Difference between revisions of "Team:NCTU Formosa/Wet Lab"

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   <title>Protein Expression</title>
 
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       <a href="https://2018.igem.org/Team:NCTU_Formosa/Wet_Lab/Growth_Curve"><img src="https://static.igem.org/mediawiki/2018/7/70/T--NCTU_Formosa--MIC.png" class="mic"></a>
 
       <a href="https://2018.igem.org/Team:NCTU_Formosa/Wet_Lab/Growth_Curve"><img src="https://static.igem.org/mediawiki/2018/7/70/T--NCTU_Formosa--MIC.png" class="mic"></a>
 
     </div>
 
     </div>
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     <div class="sec1" style="background-color:#ffffff;">
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     <div class="sec1" style="background-color:#fff;">
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       <img src="https://static.igem.org/mediawiki/2018/3/30/T--NCTU_Formosa--Experiment_design_title.png" class="title_title">
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       <img src="https://static.igem.org/mediawiki/2018/a/ad/T--NCTU_Formosa--Expression_design_title.png" class="title_title">
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      <div class="title_1"><p>Cloning</p></div>
 
       <div class="text">
 
       <div class="text">
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         <p>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Our system provided a way to regulate microbiota to an ideal balance . Since we focused our model on the excess PSB in soil, which is a large issue of agriculture in Taiwan, we chose antimicrobial peptide as the Bio-stimulator to limit the amount of PSB in soil. To express our target protein, we first designed BioBricks that contain sequences of these peptides. All the experiments we performed with BioBricks are mentioned below.
+
         <p>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;To ensure our target genes were successfully cloned into the PSB1C3 backbone, we ran the electrophoresis of Taq PCR products to check the sizes of the insert genes.</p>
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</p>
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    <div class="sec2" style="background-color:#FFFFFF;">
 
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      <div class="title"><p>BioBrick</p></div>
 
 
       <div class="text">
 
       <div class="text">
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         <p>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;The BioBrick we designed contains a T7 promoter, induced by IPTG, and RBS with our target protein behind. We also added a intein-CBD (chitin binding domain) tag behind bacteriocin to better purify our peptide.</p>
+
         <p>All of the sequences contained T7 promoter, RBS, bacteriocin, intein and CBD, were shown in Figure 1.</p>
 
       </div>
 
       </div>
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       <img src="https://static.igem.org/mediawiki/2018/a/aa/T--NCTU_Formosa--biobrick.png" class="expression" style="display:block; margin:auto;" width="700">
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       <img src="https://static.igem.org/mediawiki/2018/a/aa/T--NCTU_Formosa--biobrick.png" class="expression">
 
       <div class="explanation">
 
       <div class="explanation">
 
         <svg class="icon" aria-hidden="true" data-prefix="fas" data-icon="arrow-circle-up" class="svg-inline--fa fa-arrow-circle-up fa-w-16" role="img" xmlns="http://www.w3.org/2000/svg" viewBox="0 0 512 512"><path fill="currentColor" d="M8 256C8 119 119 8 256 8s248 111 248 248-111 248-248 248S8 393 8 256zm143.6 28.9l72.4-75.5V392c0 13.3 10.7 24 24 24h16c13.3 0 24-10.7 24-24V209.4l72.4 75.5c9.3 9.7 24.8 9.9 34.3.4l10.9-11c9.4-9.4 9.4-24.6 0-33.9L273 107.7c-9.4-9.4-24.6-9.4-33.9 0L106.3 240.4c-9.4 9.4-9.4 24.6 0 33.9l10.9 11c9.6 9.5 25.1 9.3 34.4-.4z"></path></svg>
 
