Team:SSTi-SZGD/Protocols

B. Subtilis 168 efficient chemical transformation method

Reagent preparation:

GMI: 1ml Inorganic salt solution liquor, 0.5ml yeast extract, 0.25ml Glucose, 0.2ml Casein hydrolysate, Replenish water to 10 ml.
GMI: 1ml Inorganic salt solution liquor, liquor, 0.5ml yeast extract, 0.25ml Glucose, 0.04ml Casein hydrolysate, 0.05ml Cacl 2 , 1ml Mgcl 2 , Replenish water to 10ml.

Experiment:

  1. The strains of B. Subtilis 168 LB tablet on line activation, 37℃ overnight culture.
  2. Pick a single colony in the culture dish into a solution containing 10ml of GMI culture at 37℃, 130rpm for 16h.
  3. Transfer 1ml Seed solution into 10ml fresh seed cultures of GMI, under the condition at 37℃ 160rpm for 3.5h.
  4. Transfer 1ml Seed solution into 10ml GM II, at 37℃under the condition of 160rpm cultivate for 90min.
  5. At 4℃ to 5000×g centrifuge for 10min, remove the supernatant but reserved 100ul of liquid suspended bacteria and bacteria after suspension of cells can be directly used for transformation.
  6. Take characteristic, immediately on the 37℃ dissolves in water bath for 30min, then add pAX01-HasA plasmid (~ 1 ng/μl).
  7. Coated red mildew resistance screening culture for 37℃ 160rpm to develop after 90min.

B. Subtilis 168 electroporation transformation method

Reagent preparation:

GM: 100ml LB+0.5M sorbitol(PH7.2)
RM: 10ml LB+0.5M sorbitol+0.38M mannitol
ETM: 200ml 0.5M sorbitol, 0.5M mannitol, 10% glucose

Experiment:

  1. Pick a single colony of B. subtilis 168 inoculate into 5ml of LB medium at 30°C, 130-150rpm for 12h.
  2. Take 3ml of the inoculum, transferred to 100ml of GM medium at 200 to 230rpm, and cultured at 37°C for 3.5hours.
  3. Take the bacteria solution in ice bath for 10min, then centrifuged at 4°C, 5000rpm for 8min, and discard the supernatant.
  4. Add the cold ETM 40ml 5000r 8min at 4°C centrifugation, discard the supernatant.
  5. Repeat step 4 three times
  6. Take 500ul ETM suspend the cells, and Packing 60ul of one tube.
  7. Take 1 tube add 3ul linear plasmid, ice bath for 5min.
  8. Pour into the cold electric rotor (1mm) (Bio-Red).
  9. 2.0kv 25uF 200Ω Once at a time (4.5ms).
  10. Immediately add 1ml of resuscitation medium RM at 37°C, 160rpm for 3h.
  11. Add resistant coating. Incubate at 37°C.

References: Gang-Ping Xue, Jennifer S Johnson, Brian P Dalrymple. High osmolarity improves the electro-transformation efficiency of the gram-positive bacteria Bacillus subtilis and Bacillus licheniformis. Journal of Microbiological Methods, 1999,34(3):183-191.

The fermentation manufacture

Reagent preparation:

Luria-Bertani (LB) medium: (containing 20g/L xylose, 1% glucose, 1.5g/L MgSO 4 ) was used for the cloning experiments.
Modified medium(MM): (g/L: yeast extract 20, tryptone 2, MgSO 4 1.5, 50 mM potassium phosphate buffer, sucrose 50,pH 7.0) was used for fermentation.

Experiment:

  1. Inoculate single colony into 50ml LB medium, for overnight culture as seed solution for 12h.
  2. 10% inoculation fluid medium to MM fermentation broth 50ml at 37℃ 160rpm.
  3. Xylose (20g/L) was added 2h after inoculation to induce the expression of the HasA gene.
  4. After 8h. Sucrose was exponentially fed at rates of 7.5, 7.5, 15.0, or 10.0 g/h/L from 8h to 12h. A constant feed rate of 5g/h was then maintained until the end of fermentation for 48h.

