Difference between revisions of "Team:HZAU-China/Composite Part"

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−
{{HZAU-China}}
+
{{HZAU-China/Bootstrap}}
 
<html>
 
<html>
  
 +
<head>
 +
    <meta charset="UTF-8">
 +
    <meta name="viewport" content="width=device-width, initial-scale=1.0, minimum-scale=1.0, maximum-scale=1.0, user-scalable=no">
 +
    <link href="bootstrap.min.css" rel="stylesheet">
 +
    <!-- 导航栏CSS -->
 +
    <style type="text/css">
 +
        #sideMenu,
 +
        #top_title,
 +
        #team_submenu {
 +
            display: none;
 +
        }
  
 +
        #HQ_page p {
 +
            margin: 0 !important;
 +
        }
  
−
<div class="column full_size judges-will-not-evaluate">
+
        #content {
−
<h3>★  ALERT! </h3>
+
            padding: 0px;
−
<p>This page is used by the judges to evaluate your team for the <a href="https://2018.igem.org/Judging/Medals">medal criterion</a> or <a href="https://2018.igem.org/Judging/Awards"> award listed below</a>. </p>
+
            width: 100%;
−
<p> Delete this box in order to be evaluated for this medal criterion and/or award. See more information at <a href="https://2018.igem.org/Judging/Pages_for_Awards"> Instructions for Pages for awards</a>.</p>
+
            margin-top: 0px;
−
</div>
+
            margin-left: 0px;
 +
        }
  
 +
        #bodyContent h1,
 +
        #bodyContent h2,
 +
        #bodyContent h3,
 +
        #bodyContent h4,
 +
        #bodyContent h5 {
 +
            margin-bottom: 0px;
 +
        }
  
−
<div class="clear"></div>
+
        #team_name {
 +
            display: none
 +
        }
  
 +
        .global_wrapper {
 +
            padding: 0px 0px 0px 0px;
 +
        }
  
 +
        #top_menu_14 {
 +
            height: 16px;
 +
        }
  
 +
        * {
 +
            margin: 0;
 +
            padding: 0;
 +
            list-style: none;
 +
            list-style-type: none;
 +
            text-decoration: none !important;
 +
        }
  
 +
        body {
 +
            margin: 0;
 +
            padding: 0;
 +
            background-color: #F3F3F3;
 +
            font-size: 16px;
 +
            font-family: Arial, Verdana, Sans-serif;
 +
            letter-spacing: 0;
 +
            color: #FFFFFF;
 +
        }
  
−
<div class="column full_size">
+
        .daohang {
−
<h1>Composite Parts</h1>
+
            background-color: #323643;
 +
            position: relative;
 +
            color: #ffffff;
 +
            height: 64px;
 +
            top: -2px;
 +
        }
  
 +
        .logo-daohang {
 +
            height: 64px;
 +
            left: 10%;
 +
            float: left;
 +
            position: relative;
 +
        }
  
−
<p>
+
        .daohang a {
−
A composite part is a functional unit of DNA consisting of two or more basic parts assembled together. <a href="http://parts.igem.org/wiki/index.php/Part:BBa_I13507">BBa_I13507</a> is an example of a composite part, consisting of an RBS, a protein coding region for a red fluorescent protein, and a terminator.
+
            display: block;
−
</p>
+
            text-decoration: none;
 +
        }
  
−
<p>New composite BioBrick devices can be made by combining existing BioBrick Parts (like Inverters, Amplifiers, Smell Generators, Protein Balloon Generators, Senders, Receivers, Actuators, and so on).</p>
+
        .daohang .shade {
−
</div>
+
            position: absolute;
 +
            width: 100%;
 +
            height: 160px;
 +
            top: 64px;
 +
            left: 0;
 +
            z-index: 1;
 +
            background-color: #EEEEEE;
 +
            box-shadow: 0 5px 15px #CCCCCC;
 +
            display: none;
 +
            border-bottom: #ffffff solid 1px;
 +
            border-bottom: rgba(255, 255, 255, 0.3) solid 1px;
 +
        }
  
