iGEM 2023 — Team fujian-united

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Date July 11

Detail Team meeting for experiment schedule and team slogan & team logo

![](https://static.igem.wiki/teams/4870/wiki/notebook/1-1.png)

Date July 12

Detail LB and YPD culture solution was prepared and sent to the disinfection room for sterilization.
Basic rules of use in the laboratory were understood, PCR preparation was carried out, and Escherichia coli
and yeast were inoculated.

![](https://static.igem.wiki/teams/4870/wiki/notebook/2-1.png)
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Date July 13

Detail Gel electrophoresis, spot sample, electrophoresis and blue light observation were performed. Yeast
receptive cells were prepared in the early stage, followed by yeast expansion culture. The plasmid pYES2 was
extracted and the concentration was detected after the plasmid extraction process. Clean and recover
An\_phy33 gene, and test the concentration after the glue recovery process. An\_phy33 and pYES2 plasmids were
studied.

![](https://static.igem.wiki/teams/4870/wiki/notebook/3-1.png)
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Date July 14

Detail Used ligase to reconstruct the gene fragment AppA and An\_phy33 with the plasmid pYES2. prepared
competent yeast cell. transformed the reconstructed plasmid into competent cell E. coli.

![](https://static.igem.wiki/teams/4870/wiki/notebook/4-1.png)
![](https://static.igem.wiki/teams/4870/wiki/notebook/4-2.png)

Date July 15

Detail Plasmid extration. Polymerase chain reaction. Agarose gel electrophoresis. Plasmid transformation.

![](https://static.igem.wiki/teams/4870/wiki/notebook/5-1.png)
![](https://static.igem.wiki/teams/4870/wiki/notebook/5-2.png)

Date July 16

Detail The plasmid cultured in E. coli was extracted, PCR and agarose gel electrophoresis were used to
confirm whether the culture was successful. Transform yeast receptor cells and apply.

![](https://static.igem.wiki/teams/4870/wiki/notebook/6-1.png)
![](https://static.igem.wiki/teams/4870/wiki/notebook/6-2.png)

Date July 17

Detail The yeast transformation strain carrying the specified plasmid was selected, inoculated with 4 mL
plus hygromycin into a 30 ℃ shaking table, and prepared with 800 mL NaAc solution with pH 5.0. Pre-configure
a solution with a pH of 1-8.

![](https://static.igem.wiki/teams/4870/wiki/notebook/7-1.png)
![](https://static.igem.wiki/teams/4870/wiki/notebook/7-2.png)

Date July 18

Detail Make albumen glue. Write wiki.

![](https://static.igem.wiki/teams/4870/wiki/notebook/8.png)

Date July 19

Detail Extract yeast cells then shake them up.

![](https://static.igem.wiki/teams/4870/wiki/notebook/9.png)

Date July 20

Detail Upload basic parts information and write wiki.

![](https://static.igem.wiki/teams/4870/wiki/notebook/10.png)

Date July 21

Detail One day off

Date July 22

Detail Run SDS PAGE gel.

![](https://static.igem.wiki/teams/4870/wiki/notebook/11-1.png)
![](https://static.igem.wiki/teams/4870/wiki/notebook/11-2.png)

Date July 23

Detail Phytase activity test by microplate reader

![](https://static.igem.wiki/teams/4870/wiki/notebook/12-1.png)
![](https://static.igem.wiki/teams/4870/wiki/notebook/12-2.png)

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**O****verview：**

The growing global livestock
population has led to shortages of traditional feed protein and raw materials, an urgent issue for the
livestock and breeding industry. Plants also contain antinutrient phytic acid which inhibits nutrient
absorption in animals. An alternative protein source is needed. This work aims to prepare yeast
single-cell protein with phytase activity by fusing a phytase gene to a cell wall anchor protein. Adding
this to feed could improve nutrition and absorption for livestock and poultry, benefiting feed
producers.

![](https://static.igem.wiki/teams/4870/wiki/engineering-success/figure-1.jpg)

**Figure 1 Design
diagram of this project.** **Image by LIU JINLE.**

**Cycle 1:
G****eneration****of surface-displayed
AppA of** **yeast**

**Design 1:**

We first obtained the phytase
gene *AppA* (Gene ID:946206,
NP\_415500.1) from *E. coli* at the NCBI website
(https://www.ncbi.nlm.nih.gov/). Then, using the pYES2 plasmid as a backbone, we designed an expression
frame for yeast cell surface display phytase, *i.e.*, to link the yeast promoter
GAL, MF-alpha-1 signal peptide, *AppA* gene, yeast surface anchoring
protein gene SED1, and RPL41Bt yeast (Figure
2). Finally,
through restriction endonuclease digestion and linkage, we constructed the APPA display plasmid:
pYES2-Hyg-GAL1p-α-AppA-SED1-RPL41Bt.

