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

 
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Line 280: Line 280:
 
         }
 
         }
 
     </style>
 
     </style>
 +
    <!-- 内容CSS -->
 
     <!-- 内容CSS -->
 
     <!-- 内容CSS -->
 
     <style class="text/css">
 
     <style class="text/css">
−
        .clearfix:after {
+
      .clearfix:after {
−
            content: ".";
+
          content: ".";
−
            display: block;
+
          display: block;
−
            height: 0;
+
          height: 0;
−
            clear: both;
+
          clear: both;
−
            visibility: hidden;
+
          visibility: hidden;
  
−
        }
+
      }
  
−
        .neirong {
+
      .neirong {
−
            width: 100%;
+
          width: 100%;
−
            height: auto;
+
          height: auto;
−
            background-color: #F3F3F3;
+
          background-color: #F3F3F3;
−
        }
+
      }
  
−
        .zhengwen {
+
      .zhengwen {
−
            width: 80%;
+
          width: 80%;
−
            margin: 60px auto;
+
          margin: 60px auto;
−
            padding: 20px 3%;
+
          padding: 50px 3%;
−
            background-color: #FFF;
+
          background-color: #FFF;
−
            box-shadow: 0 1px 3px #676767;
+
          box-shadow: 0 1px 3px #676767;
  
−
        }
+
      }
  
−
        .cebian {
+
      .cebian {
−
            width: 200px;
+
          width: 200px;
−
            /* height: 80vh; */
+
          /* height: 80vh; */
−
            float: left;
+
          float: left;
−
            /* left: 1vw; */
+
          /* left: 1vw; */
−
            top: 150px;
+
          top: 150px;
−
            position: fixed;
+
          position: fixed;
−
            box-shadow: 0 1px 3px #676767;
+
          box-shadow: 0 1px 3px #676767;
−
            /* border:2px solid black */
+
          /* border:2px solid black */
−
            /* background-color: #323643 */
+
          /* background-color: #323643 */
−
        }
+
      }
  
−
        .zhengwen p{
+
      .zhengwen p{
−
            text-align: justify !important;
+
          text-align: justify !important;
−
            font-family:  'Times New Roman',Helvetica,'Open Sans',  Arial, sans-serif !important;
+
          font-family:  'Times New Roman',Helvetica,'Open Sans',  Arial, sans-serif !important;
−
            color: #3B3B3B;
+
          color: #3B3B3B;
−
            font-size: 26px !important;
+
          font-size: 26px !important;
−
            padding-right: 20px;
+
          padding-right: 20px;
−
        }
+
      }
  
−
        .daimg {
+
      .daimg {
−
            width: 100%;
+
          width: 100%;
−
            height: auto;
+
          height: auto;
−
            margin: 20px 0;
+
          margin: 20px 0;
−
            box-shadow: 0 1px 3px #676767;
+
          box-shadow: 0 1px 3px #676767;
−
        }
+
      }
  
−
        .h1 {
+
      .h1 {
−
            height: 80px;
+
          line-height: 55px;
−
            line-height: 80px;
+
          font-weight: bold;
−
            font-weight: bold;
+
          height:auto;
−
            font-family: 'Product Sans', sans-serif;
+
          font-family: 'Product Sans', sans-serif;
−
            font-size: 40px;
+
          font-size: 40px;
−
            color: #3B3B3B;
+
          color: #3B3B3B;
−
            border-bottom: 2px solid #676767;
+
          border-bottom: 2px solid #676767;
−
            margin-bottom: 20px;
+
          margin-bottom: 20px;
−
        }
+
      }
  
−
        .h2 {
+
      .h2 {
−
            height: 40px;
+
          height: 40px;
−
            line-height: 40px;
+
          line-height: 40px;
−
            font-weight: bold;
+
          font-weight: bold;
−
            /* font-weight: bold; */
+
          height:auto;
−
            font-family: 'Product Sans', sans-serif;  
+
          /* font-weight: bold; */
−
            font-size: 30px;
+
          font-family: 'Product Sans', sans-serif;  
−
            color: #484848;
+
          font-size: 30px;
−
            /* margin-bottom: 20px; */
+
          color: #484848;
−
        }
+
          /* margin-bottom: 20px; */
 +
      }
  
−
        .h3 {
+
      .h3 {
−
            height: 30px;
+
          height: 30px;
−
            line-height: 30px;
+
          line-height: 30px;
−
            font-weight: bold;
+
          font-weight: bold;
−
            /* font-weight: bold; */
+
          height:auto;
−
            font-family: 'Product Sans', sans-serif;
+
          /* font-weight: bold; */
−
            font-size: 24px;
+
          font-family: 'Product Sans', sans-serif;
−
            color: #484848;
+
          font-size: 24px;
−
            /* margin-bottom: 20px; */
+
          color: #484848;
−
        }
+
          /* margin-bottom: 20px; */
−
       
+
      }
−
        table {
+
     
−
            color: #3B3B3B;
+
      table {
−
        }
+
          color: #3B3B3B;
 +
      }
  
