-like cells to simulate diabetic wounds.


Method

To induce the diabetic-like cell, the medium will supplement with 25 mM glucose and 10 nM insulin for 5 days


Materials

  • HaCaT cell
  • Glucose 25 mM
  • Insulin 10 nM

Cell viability test (CCK-8)

Purpose

Determine the number of viable cells in cell proliferation and cytotoxicity assays.


Method

  1. Adjust the cell concentration to 105 cell/mL
  2. Transferring cell suspension into a 96-well plate (100 µL/well), pre-culture the cell for 24 hours.
  3. Add the test compound in different concentrations, and let it coculture for 24 hours.
  4. Remove the culture medium, wash the cell using PBS twice, then add 100 µL of medium containing 10% of WST-8 into each well, let it react for 1~3 hours in the incubator.
  5. Measure the absorbance at 450 nm using ELISA reader

Materials

  • HaCaT cell
  • HST-8
  • 96 well plate
  • ELISA reader
  • Test compound

Viable cell test

Purpose

Examining viable cells from dead cells to decide survivability.


Method

  1. Using centrifugation to precipitate the cell, using adequate volume of DMEM to resuspend the cell.
  2. Transfer 100 µL of suspension solution into a 1.5 mL micro tube, then take the tube outside the hood.
  3. Take 10 µL of cell suspension and mix with equal volume of trypan blue.
  4. Drop 10 µL of mixture onto the hemocytometer, cover wi- th slide, then count the cell using a microscope. (Only count the four big square on the four corner)

Materials

  • HaCaT cell
  • Dulbecco's Modified Eagle Medium (DMEM)
  • Trypan blue 0.4%
  • 10 µL tip/pipet
  • Hemocytometer
  • Counter
  • Microscope

q-PCR analysis

Purpose

Quantification cells’ gene expression level after treatment.


Method

  1. The total RNA from the cell will be extracted using Trizol, the extraction steps following the manufacture suggestion.
  2. The quality of RNA would be evaluated using a UV nanodrop spectrometer.
  3. The reverse transcription was using High-Capacity cDNA Reverse Transcription Kit and followed the manufacturer's guide manual, then the cDNA was stored for q-PCR analysis.
  4. The q-PCR was using SYBR™ Green PCR Master Mix and followed the steps from the instrument manual.

Materials

  • Trizol
  • RNA sample
  • High-Capacity cDNA Reverse Transcription Kit
  • SYBR™ Green PCR Master Mix
  • q-PCR
  • nanodrop spectrometer
  • Thermal cycler

Wound healing assay

Purpose

Comparing cell adding PQQ with non-adding PQQ to test the wound healing speed.


Method

  1. Place the culture-insert into the 12-well plate
  2. Prepare the cell suspension, and adjust the cell concentration to 105 cell/mL
  3. Seed the cell in each well (70 µL/well)
  4. Culture the cell in incubator for 24 hours
  5. Check the cell density using a microscope, if the confluent cell layer is formed, gently remove the culture-insert.
  6. Wash the cell twice using PBS.
  7. Fill the cell with medium and test compound.
  8. Record the change of gap volume using microscope every 12 hours
  9. Analysis the volume using NIH ImageJ

Materials

  • Culture-Insert (ibidi)
  • HaCaT cell
  • 12-well plate
  • PQQ

UV exposure test

Purpose

Comparing the viability of diabetic HaCaT cells with PQQ from without PQQ.


Method

  1. The cell was cultured in a 96 well plate containing 10000 cells in each well for 24 hours.
  2. The cell will be washed using 1x PBS twice, then the cell will be exposed to UV light at the dosage of 100mJ.
  3. Add the compound and measure the viability after 24 hours.
  4. The analysis was using the CCK-8 kit under the manufacturer's protocol.

Materials

  • Diabetic HaCaT cell
  • PQQ
  • CCK-8 kit
  • PBS
  • UV light
  • 96 well plate