![](https://static.igem.wiki/teams/4870/wiki/engineering-success/figure-2.jpg)

**Figure 2
Expression frame of the AppA display plasmid**

**Build 1:**

To
construct the plasmid pYES2-Hyg-GAL1p-α-AppA-SED1-RPL41Bt,
we used the *E.
coli* DH5α genomic
DNA as a template to amplify the *AppA* sequence
by
PCR.
Then, the *AppA* sequence
was inserted
into the *Sph*Ⅰ and
*Avr*Ⅱ sites
of plasmid pYES2-Hyg-GAL1p-α-SED1-RPL41Bt,
by restriction endonuclease digestion and linkage.

The
plasmid was then transferred into the *E. coli* DH5α competent
cells. The result showed that transformants were successfully grown after overnight culture. After colony
PCR verification, the recombinants had the expected bands (1293
bp),
indicating the successful transformation of this plasmid.

![](https://static.igem.wiki/teams/4870/wiki/engineering-success/figure-3.jpg)

**Figure 3
Construction of the AppA display plasmid**

The
positive transformant was inoculated and plasmid was extracted using a commercial miniprep kit.
Before the
transformation, the BY4741 strain was inoculated and prepared in a competent state. Then, the lithium
acetate (LiAc) transformation method was used to transform linearized plasmid into BY4741 competent cells,
followed by incubation at 30 °C for 48 h. The colony PCR identification confirmed that the plasmid was
successfully transfected into BY4741 cells. Sequencing of the transformants verified the correct sequence
of the AppA target fragment.

![](https://static.igem.wiki/teams/4870/wiki/engineering-success/figure-4.jpg)

**Figure 4****Results of****AppA display
plasmid****transformation of
yeast cells.**

**Test 1:**

We
selected a positive transformant and cultured it to the logarithmic growth period. Galactose was then
added to induce the promoter, and
yeast cells displaying phytase on the surface were obtained. Cells were pelleted by centrifugation and
washed by resuspension in 50 mM sodium acetate buffer (pH 5.0) A portion of the suspension was sonicated
and then centrifuged to obtain lysate supernatant and precipitate containing cell wall-bound phytase. The
SDS-PAGE result showed that the AppA was successfully displayed on the yeast cell surface (Figure
5).

Then,
the activity assays were performed on yeast surface-displayed phytase and phytase lysate precipitate,
respectively. We found that the lysed precipitated phytase exhibited higher activity, so this form was
used to determine phytase activity under different pH and temperature conditions. As shown in Figure
5,
the activity of the AppA phytase initially increased with rising pH, reaching maxima at pH 5, then
declining as pH continued to rise. For reaction temperature, the activity of the AppA phytase increased
with rising temperature, with the highest activity detected at 55 °C.

![](https://static.igem.wiki/teams/4870/wiki/engineering-success/111111.png)

**Figure 5
Expression and enzyme activity test results of AppA.**

**Learn****1****:**

The
results of our experiments indicate that the AppA phytase
displayed on the surface of yeast cells is active. The highest enzyme activity was observed at pH 5, or a
reaction temperature of 55 °C. To further improve the activity of phytase, we predicted the ancestral
enzyme gene sequence of *AppA*,
*A**n**\_phy33*, which
was used for the next round of plasmid construction, protein expression, and enzyme activity
testing.

**Cycle 2:
G****eneration****of surface-displayed
An\_phy33 of** **yeast**

**Design 2:**

We obtained a series of
ancestral enzyme sequences of *E. coli* phytase AppA at the website
(https://loschmidt.chemi.muni.cz/fireprotasr/). Prediction using DeepSTABp
(https://csb-deepstabp.bio.rptu.de/) revealed that the *An\_phy33* sequence had the highest
melting temperature Tm of 87 °C compared to 51 °C for wild-type *AppA*, so we chose to use this
sequence for this round of experiments.

Similar to the previous round
of expression frames, we link the yeast promoter
GAL, MF-alpha-1 signal peptide, *An\_phy33* gene, yeast surface anchoring
protein gene SED1, and RPL41Bt yeast (Figure
6). Through restriction
endonuclease digestion and linkage, we constructed the An\_phy33 display plasmid: pYES2-Hyg-GAL1p-α-An\_phy33-SED1-RPL41Bt.