−
<div class="column full_size">
+
        .daohang .caidan {
−
<div class="highlight decoration_background">
+
            position: absolute;
−
<h3>Note</h3>
+
            top: 0;
−
<p>This page should list all the composite parts your team has made during your project. You must add all characterization information for your parts on the Registry. You should not put characterization information on this page. Remember judges will only look at the first part in the list for the Best Composite Part award, so put your best part first!</p>
+
            right: 10%;
−
</div>
+
            margin: 0;
−
</div>
+
            z-index: 2;
 +
            padding: 0;
 +
            list-style: none;
 +
            width: 730px;
 +
            float: right;
 +
            overflow: hidden;
 +
            height: 64px;
 +
        }
  
 +
        .daohang .caidan>li {
 +
            padding: 0;
 +
            float: left;
 +
            margin: 0;
 +
            text-align: center;
 +
            height: 224px;
 +
        }
  
 +
        .daohang .shortName {
 +
            width: 110px;
 +
        }
  
−
<div class="column full_size">
+
        .daohang .longName {
−
<h3>Best Composite Part Special Prize</h3>
+
            width: 170px;
 +
        }
  
−
<p>To be eligible for this award, this part must adhere to <a href="http://parts.igem.org/DNA_Submission">Registry sample submission guidelines</a> and have been sent to the Registry of Standard Biological Parts. If you have a part you wish to nominate your team for this <a href="https://2018.igem.org/Judging/Awards">special prize</a>, make sure you add your part number to your <a href="https://2018.igem.org/Judging/Judging_Form">judging form</a> and delete the box at the top of this page.
+
        .daohang .nav_head {
 +
            height: 64px;
 +
            line-height: 64px;
 +
            color: #ffffff;
 +
            font-size: 16px !important;
 +
        }
  
−
<br><br>
+
        .daohang .xsjPic {
−
<b>Please note:</b> Judges will only look at the first part number you list, so please only enter ONE (1) part number in the judging form for this prize. </p>
+
            display: inline-block;
 +
            width: 10px;
 +
            height: 10px;
 +
            vertical-align: middle;
 +
            background-size: 100% 100%;
 +
            background-image: url("https://static.igem.org/mediawiki/2018/6/63/T--HZAU-China--xsj.svg")
 +
        }
  
−
</div>
+
        .daohang .xjtPic {
 +
            display: inline-block;
 +
            width: 10px;
 +
            height: 10px;
 +
            vertical-align: middle;
 +
            background-size: 100% 100%;
 +
            background-image: url("https://static.igem.org/mediawiki/2018/8/8d/T--HZAU-China--xjt.svg")
 +
        }
 +
 
 +
        .daohang a:hover span {
 +
            transform: rotateY(180deg);
 +
            -webkit-transform: rotateY(180deg);
 +
            -moz-transform: rotateY(180deg);
 +
            -o-transform: rotateY(180deg);
 +
            -ms-transform: rotateY(180deg);
 +
            transition: all 0.5s ease-in-out;
 +
            -webkit-transition: all 0.5s ease-in-out;
 +
            -moz-transition: all 0.5s ease-in-out;
 +
            -o-transition: all 0.5s ease-in-out;
 +
        }
 +
 
 +
        .daohang .item {
 +
            font-size: 16px;
 +
            height: 40px;
 +
            line-height: 40px;
 +
            color: #333333;
 +
            position: relative;
 +
        }
 +
 
 +
        .daohang .caidan:hover {
 +
            height: 264px;
 +
            transition: height 0.3s;
 +
        }
 +
 
 +
        .daohang .caidan:hover+.shade {
 +
            display: block;
 +
        }
 +
 
 +
        .daohang .caidan>li.hiLight:hover {
 +
            background-color: #ffffff;
 +
        }
 +
 
 +
        .daohang .caidan>li:hover .nav_head {
 +
            background-color: #EA0D04;
 +
        }
 +
 