         <svg class="icon" aria-hidden="true" data-prefix="fas" data-icon="arrow-circle-up" class="svg-inline--fa fa-arrow-circle-up fa-w-16" role="img" xmlns="http://www.w3.org/2000/svg" viewBox="0 0 512 512"><path fill="currentColor" d="M8 256C8 119 119 8 256 8s248 111 248 248-111 248-248 248S8 393 8 256zm143.6 28.9l72.4-75.5V392c0 13.3 10.7 24 24 24h16c13.3 0 24-10.7 24-24V209.4l72.4 75.5c9.3 9.7 24.8 9.9 34.3.4l10.9-11c9.4-9.4 9.4-24.6 0-33.9L273 107.7c-9.4-9.4-24.6-9.4-33.9 0L106.3 240.4c-9.4 9.4-9.4 24.6 0 33.9l10.9 11c9.6 9.5 25.1 9.3 34.4-.4z"></path></svg>
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         Figure 1: BioBrick: T7 promoter + RBS + target peptide + intein-CBD
+
         Figure 1: Our BioBrick design
 
       </div>
 
       </div>
 
       <div class="text">
 
       <div class="text">
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         <p>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Intein is a protein segment that is able to excise itself from the larger protein it binds to. Therefore, it is also called “ protein introns”. We utilized intein-CBD tag to purify our peptides.<br>Although affinity tags have been widely used to purify recombinant proteins, it must removed by protease in the final purification. In contrast, intein-CBD tag will undergo the cleavage reaction with DTT (1,4-dithiothreitol) or cysteine, which will not cause the structure changes of our short peptide.
+
      <p>After amplification with PCR, all the PCR products have their length around 1200 b.p. Each directly length is in the Table 1.</p>
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</p>
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      </div>
 +
      <div class="table">
 +
         <table>
 +
        <caption>
 +
          <p class="explanation">
 +
            <svg class="icon" aria-hidden="true" data-prefix="fas" data-icon="arrow-circle-down" class="svg-inline--fa fa-arrow-circle-down fa-w-16" role="img" xmlns="http://www.w3.org/2000/svg" viewBox="0 0 512 512"><path fill="currentColor" d="M504 256c0 137-111 248-248 248S8 393 8 256 119 8 256 8s248 111 248 248zm-143.6-28.9L288 302.6V120c0-13.3-10.7-24-24-24h-16c-13.3 0-24 10.7-24 24v182.6l-72.4-75.5c-9.3-9.7-24.8-9.9-34.3-.4l-10.9 11c-9.4 9.4-9.4 24.6 0 33.9L239 404.3c9.4 9.4 24.6 9.4 33.9 0l132.7-132.7c9.4-9.4 9.4-24.6 0-33.9l-10.9-11c-9.5-9.5-25-9.3-34.3.4z"></path></svg>
 +
              Table 1: The DNA length of each Biobrick
 +
          </p>
 +
        </caption>
 +
        <thead>
 +
          <tr>
 +
            <th><p>Bacteriocin</p></th>
 +
            <th style="text-align: center;"><p>Length</p></th>
 +
            <th style="text-align: center;"><p>Length of PCR product</p></th>
 +
          </tr>
 +
        </thead>
 +
        <tbody>
 +
          <tr>
 +
            <td><p>Leucocyclicin Q</p></td>
 +
            <td style="text-align: center;"><p>186 b.p.</p></td>
 +
            <td style="text-align: center;"><p>1230 b.p.</p></td>
 +
          </tr>
 +
          <tr>
 +
            <td><p>Enterocin B</p></td>
 +
            <td style="text-align: center;"><p>210 b.p.</p></td>
 +
            <td style="text-align: center;"><p>1254 b.p.</p></td>
 +
          </tr>
 +
          <tr>
 +
            <td><p>Enterocin 96</p></td>
 +
            <td style="text-align: center;"><p>219 b.p.</p></td>
 +
            <td style="text-align: center;"><p>1263 b.p.</p></td>
 +
          </tr>
 +
          <tr>
 +
            <td><p>Lacticin Z</p></td>
 +
            <td style="text-align: center;"><p>153 b.p.</p></td>
 +
            <td style="text-align: center;"><p>1197 b.p.</p></td>
 +
          </tr>
 +
          <tr>
 +
            <td><p>Enteriocin A</p></td>
 +
            <td style="text-align: center;"><p>192 b.p.</p></td>
 +
            <td style="text-align: center;"><p>1236 b.p.</p></td>
 +
          </tr>
 +
          <tr>
 +
            <td><p>Bovicin HJ50</p></td>
 +
            <td style="text-align: center;"><p>171 b.p.</p></td>
 +
            <td style="text-align: center;"><p>1215 b.p.</p></td>
 +
          </tr>
 +
          <tr>
 +
            <td><p>Durancin TW-49M</p></td>
 +
            <td style="text-align: center;"><p>213 b.p.</p></td>
 +
            <td style="text-align: center;"><p>1257 b.p.</p></td>
 +
          </tr>
 +
          <tr>
 +
            <td><p>Subtilosin</p></td>
 +
            <td style="text-align: center;"><p>147 b.p.</p></td>
 +
            <td style="text-align: center;"><p>1291 b.p.</p></td>
 +
          </tr>
 +
        </tbody>
 +
      </table>
 +
      </div>
 +
      <div class="text">
 +
      <p>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Electrophoresis results of the PCR products with marker on the left side and target gene on the right side.<br>The length are labeled beside each band.</p>
 +
      </div>
 +
      <div class="cloning">
 +
      <img src="https://static.igem.org/mediawiki/2018/1/1d/T--NCTU_Formosa--Lu_comp.png" class="expression1">
 +
      <img src="https://static.igem.org/mediawiki/2018/4/46/T--NCTU_Formosa--B_comp.png" class="expression1">
 +
      <img src="https://static.igem.org/mediawiki/2018/1/17/T--NCTU_Formosa--Bov_comp.png" class="expression1">
 +
      <img src="https://static.igem.org/mediawiki/2018/5/5a/T--NCTU_Formosa--96_comp.png" class="expression1">
 