Separation and purification

  1. After the capsule released by the addition of 0.1% (w/v) sodium dodecyl sulfonate (SDS).
  2. The supernatant of the sample collected by centrifugation at 10,000g for 10min.
  3. HA precipitated with two volumes of ethanol and incubated at 4°C for 1h.
  4. The precipitate collected by centrifugation at 5000g for 10min.
  5. Then redissolved in an equal volume of distilled water.
  6. 3 to 5 steps performed three times.
  7. Freeze-dried to get HA powder.(easy to calculate)

HA Concentration measurement: CTAB-turbidimetric assay

  1. Prepare 0.2mol/L NaOH solution of 100ml, and use it as solvent. Add CTAB powder and mix into 2.5g/L CTAB solution.
  2. 1ml of the liquid to test taken in the colorimetric dish, and 2ml CTAB solution accurately added. The timing start when the solution added, and the shaking time was sufficient.
  3. The absorbance of 400nm wavelength was measured at 10min. 1ml water plus 2ml CTAB were used as blank controls. Test to know the concentration of the standard product as the standard curve.
  4. The formula obtained to calculate the sample concentration.
  5. Calculate the Concentration.

HA molecular weight measurement: Ubblelohde viscometer

  1. Dissolve 0.1g HA sample in 100ml of 0.2M NaCL to make HA solution.
  2. Take 10ml, 20ml, 30ml, 40ml of HA solution, and configure it into 0.02g/100ml 0.03g/100ml 0.04g/100ml 0.05g/ml.
  3. Clean the viscometer using suitable solvents, and by passing clean, dry, filtered air through the instrument to remove the final traces of solvents.
  4. Place the viscometer into the holder, and insert it into the constant temperature bath. Vertically align the viscometer in the bath if a self-aligning holder has not been use.
  5. Place a finger over tube M and apply suction to tube N until the liquid reaches the center of bulb D. Remove suction from tube N. Remove finger from tube M, and immediately place it over tube N until the sample drops away from the lower end of the capillary into bulb B. Then remove finger and measure the efflux time.
  6. To measure the efflux time, allow the liquid sample to flow freely down past mark E, measuring the time for the meniscus to pass from mark E to mark F.
  7. Under 25℃, respectively test record three times out of time.
  8. Calculate the kinematic viscosity of the sample by multiplying the efflux time by the viscometer constant.

The plasmid extraction of E.coli
  1. Take 1ml bacteria solution, centrifuge at 12000rpm for 2min, and discard supernatant.
    [The extraction of E.coli performed using TaKaRa MiniBEST Plasmid Purification Kit Ver.4.0 (Takara Co Ltd. Cat. No 9760)]
  2. Use 250μl solution I to resuspend cell pellets, make bacteria suspension.
  3. Add 250μl of solution II and flip it up and down 5-6 times until the transparent solution is formed.
  4. Add 350μl 4 ℃ pre-cooling solution III and turn it up and down for 5-6 times until it forms a solidified lump and remains for 5min.
  5. Room temperature centrifuge at 12000rpm for 10min, preserve supernatant.
  6. Place the spin column of the kit on collection tube.
  7. Transfer the supernatant from step 5 to spin column, centrifuge at 12000rpm for 1min and discard filtrate.
  8. Add 500μl buffer WA into spin column, centrifuge at 12000rpm for 30s, discard filtrate.
  9. Add 700μl buffer WB into spin column, centrifuge at 12000rpm for 30s, discard filtrate.
  10. Repeat steps 9.
  11. Place spin column in the new column, centrifuge at 12000rpm for 1min, and remove the filtrate.
  12. Add 50μl elution buffer to the center of the film and leave for 2min.
  13. Centrifuge at 12000rpm for 1min and save the filtrate at -20 ℃.
Agarose gel electrophoresis

