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

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        <title>Document</title>
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<div class="column full_size judges-will-not-evaluate">
 
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<h3>★  ALERT! </h3>
 
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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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<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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        #bodyContent h1,
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<h1>Engineering Success</h1>
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        #bodyContent h2,
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<h3> Silver Medal Criterion #1</h3>
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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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<br><br>
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        .link-https {
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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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<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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        <div style="height: 255px;width: 100%;display: flex;justify-content: space-around;">
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                <img src="https://static.igem.org/mediawiki/2021/2/27/T--Shanghai_United--homepage01.png"
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                        style="width: 173px; height: 89px;margin-right: 271px;margin-left: 120px;margin-top: 80px;display: block;" />
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                <ul class="layui-nav navbar navbar-fixed-top text-center" lay-filter=""
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                        style="background-color:white;height: 255px;line-height: 255px;">
 +
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                        <li class="layui-nav-item" style="text-align: center;">
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                                <a href="https://2021.igem.org/Team:Shanghai_United"
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                                        style="text-align: center;">Home</a>
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                        </li>
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                        <li class="layui-nav-item" style="text-align: center;">
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                                <a href="javascript:;">Project</a>
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                                <dl class="layui-nav-child">
 +
                                        <!-- 二级菜单 -->
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                                        <dd>
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                                                <a
 +
                                                        href="https://2021.igem.org/Team:Shanghai_United/Description">Description</a>
 +
                                        </dd>
 +
                                        <dd>
 +
                                                <a href="https://2021.igem.org/Team:Shanghai_United/Proof_Of_Concept">Proof
 +
                                                        of Concept</a>
 +
                                        </dd>
 +
                                        <dd>
 +
                                                <a
 +
                                                        href="https://2021.igem.org/Team:Shanghai_United/Experiments">Experiments</a>
 +
                                        </dd>
 +
                                        <dd>
 +
                                                <a
 +
                                                        href="https://2021.igem.org/Team:Shanghai_United/Notebook">Notebook</a>
 +
                                        </dd>
 +
                                        <dd>
 +
                                                <a href="https://2021.igem.org/Team:Shanghai_United/Results">Results</a>
 +
                                        </dd>
 +
                                        <dd>
 +
                                                <a href="https://2021.igem.org/Team:Shanghai_United/Safety">Safety</a>
 +
                                        </dd>
 +
 +
                                </dl>
 +
                        </li>
 +
                        <li class="layui-nav-item" style="text-align: center;">
 +
                                <a href="javascript:;">Parts</a>
 +
                                <dl class="layui-nav-child">
 +
                                        <!-- 二级菜单 -->
 +
                                        <dd>
 +
                                                <a
 +
                                                        href="https://2021.igem.org/Team:Shanghai_United/Engineering">Engineering</a>
 +
                                        </dd>
 +
                                        <dd>
 +
                                                <a href="https://2021.igem.org/Team:Shanghai_United/Parts Collection">Parts
 +
                                                        Collection</a>
 +
                                        </dd>
 +
 +
                                </dl>
 +
                        </li>
 +
                        <li class="layui-nav-item">
 +
                                <a href="javascript:;">Human Practice</a>
 +
                                <dl class="layui-nav-child">
 +
                                        <!-- 二级菜单 -->
 +
                                        <dd>
 +
                                                <a
 +
                                                        href="https://2021.igem.org/Team:Shanghai_United/Fundraising">Fundraising</a>
 +
                                        </dd>
 +
                                        <dd>
 +
                                                <a
 +
                                                        href="https://2021.igem.org/Team:Shanghai_United/Communication">Communication</a>
 +
                                        </dd>
 +
                                        <dd>
 +
                                                <a
 +
                                                        href="https://2021.igem.org/Team:Shanghai_United/Integrated_Human_Practices">Integrated Human Practices</a>
 +
                                        </dd>
 +
                                </dl>
 +
                        </li>
 +
                        <li class="layui-nav-item" style="text-align: center;">
 +
                                <a href="https://2021.igem.org/Team:Shanghai_United/Implementation">Implementation</a>
 +
                        </li>
 +
 +
                        <li class="layui-nav-item" style="text-align: center;">
 +
                                <a
 +
                                        href="https://2021.igem.org/Team:Shanghai_United/Entrepreneurship">Entrepreneurship</a>
 +
                        </li>
 +
 +
                        <li class="layui-nav-item" style="text-align: center;">
 +