       </div>
 
       </div>
 
       <div class="explanation">
 
       <div class="explanation">
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        <svg class="icon" aria-hidden="true" data-prefix="fas" data-icon="arrow-circle-up" class="svg-inline--fa fa-arrow-circle-up fa-w-16" role="img" xmlns="http://www.w3.org/2000/svg" viewBox="0 0 512 512"><path fill="currentColor" d="M8 256C8 119 119 8 256 8s248 111 248 248-111 248-248 248S8 393 8 256zm143.6 28.9l72.4-75.5V392c0 13.3 10.7 24 24 24h16c13.3 0 24-10.7 24-24V209.4l72.4 75.5c9.3 9.7 24.8 9.9 34.3.4l10.9-11c9.4-9.4 9.4-24.6 0-33.9L273 107.7c-9.4-9.4-24.6-9.4-33.9 0L106.3 240.4c-9.4 9.4-9.4 24.6 0 33.9l10.9 11c9.6 9.5 25.1 9.3 34.4-.4z"></path></svg>
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      <svg class="icon" aria-hidden="true" data-prefix="fas" data-icon="arrow-circle-up" class="svg-inline--fa fa-arrow-circle-up fa-w-16" role="img" xmlns="http://www.w3.org/2000/svg" viewBox="0 0 512 512"><path fill="currentColor" d="M8 256C8 119 119 8 256 8s248 111 248 248-111 248-248 248S8 393 8 256zm143.6 28.9l72.4-75.5V392c0 13.3 10.7 24 24 24h16c13.3 0 24-10.7 24-24V209.4l72.4 75.5c9.3 9.7 24.8 9.9 34.3.4l10.9-11c9.4-9.4 9.4-24.6 0-33.9L273 107.7c-9.4-9.4-24.6-9.4-33.9 0L106.3 240.4c-9.4 9.4-9.4 24.6 0 33.9l10.9 11c9.6 9.5 25.1 9.3 34.4-.4z"></path></svg>
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        Figure 2: Procedure of intein-mediated protein splicing
+
      Figure 2: The electrophoresis results of Leucocyclicin Q, Enteroicin B, Bovicin HJ50 and Enterocin 96.
 
