iGEM 2023 — Team subcat-hongkong

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**Day1
15/08/23**

 Team Ice-breaking

 Brainstorm Meeting on team logo, etc.

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Overview

Drinking
alcohol is enjoyed by people everywhere, however,
excessive alcohol consumption can lead to alcohol poisoning and the harmful effects of other substances.
It is alarming that such widespread activity can potentially threaten human health. To address this issue,
we have employed technological means to eliminate this threat and make drinking safer for
individuals.

In
our experiments, we introduced the AocI gene into yeast, enabling it to break down biogenic amines
produced during fermentation. We have also designed a process utilizing the FAD gene to enhance the
expression of this enzyme. As a result, our product - the amine-reducing brewing yeast - produces
alcohol-free from the threat of biogenic amines.

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PCR

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 |

l Procedure

1. Remove
three 200μL EP
tubes, add 2![](file:///C:/Users/zyj/AppData/Local/Temp/ksohtml14528/wps17.jpg)

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![](file:///C:/Users/zyj/AppData/Local/Temp/ksohtml14528/wps21.jpg)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.

Make medium (LB)-culture coli

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.

pAO815-AocI-FAD Construction

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.

Shaking bacteria

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.

DNA concentration detection

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.

Homologous recombination ( pAO815-AocI & pAO815-AocI-FAD)

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.

Heat shock

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.

Coating

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.

Colony identification by PCR

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 |

l Procedure

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.

Make YPD(1L)

l Materials

|  |  |
| --- | --- |
| Reagent | 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.

Make SDS-His(100mL)

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.

Yeast transformation

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.

Yeast PCR testing

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![](file:///C:/Users/zyj/AppData/Local/Temp/ksohtml14528/wps22.jpg)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**

l Material

|  |  |
| --- | --- |
| 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.

Methanol induction

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.

Makeup SDS-PAGE

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

Cell disruption

l Material

|  |  |
| --- | --- |
| 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**

Ni-His Protein purification

l Material

|  |  |
| --- | --- |
| Reagent | Volume |
| Nondenaturing Lysis Buffer | 500μL |
| Crude protein | 1mL |
| Elution Buffer | 500μL |

|  |  |
| --- | --- |
| Apparatus | Amount |
| Centrifugal machine | 1 |

l Procedure

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

SDS-PAGE gel electrophoresis

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.

Dyeing & decoloring

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 | 12μg/mL |
| 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.

HPLC Experiment

 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 |

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Overview

Biogenic
amines can be produced by yeast through the process of fermentation. For wine consumers, these substances
may cause headaches, intestinal problems, pseudo-allergic responses, and many other problems [1]. Thus,
lowering the amines produced in wine production is necessary for safety concerns. Previously, efforts have
been made to find better microbes and improve fermentation technic. Aiming to solve this problem more
effectively, we, the safe alcohol team, started the Biogenic amine reduction Saccharomyces cerevisiae
project, applying gene engineering methods innovatively.

Noticing
that gene fragment AocI can conduct amine oxidase production and FAD can promote expression of the enzyme,
we decided to combine them with plasmids pAO815. And we select GS115 as our vector, suitable for wine
production. Our project included three basic sections-construction of recombinant plasmids, yeast
conversion and protein expression, and enzyme function testing. Results showed that our products were
successfully made, and the gene-engineered yeasts worked well on reducing biogenic amine
production.

Construction of recombinant plasmid

**2.1 Amplification of
fragments AocI and FAD**

   Fragment
AocI-sized 2285 bp conducts amine oxidase production, and FAD-sized 2119 can facilitate the expression of
the oxidase. We mixed corresponding primers, 2xmix, and template fragments of AocI and FAD, respectively,
and put them into PCR equipment to get multiple pieces. The fluorescence fragment of the FAD product was
approximately parallel to the 2000 bp marker, while the fragment of AocI was parallel to the 3000 bp one,
which is plausible considering the size of the elements (Fig 1B). Thus, it proved that our PCR
amplification was successful, and we prepared experiment material for the following process.

![](https://static.igem.wiki/teams/4929/wiki/result/1.png)

Fig
1. PCR amplification of AocI and FAD

A:
PCR testing result of fragments FAD and AocI

B:
Graph of FAD and AocI

**2.2 Amplification and
linearization of plasmid pAO815.**

Plasmid
pAO815 is 7709 bp in size (Fig 2B), which can function in yeast. We put a template of plasmid pAO815,
2xmix, and corresponding primers together and did a PCR to amplify and linearize it. PCR testing showed
that fluorescence fragments fell approximately between 10000 and 7500 bp. Compared to the expected length
of pAO815, the result convinced us that PCR amplification and linearization processes succeeded. Thus, we
cut off the fluorescence fragments for the following gel purification procedure.

