iGEM 2023 — Team bzk

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each
day for your project.{% endblock %}
{% block page\_content %}

![](https://static.igem.wiki/teams/5024/wiki/g-out-notebook.png)

**Wet
Lab Notebook**

**Day
1 23/7/30**

1.
-Introduce the discipline in the laboratory, and review the general
process of the experiment

**.**

2.-Instructions
of operating gauges - LB Medium

：

**3.-**

Introduction
of PCR(Polymerization Chain Reaction), each group starts to create
the samples for PCR

4.-Introduction
and operation of Agarose Gel Electrophoresis

5.
Recovery of the products of PCR

**Day
2 23/7/31**

**1.-**

Introduction
about extraction of plasmids

：

2.-Introduction
of the enzyme cutting, ligation, and transformation

3.
Start the Enzyme Cutting Process. Each group creates one sample for
vector PET28a and one sample for one of three target genes: UAO,
allantoicase, and allantoinase.

\*BamHI,
XhoI, and NotI are restriction enzymes.

4.-Examine
the product of enzyme cutting for vectors by electrophoresis.

5. Production
   and Laying of LB Agar Plates

   ：

6.-Extraction
of Gel Electrophoresis Sample

7.-Start
ligation process. According to the kind of target gene each group has
made, all groups focus on constructing plasmids.

8.-Transformation
of Bacteria

![](https://static.igem.wiki/teams/5024/wiki/wechatimg60.jpeg)

**Day
3 23/8/1**

**1.-**

Start
isolated colonies validation process.

2.-Introduction
of constructing plasmid with the vector of pGex-4T-1.

3.-Start
the linearization process for both vector(pGex-4T-1) and target
genes(UAO,A-nase, and A-case). Each group creates 3 samples: two of
them are samples of vector pGex-4T-1, and the rest is the sample of
the target gene(UAO/ allantoinase/ allantoicase)

Reagents
required

：

4.-Start
to examine the product of PCR in isolated colonies validation
process via agarose gel electrophoresis.

5.-Start
shaking bacteria.

\*Those
bacteria will undergo transformation in the following procedures.
When the products of PCR have undergone homologous recombination, the
plasmids will be replicated in those bacteria.

![](https://static.igem.wiki/teams/5024/wiki/wechatimg62.jpeg)

**Day
4 23/8/2**

1.-Start
to prepare for the examination of the product of PCR on 8.1 by
electrophoresis. (Create the gel)

2.-Start
to extract the products of PCR in 8.1.

\*There
are 3 samples for each group: two for the vector,pGex-4T-1, and one
for the target gene. During the extraction process, one sample of
vector and one sample of target gene are used.

3.-Examine
the products received by electrophoresis.

4.-Start
the homologous recombination process.

5.-Extract
the plasmids from the bacteria cultured in 8.1.

6.-Start
transformation.

7.
Production and Laying of LB Agar Plates

**Day
5 23/8/3**

1. The
   tasks today are examining the products(plasmids) of the
   transformation and replication for the pGex-UAO plasmids and
   pGex-UAA plasmids, culturing and incubating the dH5alpha bacteria
   with target gene, extracting the target vectors from the incubated
   dH5alpha bacteria, transferring them to BL21 bacteria, incubating
   the bacteria, examining the vectors.

STEP
I

-Prepare
for and operate the isolated colonies validation

：

\*Reagents
for pGex-UAO

\*Reagents
for pGex-UAA

STEP
II

-Incubate
the BL21 bacteria.

STEP
III

-After
the examination for vectors pGex-UAO and pGex-UAA, start to retrieve
the vectors from the dH5alpha cells by centrifuging.

STEP
IV

-Transformation:
transfer the vectors acquired to the incubated bacteria BL21.

STEP
V

-Incubate
the BL21 samples with vectors. (acquired from the transformation
procedure)

STEP
VI

-Use
electrophoresis to examine the vector in BL21.

STEP
VII

-Carry
out IPTG Induction

![](https://static.igem.wiki/teams/5024/wiki/wechatimg63.jpeg)

**Day
6 23/8/5**

The
purposes of today’s experiments are extracting the protein
synthesized, purifying the protein synthesized to extract the
enzymes(target products), and determining the optimum concentration
for IPTG induction by measuring the concentration of IPTG used in
each group and the concentration of protein in the products.

STEP
I

-Exposes
the proteins synthesized by ultrasonication.

STEP
II

-Purify
the proteins obtained by using Ni-NTA resin.

**Day
7 23/8/6**

The
experiments today involve creating gels for SDS-PAGE and using
SDS-PAGE to separate the proteins obtained in previous procedures,
which demonstrates that the BL21 bacteria with the vector pGex-UAO
and pGex-UAA is able to synthesize Uric acid oxidase, allantoinase,
and allantoicase successfully.

STEP
I

-Introduce
the SDS-PAGE and create the gel for the electrophoresis.

STEP
II

-Prepare
for the sample used in SDS-PAGE analysis.

Step
III

-Start
the electrophoresis process.

-Construct
the solid culture medium for Nissle 1917.

Step
V

-Start
the transformation process, transform the pGex-UAO vectors to the
probiotics Nissle 1917.

![](https://static.igem.wiki/teams/5024/wiki/wechatimg64.jpeg)

**Day
8 23/8/7**

The
main tasks today are examining the enzymatic activity of the enzymes
produced by BL21 containing pGex-UAO and pGex-UAA, creating a
standard curve that refers to the relationship between the
concentration of uric acid and the effectiveness of the enzyme.

STEP
I

-Prepare
for the samples that are used to create the standard curve, which
reflects the relationship between the concentration of the Uric acid
and the absorbance of the product.

STEP
II

-Prepare
for the samples of testing UAO, construct the system of testing, and
obtain the results.

**Day**

**9**

**23/9/8**

1.
Amplification of pGEX-UAO, allantoinase, allantoicase:

2.
Homologous recombination of pGEX-UAO, allantoinase, allantoicase:

**Day**

**10**

**23/9/10**

1.
Validation of single clones and shaking culture:

2.
Plasmid extraction and sequencing

![](https://static.igem.wiki/teams/5024/wiki/wechatimg65.jpeg)

**Day
1**

**1**

**23/9/11**

**1.**

Transformation into BL21

2.
Single clone validation and shaking culture

**Day1**

**2**

**23/9/12**

1.
Scale-up cultivation

2.
Protein induction

![](https://static.igem.wiki/teams/5024/wiki/wechatimg66.jpeg)
![](https://static.igem.wiki/teams/5024/wiki/wechatimg67.jpeg)

**Day1**

**3**

**23/9/13**

1.
Protein extraction

2.
Protein purification

3.
SDS-PAGE

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a part of your project by going through at least one iteration of the
engineering design cycle.{% endblock %}
{% block page\_content %}

![](https://static.igem.wiki/teams/5024/wiki/g-out-engineering.png)

**Engineering**

**S**

**uccess**

**Overview**

Uric
acid is a waste product of the metabolism of purines in the food,
most of the uric acid is excreted through liver. However, if the body
is unable to efficiently excrete uric acid, uric acid level will
rise, forming urate crystal and deposit in the joints and surrounding
soft tissue, causing a painful and inflammatory response that leads
to joint pain, and swelling
1,2

.
Some animals use urate oxidase to catalyze the conversion of uric
acid into allantoin, which helps reduce the accumulation of uric acid
in the body( Figure 1).

Our
project aims to use probiotics Nissle 1917 to synthesize the
enzymes(UAO, allantoinase, and allantoicase) that can digest the uric
acid to work as a cure for gout. We constructed specific plasmids
pGex-4T-1-UAO (BBa\_K5024007) and pGex-4T-1-UAA (BBa\_K5024008), and
transformed them

in

to
the target probiotics Nissle 1917.

![](https://static.igem.wiki/teams/5024/wiki/wechatimg69.jpeg)

Figure
1. The engineering schematic diagram of the project

**ROUND
1 pGex-4T-1-UAO (**
[**BBa\_K5024007**](http://parts.igem.org/Part%3ABBa_K5024007)

**)**

**DESIGN**

Two
sections are shown as follow:

1. **pET28a-UAO**

We
firstly choose pET28a as the vectors for the plasmids with target
gene sections UAO. We choose pET28a for the His section it involves,
which is crucial in creating pGex-UAO plasmids via homologous
recombination and purifying the proteins synthesized with Ni-NTA(The
Nicole ions in Ni-NTA ensures that the protein synthesized with HIS
sequence can attach to it while other proteins cannot, and the target
proteins are thus retained during purification). The Obtained target
gene sections after this process thus all contain HIS sequence. The
product Uric acid oxidase and pET28a vector were digested with NEB
restriction enzymes BamHI and XhoI, and the vector fragment was
recovered by gel recovery (Figure 2).

![](https://static.igem.wiki/teams/5024/wiki/wechatimg70.jpeg)

Figure
2. The plasmid of pET28-UAO

2. **pGex-4T-1-UAO**

To
convert to Nissle1917, we replaced pET28a with pGEX-4T-1. The
pGex-4T-1 is chosen for its property of functioning stably in Nissle
1917. The next step is to construct the plasmids that are directly
used for target proteins synthesis in following processes. The
vectors are pGex-4T-1 and the target genes are HIS-UAO acquired. At
the same time, the GST tag of pGEX4T-1 can be replaced with His. We
replaced the GST tag on pGEX-4T-1 with His by homologous
recombination.

![](https://static.igem.wiki/teams/5024/wiki/wechatimg71.jpeg)

Figure 3.The picture of pGex-4T-1-UAO plasmid.

**BUILD**

1. **pET28a-UAO**

We
extract the pET28a vectors from existing bacteria solutions.
Primarily using centrifuging, we extract the vectors from the
bacteria solutions. The final products are obtained from the remnants
in the absorption column. Then, the vectors are linearized by using
restrictive enzymes. The electrophoresis is conducted to examine the
products. Cyclical vectors move faster during the electrophoresis
than the linearized vectors.

By
comparing the horizontal relationships between the stripes of target
gene sections and the marker, we have concluded that in the same
conditions, the pET28a control group moves faster during the
electrophoresis, and the pET28a samples move slower during the
electrophoresis. The length of the pET28a vector is 5369 bp, and the
control group of Figure 4 is correct. Line 1-2 is the band of pET28a
cut with BamHI and XhoI. This phenomenon in the figure indicates that
the vectors pET28a in the sample groups have been linearized.

![](https://static.igem.wiki/teams/5024/wiki/wechatimg72.jpeg)

Figure 4. The electrophoresis for testing the linearized vector pET28a.

Note

：

Line1-
2: The band of pET28a cut with BamHI and XhoI

Line
3- 4: The band of pET28a cut with BamHI and NotI

Line
5: The band of pET28a cut with SacI and XhoI

The
lengths of gene fragments UAO are 981bp. By comparing the horizontal
relationships between the stripes of target gene sections and the
marker, Figure 5 concluded that the fragment lengths are consistent
with the results. Therefore, the figure indicates that target genes
of UAO are successfully amplified.

![](https://static.igem.wiki/teams/5024/wiki/wechatimg73.jpeg)

Figure 5. The electrophoresis for the target genes

Next,
we connected UAO with pET28a by T4 ligase, and transformed the
plasmid into DH5α. Figure 6A shows that the

isolated
colonies were

successfully grown, and the

isolated
colonies were

selected for colony verification. The length of the target gene UAO
is 981 bp. Figure 6C concluded that the fragment lengths are
consistent with the results. We have successfully the vector
pET28a-UAO was transformed into DH5α, and the sequencing
results in Figure 6B showed no gene mutation. It indicates that our
plasmid has been successfully constructed.

![](https://static.igem.wiki/teams/5024/wiki/wechatimg74.jpeg)

Figure 6. The Electrophoresis of the Monoclonal Antibody in DH5

2. **pGEX-4T-1-UAO**

The
length of UAO is 981 bp. Figure 7A shows that the fragment lengths
are consistent with the results. It indicates that we have
successfully amplified the target gene UAO. We use inverse-PCR to
replicate the samples of pGex-4T-1. Inverse-PCR will replicate the
section except the section selected. Through inverse PCR, vast amount
of incomplete pGex-4T-1 can be perceived. After replication,the
results are tested by electrophoresis. The length of vector pGex-4T-1
extracted is 4328bp (Figure 7B). By comparing the horizontal
relationships between the stripes of extracted vector and the marker,
we have concluded that the vector lengths are consistent with the
results. Therefore, the figure indicates that vectors pGex-4T-1 are
successfully linearized.

![](https://static.igem.wiki/teams/5024/wiki/wechatimg75.jpeg)

Figure 7. The Electrophoresis for the target genes UAO and pGEx-4T-1 vectors

After
constructing the target plasmids pGex-4T-1-UAO, they are transferred
to the

*E.coli*

DH5α
for replication. The figure 8A and B show that the vector
pGex-4T-1-UAO was transformed into DH5α, The length of UAO is
981 bp. Figure 8B shows that the fragment lengths are consistent with
the results. It proves that the plasmid pGex-4T-1-UAO was
successfully constructed. And the sequencing results of pGex-4T-1-UAO
in Figure 6 CD showed that UAO has no gene mutation. It indicates
that our plasmid pGex-4T-1-UAO has been successfully constructed.