 +
        .daohang .caidan>li:hover .item {}
 +
 
 +
 
 +
        .daohang .shortName .item:before {
 +
            content: '';
 +
            display: block;
 +
            position: absolute;
 +
            width: 80px;
 +
            height: 1px;
 +
            bottom: 5px;
 +
            left: 15px;
 +
            background-color: #CCCCCC;
 +
        }
 +
 
 +
        .daohang .shortName .item:hover:after {
 +
            content: '';
 +
            display: block;
 +
            position: absolute;
 +
            width: 80px;
 +
            height: 2px;
 +
            bottom: 5px;
 +
            left: 15px;
 +
            background-color: #f51720;
 +
        }
 +
 
 +
        .daohang .longName .item:before {
 +
            content: '';
 +
            display: block;
 +
            position: absolute;
 +
            width: 130px;
 +
            height: 1px;
 +
            bottom: 5px;
 +
            left: 20px;
 +
            background-color: #CCCCCC;
 +
        }
 +
 
 +
        .daohang .longName .item:hover:after {
 +
            content: '';
 +
            display: block;
 +
            position: absolute;
 +
            width: 130px;
 +
            height: 2px;
 +
            bottom: 5px;
 +
            left: 20px;
 +
            background-color: #f51720;
 +
        }
 +
 
 +
        @media screen and (max-width: 1200px) {
 +
            .logo-daohang {
 +
                left: 3%;
 +
            }
 +
 
 +
            .daohangyi {
 +
                font-size: 12px;
 +
            }
 +
        }
 +
 
 +
        @media screen and (max-width: 880px) {
 +
 
 +
            .daohang .caidan {
 +
                right: 0;
 +
                width: 100%;
 +
            }
 +
 
 +
            .logo-daohang {
 +
                display: none;
 +
            }
 +
 
 +
            .daohang .shortName {
 +
                width: 15vw;
 +
            }
 +
 
 +
            .daohang .longName {
 +
                width: 25vw;
 +
            }
 +
 
 +
            .daohangyi img {
 +
                display: none;
 +
            }
 +
 
 +
            .daohang .longName .item:before {
 +
                width: 150px;
 +
                left: 20px;
 +
            }
 +
 
 +
            .daohang .longName .item:hover:after {
 +
                width: 150px;
 +
                left: 20px;
 +
            }
 +
        }
 +
    </style>
 +
    <!-- 内容CSS -->
 +
    <!-- 内容CSS -->
 +
    <style class="text/css">
 +
      .clearfix:after {
 +
          content: ".";
 +
          display: block;
 +
          height: 0;
 +
          clear: both;
 +
          visibility: hidden;
 +
 
 +
      }
 +
 
 +
      .neirong {
 +
          width: 100%;
 +
          height: auto;
 +
          background-color: #F3F3F3;
 +
      }
 +
 
 +
      .zhengwen {
 +
          width: 80%;
 +
          margin: 60px auto;
 +
          padding: 50px 3%;
 +
          background-color: #FFF;
 +
          box-shadow: 0 1px 3px #676767;
 +
 
 +
      }
 +
 
 +
      .cebian {
 +
          width: 200px;
 +
          /* height: 80vh; */
 +
          float: left;
 +
          /* left: 1vw; */
 +
          top: 150px;
 +
          position: fixed;
 +
          box-shadow: 0 1px 3px #676767;
 +
          /* border:2px solid black */
 +
          /* background-color: #323643 */
 +
      }
 +
 
 +
      .zhengwen p{
 +
          text-align: justify !important;
 +
          font-family:  'Times New Roman',Helvetica,'Open Sans',  Arial, sans-serif !important;
 +
          color: #3B3B3B;
 +
          font-size: 26px !important;
 +
          padding-right: 20px;
 +
      }
 +
 
 +
      .daimg {
 +
          width: 100%;
 +
          height: auto;
 +
          margin: 20px 0;
 +
          box-shadow: 0 1px 3px #676767;
 +
      }
 +
 
