l Materials
|
Reagent |
Volume |
|
2*mix |
25μL |
|
AocI (2285bp) |
1μL |
|
FAD (2137bp) |
1μL |
|
pAO815 plasmid (7709bp) |
1μL |
|
Forward primer |
1μL |
|
Reverse primer |
1μL |
|
ddH2O |
22μL |
* Total system is 50μL.
|
Apparatus |
Amount |
|
Centrifugal machine |
1 |
|
PCR thermal cycler |
1 |
1. Remove
three 200μL EP
tubes, add 2
mix
(25μL), AocI (1μL), forward primer (1μL), reserve primer
(1μL), ddH2O
(22μL) in the first tube. Add 2
mix
(25μL), FAD
(1μL),
forward primer (1μL), reserve primer (1μL), and
ddH2O
(22μL) in the second tube. Add 2
mix
(25μL), pAO815
plasmid (1μL),
forward primer (1μL), reserve primer (1μL), and ddH2O
(22μL) in the third tube.
2. Use a Centrifugal machine to make liquid mix together and drop to the bottom.
3. Use PCR thermal cycler to warm up at 95°C for 5 min, denaturation at 95°C for 30s, annealing at 55°C for 30 s, and extension at 72°C for 2 min 30s, 3 steps in a group, cycle 30 times.
AGE (Agarose gel electrophoresis)
l Materials
|
Reagent |
Volume/mass |
|
Agarose |
0.5g |
|
10TAE |
50mL |
|
Nucleic acid dye |
3μL |
|
Apparatus |
Amount |
|
Electrophoresis apparatus |
1 |
|
Molten metal bath |
1 |
l Procedure
1. Mix
agarose (0.5g), 10
TAE
(50mL),
Nucleic
acid dye (3μL).
2. Pour the mixture into the model to make two middle and three large glues.
3. Heat for 2 min until melted thoroughly, and add 3μL nucleic acid dye.
4. Place in the electrophoresis tank.
5. Spotting: Take the PCR solution, add Buffer, mix well, and point into the glue well.
6. After the electrophoresis, the glue is brought to ultraviolet light for irradiation to verify the stripe position.
l Materials
|
Reagent |
Volume/mass |
|
Yeast extract |
5g |
|
NaCl |
10g |
|
Tryptone |
10g |
|
Agar powder |
2g/100mL |
|
ddH2O |
500mL |
|
Apparatus |
Amount |
|
Electronic scale |
1 |
|
Graduated cylinder |
1 |
|
Erlenmeyer flask |
5 |
l Procedure
1. Mix yeast extract (5g), NaCl (10g), tryptone (10g), and ddH2O (500mL), and pour 100mL into an Erlenmeyer flask; a total of 300mL is required to make liquid LB.
2. Add agar powder (2g) per 100 mL to make a solid LB.
3. Autoclaved and refrigerated for subsequent use.
Gel extraction
l Materials
|
Reagent |
Volume/mass |
|
Buffer B2 |
300μL |
|
Wash Solution |
500μL |
|
ddH2O |
30μL |
|
Agarose gel |
0.001g |
|
Apparatus |
Amount |
|
Electronic scale |
1 |
|
Water bath |
1 |
|
Adsorption column |
4 |
|
Collecting pipe |
4 |
|
Centrifugal machine |
1 |
|
Centrifuge tube |
4 |
l Procedure
1. Cut the chunk containing the fragment of interest from the agarose gel and weigh it.
2. Add Buffer B2 (300μL) and let it melt in a 50°C water bath for 10 minutes.
3. Transfer the binding mix to the column and centrifuge at 8000X g for 30 sec. Discard the filtrate in the collection tube. Return the column to the collection.
4. Add Wash Solution (500μL), centrifuge at 9,000X g for 30 sec, and decant the liquid from the collection tube.
5. Repeat step 4 once.
6. Centrifuge the empty adsorption column at 9,000X g for 1 min.
7. Place the adsorption column in a clean 1.5 mL centrifuge tube, add ddH2O (30μL) to the center of the adsorption membrane, let stand at room temperature for 1 min, and centrifuge for 1 min. Save the DNA solution in the tube.
l Materials
|
Regent |
Volume |
|
Buffer S |
500μL |
|
Buffer SP1 |
250μL |
|
Buffer SP2 |
250μL |
|
Buffer SP3 |
350μL |
|
Buffer DW1 |
500μL |
|
Wash Solution |
500μL |
|
Apparatus |
Amount |
|
Centrifugal machine |
1 |
l Procedure
1. Add Buffer S (500μL) to the collection tube centrifuge for 1min, and dump the waste liquid.
2. Centrifuge the pure bacteria 12000X g for 1 min—centrifuge to settle the bacteria to the bottom and drain the waste liquid in the collection tube.