![](https://static.igem.wiki/teams/4870/wiki/engineering-success/figure-6.jpg)

**Figure****6****Expression frame of
the An\_phy33 display plasmid**

**Build 2:**

To
construct the plasmid pYES2-Hyg-GAL1p-α-An\_phy33-SED1-RPL41Bt, we used a synthetic plasmid
containing the *An\_phy33* sequence
as a template to amplify *An\_phy33* sequence by
PCR. Then,
the *An\_phy33* sequence
was inserted
into the *Sph*Ⅰ and
*Avr*Ⅱ sites
of plasmid pYES2-Hyg-GAL1p-α-SED1-RPL41Bt, by
restriction endonuclease digestion and linkage.

The
plasmid was transformed into the *E. coli* DH5α competent
cells. Transformants were successfully cultured overnight, and colony PCR showed the expected 1380 bp
band, confirming plasmid transformation.

![](https://static.igem.wiki/teams/4870/wiki/engineering-success/figure-7.jpg)

**Figure****7****Construction of the
An\_phy33 display plasmid**

The
plasmid was extracted from the positive transformant and linearized. It was then transformed into BY4741
yeast-competent
cells and incubated at 30 °C for 48 h. Colony PCR and sequencing verified the correct integration and
sequence of the An\_phy33 fragment in yeast.

![](https://static.igem.wiki/teams/4870/wiki/engineering-success/figure-8.jpg)

**Figure****8****Results of****An\_phy33 display
plasmid****transformation of
yeast cells.**

**Test 2:**

A
positive yeast transformant was cultured to a logarithmic growth period. Galactose induction yielded
cells displaying
phytase on the surface. Then, the cells were pelleted, washed with 50 mM sodium acetate buffer (pH 5.0),
and lysed. Lysate centrifugation gave a supernatant and precipitate containing cell wall-bound phytase.
The SDS-PAGE result showed a successful surface display of the An\_phy33 phytase (Figure
9).

Phytase
activity assays were performed on surface-displayed and cell wall-bound phytase. Lysed phytase precipitate
exhibited higher activity and was used to determine phytase activity under different pH and temperature
conditions. As shown in Figure 9, the activity of the An\_phy33 phytase is higher than that of the AppA
phytase. It reaches maximum activity at a pH value of 6 or a reaction temperature of 55 °C.

![](https://static.igem.wiki/teams/4870/wiki/engineering-success/figure-9.jpg)

**Figure****9****Expression and enzyme
activity test results of An\_phy33.**

**Learn 2:**

From the experimental results,
it is clear that the An\_phy33 phytase had higher activity than the AppA phytase, as predicted. In
addition, the optimal reaction pH for AppA was 5, while An\_phy33 was 6; and they both had the highest
enzyme activities at 55 °C.

Since this technology is
currently only performed at the laboratory level and has not been tested in actual feeds, it may result in
uncontrollable enzyme activity as a result of temperature and pH. Therefore, its practical
application needs to be tested in subsequent experimental validation.

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I. **Preparation of media**

1. LB
medium:

1) Weigh
10 g tryptone, 5 g yeast extract and 10 g NaCl using an electronic balance, then add these components to
an Erlenmeyer flask.

2) Use
a graduated cylinder to measure 1 L ddH2O, and
then add water into the Erlenmeyer flask mentioned above.

3) Dissolve
the components by shaking the flask.

4) Separate
the solution equally, label one as LB liqud and the other as LB solid.

5) Add
7.5 g agar into the LB solid medium.

6) Sterilize
by autoclaving at 121 ℃ for 20 min.

7) Allow
LB solid medium to cool to 50-60 °C after autoclaving.

8) Add
antibiotics to the solid LB medium, mix well and pour the plates in a laminar flow hood. Make it
solidify.

9) Store
prepared LB agar plates at 4 °C.

2. YPD
medium:

1) Weigh
20 g glucose and measure 500 mL ddH2O,
add in an Erlenmeyer flask labeled glucose.

2) Shake
the flask to dissolve the glucose.

3) Weigh
20 g tryptone and 10 g yeast extract using an electronic balance, measure 500 mL ddH2O,
and add to another Erlenmeyer flask.

4) Shake
the Erlenmeyer flask evenly.

5) Separate
the solution equally, label one as YPD liquid and the other as YPD solid.

6) Add
7.5 g agar into the YPD solid medium.

7) Sterilize
by autoclaving at 121 ℃ for 20 min.

8) Allow
YPD solid medium to cool to 50-60 °C after autoclaving.

