Difference between revisions of "Team:Shanghai HS United/Engineering"

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{{IGEM_TopBar}}
 
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{{Shanghai_HS_United}}
 
 
<html>
 
<html>
  
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<link rel="stylesheet" type="text/css"
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      href="https://2021.igem.org/wiki/index.php?title=Template:Shanghai_HS_United/StyleCSS&action=raw&ctype=text/css"/>
  
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<div class="column full_size judges-will-not-evaluate">
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<div class="flavone-lab-content">
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<h3>★  ALERT! </h3>
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    <!--navbar-->
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<p>This page is used by the judges to evaluate your team for the <a href="https://2021.igem.org/Judging/Medals">medal criterion</a> or <a href="https://2021.igem.org/Judging/Awards"> award listed below</a>. </p>
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    <div class="navbar">
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<p> Delete this box in order to be evaluated for this medal criterion and/or award. See more information at <a href="https://2021.igem.org/Judging/Pages_for_Awards"> Instructions for Pages for awards</a>.</p>
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</div>
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            <img src="https://static.igem.org/mediawiki/2021/c/c1/T--Shanghai_HS_United--logo.png" alt="">
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        </a>
  
 +
        <!--nav-->
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        <ul class="list">
 +
            <!--Team-->
 +
            <li class="item">
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                <a href="https://2021.igem.org/Team:Shanghai_HS_United/Team">Team</a>
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                <div class="child-nav">
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                    <ul class="child-list">
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                        <li class="child-item">
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                            <a href="https://2021.igem.org/Team:Shanghai_HS_United/Team">Team Members</a>
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                        </li>
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                        <li class="child-item">
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                            <a href="https://2021.igem.org/Team:Shanghai_HS_United/Attributions">Attributions</a>
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                        </li>
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                        <li class="child-item">
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                            <a href="https://2021.igem.org/Team:Shanghai_HS_United/Collaborations">Collaboration</a>
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                        </li>
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                    </ul>
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                </div>
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            </li>
  
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<div class="clear"></div>
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            <!--Modeling-->
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            <li class="item">
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                <a href="https://2021.igem.org/Team:Shanghai_HS_United/Model">Modeling</a>
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            </li>
  
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            <!--Entrepreneurship-->
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            <li class="item">
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                <a href="https://2021.igem.org/Team:Shanghai_HS_United/Entrepreneurship">Entrepreneurship</a>
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            </li>
  
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<div class="column full_size">
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            <!--Implementation-->
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<h1>Engineering Success</h1>
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            <li class="item">
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<h3> Silver Medal Criterion #1</h3>
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                <a href="https://2021.igem.org/Team:Shanghai_HS_United/Implementation">Implementation</a>
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            </li>
  
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<p>Demonstrate engineering success in a part of your project by going through at least one iteration of the engineering design cycle. This achievement should be distinct from your Contribution for Bronze.
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            <!--Human Practice-->
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<br><br>
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            <li class="item">
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Please see the <a href="https://2021.igem.org/Judging/Medals">2021 Medals Page</a> for more information.  
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                <a href="https://2021.igem.org/Team:Shanghai_HS_United/Human_Practices">Human Practice</a>
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</p>
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                <div class="child-nav">
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                    <ul class="child-list">
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                        <li class="child-item">
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                            <a href="https://2021.igem.org/Team:Shanghai_HS_United/Human_Practices">Integrated Human
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                                Practice</a>
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                        </li>
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                        <li class="child-item">
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                            <a href="https://2021.igem.org/Team:Shanghai_HS_United/Communication">Communication</a>
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                        </li>
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                        <li class="child-item">
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                            <a href="https://2021.igem.org/Team:Shanghai_HS_United/Fundraising">Fundraising</a>
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                        </li>
 +
                    </ul>
 +
                </div>
 +
            </li>
  
 +
            <!--Parts-->
 +
            <li class="item active">
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                <a href="https://2021.igem.org/Team:Shanghai_HS_United/PartsCollection">Parts</a>
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                    <ul class="child-list">
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                        <li class="child-item">
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                            <a href="https://2021.igem.org/Team:Shanghai_HS_United/Parts_Collection">Parts
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                                Collection</a>
 +
                        </li>
 +
                        <li class="child-item active">
 +
                            <a href="https://2021.igem.org/Team:Shanghai_HS_United/Engineering">Engineering</a>
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                        </li>
 +
                    </ul>
 +
                </div>
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            </li>
  