![](https://static.igem.wiki/teams/5024/wiki/wechatimg76.jpeg)

Figure 8. The electrophoresis of the Monoclonal Antibody in DH5

Note:

A:
Culture plates pGex-4T-1-UAO for DH5α

B:
Electrophoresis of the monoclonal Antibody pGex-4T-1- UAO

C
and D: The Sequencing results of pGex-4T-1-UAO

**TEST**

1. **Protein
   expression**

**1.
pGex-4T-1-UAO (BL21(DE3))**

In
order to improve the expression of the protein, we first tested the
protein in

*E.coil*

BL21(DE3). So we transformed the plasmid pGex-4T-1-UAO into
BL21(DE3). Figure 9A shows that the

isolated
colonies were

successfully grown, and the

isolated
colonies were

selected for colony verification. The length of UAO is 981 bp. Figure
9B shows that the fragment lengths are consistent with the results.
The vector pGex-4T-1-UAO was successfully transformed into BL21(DE3).

![](https://static.igem.wiki/teams/5024/wiki/wechatimg77.jpeg)

Figure 9. The Electrophoresis of the monoclonal antibody pGEX-UAO in
BL21(DE3)

In
order to improve the expression of UAO protein, we designed different
gradients of IPTG induction. Table 1 and Figure 10 showed that the
protein concentration of pGEX-UAO increased first and then decreased
with the increase of IPTG concentration. When the concentration of
IPTG was 0.5 mmol, the protein concentration of UAO was the highest.

Table1:
Protein concentration of UAO (mg/mL)

|  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- |
|  | **IPTG concentration (mmol/L)** | | | | |
|  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- |
| **Sample #** | **0.050** | **0.250** | **0.500** | **0.800** | **1.000** |
| **1** | 1.016 | 1.320 | 5.101 | 0.783 | 0.817 |
| **2** | 0.583 | 0.992 | 4.764 | 0.652 | 0.814 |
| **3** | 0.656 | 1.402 | 4.759 | 0.625 | 0.831 |
| **Average** | 0.752 | 0.991 | 4.875 | 0.687 | 0.821 |
| **SD** | 0.232 | 0.217 | 0.196 | 0.085 | 0.009 |

![](https://static.igem.wiki/teams/5024/wiki/wechatimg78.jpeg)

Figure
10. The relationship between the concentration of IPTG and proteins

Next,
we induced the protein UAO with 0.5 mmol IPTG. The size of the UAO
protein is 40 kDa. Figure 11 shows that the crude protein has the
target protein UAO, and the purified protein solution also has a
small amount of UAO, but the purification loses som
e
proteins.

![](https://static.igem.wiki/teams/5024/wiki/wechatimg79.jpeg)

Figure11.
The pGex-4T-1-UAO of SDS-PAGE in

*E.coil*

BL21(DE3)

**2.pGex-4T-1-UAO
(Nissle 1917)**

We
transformed the plasmid pGex-4T-1-UAO into Nissle 1917. Figure 12 B
shows that the

isolated
colonies were

successfully grown, and the

isolated
colonies were

selected for colony verification. The length of the target gene UAO
is 981 bp. Figure 12A shows that the fragment lengths are consistent
with the results. The vector pGex-4T-1-UAO was successfully
transformed into Nissle 1917.

![](https://static.igem.wiki/teams/5024/wiki/wechatimg80.jpeg)

Figure
12. Electrophoresis of the monoclonal antibody for pGex-UAO in Nissle
1917

Meanwhile,
we induced the protein UAO with 0.5 mmol IPTG in Nissle 1917. The
size of the UAO protein is 40 kDa. Figure 13 shows that the crude
protein has the target protein UAO. Nissle 1917 had no target protein
of UAO in the control group. It proved that The protein of UAO was
successfully expressed

![](https://static.igem.wiki/teams/5024/wiki/wechatimg81.jpeg)

Figure
13. The pGex-4T-1-UAO of SDS-PAGE in Nissle 1917

**B.The
enzyme activity detection of UAO**

**1.
The Standard Curve**

Firstly,
we made a standard curve for the determination of uric acid. The
table 1 is the original data of the table. We use a linear function
to fit. Figure 14 shows that the equation y = 1.1529x + 0.2129 is
satisfied, and the correlation coefficient is greater than 0.99.

Table
1.Result and curve for the Standard Curve

|  | Uric acid concentration (μmol/mL) | | | | | | |
| Sample# | 0.25 | 0.125 | 0.0625 | 0.0312 | 0.015625 | 0.007313 | 0 |
| 1 | 0.5086 | 0.3475 | 0.2824 | 0.2529 | 0.2338 | 0.2200 | 0.2110 |
| 2 | 0.5060 | 0.3469 | 0.2824 | 0.2536 | 0.2348 | 0.2199 | 0.2120 |
| 3 | 0.5052 | 0.3485 | 0.2832 | 0.2537 | 0.2353 | 0.2201 | 0.2128 |
| 4 | 0.5032 | 0.3475 | 0.2839 | 0.2536 | 0.2348 | 0.2205 | 0.2119 |
| 5 | 0.5042 | 0.3473 | 0.2843 | 0.2542 | 0.2355 | 0.2203 | 0.2129 |
| average | 0.50544 | 0.34754 | 0.28324 | 0.2536 | 0.23484 | 0.22016 | 0.21212 |
| SD | 0.002056 | 0.00059 | 0.000862 | 0.000464 | 0.000658 | 0.000241 | 0.000773 |

![](https://static.igem.wiki/teams/5024/wiki/wechatimg82.jpeg)

Figure
1

4

.
The Standard Curve of Urate Oxidase

**2.
The enzyme activity of UAO**

In
order to determine the ability of our constructed plasmid
pGex-4T-1-UAO to decompose uric acid, we used uric acid as a
substrate and added UAO protein. Table 2 and Figure 1

5

showed that with the increase of UAO protein concentration, the
content of uric acid gradually decreased, indicating that UAO is
active and can successfully decompose uric acid. And we used

*E.coil*

BL21
( DE3 ) as the control group, the uric acid content of the control
group had no obvious change trend.

Table
2. Uric acid oxidase activity of pGEX-4T-1-UAO

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| Concentration of UAO(mg) | Experimental group | SD | Control (  *E.coil*  BL21 ) | SD |
|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| 0.00000 | 0.23052 | 0.00058103 | 0.23583 | 0.002199284 |
| 0.01335 | 0.22330 | 0.00095916 | 0.23493 | 0.007408525 |
| 0.02670 | 0.18474 | 0.00102489 | 0.23918 | 0.006044871 |
| 0.04005 | 0.15558 | 0.00051146 | 0.22762 | 0.011768322 |
| 0.05340 | 0.15154 | 0.00127843 | 0.22007 | 0.014096617 |

![](https://static.igem.wiki/teams/5024/wiki/wechatimg83.jpeg)

Figure
1

5

.The
curve uric acid oxidase activity of pGEX-4T-1-UAO

**LEARN**

Analysis
of experimental skills:

| Issue/Phenomenon | analysis | solution |
| The spread of dH5alpha bacteria’s colonies on solid culture plates is not obvious: There aren’t visible spots that refer to dH5alpha bacteria colonies. | The procedure of preparing and applying the bacteria on the culture plate may involve operational mistakes: The time for shaking bacteria solutions may be inadequate, which results in inadequate amount for the dH5alpha on the plate for observation; the bacteria solution may have been applied violently, which damages the bacteria, thus induces failure for their growth. | Extend the bacteria shaking time for 2 hours; Improve the applying operation to ensure that the bacteria are evenly and mildly spread. |
| According to the results of SDS-PAGE, the stripes referring to target proteins(UAO) are not obvious. However, the stripes referring to the protein supernatant just after ultrasonication are obvious. | The problem may be induced by two reasons:   1. inadequate    time taken for the protein supernatant to attach with the    Ni-NTA. - the      excessive volume of wash buffer used for excluding the unwanted      proteins synthesized. | Using the samples preserved, operate the entire procedure again: Extend the time that the protein supernatant has contacted to the Ni-NTA resin and reduce the volume of the His-A buffer used. |

In
future studies, other iGEM teams can optimize the expression levels
of urate oxidase (UAO). You will further test the protein expression
and enzyme activity of pGEX-4T-1-UAO in Nissle 1917 to find the
optimal expression conditions. You also need to validate the ability
of urate oxidase (UAO) to degrade uric acid in large quantities.
Additionally, The role of urate oxidase in degrading uric acid in the
body is still pending verification. The safety of probiotics also
requires further validation. Our project can provide data support for
the treatment of gout. To establish a new treatment method, we think
not only for our country is very practical, but will also have a good
application prospect in the world.

**ROUND
2 pGex-4T-1-UAA (**
[**BBa\_K5024008**](http://parts.igem.org/Part%3ABBa_K5024008)

**)**

**DESIGN**

Two
sections are shown as follow:

1. **pET28a-allantoinase
   and pET28a-allantoinase**

We
firstly choose pET28a as the vectors for the plasmids with target
gene sections allantoinase and allantoicase. We choose pET28a for the
HIS section it involves, which is crucial in creating pGex-4T-1-UAA
plasmids via homologous recombination and purifying the proteins
synthesized with Ni-NTA. The obtained target gene sections after this
process thus all contain HIS sequence. The product Uric acid oxidase
and pET28a vector were digested with restriction enzymes BamHI, XhoI
and NotI, and the vector fragment was recovered by gel recovery
(Figure 1

6

).

![](https://static.igem.wiki/teams/5024/wiki/wechatimg84.png)

Figure
1

6

.
The plasmid map of pET28a-allantoinase and pET28a- allantoinase

2. **pGex-4T-1-UAA**

To
convert to Nissle1917, we replaced pET28a with pGEX-4T-1. The
pGex-4T-1 is chosen for its property of functioning stably in Nissle
1917. The next step is to construct the plasmids that are directly
used for target proteins synthesis in following processes. At the
same time, the GST tag of pGEX4T-1 can be replaced with His. We
replaced the GST tag on pGEX-4T-1 with His by homologous
recombination. We use homologous recombination to assemble
allantoinase and allantoicase into pGEX-4T-1-UAA(Figure 1

7

).

![](https://static.igem.wiki/teams/5024/wiki/wechatimg85.jpeg)

Figure
1

7

.
The plasmid map of pGEX-4T-1-UAA

**BUILD:**

1. **pET28a-
   allantoinase and pET28a- allantoicase**

We
added restriction sites BamHI and NotI to primer, and further
amplified the target gene

a

llantoinase
by PCR. The length of the target gene

a

llantoinase
is 1485 bp. Figure 17 shows that the fragment lengths are consistent
with the results. We added restriction sites SacI and XhoI to primer,
and further amplified the target gene allantoicase by PCR. The length
of the target gene allantoicase is 1288 bp. Figure 1

8

shows that the fragment lengths are consistent with the results. It
that we have successfully amplified the target gene allantoicase.

![](https://static.igem.wiki/teams/5024/wiki/wechatimg86.jpeg)

Figure
1

8

.
Electrophoresis for the target genes allantoinase and allantoicase

Then,
we cut the vector pET28a with restriction endonuclease, and used the
uncut vector as a control. Line 1-2 is the band of pET28a cut with
BamHI and XhoI in the figure 1

9

.
Line 3-4 is the band of pET28a cut with BamHI and NotI. Line 5 is the
band of pET28a cut with SacI and XhoI in the figure 18. The results
show that the plasmid has been cut into linearization.

![](https://static.igem.wiki/teams/5024/wiki/wechatimg87.jpeg)

Figure
1

9

.
The electrophoresis for testing the linearized vector pET28a

Note

：

Line1-
2: The band of pET28a cut with BamHI and XhoI

Line
3- 4: The band of pET28a cut with BamHI and NotI

Line
5: The band of pET28a cut with SacI and xhoI

Next,
we connected

a

llantoinase
and allantoicase with pET28a by T4 ligase, and transformed the
plasmid into DH5α. Figure

20

A
shows that the

isolated
colonies were

successfully
grown, and the

isolated
colonies were

selected
for colony verification. The length of the target gene

a

llantoinase
is 1485 bp. Figure

20

C
shows that the fragment lengths are consistent with the results. The
length of the target gene allantoicase is 1288 bp. Figure

20

C
shows that the fragment lengths are consistent with the results. We
have successfully the vector pET28a-UAO,

pET28a-

a

llantoinase
and

pET28a-

allantoicase
were transformed into DH5α, and the sequencing results in
Figure

20

B
showed no gene mutation. It indicates that our plasmid has been
successfully constructed.