  1. Pour enough running buffer into the electrophoresis tank (The surface should be higher than the top of the gel and not overflow).
  2. Prepare 1% agarose solution (0.2g agarose +20ml 0.5×TBE) and microwave it until melted, add 2µl of Gold-view dye when it's cooled down to 50℃.
  3. Choose the suitable gel tank, pour the fluid agarose gel and insert the comb.
  4. After a complete solidification put the agarose gel in the electrophoresis tank.
  5. Mix the sample with loading buffer sufficiently and load them into the sample lane together with the marker (usually marker in the first lane).
  6. Run at 120V, 80mA for 30 min.
  7. Put the agarose gel in an UV detector and record the picture.

Restriction Enzyme Digest

Deionized water: 12μl
Buffer H: 2μl
EcoRI: 1μl
DNA: 5μl
①After adding all reagents, close the tube lid and centrifuge for a few seconds.
②Incubate at 37℃ for 1h 20min, and then boil for 20min to inactivate the enzyme.

Colony PCR
PCR formuation:

ddH2O: 18μl
10xPCR buffer: 2.5μl
dNTP: 2μl
Primer-F: 1μl
Primer-R: 1μl
Taq enzyme: 0.5μl
DNA template

PCR cycle:

① 98℃ 3min
② 94℃ 40s
③ 55℃ 40s
④ 72℃ 1min
⑤ Repeat 2-4 steps 30 times
⑥ 72℃ 10min

Add all the reagents, take the PCR, after PCR is over, then do Agarose gel electrophoresis.

Extracellular protein separation

  1. Centrifuge the culture at 4℃, 7000rpm for 30min, collect supernatant to filter through a 0.45um microporous membrane filter.
  2. Bacterial cells are suspended in PBS for further analysis. Supernatant was collected by repeated freeze-drying to obtain crude enzyme powder.

SDS-PAGE

  1. The preparation of stacking gel and separating gel.
    (The preparation of SDS-PAGE IS performed using SDS-PAGE preparation kit [YEASEN Co Ltd. NO20328ES50])
    H2O 30%Acr-Bis Buffer 10%AP TEMED
    stacking gel 5.1ml 6ml 3.75ml 0.15ml 6μl
    separating gel 2.85ml 0.85ml 1.25ml 50μl 5μl
  2. Make sure a complete gelation of the stacking gel and take out the comb. Take the glass plates out of the casting frame and set them in the cell buffer dam. Pour the running buffer (electrophoresis buffer) into the inner chamber and keep pouring after overflow untill the buffer surface reaches the required level in the outer chamber.
  3. Prepare the samples:
    Mix your samples with sample buffer (loading buffer).
    Heat them in boiling water for 5-10min.
  4. Load prepared samples into wells.
  5. After setting the voltage of 150V and the current of 80mA, perform electrophoresis.
  6. Stop SDS-PAGE running when the downmost sign of the protein marker.

Coomassie Brilliant Blue (UV spectrophotometer)

  1. The preparation of solution
    ① Standard bovine serum protein: 1000ug/ml bovine serum protein
    ② Coomassie brilliant blue G250:0.01g is dissolved in 5ml 90% ethanol, in which 10ml phosphoric acid solution is added, and distilled water is finally applied to a volume of 1L.
    1 2 3 4 5 6 sample
    Standard/
    Enzyme liquid(ml)     		0 0.2 0.4 0.6 0.8 1 0.1
    Distilled water(ml) 1 0.8 0.6 0.4 0.2 0 0.9
    Coomassie Brilliant Blue(ml) 5 5 5 5 5 5 5
  2. After adding the enzyme solution for 2min, the uv spectrophotometer was measured at OD595nm.