                                <a href="https://2021.igem.org/Team:Shanghai_United/Model">Model</a>
 +
                        </li>
 +
 +
 +
                        <li class="layui-nav-item" style="text-align: center;">
 +
                                <a href="javascript:;">Team</a>
 +
                                <dl class="layui-nav-child">
 +
                                        <!-- 二级菜单 -->
 +
                                        <dd>
 +
                                                <a href="https://2021.igem.org/Team:Shanghai_United/Team">Team
 +
                                                        Member</a>
 +
                                        </dd>
 +
                                        <dd>
 +
                                                <a
 +
                                                        href="https://2021.igem.org/Team:Shanghai_United/Collaborations">Collaborations</a>
 +
                                        </dd>
 +
                                        <dd>
 +
                                                <a
 +
                                                        href="https://2021.igem.org/Team:Shanghai_United/Attributions">Attributions</a>
 +
                                        </dd>
 +
                                </dl>
 +
                        </li>
 +
                </ul>
 +
 +
        </div>
 +
 +
        <!--banner-->
 +
        <div style="width:100%;">
 +
                <img style="width:100%;" src="https://static.igem.org/mediawiki/2021/a/a5/T--Shanghai_United--Description00.png"></img>
 +
        </div>
 +
 +
        <!--text-->
 +
        <div style="width:100%;padding:10% 30%;">
 +
                <div>
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                        <p style="font-size:48px; line-height:200%; margin:15.6pt 0pt; orphans:0; text-align:center; widows:0"><span
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                            style="font-family:'Segoe UI', Tahoma, Geneva, Verdana, sans-serif; font-size:48px; font-weight:bold">Engineering</span></p>
 +
                        <p style="font-size:36px; line-height:200%; margin:15.6pt 0pt; orphans:0; text-align:justify; widows:0"><span
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                            style="font-family:'Segoe UI', Tahoma, Geneva, Verdana, sans-serif; font-size:36px; font-weight:bold">Introduction </span></p>
 +
                        <p style="font-size:22px; line-height:200%; margin:15.6pt 0pt; orphans:0; text-align:justify; widows:0"><span
 +
                            style="font-family:'Segoe UI', Tahoma, Geneva, Verdana, sans-serif; font-size:22px">Arsenic is widely distributed in the natural environment.
 +
                            Trace amounts of arsenic can be detected in soil, water, minerals, and plants. Among many environmental
 +
                            pollutants, arsenic ranks first on the blacklist of polluting toxic elements and is also a powerful carcinogen.
 +
                            In many countries, arsenic causes water, soil and crop pollution. We design an arsenic-induced response system
 +
                            in the cell or on the surface of the engineered probiotic bacteria to detect the content of arsenite in the
 +
                            environment, provide warnings to people, and reduce the health damage it causes to the human body. </span></p>
 +
                        <p style="font-size:36px; line-height:200%; margin:15.6pt 0pt; orphans:0; text-align:justify; widows:0"><span
 +
                            style="font-family:'Segoe UI', Tahoma, Geneva, Verdana, sans-serif; font-size:36px; font-weight:bold">Design</span><span
 +
                            style="font-family:'Segoe UI', Tahoma, Geneva, Verdana, sans-serif; font-size:22px"> </span></p>
 +
                        <p style="font-size:22px; line-height:200%; margin:15.6pt 0pt; orphans:0; text-align:justify; widows:0"><span
 +
                            style="font-family:'Segoe UI', Tahoma, Geneva, Verdana, sans-serif; font-size:22px">Three proteins ArsR, ArsD, and ArsA belong to the operon
 +
                            family. The cysteine thiolates on their amino acid sequences can form a three-coordinate complex with arsenic
 +
                            (iii), which is self-sensing to arsenic. </span></p>
 +
                        <p style="font-size:22px; line-height:200%; margin:15.6pt 0pt; orphans:0; text-align:justify; widows:0"><span
 +
                            style="font-family:'Segoe UI', Tahoma, Geneva, Verdana, sans-serif; font-size:22px">Under the induction of arsenite, ArsD gene is transcript
 +
                            and enxpressed in </span><span style="font-family:'Segoe UI', Tahoma, Geneva, Verdana, sans-serif; font-size:22px; font-style:italic">E.
 +
                            coli</span><span style="font-family:'Segoe UI', Tahoma, Geneva, Verdana, sans-serif; font-size:22px"> cells. At the same time, a reporter gene
 +
                            GFP is connected to the ArsD gene. Then, when the concentration of arsenite in the environment, the expression
 +
                            of ArsD-GFP can be detected by observing the intensity of the fluorescent signal.   </span></p>
 +
                        <p style="font-size:36px; line-height:200%; margin:15.6pt 0pt; orphans:0; text-align:justify; widows:0"><span
 +
                            style="font-family:'Segoe UI', Tahoma, Geneva, Verdana, sans-serif; font-size:36px; font-weight:bold">Build </span></p>
 +
                        <p style="font-size:22px; line-height:200%; margin:15.6pt 0pt; orphans:0; text-align:justify; widows:0"><span
 +
                            style="font-family:'Segoe UI', Tahoma, Geneva, Verdana, sans-serif; font-size:22px">Three recombinant plasmids pUC57-ArsD-amilGFP (Figure 1),
 +
                            pUC57-ArsR-amilGFP, and pUC57-ArsA-amilGFP were constructed in order to select the most sensitive sensor to
 +
                            arsenic. All the constructs are confirmed by DNA sequencing.</span></p>
 +
                        <p style="font-size:22px; line-height:200%; margin:15.6pt 0pt; orphans:0; text-align:center; widows:0"><img
 +
                            src="https://static.igem.org/mediawiki/2021/4/4e/T--Shanghai_United--Engineering01.jpg" width="500" alt=""
 +
                            style="-aw-left-pos:0pt; -aw-rel-hpos:column; -aw-rel-vpos:paragraph; -aw-top-pos:0pt; -aw-wrap-type:inline" />
 +
                        </p>
 +
                        <p style="font-size:16px; line-height:200%; margin:15.6pt 0pt; orphans:0; text-align:center; widows:0"><span
 +
                            style="font-family:'Segoe UI', Tahoma, Geneva, Verdana, sans-serif; font-size:16px">Figure 1. Schematic map of plasmids.</span></p>
 +
                        <p style="font-size:36px; line-height:200%; margin:15.6pt 0pt; orphans:0; text-align:justify; widows:0"><span
 +
                            style="font-family:'Segoe UI', Tahoma, Geneva, Verdana, sans-serif; font-size:36px; font-weight:bold">Test </span></p>
 +
                        <p style="font-size:22px; line-height:200%; margin:15.6pt 0pt; orphans:0; text-align:justify; widows:0"><span
 +
                            style="font-family:'Segoe UI', Tahoma, Geneva, Verdana, sans-serif; font-size:22px">Fluorescence intensity generated by an ARSD/amilGFP
 +
                            transformed E. coli reacting for 1 hour in C</span><span
 +