![](https://static.igem.wiki/teams/5024/wiki/wechatimg88.jpeg)

Figure

20

.
The Electrophoresis of the Monoclonal Antibody in DH5α

Note:

A
Culture plates UAO, allantoinase and allantoicase for DH5α

B.Electrophoresis
of the Monoclonal Antibody UAO, allantoinase and allantoicase

C.The
sequencing result of pET28a-UAO,pET28a-

a

llantoinase
and pET28a-allantoicase

**B.
pGex-4T-1-UAA**

The
length of the target gene

a

llantoinase
is 1485 bp. The length of the target gene allantoicase is 1074 bp.
Figure 2

1

A
shows that the fragment lengths are consistent with the results. The
length of the plasmid pGex-4T-1-UAO is 5301 bp. Figure 2

1

B
shows that the fragment lengths are consistent with the results. It
indicates that we have successfully amplified the target gene.

![](https://static.igem.wiki/teams/5024/wiki/wechatimg89.jpeg)

Figure
2

1

.
Electrophoresis for the genes

a

llantoinase,
allantoicase and pGex-4T-1-UAO

Next,
we transformed the plasmid pGex-4T-1-UAA into DH5α. Figure 2

2

AB
shows that the

isolated
colonies were

successfully
grown, and the

isolated
colonies

were
selected for colony verification. The length of the target gene

a

llantoinase
is 1485 bp. Figure 2

2

C
shows that the fragment lengths are consistent with the results. In
Figure 2

2

D,
line1,4,5 shows that the allantoicase band is approximately 1024bp.
It indicates that we have successfully translated the plasmid
pGex-4T-1-UAA to

*E.coil*

DH5α.

![](https://static.igem.wiki/teams/5024/wiki/wechatimg90.jpeg)

Figure
2

2

.
The electrophoresis of the monoclonal antibody in

*E.coil*

DH5α

Afterward,
we chose positive

isolated
colonies

1,
4, and 5 and sent them to the company for sequencing. The results
showed that the sequencing of the allantoicase and allantoinase genes
on the pGex-4T-1-UAA plasmid was normal (Figure 2

3

),
without any genetic mutations. It indicates that our plasmid
pGex-4T-1-UAA has been successfully constructed.

![](https://static.igem.wiki/teams/5024/wiki/wechatimg91.jpeg)

Figure
2

3

.
The sequencing of the allantoicase and allantoinase genes on the
pGex-4T-1-UAA plasmid

**TEST**

1. **Protein
   expression**

In
order to improve the expression of the protein, we first tested the
protein in

*E.coil*

BL21(DE3). So we transformed the plasmid pGex-4T-1-UAA into

*E.coil*

BL21(DE3). Figure 2

4

A
and B show that the

isolated
colonies were

successfully
grown, and the

isolated
colonies were

selected
for colony verification. The length of the target gene allantoinase
is 1485 bp and allantoinase is 1047bp. Figure 2

4

C
and D show that the vector pGex-4T-1-UAA was successfully transformed
into

*E.coil*

BL21(DE3).

![](https://static.igem.wiki/teams/5024/wiki/wechatimg92.jpeg)

Figure
2

4

.
The electrophoresis of the monoclonal antibody for pGex-UAA in
BL21(DE3)

Note:

Figure
23A-B: The monoclonal plate pGex-UAA in BL21(DE3)

Figure
23C: The Electrophoresis verification of allantoicase in pGex-UAA
(BL21(DE3))

Figure
23d: The Electrophoresis verification of allantoinase in pGex-UAA
(BL21(DE3))

In
order to improve the expression of UAA protein, we designed different
gradients of IPTG induction. The size of the UAA protein is 118.6
kDa. Figure 2

5

shows that the crude protein has the target protein UAA. However,
0.1mmol, 0.25 mmol, 0.5mmol ,0.7mmol and 1mmol IPTG had no
significant effect on the expression of UAA protein.

![](https://static.igem.wiki/teams/5024/wiki/wechatimg93.jpeg)

Figure
2

5

.
The pGex-4T-1-UAA of SDS-PAGE in

*E.coil*

BL21(DE3)

2. **Enzyme
   activity detection**

1.The
urate oxidase activity of UAA

In
order to determine the ability of our constructed plasmid
pGex-4T-1-UAA to decompose uric acid, we used uric acid as a
substrate and added UAO protein. Table 3 and Figure 2

6

showed that with the increase of UAA protein concentration, the
content of uric acid gradually decreased, indicating that UAA is
active and can successfully decompose uric acid. And we used

*E.coil*

BL21
(DE3) as the control group, the uric acid content of the control
group had no obvious change trend.

Table
3. The urate oxidase activity of UAA

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| Concentration of UAA(mg) | Experimental group | SD | Control | SD |
|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| 0.00000 | 0.247288 | 0.015616036 | 0.194080567 | 0.089358727 |
| 0.01335 | 0.215044 | 0.020638737 | 0.188096847 | 0.084278254 |
| 0.02670 | 0.186315 | 0.010903707 | 0.182951779 | 0.088550902 |
| 0.04005 | 0.142994 | 0.021953815 | 0.169561617 | 0.081267084 |
| 0.05340 | 0.137083 | 0.018292321 | 0.163117255 | 0.079973725 |

![](https://static.igem.wiki/teams/5024/wiki/wechatimg94.jpeg)

Figure
2

6

.
The curve of UAA activity

2.Contrast
enzyme activity of UAO and UAA

By
comparing the levels of uric acid after adding UAA and UAO
proteins(Figure 2

7

),
it was found that the uric acid content was lower with UAA compared
to UAO. This suggests that UAA has slightly higher enzymatic activity
than UAO, further indicating that UAA has a stronger ability to
degrade uric acid. However, the protein expression level of UAA can
be optimized in order to enhance its enzymatic activity in subsequent
experiments.

![](https://static.igem.wiki/teams/5024/wiki/wechatimg95.jpeg)

Figure
2

7

.
The curve of UAA and UAO activity

**LEARN**

Analysis
of experimental skills:

| Issue/Phenomenon | analysis | solution |
| There is almost no result in the electrophoresis examination for the pGex-UAA plasmids. The stripes are not evident or sheer absent. | This may be induced by two reasons:  1. the occurrence of hybrid bacterium in the incubated dH5alpha samples. These bacteria aren’t able to replicate the plasmids, or they don’t contain target plasmids.  2. the time for heat shock when transforming the plasmids to the dH5alpha bacteria is inadequate. Which induces results that plasmids are not fully transformed to the dH5alpha. | Operate the entire procedure again: select appropriate bacteria solutions of dH5alpha and extend the transformation time from 12-16 hours to 24 hours. |
| One of the sample groups shows the concentration of uric acid inconsistent with the concentrations of the proteins. | This may be induced by two reasons:   1. the    wrong proportion of different agents used for constructing the    system, which causes discrepancy between actual concentration of    proteins and the theoretical values. The values calculated thus    remain potentially unrelated to the concentration of the    proteins. - Moreover,      there may be errors when adding agents since the volumes      required are infinitesimal. Within the subtle changes in volume      when adding certain number of reagents for multiple times, the      final value may be inaccurate. | Create all the samples for the experiment again and measure the values of absorbance. When creating the mixture, notice the concentration of the uric acid and enzymes. |

In
future studies, other iGEM teams can optimize the expression levels
of fusion protein (urate oxidase, allantoinase, and allantoicase).
You will further test the protein expression and enzyme activity of
pGEX-4T-1-UAA in Nissle 1917 to find the optimal expression
conditions. You can also validate the ability of pGEX-4T-1-UAA to
degrade uric acid in large quantities. Additionally, The role of
urate oxidase in degrading uric acid in the body is still pending
verification. The safety of probiotics also requires further
validation. Our project can provide data support for the treatment of
gout. To establish a new treatment method, we think not only for our
country is very practical, but will also have a good application
prospect in the world.

**Reference**

1.

T.
Neogi, Clinical practice. Gout.

*N
Engl J Med*

**364**

,
443-452 (2011).

2.

Y.
Zhu, B. J. Pandya, H. K. Choi, Prevalence of gout and hyperuricemia
in the US general population: the National Health and Nutrition
Examination Survey 2007-2008.

*Arthritis
Rheum*

**63**

,
3136-3141 (2011).

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
{% block page\_content %}

![](https://static.igem.wiki/teams/5024/wiki/g-out-experiments.png)

**Experiments**

* **The
  plasmid construction of
  pET28a-UAO**

  **、**

  **pET28a-**

  **allantoinase**

  **、**

  **pET28a-**

  **allantoicase**

1. **Preparing
   Culture medium**

Materials:

Tryptone
, Yeast extract, ddH
2

O,
NaCl, Digital balance

Procedure:

A:
Ensure the volume of culture medium required is 500ml, and the
concentrations required for NaCl, tryptone and yeast extract are
10g/L, 10g/L and 5g/L.

| Reagent | Volume/mass |
| Tryptone | 5g |
| Yeast extract | 2.5g |
| Ddwater | 500ml |
| NaCl | 5g |

B:
Measure 100ml ddwater with measuring cylinder. Add the water
perceived into a flask.

C:
Use digital balance to measure 5g of NaCl sample. Set the sample on a
weighing paper and add solid sample to the balance via spatula. After
measuring, add the sample in the flask.

D:
repeat the procedure with measurement of 5g tryptone sample.

E:
repeat the procedure with measurement of 2.5g yeast extract sample.

F:
Seal the flask with culture medium with tape. Label it as “LB
Culture” and prepare for its further sterilization.

2. **Amplify
   the Target Gene PCR**

   **（**

   **UAO**

   **、**

   **Allantoinase
   and Allantoicase**

   **）**

**2.1
PCR**

Materials

UAO,
PCR thermal cycler, Allantoinase, Allantoicase, primer, Mix

Procedure
I

A:
Measure 10μl of mix by pipette and add it to the PCR tube.

B:
Measure 7μl of ddwater by pipette and add it to the PCR tube.

C:
Measure 1μl of primer R1 and 1μl of primer F1 by pipette to the
PCR tube.

D:
Measure 1μl of template by pipette and add it to the PCR tube.

E:
Fully mix the substances in the tube by attaching the PCR tube to the
vortex.

\*The
sequence of adding substances is arranged by their gradient in
amounts.

| UAO(981bp) | Amount | Allantoinase(1485bp) | Amount | Allantoicase(1228bp) | Amount |
| 2mix | 10μl | 2mix | 10μl | 2mix | 10μl |
| UAO-R1 | 1μl | A-nase-R1 | 1μl | A-case-R1 | 1μl |
| UAO-F1 | 1μl | A-nase-F1 | 1μl | A-case-F1 | 1μl |
| UAO template | 1μl | Allantoinase template | 1μl | Allantoicase template | 1μl |
| ddwater | 7μl | ddwater | 7μl | ddwater | 7μl |
| total | 20μl | total | 20μl | total | 20μl |

\*2
mix: Since the product possesses volume of 20 μl, the amounts of
substances in “mix”. should be doubled so that the
concentration is constant.

\*mix
contains DNA polymerase, dNTP(dATP, dCTP, dGTP, or dTTP), buffer,
Mg2+ ions.

\*bp
refers to the number of base groups contained in a DNA chain

Procedure
II:

| PCR program | Temp. (celsius degrees) | Period |
| Pre-denaturation | 95 | 3min. |
| Denaturation | 95 | 30sec. |
| Annealing | 55 | 30sec. |
| Elongation | 72 | 2min. |
| Elongation | 72 | 30sec. |

**2.2
Agarose Gel Electrophoresis**

Materials

Agarose,
TAE1, Nucleic acid dye

Procedure

A:
preparation of gel:

Ensure
that the volume required is 100ml.

Prepare
a flask. Use measuring cylinder to measure 100ml TAE and add it to
the flask. Use digital balance and weighing paper to measure 1.5g
agarose sample and add it to the flask.

Heat
the flask at high temperature for 3 minutes in microwave oven.

Add
2μl nucleic acid dye into the flask.

\*The
theoretic amount of agarose required is 1g; however, the result that
the gel fails to form solid structure and there’re regions
remained uncoagulated demonstrates that more agaroses are needed.
Consequently, we increase its mass by 50%.

B:
Add the gel into the gel box. Wait for approximately 10 minutes, when
it fully congeals and forms jelly-like substance.

C:
Use pipette to measure 7μl product from the PCR program(it
originally possesses 20μl), and add it to the well comb of the
apparatus for electrophoresis.

D:
Add 10μl DNA marker for reference of the categorization for the
molecules separated.

E:
Electrophoresis: connect the apparatus to the power supply with 180V
for 15 minutes.

F:
sever the connection to the power supply. Place the gel in the Blue
LED transilluminator

，

and
observe it under the illumination of ultraviolet radiation.

RESULT:
the signs that refer to PCR samples for each group approximately
correspond to the marker(according to the length of templates).

**2.3
Recovery of the products of PCR**

\*purpose:
the product of PCR contains several different substances, while the
replicated DNA is the required section. They are thus extracted from
the product.

