iGEM 2021 — Team Shanghai_HS_United

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# Team:Shanghai HS United/Notebook

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# Notebook

![](https://static.igem.org/mediawiki/2021/d/de/T--Shanghai_HS_United--img_notebook_1.jpg)

# July

**25th-26th** We finalized our team members and elected the team leader, design
the team name, logo, uniforms, and perimeters. We conducted laboratory safety training and brief
schedule analysis.

**28th** We made culture medium and buffer A and B, and done the PCR amplification.

**29th** We did the plasmid conversion, spread bacterial colony, and shook Escherichia
coli.

**30th** We collected the linear no-load vector, which was detected by gel electrophoresis
and recovered by gel extraction kit.

**31st** We cultured and then centrifuged E. coli, to obtain the plasmid for further
preservation.

# August

**1st** We did the restriction enzyme digestion, prepared the gel for the gel
electrophoresis, transformed the plasmid, and poured the plates.

**2nd** We did the inoculation, prepared LB culture media, and the protein induction.

**3rd** We purified the protein through Ni-chelating affinity chromatography.

**19th-27th** We conducted the function test to analyze the protein activity.

![](https://static.igem.org/mediawiki/2021/0/00/T--Shanghai_HS_United--img_notebook_2.jpg)

---

Email: xiaowenxuan0526@gmail.com; caroline.niujl@gmail.com
Wechat Official Account: FlavoneLabRouse

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# Team:Shanghai HS United/Engineering

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# Engineering

# Background

We are concerned about the digestion and absorption of flavonoid, which is a healthy compound beneficial
in blood circulation and anti-inflammation, in human’s body, thereafter, we have been making every
effort to find out possible solutions to enhance the degradation efficiency of flavonoid in human’s
body.

# Design

According to Yang, G., et al. (2021), flavone reductase (FLR) discovered from Flavonifractor plautii ATCC
49531 (originally assigned as Clostridium orbiscindens DSM 6740) plays a key step in catalyzing
flavonoid. Thus, we plan to over-express FLR in *Eco. li* (BL21) and test its function in degrading
flavonoid.

This is the general degradation process of flavonoid, in which FLR serves as a catalyst in the initial
step.

![](https://static.igem.org/mediawiki/2021/f/fa/T--Shanghai_HS_United--img_engineering_1.jpg)

This is the plasmid profile we designed to over-express FLR.

![](https://static.igem.org/mediawiki/2021/0/07/T--Shanghai_HS_United--img_engineering_2.jpg)

The composite part is transcripted by the T7 promoter and stopped by the T7 terminator. Meanwhile, a His
protein tag is inserted for future protein purification.

# Build

We carried out molecular biology experiments and successfully constructed the composite part above in the
vector of *Eco. Li (BL21)*. Also, we obtained the purified protein accordingly.

# Test

After obtaining the purified protein containing FLR enzyme, we tested its effectiveness in degrading
flavonoid.

We conducted enzyme activity tests by using 4 kinds of flavonoid samples: apigenin, chrysin, luteolin,
diosmetin with the initial concentration 10mg/L and the concentration of FLR enzyme was 1mM/L. Each
sample was guaranteed three replicates of the enzyme activity test in order to to ensure the credibility
of the test results. The test results are listed as follows:

![](https://static.igem.org/mediawiki/2021/f/ff/T--Shanghai_HS_United--img_engineering_3.jpg)
Figure 1. Bar graph of the enzyme activity test results

# Learn

According to our test results, the concentration of these 4 flavonoids remarkably decreases after 2 hours
which were degraded by the enzyme, even there were only 0.3-5% left after 6 hours. All these results
indicate that the FLR enzyme has high activity and is capable of degrading multiple flavonoid with a
certain universality. It means that the composite part pET28a-FLR is able to realize the expected
function in our design.

---

Email: xiaowenxuan0526@gmail.com; caroline.niujl@gmail.com
Wechat Official Account: FlavoneLabRouse

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# Team:Shanghai HS United/Experiments

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# Experiments

---

Email: xiaowenxuan0526@gmail.com; caroline.niujl@gmail.com
Wechat Official Account: FlavoneLabRouse

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:Shanghai HS United/Results

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# Results

# Enzyme Activity Test

After we obtained the purified protein, namely the FLR enzyme, we conducted enzyme activity tests by
using 4 kinds of flavonoids samples: apigenin, chrysin, luteolin, diosmetin with the initial
concentration 10mg/L and the concentration of FLR enzyme was 1mM/L. Each sample was guaranteed three
replicates of the enzyme activity test in order to gain more data to ensure the credibility of the
result of our experiment.

![](https://static.igem.org/mediawiki/2021/8/81/T--Shanghai_HS_United--img_result_1.jpg)
Figure 1. Bar graph of the enzyme activity test results

Figure 1 shows that the concentration of these 4 flavonoids remarkably decreases after 2 hours which were
degraded by the enzyme, even there were only 0.3-5% left after 6 hours. All these results indicate that
our enzyme has high activity and is capable of degrading multiple flavonoids with a certain
universality.

# Bacteria Activity Test

In order to analyze the performance of our engineered E. coli, we also conducted the activity test by
using the same 4 kinds of flavonoids samples: apigenin, chrysin, luteolin, diosmetin with the initial
concentration 10mg/L and the OD600 was 1.0. Each sample was guaranteed three replicates of the activity
test in order to gain more data to ensure the credibility of the result of our experiment.

![](https://static.igem.org/mediawiki/2021/5/56/T--Shanghai_HS_United--img_result_2.jpg)
Figure 2. Bar graph of the bacteria activity test results

In figure 2, you could easily tell that all these flavonoids were almost degraded after 2 hours with only
1-7% left, which indicates the strong ability of our engineered E. coli to degrade flavonoids as we
expected.

Above all, we could come to the conclusion that the FLR enzyme, as well as the engineered E. coli, is
capable of degrading various flavonoids with a certain universality and practicability. In comparison,
the degradability of FLR enzyme to these flavonoids could be ranked from greatest to least as apigenin >
chrysin > luteolin > diosmetin.

# Future Plan

Since the FLR enzyme showed a great ability to decompose flavonoids, we are looking forward to testing
how the human body would consume with and without the FLR enzyme. What’s more, if the further experiment
succeeds, we will probably try to add the FLR engineered bacteria into yogurt. Yogurt contains a large
number of lactic acid bacteria, which themselves belong to acidic substances. After use, they can
promote gastrointestinal peristalsis and food digestion. Yogurt helps in digesting food and FLR helps in
digesting flavonoids. By adding it, we could make a health-care yogurt which is great for human health
improvement.

---

Email: xiaowenxuan0526@gmail.com; caroline.niujl@gmail.com
Wechat Official Account: FlavoneLabRouse