9) Add
antibiotics to the solid YPD medium, mix well and pour the plates in a laminar flow hood. Make it
solidify.

10) Store
prepared YPD agar plates at 4 °C.

II. **Polymerase Chain Reaction (PCR)**

A. *AppA*:

1) Use
the *E. coli* DH5α genomic
DNA as template.

2) UseAppA-F
and AppA-R as
forward and reverse primers, respectively.

B. *An\_phy33*:

1) Use
the synthesized plasimd containing *An\_phy33* gene
as the template DNA.

2) Use
An\_phy33-F and An\_phy33-R as forward and reverse primers, respectively.

The
reaction system for PCR amplification are shown in the table below:

|  |  |
| --- | --- |
| Reagent | Volume (μL) |
| 2× PCR Master Mix (With Dye) | 25 |
| primer F (10 μM) | 1 |
| primer R (10 μM) | 1 |
| DNA template | 1 |
| ddH2O | 22 |
| Total | 50 |

The
reaction procedure for
PCR amplification are shown in the table below:

|  |  |  |
| --- | --- | --- |
| Step | Temperature ( ℃) | Time (sec) |
| 1 | 98 | 180 |
| 2 | 98 | 30 |
| 3 | 60 | 30 |
| 4 | 72 | 120 |
| 5 | 72 | 300 |
| 6 | 4 | forever |

Notice:
Repeat step 2 to step 4 for 28 times.

III. **I****noculation****of** ***E. coli*****and yeast**

1. Inoculation
of *E. coli*:

1) Measure
3 mL LB liquid medium, add 3 μL ampicillin to a final concentration of 50 μg/mL, and inoculate the
*E. coli* strain.

2) Incubate
the *E. coli* culture
in a 37 ℃ shaker at 220 rpm for 12-16 h.

1. Inoculation
of yeast:

1) Measure
4 mL YPD liquid medium, add 4 μL ampicillin to a final concentration of 50 μg/mL, and inoculate the
BY4741 yeast strain.

2) Incubate
the yeast culture in a 37 ℃ shaker at 220 rpm for 24-48 h.

IV. **Agarose gel electrophoresis**

1. Preparation
of agarose gel

1) Add
100 mL 1×TAE buffer and 1 g agarose to an Erlenmeyer flask, and place it in a microwave oven on high
for 2 min.

2) Take
it out after the solution turns transparent and shake it evenly.

3) Add
2 μL nucleic acid dye GelRed 10000 ×to
the molten agarose.

4) Assemble
gel tray and insert comb into mold.

5) Pour
the liquid agarose into the mold to a depth of 3-4 mm, avoiding air bubbles.

6) Wait
for solidification of the gel at room temperature.

7) Carefully
remove comb and transfer solidified
gel to electrophoresis tank.

8) Completely
submerge gel in 1 ×TAE
buffer.

2. Loading
samples

1) Add
DNA
loading buffer
into samples to dye them blue, then load into wells sequentially. Avoid contacting gel when
pipetting.

2) Add
10 μL of Trans15K DNA Marker into a lane.

3. Electrophoresis

1) Place
wells at the negative electrode, and cover tank.

2) Turn
on the power, and run the agarose gel at 180V for 20 min.

4. Visualizing
DNA bands

1) Turn
off power, and remove gel.

2) Place
the gel under the Ultra Slim LED Illuminator to visualize the results.

3) Take
out the gel, and put it under gel-imaging system to save data.

V. **Gel Extraction (with DNA Gel Extraction
Kit)**

1. Preparation

1) Check
whether anhydrous ethanol has been added to the Wash Solution.

2) Make
sure Buffer B2 does not appear to be precipitated.

3) Set
the constant temperature water bath to 50 ℃.

2. Extraction

1) Rapidly
excise the DNA band of interest under UV light using a clean scalpel.

2) Transfer
the gel slices into centrifuge tubes, and
measure the weight of the gel slices.

3) Add
3 times of sample volume of Buffer B2 into the centrifuge tubes, and mix until gel completely
dissolved.

4) Transfer
the solution into
the absorption column, and centrifuge at 8000 ×*g* for
30 sec. Pour off the filtrate in
the collection tube.

5) Add
500 μL of Wash Solution and centrifuge at 9000 ×*g* for
30 sec. Pour off the filtrate in the collection tube.

6) Repeat
wash step.

7) Centrifuge
the empty column at 9000 ×*g* for
1 min.

8) Place
the absorption column into a clean 1.5 mL centrifuge tube, add 15-40 μL Elution Buffer to the center of
the absorption membrane, and centrifuge at 9000 ×g for 1 min. Store the DNA solution in the
tube.