Materials

Buffer
B2, Product of PCR

Procedure

A.
Obtain a centrifuge tube which has a volume of 1.5ml. Use pipette to
measure 300μl buffet b2 and add it to the tube.

B.
Use pipette to measure 13μl from the product of PCR and add it to
the centrifuge tube.

\*13μl
is the volume of product of PCR remaining after electrophoresis.

C.
Transfer the mixture in the centrifuge tube to the absorption column.
Attach it to a centrifuge shield and place it in the
centrifuge(machine), start the centrifuging process with centrifugal
force of 8000 xg and time of 30 seconds. Remove the mixture in the
centrifuge shield after the process.

D.
Use pipette to add 500μl wash buffer into the absorption column
and start the centrifuging process with centrifugal force of 9000 xg
and time of 30 seconds. Remove the mixture in the centrifugal shield.

E.
Repeat procedure D

F.
Without adding any substance, start centrifuging with centrifugal
force of 9000 xg and time of 1 minute.

G.
Obtain a new centrifuge tube, add 30μl Elusion Buffer to it via
pipette, place it at rest for 1 minute, then centrifuge it with 9000
xg for 1 minute. After that, preserve the centrifuge tube.

H.
Measure the concentration of the remaining mixture: by using
spectrophotometer

-Use
ddwater to rinse the detector of the apparatus.

-add
two samples of ddwater as control group: for the situation of adding
nothing(pure solvent). The result is close to zero.

-Add
sample to the detector.

-the
concentration of five known samples from the experiment: 22.65;
24.35; 27.05; 16.35; 20.2. (unit: ng/μl)

3. **Plasmid
   extraction and enzyme digestion plasmid**

**3.1
Extraction of Plasmids**

Materials

Buffer
SP1,Buffer SP2,Buffer SP3

Procedure

A.
Use pipette to measure 2ml of bacteria solution and transfer it to a
2ml centrifuge tube(use pipette with maximum range of 1000μl and
operate twice). Place the centrifuge tube into the
centrifuge(machine), and then start centrifuging with centrifugal
force of 12000 xg and time of 1 minute.

\*purpose:
To separate the cells and medium in the bacteria solution.

B.
Use pipette to measure 250μl of buffer SP1, add it to the
centrifuge tube in procedure A. Continuously inject and retrieve the
liquid in the pipette, which separates the bacteria aggregated at the
bottom of the tube and spread them till they suspend/levitate in the
solution.

\*purpose:
To spread the bacteria in the tube.

C.
use pipette to measure 250μl of buffet SP2 and add it to the
centrifuge tube.

\*purpose:
to crack the cell membranes of the bacteria, which exposes the
plasmids to the solution.

D.
Use pipette to measure 350μl of buffet SP3 and add it to the
centrifuge tube.

\*purpose:
to neutralize buffet SP2.

E.
Centrifuge the product in procedure D with centrifugal force of 12000
xg and time of 5 minutes. After that process, it can be observed that
white, solid substances remain at the bottom of the tube.

\*purpose:
to separate the organelles and remnants of cell membranes with the
plasmids.

F.
Use pipette to measure 700μl of supernatant in the centrifuge tube
and transfer it to the absorption column, then centrifuge it with
force of 9000 xg and time of 1 minute.

G.
Use pipette to measure 500μl wash buffer and add it to the
absorption column. Centrifuge it with force of 9000 xg and time of 30
seconds. Repeat this procedure again.

H.
Centrifuge the product of procedure G without adding any agent. The
indices are 9000 xg and 1 minute.

I.
Obtain a new centrifuge tube. Use pipette to measure 30μl Elution
Buffer and add it to the absorption column. Place the tube at rest
for a minute, and then centrifuge it with indices of 9000 xg, 1
minute. The remnant in the centrifuge tube is the vector required.

**3.2
Enzyme Cutting**

Materials

pET28a,
template, Enzyme

Procedure

A.Ensure
that to aggregate volume required is 20μl or 50μl. The reagents
are added according to the gradient of their volumes.

\*the
following values are corresponding to the group which cuts
allantoinase.

B.Use
pipette to measure 10μl of pET28a sample created from vector
extraction. Transfer it to the centrifuge tube.

C.Use
pipette to measure 6μl of ddwater and transfer it to the
centrifuge tube.

D.Use
pipette to measure 2μl of Cut smart Buffer and transfer it to the
the centrifuge tube.

E.Use
pipette to measure 1μl of BamHI restriction enzyme and transfer it
to the centrifuge tube.

F.Use
pipette to measure 1μl of NotI restriction enzyme and transfer it
to the centrifuge tube.

G.Attach
the tube to the vortex.

H.Set
the tube to a 37 Celsius Degree environment for 30 minutes.

\*It’s
the optimum temperature for the restriction enzymes.

\*It’s
the procedure for enzyme cutting.

I.
Store the sample in ice.

J.
Repeat procedure A to I while replacing the pET28a vector to
allantoinase (target. gene. )

Group
of UAO:

| Vector system | Amount/μl | Target gene system | Amount/μl |
| PET28a | 10 | UAO | 10 |
| Cut smart buffer | 2 | Cut smart buffer | 2 |
| BamHI | 1 | BamHI | 1 |
| Xhol | 1 | Xhol | 1 |
| Ddwater | 6 | Ddwater | 6 |
| total | 20 | total | 20 |

Group
of a-nase:

| Vector system | Amount/μl | Target gene system | Amount/μl |
| PET28a | 10 | allantoinase | 10 |
| Cut smart buffer | 2 | Cut smart buffer | 2 |
| BamHI | 1 | BamHI | 1 |
| NotI | 1 | NotI | 1 |
| Ddwater | 6 | Ddwater | 6 |
| total | 20 | total | 20 |

Group
of a-case:

| Vector system | Amount/μl | Target gene system | Amount/μl |
| PET28a | 30 | allantoicase | 30 |
| Cut smart buffer | 5 | Cut smart buffer | 5 |
| SacI | 1 | SacI | 1 |
| Xhol | 1 | Xhol | 1 |
| Ddwater | 13 | Ddwater | 13 |
| total | 50 | total | 50 |

**3.3
Electrophoresis**

\*
The product of enzyme cutting for target genes cannot be examined,
since they can’t be separated by certain features.

Materials

Loading
buffer, Marker,Vector samples-control group

Procedure

A.Use
pipette to measure loading buffer and add it to the centrifuge tube
which contains severed pET28a. The volume required for the buffer is
10% of the total volume of the vector sample. (e.g. for allantoinase
group, since the volume of the vector is 20μl, the loading buffer
added is 2μl)

B.Add
samples to the well comb: two samples of 10μl marker, 20μl of
control group sample, and 20μl for each kind of sample.

C.Connect
to 160V electricity for approximately 20 minutes

4. **Enzyme
   ligation and transformation**

**4.1
LB Agar Plates**

Materials

Pre-prepared
liquid sterilized agar solution, Agar powder

Procedure

a)
Acquired sterilized liquid agar solution

b)
Put all materials inside of fume hood workstation; everyone working
under fume hood sterilized hands with ethanol

c)
Light an alcohol lamp within fume hood

d)
Add 3g of agar powder to liquid agar solution and mix

e)
Prepare a 50mL centrifugal tube, pour some of the prepared mixture
and add KANT sample

f)
Pour previously mentioned mixture with KANT evenly onto petri dish,
make sure it spreads throughout the dish and remove any air bubbles

g)
Set aside petri dish with lid awry to cool and solidify

**4.2
Ligation Process**

Materials

pET28a
vector,UAO/a-nase/a-case 12μl,T4 ligase

Procedure

A.Ensure
that the volume required for the product of ligation system is 20μl

B.Obtain
a centrifuge tube(1.5ml), use pipette to measure 12μl product of
target gene(undergone enzyme cutting) and transfer it to the tube.

C.use
pipette to measure 3μl product of pET28a(undergone enzyme cutting)
and transfer it to the tube.

\*The
proportion between volume of gene and vector is 4:1 or 6:1.

D.use
pipette to measure 2μl of 10xT4 ligase and transfer it to the
tube.

E.use
pipette to measure 2μl ddwater and transfer it to the tube.

F.use
pipette to measure 1μl of T4 ligase and transfer it to the tube.

G.Attach
the tube to the vortex.

H.Set
the tube at rest under room temperature for 30 minutes.

| **Reagent** | **Volume/mass** |
| pET28a vector | 3μl |
| UAO/a-nase/a-case | 12μl |
| T4 ligase | 1μl |
| 10xT4 buffer | 2μl |
| Ddwater | 2μl |

**4.3
Transformation of Bacteria**

Materials

Ligated
samples, LB Agar plates and liquid solution

Procedure

a)Ligated
samples are added to

*E.Coli*

samples

b)Placed
on ice for 20 minutes.

c)Heat
at 42 degrees Celsius for 45 seconds, then immediately put on ice for
2-3 minutes.

d)Add
900μL LB Agar solution, incubate for 30 minutes

e)Centrifuge
at 5000xg for 3 minutes, extract 900μL of supernatant then discard

f)Apply
end product to agar plates, incubate for 12-16hr at 37 degrees
Celsius.

\*Possible
mistake: It’s required to spread bacteria mildly, since harsh
physical shock will damage their structure.

**4.4
Isolated colonies validation**

\*purpose:
DH5alpha bacteria containing constructed plasmids has multiplied.

Isolated
colonies validation

is used for ensuring that the plasmids have replicated during this
process.

**Procedure**

a)Within
sterilized fume hood workstation, remove a colony of preprepared
bacteria from the agar plate and add to 1.5mL centrifuge tube (Note:
choose larger colonies but also be aware that colonies near the sides
of the petri dish are most likely to be contaminated or are undesired
bacteria and are less ideal)

b)Using
pipette, add the 10µL mixture to the PCR tube.

c)Using
pipette, add the 1µL F primer to the PCR tube.

d)Using
pipette, add the 1µL R primer to the PCR tube.

e)Using
pipette, add the 8uL of ddH2O to the PCR tube.

f)Attach
the tube to the vortex, then place it to the PCR machine.

| **PCR program** | **Temp. (celsius degrees)** | **Period** |
| Pre-denaturation | 95 | 3min. |
| Denaturation | 95 | 30sec. |
| Annealing | 55 | 30sec. |
| Elongation | 72 | 2min. |
| Repetition-Denaturation,Annealing,Elongation | / | 30times |

**Procedure**

A.
Obtain a conical flask. Use measuring cylinder to measure 100ml of
(1x)TAE solution and transfer it to the conical flask.

B.
Place the agarose sample on the weighing paper, Use digital balance
and spatula to measure 1g of agarose sample and add it to the flask.

C.
Place the flask in the microwave oven and heat at high temperature
for 2 to 3 minutes.

D.
Use pipette to measure 5μl of Nucleic acid dye(Gel red) and add it
to the flask.

E.
Add the sample in the flask to the mode of electrophoresis
instrument.

F.
Wait for approximately 20 minutes till the gel coagulates.

* **Homologous
  Recombination**

  **（**

  **pGex-4T-1-UAO**

  **、**

  **pGex-4T-1-UAA**

  **）**

**1.
PCR pGex-4T-1,**

**Uric
acid oxidase**

**、**

**allantoinase
and allantoi**

**C**

**ase**

Procedure

For
vectors pGex-4T-1:

| REAGENT | AMOUNT/μl |
| Primer start | 10 |
| pGex-4T-1 primer F | 1 |
| pGex-4T-1 primer R | 1 |
| pGex-4T-1 template | 1 |
| DdH2O | 7 |
| total | 20 |

For
target gene UAO

：

| REAGENT | AMOUNT/μl |
| 2mix | 10 |
| UAO primer F | 1 |
| UAO primer R | 1 |
| UAO template | 1 |
| DdH2O | 7 |
| total | 20 |

For
target gene Allantoinase

：

| REAGENT | AMOUNT/μl |
| 2mix | 10 |
| A-nase primer F | 1 |
| A-nase primer R | 1 |
| A-nase template | 1 |
| DdH2O | 7 |
| total | 20 |

For
target gene Allantoicase

：

| REAGENT | AMOUNT/μl |
| 2mix | 10 |
| A-case primer F | 1 |
| A-case primer R | 1 |
| A-case template | 1 |
| DdH2O | 7 |
| total | 20 |

Procedures

For
vectors pGex-4T-1:

| **PCR program** | **Temp. (celsius degrees)** | **Period** |
| Pre-denaturation | 95 | 3min. |
| Denaturation | 95 | 30sec. |
| Annealing | 55 | 30sec. |
| Elongation | 72 | 3min. |
| Repetition-Denaturation,Annealing,Elongation | / | 30times |
| Elongation | 72 | 10min. |

For
gene sections(UAO/A-nase/A-case):

| **PCR program** | **Temp. (celsius degrees)** | **Period** |
| Pre-denaturation | 95 | 3min. |
| Denaturation | 95 | 30sec. |
| Annealing | 55 | 30sec. |
| Elongation | 72 | 2min. |
| Repetition-Denaturation,Annealing,Elongation | / | 30times |
| Elongation | 72 | 10min. |

**2.
Electrophoresis**

**Procedure**

A.
According to the volume of the vectors and genes samples, use pipette
to measure certain amount of loading buffer to the PCR tubes
containing the samples.

