Difference between revisions of "Team:Shanghai HS United/Proof Of Concept"

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        <img src="https://static.igem.org/mediawiki/2021/9/97/T--Shanghai_HS_United--bg_1.png" alt="">
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        <h1>Proof of Concept</h1>
 +
    </div>
 +
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    <!--内容-->
 +
    <div class="content-small">
 +
        <section class="article p-t-54 p-b-54">
 +
            <section class="en-indent-all">
 +
                <h1 class="title">Concept</h1>
 +
                <p>Flavonoids are a large class of important plant secondary metabolites, which not only have good
 +
                    pharmacological activities but also important nutritional factors. Its absorption, metabolism and
 +
                    excretion are completed in the gastrointestinal tract. It plays a variety of physiological functions in
 +
                    the gastrointestinal tract, including antioxidant, anti-inflammatory, anti-cancer, regulating intestinal
 +
                    flora, etc... (see figure 1)</p>
 +
 +
                <div class="img-container">
 +
                    <img src="https://static.igem.org/mediawiki/2021/5/56/T--Shanghai_HS_United--img_proof_of_concept_1.jpg" alt="" style="width: 80%">
 +
                    <span class="figure">Figure. 1 Schematic diagram of how flavonoids benefit the human body</span>
 +
                </div>
 +
 +
                <p>In recent years, a large number of studies have reported the digestion and absorption law of flavonoids
 +
                    in the digestive tract, and aimed at the functional activity of flavonoids, they have been developed for
 +
                    various new foods, drugs, and health products for digestive tract diseases. Based on this aim, we
 +
                    designed to construct intestinal engineering probiotics to improve the absorption of drug and food
 +
                    homologous flavonoids. </p>
 +
 +
                <div class="img-container">
 +
                    <img src="https://static.igem.org/mediawiki/2021/6/63/T--Shanghai_HS_United--img_proof_of_concept_2.jpg" alt="" style="width: 80%">
 +
                    <span class="figure">Figure. 2 Schematic diagram of FLR degrading flavonoids</span>
 +
                </div>
 +
 +
                <p>Our project inspiration came from a paper from Nature Communication written by Weihong Jiang Research
 +
                    Group, CAS, they discovered a new alkene reductase - Flavone reductase (FLR) from Clostridium
 +
                    orbiscindens. This enzyme can not only catalyze the degradation and transformation of flavonoids but
 +
                    also has the same catalytic activity for flavonols, another kind of flavonoids. As said, this enzyme
 +
                    also comes from intestinal bacteria, so we can also ensure the product of our experiments has high
 +
                    practicability.</p>
 +
 +
                <p>In order to enhance the feasibility of our experiment, we chose chrysin, luteolin, diosmeti and apigenin
 +
                    as samples for function tests based on the substrate spectrum analysis (see figure 3). </p>
 +
 +
                <div class="img-container m-t-12">
 +
                    <img src="https://static.igem.org/mediawiki/2021/c/c2/T--Shanghai_HS_United--img_proof_of_concept_3.jpg" alt="" style="width: 80%">
 +
                    <span class="figure">Figure 3 Substrate spectrum of flavonoids</span>
 +
                </div>
 +
            </section>
 +
 +
            <section>
 +
                <h1 class="title">Design and Proof</h1>
 +
 +
                <p>1. Firstly we will combine the pET28a vector and the FLR gene to build the pET28a-FLR plasmid then, put
 +
                    it into E.coli in order to produce the engineered strain for the primary validation of our product
 +
                    concept.</p>
 +
 +
                <div class="img-container m-t-18">
 +
                    <img src="https://static.igem.org/mediawiki/2021/1/13/T--Shanghai_HS_United--img_proof_of_concept_4.jpg" alt="" style="width: 70%">
 +
                    <span class="figure">Figure. 4 Flow chart of the engineered strain design</span>
 +
                </div>
 +
 +
                <p class="en-indent">In lab, we successfully constructed the plasmid and it was proved by colony PCR and
 +
                    sequencing
 +
                    result.</p>
 +
 +
                <p>2. In order to determine the performance of the FLR enzyme and the bacteria, we conducted two groups of
 +
                    experiments with four kinds of flavonoids as previously said, chrysin, luteolin, diosmeti and apigenin.
 +
                    All groups were given the initial concentration of the flavonoids as 10 mg/L and we measured the
 +
                    concentration of these samples by hours to collect data.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.</p>
 +
 +
                <p class="en-indent">The results are given in the following: </p>
 +
 +
                <div class="img-container m-t-18">
 +
                    <img src="https://static.igem.org/mediawiki/2021/d/d3/T--Shanghai_HS_United--img_proof_of_concept_5.jpg" alt="" style="width: 80%">
 +
                    <span class="figure">Figure 5. Bar graph of the enzyme activity test results</span>
 +
                </div>
 +
 +
                <div class="img-container m-t-18">
 +
                    <img src="https://static.igem.org/mediawiki/2021/2/24/T--Shanghai_HS_United--img_proof_of_concept_6.jpg" alt="" style="width: 80%">
 +
                    <span class="figure">Figure 6. Bar graph of the bacteria activity test results</span>
 +
                </div>
 +
 +
                <p class="en-indent">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.</p>
 +
            </section>
 +
 +
            <section class="en-indent-all">
 +
                <h1 class="title">Future Plan</h1>
 +
 +
                <div class="img-container m-t-18">
 +
                    <img src="https://static.igem.org/mediawiki/2021/f/f3/T--Shanghai_HS_United--img_proof_of_concept_7.jpg" alt="" style="width: 50%">
 +
                    <span class="figure">Figure 7. Design sketch of the FLR Yogurt</span>
 +
                </div>
 +
 +
                <p>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.</p>
 +
 +
                <p>In order to achieve our final goal, there are several topics we will take into consideration, including
 +
                    food safety standards,requirement of storage, recipe design, etc.</p>
 +
            </section>
 +
 +
            <section>
 +
                <h1 class="title">Reference</h1>
 +
 +
                <ol>
 +
                    <li>
 +
                        <p>Yang G , Hong S , Yang P , et al. Discovery of an ene-reductase for initiating flavone and
 +
                            flavonol catabolism in gut bacteria[J]. Nature Communications.</p>
 +
                    </li>
 +
                </ol>
 +
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 +
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 +
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 +
 +
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 +
            <span>Email: xiaowenxuan0526@gmail.com; caroline.niujl@gmail.com</span>
 +
            <span>Wechat Official Account: FlavoneLabRouse</span>
 +
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Revision as of 01:38, 28 September 2021