![](https://static.igem.wiki/teams/4929/wiki/result/2.png)

Fig
2 Amplification and linearization of plasmid pAO815

A:
PCR testing result of pAO815 linear

B:
Graph of PAO815

**2.3 Construction of plasmid
pAO815-AocI.**

Aiming
for recombinant plasmids and preparing for the following processes, we connected the two parts through
homologous recombination and transformed them into DH5α. Then, applying the primers corresponding to
AocI, we did a PCR testing (Fig 3A, 2285
bp).
The test result showed that the size of our fragments was plausible. 100% DNA alignment (Fig 3C) further
proved that our plasmid had been finely reconstructed and could successfully replicate.

![](https://static.igem.wiki/teams/4929/wiki/result/3.png)

Fig
3 plasmid pAO815-AocI

A:
PCR testing of plasmid pAO815-AocI

B:
Graph of plasmid pAO815-AocI

C:
DNA alignment result

**2.4 Construction of plasmid
pAO815-AocI-FAD.**

After
we combined fragment FAD and the plasmid Pao815-AocI, we did a PCR test (Fig 4A).
Compared to the markers, the size of our products aligned with the size of AocI and FAD fragments. The
result showed successful conversion of the reconstructed plasmid into DH5α. DNA
alignment test showed that our plasmid completely aligned with expectations within goal sequences (Fig
4C), which further proved our conclusion. *\*The area we requested to sequence covered the target region, so
in fact the target region alignment is 100%.*

![](https://static.igem.wiki/teams/4929/wiki/result/4.png)

Fig
4. Plasmid pAO815-AocI-FAD

A:
PCR testing result of plasmid pAO815-AocI-FAD in DH5α

B:
graph of plasmid pAO815-AocI-FAD

C:
DNA alignment result

Yeast transformation and protein expression

**3.1 Yeast transformation.**

We
linearized plasmid pAO815-AocI-FAD and did a PCR test (Fig 5A) aligned with expectations, indicating that
our plasmid reconstruction was successful.

After
converting the plasmid into the yeast and conducting incubation, we tested our yeast (Fig 5C) by PCR.
Fragments of FAD and AocI were respectively 2285 bp and 2119 bp. The result aligned with the size
expected, showing that the plasmid had been converted into the cell and could replicate
successfully.

![](https://static.igem.wiki/teams/4929/wiki/result/5.png)

Fig
5.

A:
PCR testing result of plasmid pAO815-AocI-FAD linear

B:
PCR testing result of plasmid pAO815-AocI-FAD linear in GS115

C:
GS115 colonies containing plasmid pAO815-AocI-FAD in the SDS-His medium

**3.2 polyacrylamide gel
electrophoresis verified the size of protein bands**

After
product purification, we used protein electrophoresis to verify whether the major protein AocI was
successfully induced in culture and checked whether AocI was successfully expressed.

As
shown in Fig. 6, protein AocI has a size of 85kDa. There was a clear difference between the protein band
of yeast containing the AocI plasmid and the blank control group, indicating that the AocI gene was
successfully expressed.

At
the same time, the protein banding was most clearly observed at 0.50% methanol. It can be seen that the
optimal environment for AocI expression was 0.50% methanol. (Fig 6)

![](https://static.igem.wiki/teams/4929/wiki/result/6.png)

Fig
6. Result of polyacrylamide gel electrophoresis

**3.3** **Validation map of amine
oxidase functional activity**

The
graph shows changes in crude and purified AocI protein concentrations at different methanol-induced
concentrations. It can be intuitively seen from the table that the crude protein concentration was the
highest at 0.50% methanol-induced concentration. The lowest crude protein concentration was observed at
the induced concentration of 1.00% methanol. After 0.50% methanol, crude protein concentration decreased
with increasing methanol-induced engagement. There was little difference in protein concentration after
purification.

![](https://static.igem.wiki/teams/4929/wiki/result/7.png)

Fig
7. Validation map of amine oxidase functional activity

Validation of amine oxidase functional activity

**4.1** **Using the acid-base
indicator method to qualitatively compare the ability of the transformants to metabolize biogenic
amines**

Bromocresol purple, as an acid-base indicator, turns yellow in acidic
conditions and purple-red in alkaline conditions. Therefore, after adding histamine to the system, the culture
medium will exhibit a purple-red color. When histamine is hydrolyzed by oxidases, the products are
ammonium salts and aldehydes, which are weakly acidic. As a result, the alkalinity in the hydrolysis area decreases,
gradually changing to yellow. Thus, it is possible to determine whether histamine has been
oxidatively hydrolyzed by observing the color change.

In order to evaluate the activity of amine oxidase expressed by
our yeast, bromocresol purple and
different histamine concentrations were added to the PDA solid medium. For comparison, transformants
and blank control (wild strains) were
inoculated on the culture medium.

After
a certain period, the color of the bromocresol purple in the plates changed
from purple to yellow, and the color change of plates within the
transformants was more prominent. At the same time, the diameter of the transparent circle around the
transformants was longer than that of the blank control group. It
can be seen that the transformants have a more vital ability to metabolize biogenic amines.