\*Volume
of sample in PCR tube : Volume of the loading buffer is 10 : 1

Consequently,
since volume for the remaining sample of vector is 20μl and the
volume for the remaining sample of target genes is 5μl, the volume
of loading buffer added to each sample are pertinently 2μl and
0.5μl.

B.
Use pipette to measure 10μl of marker and add it to the well comb
in the electrophoresis instrument.

C.
Use pipette to measure 7μl for each sample of vector pGex-4T-1 and
target gene UAO, Allantoinase , and Allantoicase in the well combs of
the electrophoresis instrument.

D.
Connect the instrument to a 180V power source for approximately 25
minutes.

E.
Place the sample under the illumination of the ultraviolet radiation,
then observe the results.

**3.
Extraction**

**Procedure**

A.Use
pipette to measure 2 samples of 300μl Buffer B2 and transfer them
separately to two labelled centrifuge tube.

B.Use
pipette to measure 15μl of vector sample and 15μl of target
gene sample.

C.Transfer
the mixture in the centrifuge tubes to the absorption columns. Attach
them to centrifuge shields and place them in the centrifuge(machine),
start the centrifuging process with centrifugal force of 8000 xg and
time of 30 seconds. Remove the mixtures in the centrifuge shields
after the process.

D.Use
pipette to add 500μl wash buffer into the absorption columns and
start the centrifuging process with centrifugal force of 9000 xg and
time of 30 seconds. Remove the mixtures in the centrifugal shields.

E.Repeat
procedure D.

F.Without
adding any substance, start centrifuging with centrifugal force of
9000 xg and time of 1 minute.

G.Obtain
2 new centrifuge tubes, add 30μl Elusion Buffer to it via pipette,
place them at rest for 1 minute, then centrifuge them with 9000 xg
for 1 minute. After that, preserve the centrifuge tubes.

H.Measure
the concentration of the samples received.

**4.
pGex-UAO and pGex-UAA homologous recombination**

Materials

DpnI
enzyme sample,PCR product: vector pGex-4T-1,PCR product: target
genes(UAO, A-nase, and A-case)

Procedure
I

A.Ensure
the volume of each PCR sample and determine the volume of DpnI enzyme
sample required for the system. The volume of each sample in the
experiment is 30μl, indicating the volume required for enzyme is
1μl for each.

\*The
DpnI enzyme prevent the DNA molecules from methylation.

\*The
theoretical relationship of value between the sample and DpnI needed:
10μl of sample requires 1μl DpnI, 30μl of sample requires
2μl DpnI. However, since the concentration of PCR samples is
relatively low, less DpnI is needed.

B.After
adding the ppnI enzyme sample, place the PCR tube in 37 Celsius
Degree environment for 10 minutes.

C.Place
the PCR tubes in 80 Celsius Degree environment for 20 minutes.

\*Excess
DpnI will deform the structure of DNA molecules. High temperature of
80 Celsius Degrees will denature them fully.

Procedure
II

Every
group constructs a pGex-UAO plasmid and a pGex-UAA plasmid. Since the
concentration of vector samples used is inconsistent, the volume of
reagents used in each group varies. (Follow certain formula)

For
pGex-UAO:

| Reagents | amount |
| (total) | 10μl |
| DNA recombinase | 5μl |
| Vector: pGex-4T-1 | 1.5μl |
| UAO-2 | 1.0μl |
| ddwater | 2.5μl |

For
pGex-UAA:

| Reagents | amount |
| (total) | 20μl |
| DNA recombinase | 5μl |
| Vector: pGex-4T-1 | 1μl |
| UAO-2 | 1μl |
| A-nase | 2μl |
| A-case | 10μl |
| **Ddwater** | **1μl** |

Procedure
III

1.The
UAO group: Place it in 50 Celsius Degree surrounding for 5 minutes.
Then place it in 4 Celsius degree environment.

2.The
UAO group: Place it in 50 Celsius Degree surrounding for 15 minutes.
Then place it in 4 Celsius degree environment.

**5.
Transformation of Bacteria**

Procedure

a)Ligated
samples are added to

*E.Coli(DH5)*

samples

b)Placed
on ice for 20 minutes.

c)Heat
at 42 degrees Celsius for 45 seconds, then immediately put on ice for
2-3 minutes.

d)Add
900μL LB Agar solution, and incubate for 30 minutes

e)Centrifuge
at 5000xg for 3 minutes, extract 900μL of supernatant then discard

f)Apply
end product to agar plates, incubate for 12-16hr at 37 degrees
Celsius.

\*Possible
mistake: It’s required to spread bacteria mildly, since harsh
physical shock will damage their structure.

**6.
Isolated colonies validation**

\*purpose:
examine the vectors(pGex-UAO and pGex-UAA) in the bacteria DH5alpha

Procedure
I

A.
Use pipette to measure 8 of ddwater and add it to the PCR tube.

B.
Obtain template: using a 0.1~2.5μl pipette to mildly scratch the
plate with bacteria and add it to the PCR tube. (For two vectors,
pGex-UAO and pGex-UAA, operate separately )

C.
Use pipette to measure 10μl of 2mix and add it to the PCR tube.

D.
Use pipette to measure 1μl of UAO-2-F or UAA-F and add it to the
PCR tube.

E.
Use pipette to measure 1μl of UAO-2-R or UAA-R and add it to the
PCR tube.

F.
Attach the PCR tubes to the vortex.

Reagents
for pGex-UAO

| Reagents | Amount/μl |
| 2mix | 10 |
| UAO-2-F | 1 |
| UAO-2-R | 1 |
| pGex-UAO | (0) |
| ddwater | 8 |

Reagents
for pGex-UAA

| Reagents | Amount/μl |
| 2mix | 10 |
| UAA-F | 1 |
| UAA-R | 1 |
| pGex-UAA | (0) |
| ddwater | 8 |

Procedure
II-PCR process

| PCR program | Temp. (celsius degrees) | Period |
| Pre-denaturation | 95 | 3min. |
| Denaturation | 95 | 30sec. |
| Annealing | 55 | 30sec. |
| Elongation | 72 | 3min. |
| Repetition-Denaturation,Annealing,Elongation | / | 30times |
| Elongation | 72 | 10min. |

Procedure
III-electrophoresis

A.Add
samples to the well comb: two samples of 10μl marker and 13μl
of the samples in each group.

B.Connect
to 160V electricity for approximately 20 minutes.

C.Examine
the result with the illumination of ultraviolet radiation.

After
the examination, shake the bacteria of DH5alpha containing target
vectors in 37 Celsius Degree for 12~16 hours with adding LB to the
tubes.

\*Purpose:
increase the amount of target vectors and prepare for transformation.

7. **Incubate
   the BL21(DE3)**

7.1
Plasmid extraction

Procedure

A.
Use pipette to measure 2ml of bacteria solution and transfer it to a
2ml centrifuge tube. Place the centrifuge tube into the
centrifuge(machine), and then start centrifuging with centrifugal
force of 12000 xg and time of 1 minute.

B.
Use pipette to measure 250μl of buffer SP1, add it to the
centrifuge tube in procedure A. Continuously inject and retrieve the
liquid in the pipette, which separates the bacteria aggregated at the
bottom of the tube and spread them till they suspend/levitate in the
solution.

C.
use pipette to measure 250μl of buffet SP2 and add it to the
centrifuge tube.

D.
Use pipette to measure 350μl of buffet SP3 and add it to the
centrifuge tube.

E.
Centrifuge the product in procedure D with centrifugal force of 12000
xg and time of 5 minutes. After that process, it can be observed that
white, solid substances remain at the bottom of the tube.

F.
Use pipette to measure 700μl of supernatant in the centrifuge tube
and transfer it to the absorption column, then centrifuge it with
force of 9000 xg and time of 1 minute.

G.
Use pipette to measure 500μl wash buffer and add it to the
absorption column. Centrifuge it with force of 9000 xg and time of 30
seconds. Repeat this procedure again.

H.
Centrifuge the product of procedure G without adding any agent. The
indices are 9000 xg and 1 minute.

I.
Obtain a new centrifuge tube. Use pipette to measure 30μl Elution
Buffer and add it to the absorption column. Place the tube at rest
for a minute, and then centrifuge it with indices of 9000 xg, 1
minute. The remnant in the centrifuge tube is the vector required.

7.2
Transformation

\*purpose:
a necessary prerequisite for the following steps: the examination of
vectors in BL21 and the protein synthesis.

\*Method:
Heat shock

Materials

pGex-UAO,
pGex-UAA,BL21(DE3)

Procedure

a)Add
vector samples to the bacteria sample.

b)Placed
on ice for 20 minutes.

c)Heat
at 42 degrees Celsius for 45 seconds, then immediately put on ice for
2-3 minutes.

d)Add
900μL LB Agar solution, incubate for 30 minutes

e)Centrifuge
at 5000xg for 3 minutes, extract 900μL of supernatant then discard

f)Apply
end product to agar plates, incubate for 12-16hr at 37 degrees
Celsius.

**8.
Isolated colonies validation and shaking bacteria**

\*purpose:
to replicate the vector and BL21, prepare for protein synthesis

Procedure
I

A.
Pick a 1.5ml centrifuge tube, use pipette to add 8μl ddwater to
the tube.

B.
Use 0.1~2.5μl pipette to obtain BL21 from the plate and add it to
the tube.

C.
Attach the tube to the vortex to form the bacteria solution

D.
Use pipette to measure 2μl of the bacteria solution and add it to
a new, 50ml centrifuge tube.

E.
Use pipette to measure 900μl of LB culture medium and add it to
the centrifuge tube.

F.
Place the centrifuge on the shaking bed.

(The
procedure is the same as the STEP II)

Procedure
II

1.Procedure-Construct
PCR system

A.
Use pipette to measure 8μL of ddwater and add it to the PCR tube.

B.
Obtain template: Add the sample of BL21 into the PCR tube

C.
Use pipette to measure 10μl of 2mix and add it to the PCR tube.

D.
Use pipette to measure 1μl of UAO-2-F or UAA-F and add it to the
PCR tube.

E.
Use pipette to measure 1μl of UAO-2-R or UAA-R and add it to the
PCR tube.

F.
Attach the PCR tubes to the vortex.

Procedure
III-PCR process

| **PCR program** | **Temp. (celsius degrees)** | **Period** |
| Pre-denaturation | 95 | 3min. |
| Denaturation | 95 | 30sec. |
| Annealing | 55 | 30sec. |
| Elongation | 72 | 3min. |
| Repetition-Denaturation,Annealing,Elongation | / | 30times |
| Elongation | 72 | 10min. |

Procedure
VI-electrophoresis

A.Add
samples to the well comb: two samples of 10μl marker and 13μl
of the samples in each group.

B.Connect
to 160V electricity for approximately 20 minutes.

C.Examine
the result with the illumination of ultraviolet radiation.

* **IPTG
  induced protein expression**

  **、**

  **purification
  and identification**

1. **IPTG
   induced protein expression**

Materials

| **Reagent** | **Volume/mass** |
| Agar solution | 20ml |
| IPTG Mix | 50ml |
| Bacterial fluid solution | 20ml |

**Procedure**

a)Expand/transfer
the incubation container to 50mL centrifugal tube, add 20mL LB agar
solution

b)Add
20mL bacterial fluid solution to the tube

c)Add
50mL IPTG to the tube and incubate at 16 degrees Celsius for 12 to 16
hours

The
amount of IPTG added is based on the equation C1V1 = C2V2

-the
ratio of concentration of IPTG to bacterial fluid solution is 1:1000

-in
order to find the most suitable concentration of IPTG, we tested a
variety of different concentrations based on amount of IPTG:
0.05mmol, 0.25mmol, 0.5mmol, 0.8mmol, 1mmolwhen OD600=0.4-0.8

d
) 16 degrees, overnight culture 12-16 hours.

2. **Protein
   Purification**

**2.1
Ultrasonic crushing of cell membranes**

\*Essence:
with ultrasonic waves, the membrane structures of bacteria BL21 are
damaged and split. The electrical energy transfers to sound energy
during this process, and the high-frequency sound waves in water can
severely shock the bacteria.

\*Other
methods like alternating pH(e.g. alkaline splitting) present similar
outcome. However, they possess lower efficiency.

Materials

Bacteria
solution of BL21,Buffer HisA,Buffer HisB

Procedure

A.
Obtain the 50ml centrifuge tubes containing cultured BL21 bacteria.
Centrifuge it with the centrifuging force of 12000xg, the temperature
of 4 Celsius Degree, and the time of approximately 10 minutes.

B.
After centrifuging, discard the supernatant and retain the
precipitate.

\*The
ppt. contains proteins required and the bacteria BL21.