                            style="font-family:'Segoe UI', Tahoma, Geneva, Verdana, sans-serif; font-size:16px; vertical-align:sub">2</span><span
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                            style="font-family:'Segoe UI', Tahoma, Geneva, Verdana, sans-serif; font-size:22px">H</span><span
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                            style="font-family:'Segoe UI', Tahoma, Geneva, Verdana, sans-serif; font-size:16px; vertical-align:sub">6</span><span
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                            style="font-family:'Segoe UI', Tahoma, Geneva, Verdana, sans-serif; font-size:22px">AsNaO</span><span
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                            style="font-family:'Segoe UI', Tahoma, Geneva, Verdana, sans-serif; font-size:16px; vertical-align:sub">5</span><span
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                            style="font-family:'Segoe UI', Tahoma, Geneva, Verdana, sans-serif; font-size:22px"> </span><span
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                            style="font-family:'Segoe UI', Tahoma, Geneva, Verdana, sans-serif; font-size:22px">solutions. As seen in the figure, fluorescence was
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                            detected for all C</span><span
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                            style="font-family:'Segoe UI', Tahoma, Geneva, Verdana, sans-serif; font-size:16px; vertical-align:sub">2</span><span
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                            style="font-family:'Segoe UI', Tahoma, Geneva, Verdana, sans-serif; font-size:22px">H</span><span
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                            style="font-family:'Segoe UI', Tahoma, Geneva, Verdana, sans-serif; font-size:16px; vertical-align:sub">6</span><span
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                            style="font-family:'Segoe UI', Tahoma, Geneva, Verdana, sans-serif; font-size:22px">AsNaO</span><span
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                            style="font-family:'Segoe UI', Tahoma, Geneva, Verdana, sans-serif; font-size:16px; vertical-align:sub">5</span><span
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                            style="font-family:'Segoe UI', Tahoma, Geneva, Verdana, sans-serif; font-size:22px"> concentrations that are above 0, which confirms that the
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                            designed plasmid worked as intended (Figure 2).</span></p>
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                        <p style="font-size:12pt; line-height:130%; margin:3pt 0pt; orphans:0; text-align:center; widows:0"><img
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                        <p style="font-size:16px; line-height:200%; margin:15.6pt 0pt; orphans:0; text-align:center; widows:0"><span
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                            style="font-family:'Segoe UI', Tahoma, Geneva, Verdana, sans-serif; font-size:16px">Figure 2: Fluorescence Intensity of Transformed E. coli In
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                            Different Concentrations of C</span><span
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                            style="font-family:'Segoe UI', Tahoma, Geneva, Verdana, sans-serif; font-size:16px; vertical-align:sub">2</span><span
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                            style="font-family:'Segoe UI', Tahoma, Geneva, Verdana, sans-serif; font-size:16px">H</span><span
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                            style="font-family:'Segoe UI', Tahoma, Geneva, Verdana, sans-serif; font-size:16px; vertical-align:sub">6</span><span
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                            style="font-family:'Segoe UI', Tahoma, Geneva, Verdana, sans-serif; font-size:16px">AsNaO</span><span
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                            style="font-family:'Segoe UI', Tahoma, Geneva, Verdana, sans-serif; font-size:16px; vertical-align:sub">5</span><span
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                            style="font-family:'Segoe UI', Tahoma, Geneva, Verdana, sans-serif; font-size:16px"> Solutions</span></p>
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                        <p style="font-size:36px; line-height:200%; margin:15.6pt 0pt; orphans:0; text-align:justify; widows:0"><span
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                            style="font-family:'Segoe UI', Tahoma, Geneva, Verdana, sans-serif; font-size:36px; font-weight:bold">Learn</span></p>
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                        <p style="font-size:22px; line-height:200%; margin:15.6pt 0pt; orphans:0; text-align:justify; widows:0"><span
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                            style="font-family:'Segoe UI', Tahoma, Geneva, Verdana, sans-serif; font-size:22px">C</span><span
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                            style="font-family:'Segoe UI', Tahoma, Geneva, Verdana, sans-serif; font-size:16px; vertical-align:sub">2</span><span
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                            style="font-family:'Segoe UI', Tahoma, Geneva, Verdana, sans-serif; font-size:22px">H</span><span
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                            style="font-family:'Segoe UI', Tahoma, Geneva, Verdana, sans-serif; font-size:16px; vertical-align:sub">6</span><span
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                            style="font-family:'Segoe UI', Tahoma, Geneva, Verdana, sans-serif; font-size:22px">AsNaO</span><span
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                            style="font-family:'Segoe UI', Tahoma, Geneva, Verdana, sans-serif; font-size:16px; vertical-align:sub">5</span><span
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                            style="font-family:'Segoe UI', Tahoma, Geneva, Verdana, sans-serif; font-size:22px"> was used for this function test. The observed
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                            fluorescence intensity suggested that the engineered bacteria are functional. However, the fluorescence was
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                            rather minor, so we speculate that </span><span
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                            style="font-family:'Segoe UI', Tahoma, Geneva, Verdana, sans-serif; font-size:22px; font-style:italic">E. coli</span><span
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                            style="font-family:'Segoe UI', Tahoma, Geneva, Verdana, sans-serif; font-size:22px"> responds poorly to organic arsenic compounds. Further
 +
                            experiments would be conducted to test for fluorescence intensities of E. coli in inorganic arsenic solutions.  
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                          </span></p>
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Revision as of 10:56, 12 October 2021

