Protocols
E. coli
Transformation of E. coli
  1. Take 100 µL of melted receptor cells on ice, add 10 μL of the target plasmid, mix gently and leave on ice for 30 min.
  2. Heat stimulate at 42°C for 45-60 s in a water bath, transfer to an ice bath and leave for 2 min.
  3. Add 700 µL of sterile liquid LB medium containing the antibiotic Amp to the centrifuge tube, mix well and recover at 37°C for 60 min at 220 rpm.
  4. Pipette 20 μL of the recovery solution onto the LB solid medium containing Amp and place the plate upside down in an incubator at 37°C for overnight incubation.
Determining the optimum concentration of iptg
  1. Rejuvenation of the bacterial broth
    Take out the bacterial broth from the fridge and incubate it in a small volume for re-strengthening
    Overnight at 37°C and 220rpm.
  2. Expand the culture
    Inoculate the re-strengthened bacterium into 25 mL of LB (amp+) and incubate in a shaker at 37 degrees and 220rpm.
  3. Induction of iptg
    After step 2, the obtained bacterial broth was divided into 5 groups of about 5 mL each and numbered. 1 group continued to be incubated on a shaker at 37 degrees, 220rpm. The OD600 of the bacterial broth was measured and iptg (final concentration 0.5mM) was added to groups 2, 3, 4 and 5 at OD600=0.6, 0.8, 1 and 1.2 respectively. 3-4h incubation at 37 degrees, 220rpm.
  4. SDS-PAGE
Induction of iptg
  1. Pick a single colony from each plate into 4 LB (4 mL) tubes corresponding to the resistance, numbered "0" "1" "2" "3 ".
    Place the tubes in a shaker (37°C; 220r), incubate for a period of time and measure the OD value.
  2. Add 700 μL of suspension from tube "0" to 100 μL (C=80%) of seed preserving glycerol, shake well and store in the freezer (-20°C).
  3. Add 2 μL of IPTG to tubes "1", "2" and "3" of each group of bacteria, at a final concentration of 0.5 mM.
  4. Expression at 37°C for 3h to 4h.
Crude protein extraction
  1. Transfer E. coli to 2 mL centrifuge tubes (50 mL), 4°C, 12000rpm, 25min, discard supernatant and collect bacteriophage.
  2. 400 μL (2 mL of bacteriophage premise) PBS was used to wash the bacteriophage, followed by centrifugation at 4°C, 4000rpm, 5min, discard the PBS supernatant and collect the bacteriophage.
  3. Resuspend the bacterium using 1 mL PBS.
  4. The bacterial solution was placed in an ultrasonic cell crusher with 150W power, 1s working, 2s gap, temperature alarm set to 30°C and ultrasonic crushing in an ice bath. The total working time is 10-20 min, depending on the volume.
  5. After crushing, the sample is centrifuged at 4°C for 25min at 12000rpm and the supernatant is discarded and stored as the crude product.
SDS-PAGE
  1. Cleaning the glass plate
    Hold the glass plate firmly with one hand and gently scrub it with the other hand with a little washing powder. Rinse with tap water after scrubbing both sides, then rinse with distilled water and leave to dry in the basket.
  2. Gluing and sampling
    1. Align the glass panels and place them in the clamps to be stuck tightly. Then snap vertically onto the shelf ready for gluing. (The operation should be done so that the two glasses are aligned so as not to leak glue.)
    2. Preparation of the separating glue and glue concentrate: Configure 12% of the separating glue and 5% of the glue concentrate in the order and proportion of the solutions in the table below.
      When there is a fold line between the water and the glue, the glue has set. Wait 3 min for the gum to solidify before pouring off the top layer of water and blotting up the water with absorbent paper.
    3. Rinse the gel concentrate with water and place it in the electrophoresis bath. (Small glass plate facing inwards, large glass plate facing outwards. If you are running only one piece of glue, the other side of the tank should be padded with a plastic plate and the side with the letters should face outwards.)
  3. Electrophoresis
    After the sample is added, cover the top lid, connect the electrophoresis instrument, turn on the switch of the electrophoresis instrument, control the voltage at 100-200V before the sample enters the glue, about 15-20min; after the bromophenol blue indicator in the sample reaches the separation glue, the voltage rises to 200V, keep the voltage stable during electrophoresis. The electrophoresis was stopped when the bromophenol blue indicator migrated to 1~2cm from the front, about 0.5-1hour.
  4. Staining and decolorization
    After electrophoresis, turn off the power, take out the glass plate, pry it gently with a knife in the gap between the lower corners of the short and long glass plates to separate the adhesive surface from one glass plate, then gently lift the film, insert a copper wire in the center of the indicator zone as a marker and put it into a large petri dish for staining, using 0.25% Kormas Brilliant Blue staining solution, stain for 2-4h, or overnight if necessary.
    Discard the staining solution and rinse the gel surface several times with distilled water, then add decolorizing solution and perform diffusion decolorization, changing the decolorizing solution frequently until the protein band is clear.