C.
Use pipette to measure 200μl of buffer HisA and add it to the
centrifuge tube. Use the pipette to inject and retrieve the mixture
in the tube continuously to float the precipitate (bacteria)in the
solution.

D.
Place the tube on the ice for ultrasonication with the lid uncovered.
The process includes repetition of breaking for 3 seconds and stop
for 3 seconds. The entire process lasts for about 15 minutes.

**2.2
Purify the proteins**

\*Essence:
during the design of the product vector, the DNA sections that form
target protein all contain His group. In the proteins formed, His
group can attach to the Nicole cations in Ni-NTA resin while other
proteins can’t. The target enzymes and other proteins are thus
separated.

\*The
result is also used for determining the optimum concentration of IPTG
in induction.

Materials

Ni-NTA
resin,His-A buffer,His-B buffer

Procedure

A.
Obtain the product of ultrasonication, which is in a 50ml centrifuge
tube. Centrifuge the product with centrifugal force of 8000xg and
time of 5 minutes.

B.
Obtain a 15ml centrifuge tube, a 50ml centrifuge tube ,and a 2ml
centrifuge tube.

C.
add all the supernatant from the centrifuging process to the 50ml
centrifuge tube.

D.
Use pipette to measure 200μl of Ni-NTA risen into the gravity
column. Attach a 2ml centrifuge tube under the gravity column.

E.
Use pipette to measure 100μl of His-A buffer into the gravity
column to wash and filter the mixture in the gravity column. Discard
the filtrate collected in the 2ml centrifuge tube.

F.
Use pipette to add 100μl ddwater into the gravity column, wash and
filter the mixture. Discard the mixture acquired in the 2ml
centrifuge tube.

G.
Add 5ml of supernatant into the gravity column. Place the tube for
filtering.

H.
add 3ml of His-A into the gravity column. Discard the filtrate.

\*purpose:
to wash the unwanted proteins out of the mixture

I.
Add 3ml of His-B into the gravity column. Retain the filtrate in the
centrifuge tube.

3. **SDS-PAGE**

\*the
gel required for the SDS-PAGE contains two sections: stacking gel and
separating gel. The former aggregates protein molecules together, and
the latter separates molecules like common electrophoresis.

\*The
reagents include β-mercaptoethanol, which breaks the disulfide
bonds and eliminate the tertiary structure for the enzymes, and the
SDS, which breaks the hydrogen bonds in the protein structure.

\*The
electrophoresis instrument requires specific procedures for operation

Materials

| Reagent | Volume/mass |
| \* Separating gel: | μL |
| 2x Separating solution(10%) |  |
| Separating buffer |  |
| Coagulant |  |
| \* Stacking gel: |  |
| 2x stained stacking solution |  |
| Stacking buffer |  |
| ddwater | 1000μL |

Procedure

A.
Pick the base and set it on a horizontal table. Let the side with a
light-red stripe face to the person. Attach the gel model to the
base.

B.
Obtain the glass plates. Attach it to the model and ensure that the
higher glass plate is placed toward the operator.

C.
Examine that the model for gel is sealed. Use pipette to measure
1000μl ddwater and add it from the aperture between the two glass
plates. Place the instrument at rest and observe whether water leaks
from the bottom of the water or not. If the water doesn’t
penetrate through the base, the model is sealed.

For
separating gel:

A.Pick
a plastic cup. Use pipette to measure 2.7ml of separating buffer and
add it to the container.

B.Use
pipette to measure 2.7ml of (2x) 10% separating solution and transfer
it to the container.

C.Use
pipette to measure 55μl of coagulant and add it to the container.

D.Stir
the mixture to separate the three reagents evenly.

E.Quickly
transfer the mixture to the electrophoresis instrument by using
pipette. When the edge of the liquid observed has reached the while
line indicated on the larger glass plate, stop adding the mixture.

\*The
mixture must be added quickly: the added sample will coagulate
rapidly if the remaining samples are added slowly.

F.Use
pipette to measure 1000μl of ddwater and add it to the model of
electrophoresis.

G.Place
the instrument at rest. Observe if the mixture has penetrated the
base and leaked out. If this circumstance happens, operate the entire
procedure again.

For
stacking gel:

A.Pick
a plastic cup. Use pipette to measure 750μl of (2x) stained
stacking solution and add it to the container.

B.Use
pipette to measure 750μl of stacking buffer and add it to the
container.

C.Use
pipette to measure 15μl of coagulant and add it to the container.

D.Stir
the mixture.

E.Use
pipette to transfer the mixture in the cup to the electrophoresis
instrument by adding the mixture into the aperture between two glass
plates. Continue to add the mixture until the model is full .

F.Place
the instrument at rest until the stacking gel and separating gel have
all coagulated.

* **Enzyme
  activity determination**

**1.
Prepare for creating the standard curve**

\*Standard
Curve is significant since it can transfer the result of
absorbance(which reflects chromatic traits) to the concentration of
uric acid remaining in the mixture, indicating that it can directly
infer the efficiency of UAO from the indices acquired.

\*Essence:

The
difference in the concentration of Uric acid is directly related to
the amount of Uric acid that has been digested by UAO, which is
further related to the H2O2 formed and the absorbance of the
products. The proportionality between Uric acid concentration and
absorbance can thus be determined and a linear function can be
deduced.

Materials:

Standard
Uric acid solution,

UAO
and UAA sample

Procedure:

1. Ensure
   that the experiment need to create specified samples that will show
   a linear relationship between the absorbance(Final examination
   result of the decomposition for uric acid) and the concentration of
   the uric acid. The volume required for each sample is 250μl(Uric
   acid and water)+750μl(testing agent).

   - The
     volume for each group are thus as follow:

|  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Sample\conc. of acid(μmol/ml) | 0.25 | 0.125 | 0.0625 | 0.03125 | 0.015625 | 0.0073125 | 0 |
|  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Standard Uric acid solution(μl) | 25 | 12.5 | 6.25 | 3.13 | 1.56 | 0.73 | 0 |
| Ddwater(μl) | 225 | 237.5 | 243.75 | 246.87 | 248.44 | 249.27 | 250 |
| Testing agent(μl) | 750 | 750 | 750 | 750 | 750 | 750 | 750 |

4. Pix
   6 1.5ml centrifuge tubes.

   - Use
     pipette to measure the standard uric acid solution required for each
     group and add them to the centrifuge tubes. The volumes are
     corresponded to the graph shown.

     - Use
       pipette to measure the ddwater required for each group and add them
       to the centrifuge tubes. The volumes are corresponded to the chart
       shown above.

       - Use
         pipette to measure 750μl testing agent with UAO,UAA and add it to
         the centrifuge tube in group 1. Operate the same procedure to the
         other 6 tubes.

         - Put
           the 7 centrifuge tubes in the 37 Celsius Degree environment for 30
           minutes.

           - After
             reacting, examine the absorbance of the products by using
             ultraviolet spectrophotometer. Operate the measurement procedure for
             5 times and calculate the average value.

2. **Construct
   the system of testing**

\*
Each group creates 6 testing groups with different concentrations of
UAO and UAA.

\*
Essence:

The
route of decomposing uric acid by UAO is:

Uric
acid(+UAO)allantoin+H
2

O
2

+CO
2

The
examination focuses on testing the H
2

O
2

produced. If the amount of the product acquired in the testing of
H
2

O
2

is high, the amount of H
2

O
2

formed in the decomposition reaction is high; it can thus be
confirmed that the enzymatic activity for the UAO sample is high.

\*H
2

O
2

possesses reducibility. The reagents used to test H
2

O
2

contains Fe
2+

cations. These ions are oxidized during the reaction and are
transferred to Fe
3+

cations, which forms red brown quinone when combining with
4-Aminoantiprymine and phenol. The color is used for the chromatic
examination, which determines the activity of UAO.

Materials

| **Reagent** | **Volume/mass** |
| Ddwater | / |
| Standard Uric acid solution  (conc. = 2.5μmol/ml) | 50μL |
| UAO sample  (The supernatant of the mixture in the centrifuging and ultrasonication yesterday is used, since the extracted sample possesses low concentration) | / |
| The testing agents  (Contains UAO,   4-Aminoantiprymine, and Potassium ferrocyanide oxide  ). | 750μL |

Procedure

A.
pre-Information:

\*Ensure
the concentration of Uric acid required is 0.25

μmol/ml,
the volume of the uric acid sample needed is 100μl, and the total
volume for the sample is 300μl(enzyme+uric acid
sample)+750μl(testing agents)

\*Ensure
that the concentration of UAO needs to be altered, and the amount of
uric acid needs to be constant.

\*The
volumes of reagents required in different groups thus are:

|  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- |
| Reagents\group | 1 | 2 | 3 | 4 | 5 | 6(control group) |
|  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- |
| Uric acid sample(  0.25  μmol/ml) | 100 | 100 | 100 | 100 | 100 | 0 |
| UAO sample   （  μl   ） | 0 | 50 | 100 | 150 | 200 | 300 |
| Testing reagents(μl) | 750 | 750 | 750 | 750 | 750 | 750 |
| Ddwater | 200 | 150 | 100 | 50 | 0 | 0 |

B.
Pick six 1.5ml centrifuge tubes and label them 1~6.

C.
Pick one 2ml centrifuge tube(the tube is used for preparing the uric
acid sample). \*Since the total volume required for uric acid sample
is 500μl and the concentration of uric acid is 0.25μmol/ml, the
ratio between standard uric acid sample(conc. : 2.5μl) and the
ddwater is 1 : 9.

Use
pipette to measure 50μl of standard uric acid sample and add it to
the 2ml centrifuge tube. Use pipette to measure 450μl of ddwater
and add it to the 2ml centrifuge tube. Attach the tube to the vortex.

D.
Use pipette to measure 50μl, 100μl, 150μl, 200μl, and
300μl of UAO sample(supernatant) and add them correspondingly to
the 1.5ml centrifuge tubes labelled 2,3,4, and 5. Attach the tubes to
the vortex and directly place them on ice.

E.
use pipette to measure 750μl of testing agents and add it to the
centrifuge tube labelled 1. Repeat the procedure to other 5 tubes as
well.

F.
After adding testing agents, place the centrifuge tubes on ice for
approximately 10 minutes, and then transfer them to the 37 Celsius
Degree environment for 30 minutes.

\*This
is the procedure for the UAO to decompose the Uric acid.

G.
After 30 minutes, use pipette to measure 200μl, 150μl, 100μl,
and 50μl of ddwater and add them correspondingly to the centrifuge
tubes labelled 1,2,3,and 4. Attach all 6 centrifuge tubes to the
vortex.

\*This
procedure must be operated after the uric acid has been fully
digested by UAO. If it is added earlier, the concentrations of the
enzyme in different groups will be affected.

H.
Add the samples to the Coated Wells: 3 groups refer to 3 lines
labelled B,C, and D.

I.
examine the absorbance of the products by using ultraviolet
spectrophotometer. Operate the measurement procedure for 5 times and
calculate the average value.

\*Absorbance
reflects the amount of the uric acids that have been decomposed by
UAO.

* **Transformation
  1917**

1

**. Transformation**

Materials

| Apparatus | Amount |
| Pipette (range:100-1000μL) | 1 |
| Centrifuge | 1 |
| Pipette (range:2-20μL) | 1 |
| Incubator | 1 |
| Rod | 2 |
| Culture plate | 2 |

Procedure

A.Add
the Nissle 1917 bacteria solution to the samples of vectors pGex-UAA
and pGex-UAO. Flick the tube lightly.

B.Placed
the sample on ice for 20 minutes.

C.Heat
at 42 degrees Celsius for 45 seconds, then immediately put on ice for
2-3 minutes.

D.Add
900μL LB Agar solution, incubate for 30 minutes

E.Centrifuge
at 5000xg for 4 minutes, extract 900μL of supernatant then discard

F.Continuously
inject and retrieve the mixture in the centrifuge tube by using a
pipette to float the bacteria in the centrifuge tube.

G.Use
pipette to measure and apply 10μl product to agar plates, then use
a rod to spread the bacteria solution evenly on the plate.

H.Use
tapes to seal the culture plate. Incubate the plate for 12-16hr at 37
degrees Celsius. (The plates are placed upside down)

**2.
Isolated colonies validation**

A.
PCR system

：

| Uric acid oxidase  (  981  bp) | Volume (ul) |
| 2mix | 10 |
| Uric acid oxidase-R1 | 1 |
| Uric acid oxidaseF1 | 1 |
| pGEX-4T-1-UAO | 1 |
| ddH2O | 7 |
| Total | 20 |

PCR
procedure

：

| procedure | temperature   （℃） | Time |
| 1 | 95 | 2 min |
| 2 | 95 | 30 sec |
| 3 | 55 | 30 sec |
| 4 | 72 | 2 min |
| 5 | 72 | 10 min |
| 6 | 4 | forever |

2. A

   **garose
   gel electrophoresis**

1.Preparation
of agarose gel : 100 mL 1 × TAE buffer was added to a conical
flask, and 1 g agarose was weighed and placed in a conical flask,
heated in a microwave oven for 2 min to completely melt, and shaken
well.5 μL of nucleic acid dye Gel Red was added to the agarose
gel, which was 1 % agarose gel containing nucleic acid dye. Install
the gel plate and insert the comb. Pour the liquid gel into the gel
plate, 3-4 mm high, avoid bubbles, and solidify at room temperature.
After complete solidification, the comb was pulled out, the solid gel
was transferred to the electrophoresis tank, 1 × TAE buffer was
added, and the gel was completely immersed.