PDG1730-HAase Coomassie Brilliant Blue (enzyme-labeled instrument)

  1. The preparation of solution
    ① Standard bovine serum protein: 1000ug/ml bovine serum protein.
    ② Coomassie brilliant blue G250: 0.01g is dissolved in 5ml 90% ethanol, in which 10ml phosphoric acid solution is added, and distilled water is finally applied to a volume of 1L.
  2. Different levels of the standard protein were diluted first
    1 2 3 4 5 6 sample
    Standard/
    Enzyme liquid(ml)     		0ug/ml
    25μl     		20ug/ml
    25μl     		60ug/ml
    25μl     		100ug/ml
    25μl     		150ug/ml
    25μl     		200ug/ml
    25μl     		25μl
    Coomassie Brilliant Blue (μl) 125 125 125 125 125 125 125
  3. After adding the enzyme solution for 2min, Use the fact-labeled instrument test OD595nm

PDG1730-HAase (DNS method)

1 2 3 4 5 6
PBS 100μl 100μl 100μl 100μl 100μl 100μl
Glucose 0μl 30μl 50μl 75μl 100μl 125μl
H2O 600μl 570μl 550μl 525μl 500μl 475μl
DNS 300μl 300μl 300μl 300μl 300μl 300μl
  1. Add all reagents expect DNS, incubate at 38℃water bath for 10 min.
  2. Add all the standard curves (except DNS) and boil the water with the samples after bathing for 2min.
  3. Cool them to room temperature. Add DNS and boil for 15 min for color changing.
  4. Cool to room temperature, add 200μl of each to a 96-well plate, and measure the absorbance at 540nm on a plate reader.

PDG1730 - HAase purification（Ni-NTA spin columns）

(The purification of HAase is performed using NI-NTA spin kit [QIAGEN Co Ltd.Cat NO31314])

① sample preparation:

  1. enzyme liquid 4℃, centrifuge at 1000 rpm for 1min, take with 0.45 um microporous membrane filter
  2. Adjust its NaCl concentration to 0.5M and PH 7-8.
  3. Add the same concentration of imidazole to the sample as to the binding buffer.

② Filler material preparation:

  1. Take 10ml of agarose beads (the filling materials), 4℃, centrifuge at 500g for 5min, the supernatant.
  2. Add distilled water of triploid product of material, mix well and shake, centrifuge discard supernatant (repeat once time).
  3. Add binding buffer of diploid product of material, wash, centrifuge discard and superclean. (repeat once time)
  4. Add the binding buffer with the same volume of material to make 50% solution.

③ column preparation:

  1. Wash with 20% ethanol 2- 3 column volume.
  2. Then wash with 2 to 3 times the volume of sterilized water.

④ sample mixed with materials, pour into centrifuge tube, incubate at 4℃ for 2h, stir once every 20 min.

⑤ Incubate liquid over column, filter out and retain the flowthrough.

⑥ The agarose beads are then washed with binding buffer with different concentrations of imidazole (0mM, 10mM, 20mM, 30mM, 40mM , 50mM) and collect the flowthrough separately.

⑦ The agarose beads are then eluted with elution buffer containing different concentration of imidazole (250mM, 500mM), and collect the flowthrough.

⑧ Dialysis:

  1. Cut the appropriate length of the dialysis bag and boil it in boiling water for 10min before use.
  2. Dialysis with a 50mM phosphate buffer of 100X the volume of the sample.
  3. Replace the buffer solution every 4h, about three times.

⑨ freeze-dried.

Plate method

  1. Prepare solutions: 50mM citric buffer, HA solution (1mg/ml).
  2. Prepared plates with HA, agarose and citric buffer (50ml).
  3. Punch holes in the plate.
  4. Add 150ul of the sample, or commercial enzyme solution, or water to each hole on the plate, incubate at 37℃ for 10h.
  5. After incubation, a layer of cetylpyridinium chlorid solution was poured onto the plate to cover the entire gel, incubate for 30min, and observe the formation of clear circles.