Enzyme activity determination
  1. Standard curve preparation
    1. L-Tyrosine Standard Solution: This can be prepared by referring to the table below. (Note: L-Tyrosine dilutions should be measured immediately after dilution)
      No. Concentration of tyrosine standard solution/(μg/mL) Volume of tyrosine standard stock solution/mL Volume of water/mL
      Group Concentration of tyrosine standard solutions /(μg/mL) Volume of tyrosine standard stock solution /mL H2O/mL
      0 0 0 10
      1 10 1 9
      2 20 2 8
      3 30 3 7
      4 40 4 6
      5 50 5 5
    2. Take 1.00mL of each of the above solutions (parallel test must be done), add 5.00mL of sodium carbonate solution and 1.00mL of forint reagent solution, shake well, place in a water bath at 40℃±0.2℃ for 20min, remove and measure the absorbance of each solution with a spectrophotometer at a wavelength of 680nm, 10mm cuvette, using a 0 tube without tyrosine as a blank. The absorbance A was used as the vertical coordinate and the concentration c of tyrosine was used as the horizontal coordinate to draw the standard curve.
    3. Using the regression equation, calculate the amount of tyrosine when the absorbance is 1, which is the value of the absorbance constant K. The K value should be in the range of 95 to 100. (If it is not, the reagents need to be reconstituted and the test carried out.)
  2. Sample determination
    1. Preparation of the enzyme solution to be tested
      Weigh 1g~2g of enzyme sample, accurate to 0.0002g, then dissolve and dilute to a certain concentration with the appropriate buffer, the recommended concentration range is 10 U/mL~15 U/mL. For powdered samples, dissolve with the appropriate buffer, then take slow qualitative filter paper and dilute to the appropriate concentration.
    2. Assay procedure (for reference only)
      The casein solution is first placed in a constant temperature water bath at 40°C ± 0.2°C and preheated for 5 min.
  3. Activity calculation
    Read the enzyme activity of the final dilution of the sample from the standard curve in U/mL. The enzyme activity of the sample is calculated according to the following formula:
    • X=(A_1×V_1×V_0×n)/m_0 ×1/10
    In the formula:
    • X → enzyme activity of the sample U/g;
    • A1 → the activity U/mL of the final dilution of the sample derived from the standard curve;
    • V1 → volume of the volumetric flask used to dissolve the sample mL;
    • V0 → the total volume of reaction reagent mL;
    • n → the number of dilutions of the sample;
    • m0 → the mass of the sample g;
    • 1/10 → reaction time of 10 min, measured in 1 min.
    The results shown are taken approximately to the nearest whole number.
Soybean meal experiment
  1. Preparation of pineapple protease treated feed samples
    1. Experimental group:
      Soya bean meal powder (4 g) was added with 50 mL (0.5%) of pineapple protease and incubated in a shaker water bath at 50°C for 3 hours. After protein hydrolysis, 20% (w/v) TCA (trichloroacetic acid) at a volume ratio of 1:1 was added to the protein hydrolysate to give 10% TCA soluble material. The mixture was allowed to stand for 20 minutes to allow precipitation and then diluted to a volume of 100 mL. The sample (35 mL) was placed in a conical tube and the supernatant (1.5 mL) was analysed for protein by centrifugation for 20 minutes.
    2. Control group:
      Soya bean meal powder (4 g) was added with 50 mL of distilled water and incubated for 3 hours at 50°C in a shaker water bath.
      The W5 solution was not used as the soybean meal had destroyed the cell structure.
      A 1.5 mL sample was mixed before analysis.
  2. SDS-PAGE
Bacillus subtilis
Resuscitation of Bacillus subtilis
  1. Wipe the outer tube with cotton moistened with 70% alcohol and heat the tip of the tube over a flame.
  2. Apply a few drops of sterile water to the heated area to break the outer tube and then crack the tip with a stiff stick.
  3. Remove the insulating paper and the inner tube and use sterilised forceps to remove the cotton plug from the inner tube.
  4. Pipette 0.3-0.5 ml of the specified liquid medium into the inner tube and mix well. After the dry powder has dissolved, pipette with a sterile tip into a tube containing approximately 5 ml of sterile LB medium and incubate for 24 h.
Preservation of Bacillus subtilis
  1. Add glycerol 1:1 to the fully heated and mixed Bacillus subtilis solution and dispense into EP tubes (2 mL or so).
Transformation of Bacillus subtilis
  1. Prepare a fresh 168 monoclonal plate, take a LB plate full of Bacillus subtilis glycerol and incubate for 12 h at 37°C.
  2. The day before the transformation, pick a single colony in the evening and incubate it in 3 ml LB medium at 37°C, 250 r/min overnight.
  3. The next morning, transfer 160 μL of culture to 8 ml of SPI medium and incubate at 37°C, 250 r/min until the end of logarithmic growth (168 for about 4-5 h).
  4. Transfer 0.2 ml of the end of logarithmic growth to 2 ml of SPII medium and incubate at 37 °C for 90 min at 100 r/min.
  5. Add 20 μL of 10mmol/L EGTA to the SPII medium and incubate at 37°C for 10 minutes at 100 r/min.
  6. Add 5 μL of DNA to each 0.5 ml tube (the amount of DNA should not exceed 5 ug) and incubate at 37 ℃, 250 r/min for 90 minutes.