 +
      .h1 {
 +
          line-height: 55px;
 +
          font-weight: bold;
 +
          height:auto;
 +
          font-family: 'Product Sans', sans-serif;
 +
          font-size: 40px;
 +
          color: #3B3B3B;
 +
          border-bottom: 2px solid #676767;
 +
          margin-bottom: 20px;
 +
      }
 +
 
 +
      .h2 {
 +
          height: 40px;
 +
          line-height: 40px;
 +
          font-weight: bold;
 +
          height:auto;
 +
          /* font-weight: bold; */
 +
          font-family: 'Product Sans', sans-serif;
 +
          font-size: 30px;
 +
          color: #484848;
 +
          /* margin-bottom: 20px; */
 +
      }
 +
 
 +
      .h3 {
 +
          height: 30px;
 +
          line-height: 30px;
 +
          font-weight: bold;
 +
          height:auto;
 +
          /* font-weight: bold; */
 +
          font-family: 'Product Sans', sans-serif;
 +
          font-size: 24px;
 +
          color: #484848;
 +
          /* margin-bottom: 20px; */
 +
      }
 +
     
 +
      table {
 +
          color: #3B3B3B;
 +
      }
 +
 
 +
      .zhengwen .tuandui_list {
 +
          position: relative;
 +
          margin-top: 40px;
 +
          height: 520px;
 +
          overflow: hidden;
 +
      }
 +
 
 +
      .tuandui_list>li {
 +
          padding: 0;
 +
          width: 20%;
 +
          float: left;
 +
          position: relative;
 +
          margin: 0 2.4%;
 +
          text-align: center;
 +
      }
 +
 
 +
      .zhengwen .laoshi_list {
 +
          position: relative;
 +
          margin-top: 40px;
 +
          height: 430px;
 +
      }
 +
 
 +
      .laoshi_list>li {
 +
          padding: 0;
 +
          width: 20%;
 +
          float: left;
 +
          position: relative;
 +
          margin: 0 2.4%;
 +
          text-align: center;
 +
      }
 +
 
 +
      .duiyuan {
 +
          margin: 20px 0;
 +
          /* border: 1px solid red; */
 +
      }
 +
 
 +
      .laoshi {
 +
          margin: 20px 0;
 +
      }
 +
 
 +
      .duiyuan img {
 +
          width: 100%;
 +
          height: auto;
 +
          border-top: 3px solid #323643;
 +
          border-left: 3px solid #323643;
 +
          border-right: 3px solid #323643;
 +
      }
 +
 
 +
      .laoshi img {
 +
          width: 100%;
 +
          height: 50vh;
 +
          border-top: 3px solid #323643;
 +
          border-left: 3px solid #323643;
 +
          border-right: 3px solid #323643;
 +
      }
 +
 
 +
      .duiyuan .xingming {
 +
          width: 100%;
 +
          height: 50px;
 +
          line-height: 50px;
 +
          background-color: #323643;
 +
          border-left: 3px solid #323643;
 +
          border-right: 3px solid #323643;
 +
          font-size: 16px;
 +
          color: white;
 +
          box-shadow: 0 1px 3px #CCCCCC;
 +
          font-family: Cambria, Cochin, Georgia, Times, 'Times New Roman', serif;
 +
          font-size: 24px;
 +
          font-weight: bold;
 +
          text-align: center;
 +
      }
 +
 
 +
      .laoshi .xingming {
 +
          width: 100%;
 +
          height: 50px;
 +
          line-height: 50px;
 +
          background-color: #323643;
 +
          border-left: 3px solid #323643;
 +
          border-right: 3px solid #323643;
 +
          font-size: 16px;
 +
          color: white;
 +
          box-shadow: 0 1px 3px #CCCCCC;
 +
          font-family: Cambria, Cochin, Georgia, Times, 'Times New Roman', serif;
 +
          font-size: 24px;
 +
          font-weight: bold;
 +
          text-align: center;
 +
      }
 +
 