PDG1730-HAase(ELISA)

(The testing of HAase is performed using Microorganism HA ELISA [SHANGHAI TONGWEI Co Ltd.Cat NO.tw045410])

  1. Prepare all reagents before starting assay procedure. It is recommended that all Standards and Samples be added in duplicate to the Microtiterplate.
  2. Add 50μl of Standard or Sample to the appropriate wells. Blank well doesn’t add anyting.
  3. Add 100μl of Enzymeconjugate to standard wells and sample wells except the blank well, cover with an adhesive strip and incubate for 60 minutes at 37°C.
  4. Wash the Microtiter Plate 4 times.
  5. Add Substrate A 50μl and Substrate B 50μl to each well. Gently mix and incubate for 15 minutes at 37°C
  6. Add 50μl Stop Solution to each well. The color in the wells should change from blue to yellow. If the color in the wells is green or the color change does not appear uniform, gently tap the plate to ensure thorough mixing.
  7. Read the Optical Density (OD) at 450nm using a microtiter plate reader within 15min.

Reverse transcription-polymerase chain reaction (RT-PCR)

  1. Prepare the following reaction mixture in Microtube.
    [The RT-PCR is using PrimeScript™ II 1st Strand cDNA Synthesis Kit(Takara Co Ltd. Cat. No6210A )]
    Reagent Consumption
    Random 6 mers (50μM) 1μl
    dNTP Mixture (10mM each) 1μl
    RNA template 1μl
    RNase Free dH2O Up to 10μl
  2. When the water bath is kept at 65 C for 5 min, the ice will quickly cool down.
  3. The following reverse transcription reaction solution was prepared in the above Microtube tube, with a total volume of 20μl.
    formuation
    Above denatured reaction solution(from step 2) 10μl
    5×PrimeScript II Buffer 4μl
    RNase Inhibitor (40 U/μl) 0.5μl
    PrimeScript II RTase (200 U/μl) 1μl
    RNase Free dH2O Up to 20μl
  4. 30℃ 10min, 47℃ 45min, 75℃ 15min, 95℃ 5min, the ice will quickly cool down.
  5. PCR
    PCR formuation
    Above denatured reaction solution(from step 4) 1μl
    10×PCR Buffer 2.5μl
    dNTP Mixture 2μl
    PDG primer-F 0.5μl
    PDG primer-R 0.5μl
    TaKaRa Taq 0.5μl
    dH2O 0.5μl
  6. It was detected by agarose gel electrophoresis.

HA Microneedle Experiment

Reagent preparation:

BDDE(1,4-butanediol diglycidyl ether，MW = 202.25Da);
0.25 mol/L NaOH (pH 13) ; HA (MW = 447.37Da);
ultrapure water

Fabrication of cHA hydrogel particles:

  1. Cross-linking HA (cHA) reagent solution was prepared by mixing 200μL of 1,4-butanediol diglycidyl ether (BDDE) into 9.8 mL of 0.25 mol/L NaOH, (pH 13).
  2. Approximately 1.0g of HA powder was added to the cHA reagent solution and stirred well, and thoroughly mixed at 40℃ for 5 hours.
  3. The prepared hydrogel was ground, squeezed out and screened with a 170 mesh sieve to get particles having a diameter of less than 90 μm.

Fabrication of HA-cHA-micro-needles(MN):
(1)We prepare HA-cHA-MNs by mixing different proportions of HA-cHA mixture, so we will use three ratios to compare. (1:0,1:1,5:1)
(2)The mixtures were dissolved in ultrapure water and a 20% (w/v) viscous polymer solution was stirred at room temperature under a magnetic stirrer for the production of homogenous suspension.
(3)The polymer solution was poured into a mold, placed in a centrifuge and centrifuged at 10,000rpm for 10min, and then taken out (this step was repeated twice), and then the polymer solution was again poured into the mold.
(4)After curing overnight at room temperature, the cured MN was successfully prepared by separating from the mold using a blade.

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Copyright © 2018 SSTi-SZGD All Rights Reserved.