−
        .zhengwen .tuandui_list {
+
      .zhengwen .tuandui_list {
−
            position: relative;
+
          position: relative;
−
            margin-top: 40px;
+
          margin-top: 40px;
−
            height: 520px;
+
          height: 520px;
−
            overflow: hidden;
+
          overflow: hidden;
−
        }
+
      }
  
−
        .tuandui_list>li {
+
      .tuandui_list>li {
−
            padding: 0;
+
          padding: 0;
−
            width: 20%;
+
          width: 20%;
−
            float: left;
+
          float: left;
−
            position: relative;
+
          position: relative;
−
            margin: 0 2.4%;
+
          margin: 0 2.4%;
−
            text-align: center;
+
          text-align: center;
−
        }
+
      }
  
−
        .zhengwen .laoshi_list {
+
      .zhengwen .laoshi_list {
−
            position: relative;
+
          position: relative;
−
            margin-top: 40px;
+
          margin-top: 40px;
−
            height: 430px;
+
          height: 430px;
−
        }
+
      }
  
−
        .laoshi_list>li {
+
      .laoshi_list>li {
−
            padding: 0;
+
          padding: 0;
−
            width: 20%;
+
          width: 20%;
−
            float: left;
+
          float: left;
−
            position: relative;
+
          position: relative;
−
            margin: 0 2.4%;
+
          margin: 0 2.4%;
−
            text-align: center;
+
          text-align: center;
−
        }
+
      }
  
−
        .duiyuan {
+
      .duiyuan {
−
            margin: 20px 0;
+
          margin: 20px 0;
−
            /* border: 1px solid red; */
+
          /* border: 1px solid red; */
−
        }
+
      }
  
−
        .laoshi {
+
      .laoshi {
−
            margin: 20px 0;
+
          margin: 20px 0;
−
        }
+
      }
  
−
        .duiyuan img {
+
      .duiyuan img {
−
            width: 100%;
+
          width: 100%;
−
            height: auto;
+
          height: auto;
−
            border-top: 3px solid #323643;
+
          border-top: 3px solid #323643;
−
            border-left: 3px solid #323643;
+
          border-left: 3px solid #323643;
−
            border-right: 3px solid #323643;
+
          border-right: 3px solid #323643;
−
        }
+
      }
  
−
        .laoshi img {
+
      .laoshi img {
−
            width: 100%;
+
          width: 100%;
−
            height: 50vh;
+
          height: 50vh;
−
            border-top: 3px solid #323643;
+
          border-top: 3px solid #323643;
−
            border-left: 3px solid #323643;
+
          border-left: 3px solid #323643;
−
            border-right: 3px solid #323643;
+
          border-right: 3px solid #323643;
−
        }
+
      }
  
−
        .duiyuan .xingming {
+
      .duiyuan .xingming {
−
            width: 100%;
+
          width: 100%;
−
            height: 50px;
+
          height: 50px;
−
            line-height: 50px;
+
          line-height: 50px;
−
            background-color: #323643;
+
          background-color: #323643;
−
            border-left: 3px solid #323643;
+
          border-left: 3px solid #323643;
−
            border-right: 3px solid #323643;
+
          border-right: 3px solid #323643;
−
            font-size: 16px;
+
          font-size: 16px;
−
            color: white;
+
          color: white;
−
            box-shadow: 0 1px 3px #CCCCCC;
+
          box-shadow: 0 1px 3px #CCCCCC;
−
            font-family: Cambria, Cochin, Georgia, Times, 'Times New Roman', serif;
+
          font-family: Cambria, Cochin, Georgia, Times, 'Times New Roman', serif;
−
            font-size: 24px;
+
          font-size: 24px;
−
            font-weight: bold;
+
          font-weight: bold;
−
            text-align: center;
+
          text-align: center;
−
        }
+
      }
  
−
        .laoshi .xingming {
+
      .laoshi .xingming {
−
            width: 100%;
+
          width: 100%;
−
            height: 50px;
+
          height: 50px;
−
            line-height: 50px;
+
          line-height: 50px;
−
            background-color: #323643;
+
          background-color: #323643;
−
            border-left: 3px solid #323643;
+
          border-left: 3px solid #323643;
−
            border-right: 3px solid #323643;
+
          border-right: 3px solid #323643;
−
            font-size: 16px;
+
          font-size: 16px;
−
            color: white;
+
          color: white;
−
            box-shadow: 0 1px 3px #CCCCCC;
+
          box-shadow: 0 1px 3px #CCCCCC;
−
            font-family: Cambria, Cochin, Georgia, Times, 'Times New Roman', serif;
+
          font-family: Cambria, Cochin, Georgia, Times, 'Times New Roman', serif;
−
            font-size: 24px;
+
          font-size: 24px;
−
            font-weight: bold;
+
          font-weight: bold;
−
            text-align: center;
+
          text-align: center;
−
        }
+
      }
  