 +
      .duiyuan .jieshao {
 +
          width: 100%;
 +
          height: 200px;
 +
          background-color: #f6eee0;
 +
          border-bottom: 3px solid #323643;
 +
          border-left: 3px solid #323643;
 +
          border-right: 3px solid #323643;
 +
          /* border-bottom-left-radius: 5px;
 +
          border-bottom-right-radius: 5px; */
 +
      }
 +
 
 +
      .duiyuan .jieshao .saohua {
 +
          padding: 10px 4px 0px 4px !important;
 +
          color: black;
 +
          font-family: Arial;
 +
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    <!-- 内容 -->
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    <div class="neirong clearfix">
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        <!-- 正文 -->
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        <div class="zhengwen">
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            <div id="float01" class="cur">
 +
                <div class="h1">Composite Part</div>
 +
                <div style="width: 85%; margin: 30px auto">
 +
                    <table class="table table-bordered table-hover">
 +
                        <thead>
 +
                            <th>Name</th>
 +
                            <th>Type</th>
 +
                            <th>Description</th>
 +
                            <th>Designer</th>
 +
                            <th>Length(bp)</th>
 +
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 +
                        </thead>
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                        <tbody>
 +
                            <tr>
 +
                                <td><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2632008">BBa_K2632008</a></td>
 +
                                <td>Promoter</td>
 +
                                <td>Plac-Lpp-OmpA-RGD</td>
 +
                                <td>Lingyu Zhong</td>
 +
                                <td>619</td>
 +
                            </tr>
 +
                        </tbody>
 +
                    </table>
 +
                </div>
 +
              <p>We gave the surface display system a new function through displaying a RGD motif on
 +
                    the Lpp-OmpA which contains a signal sequence, the N-terminal of the lipoprotein (Lpp) and the residual 46-159 amino acids of the OmpA. In addition, lipoprotein performs the function of targeting to the outer membrane and OmpA constructs an anchor on the outer membrane. RGD motif can specifically bind to αvβ3, a biomarker of cancer cells such as melanoma, neuroblastoma, glioma and adenocarcinoma<sup>1</sup>. We determined the surface display site on the third loop of Lpp-OmpA through homology modelling (<b>Figure 1</b>). This part is under the control of <i>lac</i> promoter. </p>
 +
                <div style="width: 30%; margin: 0px auto">
 +
                    <img src="https://static.igem.org/mediawiki/2018/5/5f/T--HZAU-China--Improve1.png" width=100% alt="">
 +
                </div>
 +
                <p><b>Figure 1</b>. The homology modelling result of Lpp-OmpA-RGD. Red arrow shows the location of RGD motif. </p><br>
 +
            </div>
 +
            <div id="float02">
 +
                <p>Microscopy shows that Lpp-OmpA-RGD overexpressed in <i>E. coli</i> induced by 0.1mM IPTG can bind to αvβ3-positive MDA-MB-231 cell (<b>Figure 2</b>), but cannot bind to αvβ3-negative MCF7 cell (<b>Figure 3</b>). We also use BBa_J36850 as a control. This strain cannot bind to Vβ3-positive MDA-MB-231 cell line (<b>Figure 4</b>) and αvβ3-negative MCF7 cell line (<b>Figure 5</b>). These results suggest that we successfully improved the part <a href="http://parts.igem.org/Part:BBa_J36850">BBa_J36850</a>. </p>
 +
                <div style="width: 90%; margin: 0px auto">
 +