3. Add Buffer SP1 (250μL) and centrifuge.
4. Add Buffer SP2 (250μL), gently shake the centrifuge tube 10 times, and let stand for 2 min.
5. Add Buffer SP3 (350μL) and gently shake the centrifuge tube 10 times.
6. Add Buffer DW1 (500μL) and centrifuge for 30 s; add Wash Solution (500μL) and centrifuge for 30 sec, then drain the waste from the collection tube and repeat. Aspirate the liquid from the collection tube and re-drip it.
7. Place the adsorption column in a clean 1.5 mL centrifuge tube, add ddH2O (50μL) to the center of the adsorption membrane, let stand at room temperature for 2 min, and centrifuge for 2 min. Save the DNA solution in the tube.
l Materials
|
Reagent |
Volume |
|
Medium (LB) |
5mL |
|
Amp antibiotics |
5μL |
|
Apparatus |
Amount |
|
Centrifuge tubes |
6 |
|
Petri dish |
6 |
l Procedure
1. Add 5 ml of LB liquid medium to a sterile centrifuge tube, add 5μl Amp antibiotic, and pick up single colonies into the medium.
2. Place in a constant temperature shaker at 37 Celsius for 16 hours until the liquid becomes turbid.
l Materials
|
Reagent |
Volume |
|
ddH2O |
130μL |
|
pAO815-AocI |
50μL |
|
pAO815-AocI-FAD |
50μL |
|
Apparatus |
Amount |
|
Concentration detector |
1 |
l Procedure
1. Wash the metal sheet of the concentration detector and dry it three times.
2. Aspirate three tubes of pAO815-AocI (10μL) samples and three tubes of pAO815-AocI-FAD (10μL) samples sequentially and dropwise to the middle of the metal sheet.
3. Tested on a computer, concentration and purity are recorded on sample tubes.
4. After the test, the metal sheet of the instrument is washed and dried three times.
l Materials
|
Reagent ( pAO815-AocI) |
Volume |
|
Clone mix |
5μL |
|
pAO815 |
4μL |
|
AocI |
1μL |
|
Reagent ( pAO815-AocI-FAD) |
Volume |
|
Clone mix |
5μL |
|
pAO815 |
4μL |
|
AocI |
1μL |
|
FAD |
1μL |
|
Apparatus |
Amount |
|
EP tube |
2 |
|
PCR thermal cycler |
1 |
l Procedure
1. Take two centrifuge tubes (1.5mL), one labeled pAO815-AocI, and add clone mix (5μL), pAO815 sample (4μL), and AocI (1μL) in the tube. Label pAO815-AocI-FAD on another tube and add clone mix (5μL), pAO815 (4μL), AocI (1μL), and FAD (1μL).
2. Place both tubes into the PCR thermal cycler at 55 °C for 20 min and immediately lower it to 4 °C.
l Materials
|
Reagent |
Volume |
|
DH5ɑ( pAO815-AocI) |
100μL |
|
DH5ɑ( pAO815-AocI-FAD) |
100μL |
|
Plasmid |
10μL |
|
LB medium |
700μL |
|
Apparatus |
Amount |
|
EP tube |
2 |
|
Metal bath |
1 |
|
Orbital Shaker |
1 |
l Procedure
1. Take out the EP tube containing the plasmid, take the DH5α competent two tubes from the refrigerator, label pAO815-AocI and pAO815-AocI-FAD, and put them in ice.
2. Place the DH5ɑ in ice to thaw, and after the solid in the tube is melted, add plasmid (10μL) and pAO815-AocI (100μL) to the first EP tube, add plasmid (10μL) and pAO815-AocI-FAD (100μL) in the second tube, and insert the EP tube into ice for 30 min.
3. Remove the tube from ice and put it in the metal bath at 42 °C for 45 s.
4. Remove the tube and insert it into ice for 2 min.
5. add LB medium to the tube (700μL) in the ultra-clean stage.
6. Place the tubes on a shaker at 37 °C at 200 rpm for 1 h.
l Materials
|
Reagent |
Volume |
|
Bacterial solution |
200μL |
|
Apparatus |
Amount |
|
Petri dish |
8 |
l Procedure
1. Centrifuge bacterial solution 12,000X g for 30 sec.
2. Take bacterial solution (50μL) on the ultra-clean stage, discard the remaining bacterial solution, beat the bacterial solution in the pipette gun back to the original test tube, and pipette evenly.