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<p>If you plan to show engineering success by creating a new Part that has been shown to work as expected, you must document your contribution on the Part's Main Page on the <a href="http://parts.igem.org/Main_Page">Registry</a> for your team to be eligible for this criteria.</p>
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            <!--Project-->
 +
            <li class="item">
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                <a href="https://2021.igem.org/Team:Shanghai_HS_United/Description">Project</a>
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                    <ul class="child-list">
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                        <li class="child-item">
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                            <a href="https://2021.igem.org/Team:Shanghai_HS_United/Description">Description</a>
 +
                        </li>
 +
                        <li class="child-item">
 +
                            <a href="https://2021.igem.org/Team:Shanghai_HS_United/Experiments">Experiments</a>
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                        </li>
 +
                        <li class="child-item">
 +
                            <a href="https://2021.igem.org/Team:Shanghai_HS_United/Results">Result</a>
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                        </li>
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                        <li class="child-item">
 +
                            <a href="https://2021.igem.org/Team:Shanghai_HS_United/Proof_Of_Concept">Proof of Concept</a>
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                        </li>
 +
                        <li class="child-item">
 +
                            <a href="https://2021.igem.org/Team:Shanghai_HS_United/Notebook">Notebook</a>
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                        </li>
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                        <li class="child-item">
 +
                            <a href="https://2021.igem.org/Team:Shanghai_HS_United/Safety">Safety</a>
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                        </li>
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                    </ul>
 +
                </div>
 +
            </li>
  
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</div>
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            <!--Home-->
 +
            <li class="item">
 +
                <a href="https://2021.igem.org/Team:Shanghai_HS_United">Home</a>
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            </li>
 +
        </ul>
 +
    </div>
  
 +
    <!--图片-->
 +
    <div class="page-header">
 +
        <img src="https://static.igem.org/mediawiki/2021/9/97/T--Shanghai_HS_United--bg_1.png" alt="">
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        <h1></h1>
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    </div>
  
 +
    <!--内容-->
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    <div class="content-small">
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        <section class="article p-t-54 p-b-54">
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            <section class="en-indent-all">
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                <h1 class="title">Background</h1>
 +
 +
                <p>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.</p>
 +
            </section>
 +
 +
            <section class="en-indent-all">
 +
                <h1 class="title">Design-Build-Test-Learn</h1>
 +
 +
                <p>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 <i>Eco. li</i> (BL21) and test its function in degrading
 +
                    flavonoid. </p>
 +
 +
                <p>This is the general degradation process of flavonoid, in which FLR serves as a catalyst in the initial
 +
                    step.</p>
 +
 +
                <img src="https://static.igem.org/mediawiki/2021/f/fa/T--Shanghai_HS_United--img_engineering_1.jpg" alt="" style="width: 80%">
 +
 +
                <p>This is the plasmid profile we designed to over-express FLR.</p>
 +
 +
                <img src="https://static.igem.org/mediawiki/2021/0/07/T--Shanghai_HS_United--img_engineering_2.jpg" alt="" style="width: 80%">
 +
 +
                <p>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. </p>
 +
            </section>
 +
 +
            <section>
 +
                <h1 class="title">Build</h1>
 +
 +
                <p>We carried out molecular biology experiments and successfully constructed the composite part above in the
 +
                    vector of <i>Eco. Li (BL21)</i>. Also, we obtained the purified protein accordingly. </p>
 +
            </section>
 +
 +
            <section>
 +
                <h1 class="title">Test</h1>
 +
 +
                <p>After obtaining the purified protein containing FLR enzyme, we tested its effectiveness in degrading
 +
                    flavonoid. </p>
 +
 +
                <p>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:</p>
 +
 +
                <div class="img-container">
 +
                    <img src="https://static.igem.org/mediawiki/2021/f/ff/T--Shanghai_HS_United--img_engineering_3.jpg" alt="" style="width: 90%">
 +
                    <span class="figure">Figure 1. Bar graph of the enzyme activity test results</span>
 +
                </div>
 +
            </section>
 +
 +
            <section>
 +
                <h1 class="title">Learn</h1>
 +
 +
                <p>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. </p>
 +
            </section>
 +
        </section>
 +
    </div>
 +
 +
    <!--Footer-->
 +
    <div class="footer">
 +
        <div class="content">
 +
            <hr>
 +
            <span>Email: xiaowenxuan0526@gmail.com; caroline.niujl@gmail.com</span>
 +
            <span>Wechat Official Account: FlavoneLabRouse</span>
 +
        </div>
 +
    </div>
 +
</div>
 
</html>
 
</html>

Revision as of 08:48, 11 October 2021

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-Build-Test-Learn

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.

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

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:

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