VI. **Plasmid extraction (Plasmid Mini-PREPS
Kit)**

1. Preparation

1) Make
sure RNase A has been added to Buffer SP1, anhydrous ethanol has been added to Wash Solution, and no
precipitation appears in Buffer SP2 and SP3.

2. Equilibrate
the adsorption column

1) Add
500 μL of Buffer S to the adsorption column and place a collection tube below it.

2) Centrifuge
at 12,000 ×*g* for
1 min, discard the waste liquid in the collection tube and put the adsorption column back into the
collection tube.

3. Transfer
all the *E. coli* culture into
a 2 mL centrifuge tube, centrifuge at 12,000 ×*g* for
2 min to collect the bacteria. Discard the supernatant and retain the precipitate.

4. Add
250 μL of Buffer SP1 to the 2 mL centrifuge tube, and thoroughly suspend the precipitate with
pipette.

5. Add
250 μL of Buffer SP2, and gently invert the tube 5-10 times to mix the solution.

6. Add
350 μL of Buffer SP3 and mix the solution by gently inverting the tube 5-10 times.

7. Centrifuge
at 12,000 ×*g* for
5-10 min. Transfer 700 μL of the supernatant to the adsorption column, centrifuge at 8,000 ×*g* for
30 sec, and discard the filtrate in
the collection tube.

8. Add
500 μL of Wash Solution, centrifuge at 9,000 ×*g* for
30 sec, and discard the filtrate in
the collection tube.

9. Repeat
wash step.

10. Centrifuge
the empty column at 9,000 ×*g* for
1 min.

11. Place
the column into a clean 1.5 mL centrifuge tube, add 25 μL of Elution Buffer to the center of the
adsorption membrane. Incubate
for 1 min at room temperature,
centrifuge at 9,000 ×*g* for
1 min, and keep the DNA solution in the tube.

VII. **Double enzyme digestion of fragments and
vectors**

1. The
double digestion system for PCR products is shown in the table below:

|  |  |
| --- | --- |
| Reagent | Volume (μL) |
| PCR products (*AppA / An\_phy33*) | 20 |
| *Sph*Ⅰ | 1 |
| *Avr*Ⅱ | 1 |
| Buffer (CutSmart) | 5 |
| ddH2O | 23 |
| Total | 50 |

2. The
double digestion system for vector is shown in the table below:

|  |  |
| --- | --- |
| Reagent | Volume (μL) |
| plasmid  (pYES2-Hyg-GAL1p-α-SED1-RPL41Bt) | 30 |
| *Sph*Ⅰ | 1 |
| *Avr*Ⅱ | 1 |
| Buffer (CutSmart) | 5 |
| ddH2O | 13 |
| Total | 50 |

The
enzyme digestion systems are incubated at 37 ℃ for 3 h.

VIII. **Ligation** **of recombinant plasmids**

1. Plasmid
1: pYES2-Hyg-GAL1p-α-**AppA**-SED1-RPL41Bt

|  |  |
| --- | --- |
| Reagent | Volume (μL） |
| plasmid  (pYES2-Hyg-GAL1p-α-SED1-RPL41Bt) | 13.6 |
| *AppA* | 3.4 |
| *T4 ligase* | *1* |
| T4 ligase buffer (10×) | 2 |
| Total | 20 |

2. Plasmid
2: pYES2-Hyg-GAL1p-α-**An\_phy33**-SED1-RPL41Bt

|  |  |
| --- | --- |
| Reagent | Volume (μL） |
| plasmid  (pYES2-Hyg-GAL1p-α-SED1-RPL41Bt) | 15 |
| *An\_ phy33* | 2 |
| T4 ligase | 1 |
| T4 ligase buffer (10×) | 2 |
| Total | 20 |

The
ligation systems are incubated at 16 ℃ for 16 h, followed
by 5 min at 65 ℃ to inactivate the ligase.

IX. **Transformation** **of recombinant
plasmids****to*****E. coli*****DH5α**

1. Add
5 μL of pYES2-Hyg-GAL1p-α-AppA-SED1-RPL41Bt plasmid
and pYES2-Hyg-GAL1p-α-An\_phy33-SED1-RPL41Bt plasmid
into 50 μL of *E. coli*DH5α
competent cells, respectively. Incubate
the mixture on
ice for 30 min.