2.Spotting
: each sample was added to the spotting hole of the gel in turn, and
finally 10μL DNA Maker was added ( the gel around the sample hole
should be prevented from being damaged and penetrating the bottom of
the gel ) ;

3.Electrophoresis
: cover the electrophoresis tank, the sample hole is located in the
negative electrode of the electric field, turn on the power supply,
180V, 15min ;

4.Blue
light observation : turn off the power supply, take out the gel,
observe under the blue light glue instrument, the red fluorescent
substance is DNA.

**3.IPTG
induction and fragmentation**

When
the bacteria were cultured to OD = 0.6, 0.5 mmol IPTG16 was added for
overnight.

4.

**Ultrasonic
crushing of cell membranes**

\*Essence:
with ultrasonic waves, the membrane structures of bacteria Nissle
1917 are damaged and split. The electrical energy transfers to sound
energy during this process, and the high-frequency sound waves in
water can severely shock the bacteria.

\*Other
methods like alternating pH(e.g. alkaline splitting) present similar
outcome. However, they possess lower efficiency.

Materials

Bacteria
solution of Nissle 1917 ,Buffer HisA,Buffer HisB

Procedure

A.
Obtain the 50ml centrifuge tubes containing cultured Nissle 1917
bacteria. Centrifuge it with the centrifuging force of 12000xg, the
temperature of 4 Celsius Degree, and the time of approximately 10
minutes.

B.
After centrifuging, discard the supernatant and retain the
precipitate.

\*The
ppt. contains proteins required and the bacteria Nissle 1917 .

C.
Use pipette to measure 200μl of buffer HisA and add it to the
centrifuge tube. Use the pipette to inject and retrieve the mixture
in the tube continuously to float the precipitate (bacteria)in the
solution.

D.
Place the tube on the ice for ultrasonication with the lid uncovered.
The process includes repetition of breaking for 3 seconds and stop
for 3 seconds. The entire process lasts for about 15 minutes.

**2.2
Purify the proteins**

\*Essence:
during the design of the product vector, the DNA sections that form
target protein all contain His group. In the proteins formed, His
group can attach to the Nicole cations in Ni-NTA resin while other
proteins can’t. The target enzymes and other proteins are thus
separated.

\*The
result is also used for determining the optimum concentration of IPTG
in induction.

Materials

Ni-NTA
resin,His-A buffer,His-B buffer

Procedure

A.
Obtain the product of ultrasonication, which is in a 50ml centrifuge
tube. Centrifuge the product with centrifugal force of 8000xg and
time of 5 minutes.

B.
Obtain a 15ml centrifuge tube, a 50ml centrifuge tube ,and a 2ml
centrifuge tube.

C.
add all the supernatant from the centrifuging process to the 50ml
centrifuge tube.

D.
Use pipette to measure 200μl of Ni-NTA risen into the gravity
column. Attach a 2ml centrifuge tube under the gravity column.

E.
Use pipette to measure 100μl of His-A buffer into the gravity
column to wash and filter the mixture in the gravity column. Discard
the filtrate collected in the 2ml centrifuge tube.

F.
Use pipette to add 100μl ddwater into the gravity column, wash and
filter the mixture. Discard the mixture acquired in the 2ml
centrifuge tube.

G.
Add 5ml of supernatant into the gravity column. Place the tube for
filtering.

H.
add 3ml of His-A into the gravity column. Discard the filtrate.

\*purpose:
to wash the unwanted proteins out of the mixture

I.
Add 3ml of His-B into the gravity column. Retain the filtrate in the
centrifuge tube.

4. **SDS-PAGE**

\*the
gel required for the SDS-PAGE contains two sections: stacking gel and
separating gel. The former aggregates protein molecules together, and
the latter separates molecules like common electrophoresis.

\*The
reagents include β-mercaptoethanol, which breaks the disulfide
bonds and eliminate the tertiary structure for the enzymes, and the
SDS, which breaks the hydrogen bonds in the protein structure.

\*The
electrophoresis instrument requires specific procedures for operation

Materials

| Reagent | Volume/mass |
| \* Separating gel: | μL |
| 2x Separating solution(10%) |  |
| Separating buffer |  |
| Coagulant |  |
| \* Stacking gel: |  |
| 2x stained stacking solution |  |
| Stacking buffer |  |
| ddwater | 1000μL |

Procedure

A.
Pick the base and set it on a horizontal table. Let the side with a
light-red stripe face to the person. Attach the gel model to the
base.

B.
Obtain the glass plates. Attach it to the model and ensure that the
higher glass plate is placed toward the operator.

C.
Examine that the model for gel is sealed. Use pipette to measure
1000μl ddwater and add it from the aperture between the two glass
plates. Place the instrument at rest and observe whether water leaks
from the bottom of the water or not. If the water doesn’t
penetrate through the base, the model is sealed.

For
separating gel:

A.Pick
a plastic cup. Use pipette to measure 2.7ml of separating buffer and
add it to the container.

B.Use
pipette to measure 2.7ml of (2x) 10% separating solution and transfer
it to the container.

C.Use
pipette to measure 55μl of coagulant and add it to the container.

D.Stir
the mixture to separate the three reagents evenly.

E.Quickly
transfer the mixture to the electrophoresis instrument by using
pipette. When the edge of the liquid observed has reached the while
line indicated on the larger glass plate, stop adding the mixture.

\*The
mixture must be added quickly: the added sample will coagulate
rapidly if the remaining samples are added slowly.

F.Use
pipette to measure 1000μl of ddwater and add it to the model of
electrophoresis.

G.Place
the instrument at rest. Observe if the mixture has penetrated the
base and leaked out. If this circumstance happens, operate the entire
procedure again.

For
stacking gel:

A.Pick
a plastic cup. Use pipette to measure 750μl of (2x) stained
stacking solution and add it to the container.

B.Use
pipette to measure 750μl of stacking buffer and add it to the
container.

C.Use
pipette to measure 15μl of coagulant and add it to the container.

D.Stir
the mixture.

E.Use
pipette to transfer the mixture in the cup to the electrophoresis
instrument by adding the mixture into the aperture between two glass
plates. Continue to add the mixture until the model is full .

F.Place
the instrument at rest until the stacking gel and separating gel have
all coagulated.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
{% block page\_content %}

![](https://static.igem.wiki/teams/5024/wiki/g-out-results.png)

**Overview**

Uric
acid is a waste product of the metabolism of purines in the food,
most of the uric acid is excreted through liver. However, if the body
is unable to efficiently excrete uric acid, uric acid level will
rise, forming urate crystal and deposit in the joints and surrounding
soft tissue, causing a painful and inflammatory response that leads
to joint pain, and swelling
1,2

.
Some animals use urate oxidase to catalyze the conversion of uric
acid into allantoin, which helps reduce the accumulation of uric acid
in the body( Figure 1). Therefore, this project uses probiotics to
express enzymes such as urate oxidase(UAO), a

llantoinase,
and allantoicase,

to
degrade excess uric acid in the body, thereby reducing the risk of
gout.

![](https://static.igem.wiki/teams/5024/wiki/wechatimg40.jpeg)

Figure
1. The engineering schematic diagram of the project

**Construct
plasmid**

Goal:
In order to replace the GST tag of pGEX-4T-1 vector with His, we
first constructed pET28a-UAO, pET28a-

llantoinase
and

pET28a-

allantoicase

.

**pET28a-UAO,
pET28a-**

**a**

**llantoinase
and pET28a- allantoicase**

We
added restriction sites BamHI and XhoI to primer, and further
amplified the target gene UAO by PCR. The length of the target gene
UAO is 981 bp. Figure 2 shows that the fragment lengths are
consistent with the results. We added restriction sites BamHI and
NotI to primer, and further amplified the target gene

a

llantoinase
by PCR. The length of the target gene

a

llantoinase
is 1485 bp. Figure 2 shows that the fragment lengths are consistent
with the results. We added restriction sites SacI and XhoI to primer,
and further amplified the target gene allantoicase by PCR. The length
of the target gene allantoicase is 1288 bp. Figure 2 shows that the
fragment lengths are consistent with the results. It indicates that
we have successfully amplified the target gene allantoicase,
allantoinase and UAO.

![](https://static.igem.wiki/teams/5024/wiki/wechatimg41.jpeg)

Figure
2. Electrophoresis for the target genes UAO, allantoinase and
allantoicase

Then,
we cut the vector pET28a with restriction endonuclease, and used the
uncut vector as a control. The length of the pET28a vector is 5369
bp, and the control group of Figure 3 is correct. Line 1-2 is the
band of pET28a cut with BamHI and XhoI. Line 3-4 is the band of
pET28a cut with BamHI and NotI. Line 5 is the band of pET28a cut with
SacI and XhoI. The results show that the plasmid has been cut into
linearization.

![](https://static.igem.wiki/teams/5024/wiki/wechatimg42.jpeg)

Figure
3. The electrophoresis for testing the linearized vector pET28a

Note

：

Line1-
2: The band of pET28a cut with BamHI and XhoI

Line
3- 4: The band of pET28a cut with BamHI and NotI

Line
5: The band of pET28a cut with SacI and xhoI

Next,
we connect UAO,

a

llantoinase
and allantoicase with pET28a by T4 ligase, and transformed the
plasmid into DH5α. Figure 4A shows that the

isolated
colonies were

successfully grown, and the

isolated
colonies were

selected for colony verification. The length of the target gene UAO
is 981 bp. The length of the target gene

a

llantoinase
is 1485 bp. The length of the target gene allantoicase is 1288 bp.
Figure 4C shows that the fragment lengths are consistent with the
results. We have successfully the vector pET28a-UAO,

pET28a-

a

llantoinase
and

pET28a-

allantoicase
were transformed into DH5α, and the sequencing results in
Figure 4B showed no gene mutation. It indicates that our plasmid has
been successfully constructed.

![](https://static.igem.wiki/teams/5024/wiki/wechatimg43.jpeg)

Figure
4. The Electrophoresis of the Monoclonal Antibody in DH5α

Note:

A
Culture plates UAO, allantoinase and allantoicase for DH5α

B.Electrophoresis
of the Monoclonal Antibody UAO, allantoinase and allantoicase

C.The
sequencing result of pET28a-UAO,pET28a-

a

llantoinase
and pET28a-allantoicase

**pGex-4T-1-UAO
and pGex-4T-1-UAA**

We
used pET28a-UAO, pET28a-allantoinase and pET28a-allantoicase as
templates for the following experiments.

**2.1
pGex-4T-1-UAO**

We
wanted to use homologous recombination for constructing
pGex-4T-1-UAO, so we added homologous arms at both ends of the
primer, and amplified UAO and pGex-4T-1 by PCR. The length of UAO is
981 bp. Figure 5A shows that the fragment lengths are consistent with
the results. It indicates that we have successfully amplified the
target gene UAO. The length of the plasmid pGex-4T-1 is 4328 bp.
Figure 5B shows that the pGex-4T-1 band is about 4500 bp. It
indicates that we have successfully amplified the plasmid.

![](https://static.igem.wiki/teams/5024/wiki/wechatimg44.jpeg)

Figure
5. The electrophoresis for the target genes UAO and pGEx-4T-1 vectors

Next,
we transformed the plasmid pGex-4T-1-UAO into DH5α. Figure 6A
shows that the

isolated
colonies were

successfully grown, and the

isolated
colonies were

selected for colony verification. The length of the target gene UAO
is 981 bp. Figure 6B shows that the UAO band is about 1000 bp. The
results show that the vector pGex-4T-1-UAO was transformed into DH5α,
and the sequencing results of pGex-4T-1-UAO in Figure 6 CD showed
that UAO has no gene mutation. It proves that the plasmid
pGex-4T-1-UAO was successfully constructed. It indicates that the
plasmid pGex-4T-1-UAO has been successfully constructed.

![](https://static.igem.wiki/teams/5024/wiki/wechatimg45.jpeg)

Figure
6. Culture plates for DH5α and Electrophoresis of the
Monoclonal Antibody

Note:

A:
Culture plates pGex-4T-1-UAO for DH5α

B:
Electrophoresis of the monoclonal Antibody pGex-4T-1- UAO

C
and D: The Sequencing results of pGex-4T-1-UAO

**2
pGex-4T-1-UAA**

We
used homologous recombination to construct pGex-4T-1-UAA, and add
homologous arms at both ends of the primer and amplified

a

llantoinase,
allantoicase and pGex-4T-1-UAO by PCR. The length of

a

llantoinase
is 1485 bp. The length of the target gene allantoicase is 1288 bp.
Figure 7 A shows that tthe fragment lengths are consistent with the
results. It indicates that we have successfully amplified the target
gene. The length of the plasmid pGex-4T-1-UAO is 5301 bp. Figure 7B
shows that the fragment lengths are consistent with the results. It
indicates that we have successfully amplified the plasmid(Figure 7C).