![](https://static.igem.wiki/teams/4929/wiki/result/8.png)

***Fig 8. results of the
transparent circle experiment and the control group.***

**4****.2
Determination of histamine content using High-Performance Liquid Chromatography (HPLC)**

High-performance
liquid chromatography, or HPLC (High-performance liquid chromatography) is a widely used separation and
detection technique in analytical and preparative chemistry. It uses a mobile phase to separate and detect
chemical samples from a stationary phase.

To
further investigate the ability of the strain to degrade histamine, we also performed an HPLC assay
(Figure
9). Histamine concentrations were measured at five different temperatures (20, 25, 30, 37, 45℃) and
different sampling times (12, 24, 36, 48, 72 h).

We
first incubated the bacterial solution to OD 0.6-0.8 to give the bacteria the highest viability. Then
histamine was added and the initial concentration of histamine was 200ug/ml. changes in histamine content
were detected using HPLC. As
can be seen from the chromatogram in Fig 9, compared with the control group GS115, pAO815-AocI, and
pAO815-AocI-FAD, the measured time of histamine peak was around 18-19 min. According to the calculations,
it was learned that the concentration of histamine was significantly decreased after 72h of continuous
cultivation, and the pAO815-AocI-FAD strain had a more significant decrease in histamine concentration
than the pAO815-AocI strain, which indicated that the pAO815-AocI-FAD strain had a better ability to
degrade histamine, and this is in line with the result of the previous clear circle experiments.

![](https://static.igem.wiki/teams/4929/wiki/result/n-9.jpg)

***Fig 9.
Results of the HPLC.***

***A:*** ***Chromatograms
of GS115, pAO815-AocI and pAO815-AocI-FAD at 12h***

***B******:
Chromatograms of GS115, pAO815-AocI and pAO815-AocI-FAD at 72h***

According
to the image results, the content of histamine decreased gradually with time, and the fastest degradation
of histamine was observed at 30℃. The lowest content of histamine was observed at 72h. A
pattern can be observed: all show monotonically decreasing, but obviously, the rate of decrease at 30℃ is
accelerated, and also after 73h of experiment, the lowest values.

![](https://static.igem.wiki/teams/4929/wiki/result/n-10.jpg)

***Fig 10.
Comparison of
histamine content values at different temperatures.***

5. **Next Experiment Plan**

The
yeast we used in this experiment is not the common Saccharomyces cerevisiae, so we will use the more
common Saccharomyces cerevisiae for further investigations. With our instructor’s assistance, we drew a preliminary experiment plan to construct
the recombinant Saccharomyces
cerevisiae.

**5.1 Plasmid Construction**

We
chose pYES as the vector because this Saccharomyces cerevisiae is more commonly used. Then, the three
essential parts of the experiments, namely recombinant plasmid construction, yeast transformation and
protein expression, and enzyme function detection, were carried out again.

Plasmid
pYES is 5857 bp in size (Fig 9), which can function in yeast. We put a template of plasmid pYES, 2xmix,
and corresponding primers together and did a PCR to amplify and linearize it.

![](https://static.igem.wiki/teams/4929/wiki/result/9.png)

***Fig 11. Graph of
pYES2***

Aiming
for recombinant plasmids and preparing for the following processes, we connected the two parts through
homologous recombination and transformed them into AQ. Then, applying the primers corresponding to AocI,
we did a PCR testing (Fig 10).

![](https://static.igem.wiki/teams/4929/wiki/result/10.png)

***Fig 12. Graph of plasmid
pYES-AocI***

After
we combined fragment FAD and the plasmid pYES-AocI, we did a PCR test (Fig 11). Compared to the markers,
the size of our products aligned with the size of AocI and FAD fragments.

![](https://static.igem.wiki/teams/4929/wiki/result/11.png)

***Fig 13. Graph of plasmid
pAO815-AocI-FAD***

**5.2 Amine Oxidase Activity Test**

In
the end, we will validate
amine oxidase functional activity again since we change the host.

**5.3 Fermentation Test**

In order to simulate the real application context, we will conduct
a mini fermentation experiment with our recombinant Saccharomyces cerevisiae to produce alcohol.
 In this part, we will evaluate the alcohol production ability of our recombinant Saccharomyces
cerevisiae for commercial consideration and the amine level during the fermentation compared to the
wild.

**5.4 Future Plan**

Biogenic amine-reducing Saccharomyces cerevisiae will be extended to other fermented foods
with high biogenic amines, such as canned fish products, fermented sausage, and cheese, to broaden its
application scope further. Besides, we will also dig into investigating amine oxidase's
biological activity and biogenic amine's degradation mechanism to provide a theoretical basis for
applying amine oxidase[1].

References

[1]
Niu Tian Jiao. THE MICROBIAL FLORA STRUCTURE AND DEGRADATION OF BIOGENIC AMINES DURING RICE WINE BREWING
[D]Harbin Institute of Technology,2020

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