2. Heat
shock at 42 °C for 90 sec, and put the mixture to the ice for 4-5
min.

3. Add
the LB culture into the tubes to 1 mL, and shake the tubes at 37 ℃, 220 rpm for 30-60 min.

4. Centrifuge
it at 9000 ×*g* for
1 min, remove 800
μL of supernatant, and resuspend the cells in the remaining LB culture.

5. Spread
50 μL cells onto LB agar plates containing ampicillin
and incubate overnight at
37 ℃.

X. **PCR identification of
transformants**

1. Place
50 μL of Lysis Buffer for Microorganism to Direct PCR in a sterilized microtube. Pick a single colony
with a sterilized toothpick or lance tip, place it in the microtube and stir a few times before removing
it. (Note: Do not leave the toothpick in the microtube for too long to prevent affecting the volume of
lysate and PCR amplification.)

2. After
heat denaturation at 80 °C for 15 min, centrifuge at low speed and take 1~5 μL of supernatant after
lysis as PCR template.

The
PCR reaction system is shown in the table below:

|  |  |
| --- | --- |
| Reagent | Volume (μL) |
| Lysis supernatant | 1 |
| Premix Taq (Ex Taq Version 2.0) | 10 |
| Forward Primer (10 μM  ) | 1 |
| Reverse Primer (10 μM) | 1 |
| sterilized water | 7 |

The
PCR reaction condition is shown in the table below:

|  |  |  |
| --- | --- | --- |
| Step | Temperature (˚C) | Time (sec) |
| 1 | 94 | 600 |
| 2 | 94 | 30 |
| 3 | 55 | 30 |
| 4 | 72 | 120 |
| 5 | 72 | 300 |
| 6 | 4 | forever |

Notice:
Repeat step 2 to step 4 for 28 times.

3. Take
positive transformants and add 3 mL LB culture medium, add 3 μL ampicillin, shake bacteria, and
incubate at 37 ℃ at 220 rpm for 12-16 h

4. Inoculate
the positive transformants in 3 mL LB culture medium containing 50 μg/mL ampicillin at a 37 ℃ shaker
with 220 rpm for 12-16 h.

5. Extraction
the plasmid from overnight cultures for yeast transformation.

XI. **Preparation of*****Saccharomyces
cerevisiae*****Competent Cell**

1. Select
single bacterial colony from YPD plate, and inoculate it
into YPD liquid medium for 12-24 h.

2. Measure
the OD600 value
of the overnight culture (between 4.0 and 5.0). Notably, a 10-20 dilution of the culture solution is
required for the measurement.

3. Dilute
the overnight culture to OD600 value
of 0.2~0.4, and then transfer
into shaker.

4. The
diluted culture is continued to incubate in a shaker at 30 ℃, 220
rpm for
4-5 h,
until OD600 reaches
0.8-1.0.

5. Centrifuge
the culture at
3000
×*g* at
20 ℃ for 5 min, and discard
the supernatant.

6. Resuspend
the pellet in
ddH2O,
and centrifuge
at 3000
×*g* at
20 ℃ for 5 min. Discard the supernatant.

7. Repeat
water wash step.

8. Resuspend
the pellet in
1 mL ddH2O
into 1.5 mL centrifuge tube.
Centrifuge
at 12000
×*g* for
30 sec for
5 min, and discard
the supernatant.

9. Resuspend
the pellet in
1mL Cell Freezing Medium. Aliquot 100 μL per tube, and freeze at -80°C.

XII. **Plasmid Transformation to*****Saccharomyces
cerevisiae*** **BY4741**

1. Place
the plasmid in a 95 ℃ water bath for 10 min, and then transfer it onto ice.

2. Take
out 100 μL yeast competent cells and place them in a 37 ℃ water bath for 20 sec. Centrifuge it at 12000
×*g* for
30 sec and remove the supernatant.

3. Add
36 μL of 1.0 M LiAc, 50 μL linearized plasmid DNA, and 260 μL PEG 3350 into the tube with yeast
competent cell precipitate. Mix it thoroughly with pipette.

4. Heat
shock the cells at 42 °C for
25 min.

5. Centrifuge
at 12000 ×*g* for
30 sec, remove the supernatant and place it on ice for 5 min.

6. Resuspend
the pellet in 1 mL ddH2O.

7. Spread 100
μL of the mixture on the YPD culture containing hygromycin (100 μg/mL), and incubate at 30 ℃ for 48
h.

XIII. **PCR identification
of positive** **transformants**

1. Place
50 μL of Lysis Buffer for Microorganism to Direct PCR in a sterilized microtube. Pick a single colony
with a sterilized toothpick or lance tip, place it in the microtube, and stir a few times before removing
it. (Note: Do not leave the toothpick in the microtube for too long to prevent affecting the volume of
lysate and PCR amplification.)