       </div>
 
       </div>
−
       <div class="title_1"><p>1. Protein expression</p></div>
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       <div class="cloning">
−
      <div class="title_2"><p>Cloning:</p></div>
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      <img src="https://static.igem.org/mediawiki/2018/2/24/T--NCTU_Formosa--Lac_comp.png" class="expression1">
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      <div class="textnew">
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      <img src="https://static.igem.org/mediawiki/2018/2/23/T--NCTU_Formosa--A_comp.png" class="expression1">
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        <p>We cloned the insert gene into pSB1C3 backbone and transform into <i>E. coli</i> DH5α. <a href="https://2018.igem.org/Team:NCTU_Formosa/Parts parts"><br>These</a> have been submitted to iGEM.</p>
+
      <img src="https://static.igem.org/mediawiki/2018/d/d8/T--NCTU_Formosa--Dur_comp.png" class="expression1">
 +
      <img src="https://static.igem.org/mediawiki/2018/0/08/T--NCTU_Formosa--Sub_comp.png" class="expression1">
 
       </div>
 
       </div>
−
       <div class="title_2"><p>Expression:</p></div>
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       <div class="explanation">
−
       <div class="textnew">
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       <svg class="icon" aria-hidden="true" data-prefix="fas" data-icon="arrow-circle-up" class="svg-inline--fa fa-arrow-circle-up fa-w-16" role="img" xmlns="http://www.w3.org/2000/svg" viewBox="0 0 512 512"><path fill="currentColor" d="M8 256C8 119 119 8 256 8s248 111 248 248-111 248-248 248S8 393 8 256zm143.6 28.9l72.4-75.5V392c0 13.3 10.7 24 24 24h16c13.3 0 24-10.7 24-24V209.4l72.4 75.5c9.3 9.7 24.8 9.9 34.3.4l10.9-11c9.4-9.4 9.4-24.6 0-33.9L273 107.7c-9.4-9.4-24.6-9.4-33.9 0L106.3 240.4c-9.4 9.4-9.4 24.6 0 33.9l10.9 11c9.6 9.5 25.1 9.3 34.4-.4z"></path></svg>
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        <p>We expressed the proteins by <i>E. coli</i> ER2566 and <i>E. coli</i> BL21 Rosetta-Gami.</p>
+
      Figure 3: The electrophoresis results of Lacticin Z, Enteroicin A, Durancin and Subtilosin.
 