                    <img src="https://static.igem.org/mediawiki/2018/b/b1/T--HZAU-China--Improve2.png" width=100% alt="">
 +
                </div>
 +
                <p><b>Figure 2</b>. αvβ3-positive MDA-MB-231 cell line was incubated with <i>E. coli</i> which constitutively expressed RFP and contained BBa_J36850. This improved part expressed RGD motif under the control of
 +
                    <i>lac</i> promoter. </p>
 +
                <div style="width: 90%; margin: 0px auto">
 +
                    <img src="https://static.igem.org/mediawiki/2018/c/cf/T--HZAU-China--Improve3.png" width=100% alt="">
 +
                </div>
 +
                <p><b>Figure 3</b>. αvβ3-negative MCF7 cell line was incubated with <i>E. coli</i> which constitutively expressed RFP and contained BBa_J36850. This improved part expressed RGD motif under the control of <i>lac</i> promoter.</p>
 +
                <div style="width: 90%; margin: 0px auto">
 +
                    <img src="https://static.igem.org/mediawiki/2018/f/f7/T--HZAU-China--Improve4.png" width=100% alt="">
 +
                </div>
 +
                <p><b>Figure 4</b>. αvβ3-positive MDA-MB-231 cell line was incubated with <i>E. coli</i> which constitutively expressed RFP and contained BBa_J36850.</p>
 +
                <div style="width: 90%; margin: 0px auto">
 +
                    <img src="https://static.igem.org/mediawiki/2018/c/ce/T--HZAU-China--Improve5.png" width=100% alt="">
 +
                </div>
 +
                <p><b>Figure 5</b>. αvβ3-negative MCF7 cell line was incubated with <i>E. coli</i> which constitutively expressed RFP and contained BBa_J36850. </p>
 +
                <div class="collapseDiv">
 +
                    <label for="zhedie-toggle3">Method</label>
 +
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 +
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 +
                        <b>Preparation of Cells for Infection</b><br>
 +
                        1. Grow Hela GSDMD KO cells in a humidified 37 °C, 5% CO<sub>2</sub> tissue-culture incubator.<br>
 +
                        2. Count the cells using a hemocytometer. Seed in 24-well (5 × 10^4 per well) and grow
 +
                        overnight.<br>
 +
                        <b>Preparation of Bacteria</b><br>
 +
                        1. Grow bacterial cells overnight 16 h in 2 mL LB in a 15-mL tube. Incubate at 37 °C in a shaking
 +
                        incubator (200 rpm).<br>
 +
                        2. Subculture bacterial cells by transferring 300 μL of the overnight culture into 5 mL of LB in a
 +
                        loosely capped 50-mL tube. Incubate at 37 °C in a shaking incubator (200 rpm) to late log
 +
                        phase.<br>
 +
                        3. Pellet 1 mL of the bacterial cells subculture by centrifugation at 1000 g in a microfuge for 2
 +
                        min at room temperature.<br>
 +
                        4. Remove 900 μL of supernatant and gently resuspend the pellet in 900 μL PBS.<br>
 +
                        <b>Infection</b><br>
 +
                        1. Aspirate media and rinse the monolayer twice with PBS.<br>
 +
                        2. Inoculate cells with bacteria (MOI = 100) by adding bacteria directly to the cell-culture
 +
                        supernatant.<br>
 +
                        3. Incubate for 2 h at 37 °C in 5% CO<sub>2</sub>.<br>
 +
                        4. Aspirate media and wash<br>
 +
                        5. Add fresh GM containing 100 μg/mL gentamicin and 16 μg/mL incubate at 37 °C in 5% CO<sub>2</sub> for 2
 +
                        h.<br>
 +
                        6. Replace GM with fresh GM containing 20 μg/mL gentamicin for 1 h.<br>
 +
                        7.Add 16 μg/mL ATc for remainder of experiment.<br>
 +
                        Observation is taken after 5 min, 30 min, 90 min.<br><br>
 +
 