2. After
heat denaturation at 80 °C for 15 min, centrifuge at low speed and take 1~5 μL of supernatant after
lysis as a template for PCR reaction.

The
PCR reaction system is shown in the table below:

|  |  |
| --- | --- |
| Reagent | Volume (μL) |
| Lysis supernatant | 1 |
| Premix Taq (Ex Taq Version 2.0) | 10 |
| Forward Primer (10 μM) | 1 |
| Reverse Primer (10 μM) | 1 |
| sterilized water | 7 |

The
PCR reaction condition is shown in the table below:

|  |  |  |
| --- | --- | --- |
| Step | Temperature (˚C) | Time (sec) |
| 1 | 94 | 600 |
| 2 | 94 | 30 |
| 3 | 55 | 30 |
| 4 | 72 | 120 |
| 5 | 72 | 300 |
| 6 | 4 | forever |

Notice:
Repeat step 2 to step 4 for 28 times.

XIV. **Protein
expression**

1. Take
a positive transformant and add 3 mL YPD culture medium containing 100 μg/mL hygromycin. Grow at
30°C, 240 rpm to OD600 0.4-0.6
(∼24
h).

2. Centrifuge
the yeast culture and remove the supernatant.

3. Resuspend
the pellet with
sterile water to remove glucose and transfer it to a 50 mL YPD culture medium
containing 100 μg/mL hygromycin.

4. Add
galactose to induce promoter and express surface-displayed phytase for 24-48 h.

XV. **Preparation of samples**

1. Preparation
of yeast surface-displayed phytase

a. Collect
yeast by centrifugation at 8000 rpm for 5 min and measure the wet weight.

b. Washing
the yeast cells twice with 50 mM sodium acetate buffer (pH 5.0). Retain for the determination of surface
phytase activity.

2. Preparation
of yeast phytase from the yeast cell wall

a. Collect
yeast by centrifugation at 8000 rpm for 5 min and measure the wet weight.

b. Washing
the yeast cells twice with 50 mM sodium acetate buffer (pH 5.0).

c. Sonicate
cells at 30% power for 5 min at 0°C, and
the lysate was centrifuged at 4 °C, 8000 rpm for 5 min.

d. Washed
the lysate precipitation twice with 50 mM sodium acetate buffer (pH 5.0). Retain for the determination of
cell wall phytase activity.

XVI. **Phytase
activity**

1. Prepare
substrate solution (500 mM sodium phytate, 50 mM acetic acid/sodium acetate buffer, pH 5.0) and
termination reaction solution (15% (w/v) trichloroacetic acid).

2. Mix
200 μL phytase-displayed yeast with 200 μL substrate solution. Incubate 30 min at 37 °C.

3. Terminate
the reaction by adding 100 μL of the termination reaction solution and measure the absorbance value at
700 nm spectrophotometrically.

4. Generate
standard curve using K2HPO4 standards
from 0-1.0 mg/mL. Calculate free phosphate released.

5. A
phytase unit activity (U) was defined as the amount of enzyme required to release 1.0 μmol/min of
inorganic phosphorus from 5 mmol/L sodium phytate under optimal conditions.

6. Determination
of phytase under
different pH:

Prepare
phytase-displayed yeast cell suspensions with pH ranging from 1-8. Carry out the enzyme activity assay
according to the above steps.

7. Determination
of phytase under
different temperature:

Incubate
the phytase-displayed yeast cell suspension and the reaction substrate at different temperatures (30-60
℃). Carry out the enzyme activity measurement according to the above steps.

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![](https://static.igem.wiki/teams/4870/wiki/results.png)

**Overview**

Our
experimental results mainly include:

(1)
Construction of plasmids for surface display of phytase,

(2)
Obtaining surface-displayed phytase in *Saccharomyces
cerevisiae*,

(3)
Determining phytase activity under different pH and temperature conditions.

1. **Construction of plasmids for surface display of
phytase**

We
first constructed
the following two plasmids:

Plasmid
1: pYES2-Hyg-GAL1p-α-AppA-SED1-RPL41Bt,

Plasmid
2: pYES2-Hyg-GAL1p-α-An\_phy33-SED1-RPL41Bt.

To
construct plasmid 1, we used the *E. coli* DH5α genomic
DNA as a template to amplify the *AppA* sequence
by
polymerase chain reaction (PCR) (Figure 1A).
Then, the *AppA* sequence
was inserted
into the *Sph*Ⅰ and
*Avr*Ⅱ sites
of plasmid pYES2-Hyg-GAL1p-α-SED1-RPL41Bt, by
restriction endonuclease digestion and linkage.