Proof of Concept

Concept

Flavonoids are a large class of important plant secondary metabolites, which not only have good pharmacological activities but also important nutritional factors. Its absorption, metabolism and excretion are completed in the gastrointestinal tract. It plays a variety of physiological functions in the gastrointestinal tract, including antioxidant, anti-inflammatory, anti-cancer, regulating intestinal flora, etc... (see figure 1)

Figure. 1 Schematic diagram of how flavonoids benefit the human body

In recent years, a large number of studies have reported the digestion and absorption law of flavonoids in the digestive tract, and aimed at the functional activity of flavonoids, they have been developed for various new foods, drugs, and health products for digestive tract diseases. Based on this aim, we designed to construct intestinal engineering probiotics to improve the absorption of drug and food homologous flavonoids.

Figure. 2 Schematic diagram of FLR degrading flavonoids

Our project inspiration came from a paper from Nature Communication written by Weihong Jiang Research Group, CAS, they discovered a new alkene reductase - Flavone reductase (FLR) from Clostridium orbiscindens. This enzyme can not only catalyze the degradation and transformation of flavonoids but also has the same catalytic activity for flavonols, another kind of flavonoids. As said, this enzyme also comes from intestinal bacteria, so we can also ensure the product of our experiments has high practicability.

In order to enhance the feasibility of our experiment, we chose chrysin, luteolin, diosmeti and apigenin as samples for function tests based on the substrate spectrum analysis (see figure 3).

Figure 3 Substrate spectrum of flavonoids

Design and Proof

1. Firstly we will combine the pET28a vector and the FLR gene to build the pET28a-FLR plasmid then, put it into E.coli in order to produce the engineered strain for the primary validation of our product concept.

Figure. 4 Flow chart of the engineered strain design

In lab, we successfully constructed the plasmid and it was proved by colony PCR and sequencing result.

2. In order to determine the performance of the FLR enzyme and the bacteria, we conducted two groups of experiments with four kinds of flavonoids as previously said, chrysin, luteolin, diosmeti and apigenin. All groups were given the initial concentration of the flavonoids as 10 mg/L and we measured the concentration of these samples by hours to collect data.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.

The results are given in the following:

Figure 5. Bar graph of the enzyme activity test results
Figure 6. Bar graph of the bacteria activity test results

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

Figure 7. Design sketch of the FLR Yogurt

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.

In order to achieve our final goal, there are several topics we will take into consideration, including food safety standards,requirement of storage, recipe design, etc.

Reference

  1. Yang G , Hong S , Yang P , et al. Discovery of an ene-reductase for initiating flavone and flavonol catabolism in gut bacteria[J]. Nature Communications.

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