3. Transfer bacterial solution (20μL) to the medium of the Petri dish and apply evenly with a coater. Cover the Petri dish and label pAO815-AocI and pAO815-AocI-FAD.
l Materials
|
Reagent |
Volume |
|
2*mix |
10μL |
|
Forward primer |
1μL |
|
Reverse primer |
1μL |
|
dd H2O |
8μL |
|
Apparatus |
Amount |
|
Molten metal bath |
1 |
|
Collecting tube |
1 |
|
Centrifugal machine |
1 |
1. Mix 2*mix (10μl), forward primer (1μl), reverse primer (1μl), dd H2O (8μl)
2. Centrifuge mixing
3. Put the bacteria into the collection tube
4. Centrifuge for 30s
5. Heating by Molten metal bath at 72℃ for 2min30s.
l Materials
|
mass |
|
|
Tryptone |
20g |
|
Yeast extract |
10g |
|
Glucose |
20g |
l Procedure
1. Mix Tryptone(20g), yeast extract(10g), glucose(20g).
2. Adjust the pH value to pH 6.5 for the YPD solution.
l Materials
|
Reagent |
Volume/mass |
|
Distilled water |
95mL |
|
Agar powder |
2.5g |
|
40% glucose solution |
5mL |
l Procedure
1. Mix distilled water (95ml), Agar powder (2.5g), and 40% glucose(5ml).
2. Adjust pH value to pH6.0 for SDS-His solution.
3. High-temperature sterilization 121℃ for 20min.
l Materials
|
Reagent |
Volume |
|
Plasmid pAO815-AocⅠ-FAD |
15μL |
|
pAO815-p1-up |
1μL |
|
pAO815-p2-dn |
1μL |
|
Yeast conversion promoter |
5μL |
|
Melting GS115 receptor cells on ice |
100μL |
|
Pichia pastoris transformation liquid |
500μL |
|
0.9%NaCl |
100μL |
|
Apparatus |
Amount |
|
Sterile 1.5ml EP tub |
1 |
|
Water bath |
1 |
|
Centrifugal machine |
1 |
l Procedure
1. Label four 1.5ml centrifuge tubes pAO815-AocI/1, pAO815-AocI/2, pAO815-AocI-FAD/1 and pAO815-AocI-FAD/2, respectively
2. Each tube added high-fidelity enzyme mixture(25μL), forward primer(1μL), reverse primer(1μL), corresponding DNA, and distilled water(22μL).
3. Use the centrifuge to teleport to the centrifuge tube low.
4. Put them in the PCR thermal cycler, then set time and temperature as follows: 98℃ for the 30s, 98℃ for the 30s, 54℃ for 5s, 72℃ for 2 min,72℃ for 1 min,12℃ to until next step.
l Materials
|
Reagent |
Volume |
|
Linear plasmid |
15μL |
|
Coarse reformer |
5μL |
|
GS115 |
100mL |
|
Invert fluid |
500mL |
|
0.9% NaCl |
100μL |
|
Apparatus |
Amount |
|
Aseptic room |
1 |
|
Molten metal bath |
1 |
|
Centrifugal machine |
1 |
l Procedure
1. The plasmid pAO815-AocI-FAD to be transformed was taken, and PCR amplification was performed with primers pAO815-p1-up and pAO815-p2-dn (system 50μl). The PCR products were purified and recovered, and the amount of linear plasmid to be added was calculated after nucleic acid concentration was measured.
2. A sterile 1.5ml EP tube was taken, and precooled linear plasmid 1-5μg (volume no higher than 15μl), yeast transformation promoter 5μl, 100μl ice-melted GS115 receptive cells, and Pichia Pastoris transformation solution 500μl were added in sequence, then gently turned and mixed 6-8 times.
3.Water bath at 30℃ for 30min, gently turn and mix 6-8 times every 15min.
4.Water bath at 42℃ for 15min, gently turn and mix 6-8 times every 7.5min.
The supernatant was temporarily centrifuged at 5.12000rpm, re-suspended with 0.9% NaCl, coated with SDS-His medium, and cultured at 30℃ for 3-7 days.
l Material
|
Reagent |
Volume |
|
2*mix |
10μL |
|
Yeast DNA |
2μL |
|
Forward primer- pAO815-AocI-FAD |
1μL |
|
Reverse primer- pAO815-AocI-FAD |
1μL |
|
ddH2O |
6μL |
|
Loading buffer |
2μL |
|
Marker |
10μL |
* Total system is 20μL.
|
Apparatus |
Amount |
|
Centrifugal machine |
1 |
|
PCR thermal cycler |
1 |
l Procedure
1. Add
2
mix
(10μL), yeast DNA(2μL), forward primer (1μL), reserve primer
(1μL), ddH2O
(6μL) in the tube.