To
construct plasmid 2, we used a synthetic plasmid containing the *An\_phy33* sequence
as a template to amplify the *An\_phy33* sequence by
PCR (Figure 1A).
Then, the *An\_phy33* sequence
was inserted
into the *Sph*Ⅰ and
*Avr*Ⅱ sites
of plasmid pYES2-Hyg-GAL1p-α-SED1-RPL41Bt, by
restriction endonuclease digestion and linkage.

(Note:
The plasmid pYES2-Hyg-GAL1p-α-SED1-RPL41Bt contains GAL1p yeast promoter, MF-alpha-1
signal peptide, yeast surface anchoring protein gene SED1, and RPL41Bt yeast terminator.)

Subsequently,
the plasmids 1 and 2 were transferred into the *E. coli* DH5α competent
cells, respectively. As shown in Figure 1B,
transformants were successfully grown after overnight culture. The mapping of the recombinant plasmids is
illustrated in Figure 1C-D.
After colony PCR verification, the recombinants had the expected bands (1293
bp for plasmid
1, and 1380 bp
for plasmid
2), indicating successful transformation of both plasmids (Figure 1E).

![](https://static.igem.wiki/teams/4870/wiki/results/figure-1.jpg)

**Figure 1 Construction and
transformation of the plasmids.**

2. **Obtaining surface-displayed phytase in*****Saccharomyces
cerevisiae*** **BY4741**

Positive
transformants were inoculated and plasmids were extracted using a commercial miniprep kit for subsequent
transformation of *Saccharomyces
cerevisiae* competent
cells. Before the
transformation, the BY4741 strain was inoculated and prepared in a competent state. Then, the lithium
acetate (LiAc) transformation method was used to transform linearized plasmids 1 and 2 into BY4741
competent cells, respectively, followed by incubation at 30°C for 48 h.

As
shown in Figure 2A, transformants were successfully cultured on YPD plates. After colony PCR
identification, the two transformants had the expected bands (1293
bp for plasmid
1, and 1380 bp
for plasmid
2), indicating that the plasmids were successfully transfected into BY4741 cells (Figure 2B). These
transformants were then sequenced, confirming the *AppA* and
*An\_phy33* target
fragment sequences were correct (Figure 2C-D).

![](https://static.igem.wiki/teams/4870/wiki/results/figure-2.jpg)

**Figure 2 Results of plasmid
transformation of yeast cells.**

We
selected positive transformants and cultured them to the logarithmic growth period. Galactose was then
added to induce the promoter, and
yeast cells displaying phytase on the surface were obtained. Cells were pelleted by centrifugation and
washed by resuspension in 50 mM sodium acetate buffer (pH 5.0) A portion of the suspension was sonicated
and then centrifuged to obtain lysate supernatant and precipitate containing cell wall-bound phytase. As
shown in Figure 3, both AppA and An\_phy33 were successfully displayed on the yeast cell surface for
subsequent activity assays.

![](https://static.igem.wiki/teams/4870/wiki/results/figure-3.jpg)

**Figure 3 SDS-PAGE results of
APPA and An\_phy33** **expression****.**

3. **Determining phytase activity under different pH
and temperature conditions.**

We
performed enzyme activity assays on yeast surface-displayed phytase and phytase lysate precipitate,
respectively. We found that the lysed precipitated phytase exhibited higher activity, so this form was
used to determine phytase activity under different pH and temperature conditions. As shown in Figure 4, in
the pH range of 1-8, the activities of the two phytases initially increased with rising pH, reaching
maxima at pH 5-6, then declining as pH continued to rise. AppA phytase showed peak activity at pH 5, while
An\_phy33 phytase had maximal activity at pH 6. For reaction temperature, the activities of both phytases
increased with rising temperature, with the highest activities for both enzymes detected at 55
°C.

![](https://static.igem.wiki/teams/4870/wiki/results/figure-4.jpg)

**Figure 4 Phytase
activity** **under different pH and
temperature conditions.**

In
summary, we successfully constructed two plasmids that can display phytase on the surface of yeast cells,
obtained surface-displayed phytase in *Saccharomyces
cerevisiae*
BY4741, and tested the activity of phytase under different pH and temperature conditions. Our experimental
results confirmed fusion expression of the phytase gene with a yeast cell wall anchor protein gene enables
the successful display of active phytase on the yeast cell surface.

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