−
        .duiyuan .jieshao {
+
      .duiyuan .jieshao {
−
            width: 100%;
+
          width: 100%;
−
            height: 200px;
+
          height: 200px;
−
            background-color: #f6eee0;
+
          background-color: #f6eee0;
−
            border-bottom: 3px solid #323643;
+
          border-bottom: 3px solid #323643;
−
            border-left: 3px solid #323643;
+
          border-left: 3px solid #323643;
−
            border-right: 3px solid #323643;
+
          border-right: 3px solid #323643;
−
            /* border-bottom-left-radius: 5px;
+
          /* border-bottom-left-radius: 5px;
−
            border-bottom-right-radius: 5px; */
+
          border-bottom-right-radius: 5px; */
−
        }
+
      }
  
−
        .duiyuan .jieshao .saohua {
+
      .duiyuan .jieshao .saohua {
−
            padding: 10px 4px 0px 4px !important;
+
          padding: 10px 4px 0px 4px !important;
−
            color: black;
+
          color: black;
−
            font-family: Arial;
+
          font-family: Arial;
−
            font-style: italic;
+
          font-style: italic;
−
            font-size: 15px !important;
+
          font-size: 15px !important;
−
            list-style-type: none;
+
          list-style-type: none;
−
            text-align: justify;
+
          text-align: justify;
−
            line-height: 18px;
+
          line-height: 18px;
−
         }
+
      }
 +
  </style>
 +
    <!-- 折叠框CSS -->
 +
    <style>
 +
         .collapseDiv {
 +
              color: #333;
 +
              border-radius: 4px;
 +
              background-color: #F3F3F3;
 +
              border: 1px solid transparent;
 +
              border-color: #ddd;
 +
              box-shadow: 0 1px 1px #F3F3F3;
 +
              margin-top: 5px;
 +
              margin-bottom: 0;
 +
            }
 +
            .collapseDiv label {
 +
              cursor: pointer;
 +
              font-size: 24px  !important;
 +
              /* font-weight: bold; */
 +
              color: green  !important;
 +
              border-color: #F3F3F3  !important;
 +
              padding: 5px 15px 5px 18px  !important;
 +
              background-color: #f5f5f5  !important;
 +
              margin: 0  !important;
 +
              border-radius: 5px  !important;
 +
            }
 +
   
 +
            #zhedie-toggle1,
 +
            #zhedie-toggle2,
 +
            #zhedie-toggle3,
 +
            #zhedie-toggle4 {
 +
                display: none;
 +
            }
 +
   
 +
            #zhedie1,
 +
            #zhedie2,
 +
            #zhedie3,
 +
            #zhedie4 {
 +
                display: none;
 +
                font-size: 18px;
 +
                padding: 0 50px 0 50px;
 +
                width: 100%;
 +
                margin: 0;
 +
                color: black;
 +
            }
 +
   
 +
            #zhedie-toggle1:checked+#zhedie1,
 +
            #zhedie-toggle2:checked+#zhedie2,
 +
            #zhedie-toggle3:checked+#zhedie3,
 +
            #zhedie-toggle4:checked+#zhedie4 {
 +
                display: block;
 +
            }
 +
   
 +
           
 +
   
 +
   
 +
            .collapse_body {
 +
              background-color: #fff;
 +
              position: relative;
 +
              height: 0;
 +
              overflow: hidden;
 +
              -webkit-transition-timing-function: ease;
 +
              -o-transition-timing-function: ease;
 +
              transition-timing-function: ease;
 +
              -webkit-transition-duration: .35s;
 +
              -o-transition-duration: .35s;
 +
              transition-duration: .35s;
 +
              -webkit-transition-property: height, visibility;
 +
              -o-transition-property: height, visibility;
 +
              transition-property: height, visibility
 +
            }
 +
            .collapse_content {
 +
              font-size: 20px;
 +
              border-top: 1px solid #ddd;
 +
              background-color: #fff;
 +
              padding: 15px;
 +
            }
 
     </style>
 
     </style>
−
 
  
 
</head>
 
</head>
Line 552: Line 629:
 
         <!-- 正文 -->
 
         <!-- 正文 -->
 
         <div class="zhengwen">
 
         <div class="zhengwen">
−
 
+
             <div id="float01" class="cur">
−
             <div id="float01">
+
                 <div class="h1">Composite Part</div>
−
                 <div class="h1">Composite Part Table</div>
+
 
                 <div style="width: 85%; margin: 30px auto">
 
                 <div style="width: 85%; margin: 30px auto">
 
                     <table class="table table-bordered table-hover">
 
                     <table class="table table-bordered table-hover">
Line 576: Line 652:
 
                     </table>
 
                     </table>
 
                 </div>
 
                 </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>
 +
                    <input type="checkbox" id="zhedie-toggle3" />
 +
                    <div id="zhedie3" class="text-success text-left">
 +
                        <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>
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                </div>
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            </div>
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            <div id="float03">
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                <div class="h1">Reference</div>
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                <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).
 +
 +
 +
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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).