2. Add loading buffer (2μL ) in EP tube
3. Use a Centrifugal machine to make liquid mix together and drop to the bottom; add Marker (10μL) and sample in the gel.
4. Use PCR thermal cycler to warm up at 95°C for 5min, denaturation at 95°C for 30s, annealing at 55°C for 30s, and extension at 72°C for 2min30s, 3 steps in a group, cycle 30 times.
Enlarged incubation
|
Reagent |
Volume |
|
pAO815-AocI (after shaker) |
1mL |
|
pAO815-AocI-FAD (after shaker) |
1mL |
|
GS115 (after shaker) |
1mL |
|
YPD (include amp+) |
100mL |
|
Apparatus |
Amount |
|
Thermostatic oscillator |
1 |
l Procedure
1. Add pAO815-AocI-FAD, pAO815-AocI and GS115 after shaking to YPD liquid (100mL) containing Amp+.
2. Put it in a shaker at 30℃ for about 3-4 hours until it reaches OD600 into 0.6-0.8.
l Material
|
Reagent |
Volume |
|
Methanol*0% |
0μL |
|
Methanol*0.25% |
250μL |
|
Methanol*0.5% |
500μL |
|
Methanol*0.75% |
750μL |
|
Methanol*1% |
1mL |
l Procedure
1. 0%, 0.25%, 0.5%, 0.75% and 1% methanol were added to the YPD respectively
2. Induce protein expression.
l Material
|
Reagent (lower layer gel) |
Volume |
|
Distilled water |
4.7mL |
|
30% Acr-Bis (29:1) |
2.7mL |
|
Gel Buffer A |
2.5mL |
|
10% APS |
0.1mL |
|
TEMED |
0.005mL |
|
Reagent (upper layer gel) |
Volume |
|
Distilled water |
1.0mL |
|
30% Acr-Bis (29:1) |
0.5mL |
|
Gel Buffer B |
1.5mL |
|
10% APS |
0.03mL |
|
TEMED |
0.003mL |
|
Apparatus |
Amount |
|
Protein electrophoresis apparatus |
2 |
l Procedure
1. Add the reagents in the table above to the protein electrophoresis apparatus in turn
2. Repeat step 1
|
Reagent |
Volume |
|
Yeast culture |
50mL |
|
Protein extract solution |
15mL |
|
Apparatus |
Amount |
|
Centrifugal machine |
1 |
|
Ultrasonic Cell Disruptor |
1 |
l Procedure
1. Transfer the yeast culture into a 50mL centrifuge tube, centrifuge at 8000 rpm for 5 minutes, pour off the supernatant, add the culture to the residue, continue centrifugation, and collect all the cultures together.
2. Add 15 ml of protein extract solution (100 mmol/L NaCl, 10 mmol/L EDTA, pH 8.0) to each tube and blow with a gun to suspend the sediment.
3. Put the centrifuge tube on a small test tube rack, insert the metal head of the ultrasonic crusher into the centrifuge tube, close the door of the ultrasonic crusher after adjusting the position of the test tube, turn on the power supply of the instrument, and ultrasonically crush the bacteria in each centrifuge tube. Conditions: power 80 watts, working for 2 seconds, interval of 2 seconds, 5 cycles at a time.
4. After 6 times of treatment, centrifuge at 8000 rpm for 5min minutes.
5. Take out the centrifuge tube, carefully suck out the supernatant, protein crude body fluid, and put it in a new centrifuge tube.
Ni-His Protein purification
|
Reagent |
Volume |
|
Nondenaturing Lysis Buffer |
500μL |
|
Crude protein |
1mL |
|
Elution Buffer |
500μL |
|
Apparatus |
Amount |
|
Centrifugal machine |
1 |
1. Adding a nickel column and centrifuging at 12000rpm at 4 ℃ for 1 min.
2. Discard the supernatant, add nondenaturing lysis buffer (500μL ), and centrifuge at 12000 rpm at 4℃ for 1min.
3. Repeat step 2
4. Add nondenaturing lysis buffer (500μL, blow and mix evenly, and let it stand until the nickel column sinks.
5. Add 1mL of crude protein to the nickel column, add crude protein again after the liquid drops, and move the filtered crude protein to a new test tube.