![](https://static.igem.wiki/teams/5024/wiki/wechatimg46.jpeg)

Figure
7. Electrophoresis for the genes

a

llantoinase,
allantoicase and pGex-4T-1-UAO

Next,
we transformed the plasmid pGex-4T-1-UAA into DH5α. Figure 8AB
shows that the

isolated
colonies were

successfully grown, and the

isolated
colonies

were selected for colony verification. The length of the target gene
allantoicase is 1074 bp. In Figure 8C, line1-5 shows that the
fragment lengths are consistent with the results. The

a

llantoinase
is approximately about 1485bp. In Figure 8D, line1,4, 5 shows that
the fragment lengths are consistent with the results. It indicates
that we have successfully amplified the plasmid pGex-4T-1-UAA.

![](https://static.igem.wiki/teams/5024/wiki/wechatimg47.jpeg)

Figure
8. The electrophoresis of the monoclonal antibody of pGEX-UAA in

*E.coil*

DH5α

Afterward,
we chose positive

isolated
colonies

1, 4, and 5 and sent them to the company for sequencing. The results
showed that the sequencing of the allantoicase and allantoinase genes
on the pGex-4T-1-UAA plasmid was normal (Figure 9), without any
genetic mutations. It indicates that our plasmid pGex-4T-1-UAA has
been successfully constructed.

![](https://static.igem.wiki/teams/5024/wiki/wechatimg48.jpeg)

Figure
9. The sequencing of the allantoicase and allantoinase genes on the
pGex-4T-1-UAA plasmid

**Protein
expression and purification**

**pGex-4T-1-UAO**

**1.1pGex-4T-1-UAO(BL21(DE3))**

In
order to improve the expression of the protein, we first tested the
protein in

*E.coil*

BL21(DE3). So we transformed the plasmid pGex-4T-1-UAO into

*E.coil*

BL21(DE3).
Figure 9A shows that the

isolated
colonies were

successfully grown, and the

isolated
colonies were

selected
for colony verification. The length of the target gene UAO is 981 bp.
Figure

10

B
shows that the UAO band is about 1000 bp. The vector pGex-4T-1-UAO
was successfully transformed into

*E.coil*

BL21(DE3).

![](https://static.igem.wiki/teams/5024/wiki/wechatimg49.jpeg)

Figure

10

.
Electrophoresis of the Monoclonal Antibody Validation process for
pGex-UAO in BL21(DE3)

In
order to improve the expression of UAO protein, we designed different
gradients of IPTG induction. Table 1 and Figure 1

1

showed that the protein concentration of pGex-UAO increased first and
then decreased with the increase of IPTG concentration. When the
concentration of IPTG was 0.5 mmol, the protein concentration of UAO
was the highest.

Table1:
Protein concentration of UAO (mg/mL)

|  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- |
|  | **IPTG concentration (mmol/L)** | | | | |
|  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- |
| **Sample #** | **0.050** | **0.250** | **0.500** | **0.800** | **1.000** |
| **1** | 1.016 | 1.320 | 5.101 | 0.783 | 0.817 |
| **2** | 0.583 | 0.992 | 4.764 | 0.652 | 0.814 |
| **3** | 0.656 | 1.402 | 4.759 | 0.625 | 0.831 |
| **Average** | 0.752 | 0.991 | 4.875 | 0.687 | 0.821 |
| **SD** | 0.232 | 0.217 | 0.196 | 0.085 | 0.009 |

![](https://static.igem.wiki/teams/5024/wiki/wechatimg59.jpeg)

Figure
1

1

.
The relationship between the concentration of IPTG and the
concentration of proteins

Next,
we induced the protein UAO with 0.5 mmol IPTG. The size of the UAO
protein is 40 kDa. Figure 1

2

shows that the crude protein has the target protein UAO, and the
purified protein solution also has a small amount of UAO, but the
purification loses som
e
proteins.

![](https://static.igem.wiki/teams/5024/wiki/wechatimg50.jpeg)

Figure1

2

.
The pGex-4T-1-UAO of SDS-PAGE in BL21(DE3)

**1.2pGex-4T-1-UAO(Nissle
1917)**

Finally

,
we transformed the plasmid pGex-4T-1-UAO into Nissle 1917. Figure 1

3

B
shows that the

isolated
colonies were

successfully
grown, and the

isolated
colonies were

selected
for colony verification. The length of the target gene UAO is 981 bp.
Figure 1

3

A
shows that the UAO band is about 1000 bp. The vector pGex-4T-1-UAO
was successfully transformed into Nissle 1917.

![](https://static.igem.wiki/teams/5024/wiki/wechatimg51.jpeg)

Figure
1

3

.
Electrophoresis of the monoclonal antibody for pGex-UAO in Nissle
1917

Meanwhile,
we induced the protein UAO with 0.5 mmol IPTG in Nissle 1917. The
size of the UAO protein is 40 kDa. Figure 1

4

shows that the crude protein has the target protein UAO. In the
control group, Nissle 1917 had no target protein of UAO.

![](https://static.igem.wiki/teams/5024/wiki/wechatimg52.jpeg)

Figure
1

4

.
The pGex-4T-1-UAO of SDS-PAGE in Nissle 1917

**2.
pGex-4T-1-UAA**

In
order to improve the expression of the protein, we first tested the
protein in

*E.coil*

BL21(DE3).
So we transformed the plasmid pGex-4T-1-UAA into

*E.coil*

BL21(DE3).
Figure 14A and B show that the

isolated
colonies were

successfully
grown, and the

isolated
colonies were

selected
for colony verification. The length of the target gene allantoinase
is 1485 bp and allantoinase is 1047bp. Figure 1

5

C
and

15

D
show that the fragment lengths are consistent with the results, we
have successfully the vector pGex-4T-1-UAA was transformed into

*E.coil*

BL21(DE3).

![](https://static.igem.wiki/teams/5024/wiki/wechatimg53.jpeg)

Figure
1

5

.
The electrophoresis of the monoclonal antibody validation process for
pGex-UAA in

*E.coil*

BL21(DE3)

Note:

Figure
1

5

A-B:
The monoclonal plate pGex-UAA in BL21(DE3)

Figure
1

5

C:
The Electrophoresis verification of allantoicase in pGex-UAA
(BL21(DE3))

Figure
1

5

d:
The Electrophoresis verification of allantoinase in pGex-UAA
(BL21(DE3))

In
order to improve the expression of UAA protein, we designed different
gradients of IPTG induction. The size of the UAA protein is 118.6
kDa. Figure 1

6

shows that the crude protein has the target protein UAA. However,
0.1mmol, 0.25 mmol, 0.5mmol,0.7mmol and 1mmol IPTG had no significant
effect on the expression of UAA protein.

![](https://static.igem.wiki/teams/5024/wiki/wechatimg54.jpeg)

Figure
1

6

.
The pGex-4T-1-UAA of SDS-PAGE in (BL21(DE3))

**Enzyme
activity detection**

**pGex-4T-1-UAO**

**A.The
Standard Curve**

Firstly,
we made a standard curve for the determination of uric acid. The
second table is the original data of the table. We use a linear
function to fit. Figure 1

7

shows that the equation y = 1.1529x + 0.2129 is satisfied, and the
correlation coefficient is greater than 0.99. The table fits
well(Table 1).

Table
1. Result and curve for the Standard Curve

|  | Uric acid concentration (μmol/mL) | | | | | | |
| Sample# | 0.25 | 0.125 | 0.0625 | 0.0312 | 0.015625 | 0.007313 | 0 |
| 1 | 0.5086 | 0.3475 | 0.2824 | 0.2529 | 0.2338 | 0.2200 | 0.2110 |
| 2 | 0.5060 | 0.3469 | 0.2824 | 0.2536 | 0.2348 | 0.2199 | 0.2120 |
| 3 | 0.5052 | 0.3485 | 0.2832 | 0.2537 | 0.2353 | 0.2201 | 0.2128 |
| 4 | 0.5032 | 0.3475 | 0.2839 | 0.2536 | 0.2348 | 0.2205 | 0.2119 |
| 5 | 0.5042 | 0.3473 | 0.2843 | 0.2542 | 0.2355 | 0.2203 | 0.2129 |
| average | 0.50544 | 0.34754 | 0.28324 | 0.2536 | 0.23484 | 0.22016 | 0.21212 |
| SD | 0.002056 | 0.00059 | 0.000862 | 0.000464 | 0.000658 | 0.000241 | 0.000773 |

![](https://static.igem.wiki/teams/5024/wiki/wechatimg55.jpeg)

Figure1

7

.
The Standard Curve of Urate Oxidase

**The
activity of urate oxidase**

In
order to determine the ability of our constructed plasmid
pGex-4T-1-UAO to decompose uric acid, we used uric acid as a
substrate and added UAO protein. Table 2 and Figure 1

8

showed that with the increase of UAO protein concentration, the
content of uric acid gradually decreased, indicating that UAO is
active and can successfully decompose uric acid. And we used

*E.coil*

BL21
( DE3 ) as the control group, the uric acid content of the control
group had no obvious change trend.

Table
2. The activity of urate oxidase

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| Concentration of UAO(mg) | Experimental group | SD | Control | SD |
|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| 0.00000 | 0.23052 | 0.00058103 | 0.23583 | 0.002199284 |
| 0.01335 | 0.22330 | 0.00095916 | 0.23493 | 0.007408525 |
| 0.02670 | 0.18474 | 0.00102489 | 0.23918 | 0.006044871 |
| 0.04005 | 0.15558 | 0.00051146 | 0.22762 | 0.011768322 |
| 0.05340 | 0.15154 | 0.00127843 | 0.22007 | 0.014096617 |

![](https://static.igem.wiki/teams/5024/wiki/result-date.png)

Figure
1

8

.
The curve of UAO activity

**pGex-4T-1-UAA**

**A.**

**The
activity of urate oxidase**

In
order to determine the ability of our constructed plasmid
pGex-4T-1-UAA to decompose uric acid, we used uric acid as a
substrate and added UAO protein. Table 3 and Figure 1

9

showed that with the increase of UAA protein concentration, the
content of uric acid gradually decreased, indicating that UAA is
active and can successfully decompose uric acid. And we used

*E.coil*

BL21
( DE3 ) as the control group, the uric acid content of the control
group had no obvious change trend.

Table
3. The activity urate oxidase

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| Concentration of UAA(mg) | Experimental group | SD | Control | SD |
|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| 0.00000 | 0.247288 | 0.015616036 | 0.194080567 | 0.089358727 |
| 0.01335 | 0.215044 | 0.020638737 | 0.188096847 | 0.084278254 |
| 0.02670 | 0.186315 | 0.010903707 | 0.182951779 | 0.088550902 |
| 0.04005 | 0.142994 | 0.021953815 | 0.169561617 | 0.081267084 |
| 0.05340 | 0.137083 | 0.018292321 | 0.163117255 | 0.079973725 |

![](https://static.igem.wiki/teams/5024/wiki/result-date02.png)

Figure

19

.
The curve of UAA activity

**Contrast
enzyme activity of UAO and UAA**

By
comparing the levels of uric acid after adding UAA and UAO
proteins(Figure

20

),
it was found that the uric acid content was lower with UAA compared
to UAO. This suggests that UAA has slightly higher enzymatic activity
than UAO, further indicating that UAA has a stronger ability to
degrade uric acid. However, the protein expression level of UAA can
be optimized in order to enhance its enzymatic activity in subsequent
experiments.

![](https://static.igem.wiki/teams/5024/wiki/result-date01.png)

Figure

20

.
The curve of UAA and UAO activity

**Future
plans**

In
future studies, we can optimize the protein expression levels of
urate oxidase (UAO), allantoinase, and allantoicase. We will further
test the protein expression and enzyme activity of pGEX-4T-1-UAA in
Nissle 1917 to find the optimal expression conditions. We also need
to validate the ability of urate oxidase (UAO), allantoinase, and
allantoicase to degrade uric acid in large quantities. Additionally,
it is worth considering how to enhance their degradation
capabilities. Apart from that, the role of urate oxidase in degrading
uric acid in the body is still pending verification. The safety of
probiotics also requires further validation.

**Reference**

1.

T.
Neogi, Clinical practice. Gout.

*N
Engl J Med*

**364**

,
443-452 (2011).

2.

Y.
Zhu, B. J. Pandya, H. K. Choi, Prevalence of gout and hyperuricemia
in the US general population: the National Health and Nutrition
Examination Survey 2007-2008.

*Arthritis
Rheum*

**63**

,
3136-3141 (2011).

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