Document

Engineering

Introduction

Arsenic is widely distributed in the natural environment. Trace amounts of arsenic can be detected in soil, water, minerals, and plants. Among many environmental pollutants, arsenic ranks first on the blacklist of polluting toxic elements and is also a powerful carcinogen. In many countries, arsenic causes water, soil and crop pollution. We design an arsenic-induced response system in the cell or on the surface of the engineered probiotic bacteria to detect the content of arsenite in the environment, provide warnings to people, and reduce the health damage it causes to the human body.

Design

Three proteins ArsR, ArsD, and ArsA belong to the operon family. The cysteine thiolates on their amino acid sequences can form a three-coordinate complex with arsenic (iii), which is self-sensing to arsenic.

Under the induction of arsenite, ArsD gene is transcript and enxpressed in E. coli cells. At the same time, a reporter gene GFP is connected to the ArsD gene. Then, when the concentration of arsenite in the environment, the expression of ArsD-GFP can be detected by observing the intensity of the fluorescent signal.  

Build

Three recombinant plasmids pUC57-ArsD-amilGFP (Figure 1), pUC57-ArsR-amilGFP, and pUC57-ArsA-amilGFP were constructed in order to select the most sensitive sensor to arsenic. All the constructs are confirmed by DNA sequencing.

Figure 1. Schematic map of plasmids.

Test

Fluorescence intensity generated by an ARSD/amilGFP transformed E. coli reacting for 1 hour in C2H6AsNaO5 solutions. As seen in the figure, fluorescence was detected for all C2H6AsNaO5 concentrations that are above 0, which confirms that the designed plasmid worked as intended (Figure 2).

Figure 2: Fluorescence Intensity of Transformed E. coli In Different Concentrations of C2H6AsNaO5 Solutions

Learn

C2H6AsNaO5 was used for this function test. The observed fluorescence intensity suggested that the engineered bacteria are functional. However, the fluorescence was rather minor, so we speculate that E. coli responds poorly to organic arsenic compounds. Further experiments would be conducted to test for fluorescence intensities of E. coli in inorganic arsenic solutions.  

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