6. Filter the crude protein three times according to step 5.
7. Take out the nickel column and put it on a new test tube.
8. Add 500 mL of washing liquid and wait for the liquid to drip completely.
9. Repeat step 8 three times.
10. Take out the nickel column and put it on a new test tube.
11. Add 500 mL of eluent until the liquid drops completely.
12. Repeat step 11 three times
l Material
|
Reagent |
Volume |
|
Bromophenol blue |
500μL |
|
Apparatus |
Amount |
|
Electrophoresis apparatus |
1 |
l Procedure
1. Add Bromophenol blue dye to the sample.
2. Sample in the prepared gel.
3. 80V gel electrophoresis for 15min so that the Bromophenol blue mark moves to the edge of the concentrated glue.
4.120V, gel electrophoresis for 1 hour.
l Material
|
Reagent- dyeing |
Volume |
|
Coomassie Brilliant Blue |
400mL |
|
Reagent- decoloring |
Volume |
|
Ethanol |
400mL |
|
Glacial acetic acid |
200mL |
|
Distilled water |
400mL |
l Procedure
1. Add Coomassie Brilliant Blue (400mL) in a container.
2. Wait four hours for coloring.
3. Add ethanol, glacial acetic acid, and distilled water and mix with them.
4. Take out the lustful sample and put it in the decoloring solution.
5. Leave it for one night to decolorize the sample completely.
‘Transparent circle’ Experiment
l Material
|
Reagent |
Volume |
|
Bromocresol purple |
|
|
Histamine |
0μg/mL |
|
20μg/mL |
|
|
50μg/mL |
|
|
100μg/mL |
|
|
200μg/mL |
|
|
Amp+ |
25μL |
l Procedure
1. Prepare the concentration of histamine and bromocresol purple.
2. Prepare five Petri dishes and draw four areas on them with a mark, marking two areas as GS115 blank and the other two areas as pAO815-AocI-FAD.
3. Bromocresol purple, Amp+, and histamine were added to each Petri dish, and the concentration of histamine was different in each Petri dish, which was 0μg/mL, 20μg/mL, 50μg/mL, 100μg/mL, and 200μg/mL respectively.
To test whether the transformants were successful in acquiring the ability to degrade biogenic amines as expected, the concentration of biogenic amines in the fermentation products was quantified using high-performance liquid chromatography (HPLC) after fermentation for a period of time using two different strains under the same fermentation conditions. Firstly, the bacterial broth was incubated at 30°C until OD=0.6, followed by the addition of 200ug/ml histamine solution and setting different incubation temperatures, i.e., 20°C, 25°C, 30°C, 37°C, and 45°C. After 12h,24h,36h,48h,72h of fermentation, 50mL of supernatant was taken and sent to the company to determine the histamine concentration using HPLC. Determination of biogenic amines in samples with reference to the method of GB 5009.208-2016 "Determination of biogenic amines in food".
|
Number |
Time-pAO815-AocI |
Temperature |
Number |
Time-pAO815-AocI-FAD |
Temperature |
|
1 |
12h |
20℃ |
26 |
12h |
20℃ |
|
2 |
24h |
27 |
24h |
||
|
3 |
36h |
28 |
36h |
||
|
4 |
48h |
29 |
48h |
||
|
5 |
72h |
30 |
72h |
||
|
6 |
12h |
25℃ |
31 |
12h |
25℃ |
|
7 |
24h |
32 |
24h |
||
|
8 |
36h |
33 |
36h |
||
|
9 |
48h |
34 |
48h |
||
|
10 |
72h |
35 |
72h |
||
|
11 |
12h |
30℃ |
36 |
12h |
30℃ |
|
12 |
24h |
37 |
24h |
||
|
13 |
36h |
38 |
36h |
||
|
14 |
48h |
39 |
48h |
||
|
15 |
72h |
40 |
72h |
||
|
16 |
12h |
37℃ |
41 |
12h |
37℃ |
|
17 |
24h |
42 |
24h |
||
|
18 |
36h |
43 |
36h |
||
|
19 |
48h |
44 |
48h |
||
|
20 |
72h |
45 |
72h |
||
|
21 |
12h |
45℃ |
46 |
12h |
45℃ |
|
22 |
24h |
47 |
24h |
||
|
23 |
36h |
48 |
36h |
||
|
24 |
48h |
49 |
48h |
||
|
25 |
72h |
50 |
72h |
||
|
51 |
0h |
30℃ |
52 |
0h |
30℃ |
|
53 |
12h GS115 |
54 |
72h GS115 |
||
|
55 |
72h |
56 |
72h |