 +
                    </div>
 +
                </div>
 +
            </div>
 +
            <div id="float03">
 +
                <div class="h1">Reference</div>
 +
                <p>1. Park, S. H. et al. RGD Peptide Cell-Surface Display Enhances the Targeting and Therapeutic
 +
                    Efficacy of Attenuated Salmonella-mediated Cancer Therapy. Theranostics 6, 1672-1682,
 +
                    doi:10.7150/thno.16135 (2016).
 +
 
 +
 
 +
                </p>
 +
 
 +
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 +
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 +
 
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Latest revision as of 03:39, 18 October 2018

Composite Part
Name Type Description Designer Length(bp)
BBa_K2632008 Promoter Plac-Lpp-OmpA-RGD Lingyu Zhong 619

We gave the surface display system a new function through displaying a RGD motif on the Lpp-OmpA which contains a signal sequence, the N-terminal of the lipoprotein (Lpp) and the residual 46-159 amino acids of the OmpA. In addition, lipoprotein performs the function of targeting to the outer membrane and OmpA constructs an anchor on the outer membrane. RGD motif can specifically bind to αvβ3, a biomarker of cancer cells such as melanoma, neuroblastoma, glioma and adenocarcinoma1. We determined the surface display site on the third loop of Lpp-OmpA through homology modelling (Figure 1). This part is under the control of lac promoter.

Figure 1. The homology modelling result of Lpp-OmpA-RGD. Red arrow shows the location of RGD motif.


Microscopy shows that Lpp-OmpA-RGD overexpressed in E. coli induced by 0.1mM IPTG can bind to αvβ3-positive MDA-MB-231 cell (Figure 2), but cannot bind to αvβ3-negative MCF7 cell (Figure 3). We also use BBa_J36850 as a control. This strain cannot bind to Vβ3-positive MDA-MB-231 cell line (Figure 4) and αvβ3-negative MCF7 cell line (Figure 5). These results suggest that we successfully improved the part BBa_J36850.

Figure 2. αvβ3-positive MDA-MB-231 cell line was incubated with E. coli which constitutively expressed RFP and contained BBa_J36850. This improved part expressed RGD motif under the control of lac promoter.

Figure 3. αvβ3-negative MCF7 cell line was incubated with E. coli which constitutively expressed RFP and contained BBa_J36850. This improved part expressed RGD motif under the control of lac promoter.

Figure 4. αvβ3-positive MDA-MB-231 cell line was incubated with E. coli which constitutively expressed RFP and contained BBa_J36850.

Figure 5. αvβ3-negative MCF7 cell line was incubated with E. coli which constitutively expressed RFP and contained BBa_J36850.

Preparation of Cells for Infection
1. Grow Hela GSDMD KO cells in a humidified 37 °C, 5% CO2 tissue-culture incubator.
2. Count the cells using a hemocytometer. Seed in 24-well (5 × 10^4 per well) and grow overnight.
Preparation of Bacteria
1. Grow bacterial cells overnight 16 h in 2 mL LB in a 15-mL tube. Incubate at 37 °C in a shaking incubator (200 rpm).
2. Subculture bacterial cells by transferring 300 μL of the overnight culture into 5 mL of LB in a loosely capped 50-mL tube. Incubate at 37 °C in a shaking incubator (200 rpm) to late log phase.
3. Pellet 1 mL of the bacterial cells subculture by centrifugation at 1000 g in a microfuge for 2 min at room temperature.
4. Remove 900 μL of supernatant and gently resuspend the pellet in 900 μL PBS.
Infection
1. Aspirate media and rinse the monolayer twice with PBS.
2. Inoculate cells with bacteria (MOI = 100) by adding bacteria directly to the cell-culture supernatant.
3. Incubate for 2 h at 37 °C in 5% CO2.
4. Aspirate media and wash
5. Add fresh GM containing 100 μg/mL gentamicin and 16 μg/mL incubate at 37 °C in 5% CO2 for 2 h.
6. Replace GM with fresh GM containing 20 μg/mL gentamicin for 1 h.
7.Add 16 μg/mL ATc for remainder of experiment.
Observation is taken after 5 min, 30 min, 90 min.

Reference

1. Park, S. H. et al. RGD Peptide Cell-Surface Display Enhances the Targeting and Therapeutic Efficacy of Attenuated Salmonella-mediated Cancer Therapy. Theranostics 6, 1672-1682, doi:10.7150/thno.16135 (2016).