       </div>
 
       </div>
−
       <div class="title_2"><p>SDS-PAGE:</p></div>
+
    </div>
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       <div class="textnew">
+
    <div class="sec2" style="background-color:#ffffff;">
−
         <p>We checked the expression result through SDS-PAGE.</p>
+
       <div class="title_1"><p>Protein Expression</p></div>
 +
      <div class="text">
 +
      <p>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;After the expression from <i>E. coli</i> ER2566, we have to check whether the proteins are successfully expressed. We sonicate <i>E. coli</i> and run SDS-PAGE to make sure the correct sizes. The mass of each proteins are in Table 2.</p>
 +
      </div>
 +
      <div class="table">
 +
        <table>
 +
        <caption>
 +
          <p class="explanation">
 +
            <svg class="icon" aria-hidden="true" data-prefix="fas" data-icon="arrow-circle-down" class="svg-inline--fa fa-arrow-circle-down fa-w-16" role="img" xmlns="http://www.w3.org/2000/svg" viewBox="0 0 512 512"><path fill="currentColor" d="M504 256c0 137-111 248-248 248S8 393 8 256 119 8 256 8s248 111 248 248zm-143.6-28.9L288 302.6V120c0-13.3-10.7-24-24-24h-16c-13.3 0-24 10.7-24 24v182.6l-72.4-75.5c-9.3-9.7-24.8-9.9-34.3-.4l-10.9 11c-9.4 9.4-9.4 24.6 0 33.9L239 404.3c9.4 9.4 24.6 9.4 33.9 0l132.7-132.7c9.4-9.4 9.4-24.6 0-33.9l-10.9-11c-9.5-9.5-25-9.3-34.3.4z"></path></svg>
 +
              Table 2. Mass of the bacteriocins
 +
          </p>
 +
        </caption>
 +
        <thead>
 +
          <tr>
 +
            <th><p>Bacteriocin</p></th>
 +
            <th><p style="white-space: nowrap;">Mass (kDa)</p></th>
 +
            <th><p style="white-space: nowrap;">Mass of peptides with intein</p></th>
 +
          </tr>
 +
        </thead>
 +
        <tbody>
 +
          <tr>
 +
            <td><p style="white-space: nowrap;">Leucocyclicin Q</p></td>
 +
            <td style="text-align: center;"><p>6.4</p></td>
 +
            <td style="text-align: center;"><p>34.4</p></td>
 +
          </tr>
 +
          <tr>
 +
            <td><p>Enterocin B</p></td>
 +
            <td style="text-align: center;"><p>7.5</p></td>
 +
            <td style="text-align: center;"><p>35.5</p></td>
 +
          </tr>
 +
          <tr>
 +
            <td><p>Bovicin HJ50</p></td>
 +
            <td style="text-align: center;"><p>6.25</p></td>
 +
            <td style="text-align: center;"><p>34.25</p></td>
 +
          </tr>
 +
          <tr>
 +
            <td><p>Enterocin 96</p></td>
 +
            <td style="text-align: center;"><p>7.9</p></td>
 +
            <td style="text-align: center;"><p>35.9</p></td>
 +
          </tr>
 +
          <tr>
 +
            <td><p>Lacticin Z</p></td>
 +
            <td style="text-align: center;"><p>5.9</p></td>
 +
            <td style="text-align: center;"><p>33.9</p></td>
 +
          </tr>
 +
          <tr>
 +
            <td><p style="white-space: nowrap;">Durancin TW-49M</p></td>
 +
            <td style="text-align: center;"><p>7.3</p></td>
 +
            <td style="text-align: center;"><p>35.3</p></td>
 +
          </tr>
 +
        </tbody>
 +
      </table>
 +
      </div>
 +
       <div class="text">
 +
         <p>
 +
          &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;The gel of SDS-Page result are shown below. The mass of intein-CBD tag is 28 kDa, therefore, all the result shows the initial mass of each bacteriocin plus the mass of intein-CBD tag. From each SDS-Page result, we can confirm the production of target peptides.
 +
        </p>
 +
      </div>
 +
      <img src="https://static.igem.org/mediawiki/2018/0/08/T--NCTU_Formosa--Lu_SDS.png" class="sds_1">
 +
      <img src="https://static.igem.org/mediawiki/2018/5/59/T--NCTU_Formosa--Dur_SDS.png" class="sds_2">
 +
      <div class="explanation">
 +
        <svg class="icon" aria-hidden="true" data-prefix="fas" data-icon="arrow-circle-up" class="svg-inline--fa fa-arrow-circle-up fa-w-16" role="img" xmlns="http://www.w3.org/2000/svg" viewBox="0 0 512 512"><path fill="currentColor" d="M8 256C8 119 119 8 256 8s248 111 248 248-111 248-248 248S8 393 8 256zm143.6 28.9l72.4-75.5V392c0 13.3 10.7 24 24 24h16c13.3 0 24-10.7 24-24V209.4l72.4 75.5c9.3 9.7 24.8 9.9 34.3.4l10.9-11c9.4-9.4 9.4-24.6 0-33.9L273 107.7c-9.4-9.4-24.6-9.4-33.9 0L106.3 240.4c-9.4 9.4-9.4 24.6 0 33.9l10.9 11c9.6 9.5 25.1 9.3 34.4-.4z"></path></svg>
 +
          Figure 4: SDS-PAGE result of the bacteriocin.<br>
 +
          <p style="width: 70%; text-align: center; margin-left: 15%;">
 +
            ER2566: E. coli ER2566 without plasmid; pTXB1: E. coli ER2566 with empty plasmid pTXB1;<br> Leu: Leucocyclicin Q+intein+CBD(34.4kDa); Dur: Durancin +intein+CBD(35.3kDa)
 +
          </p>
 +
      </div>
 +
      <img src="https://static.igem.org/mediawiki/2018/c/c5/T--NCTU_Formosa--96_SDS.png" class="sds_1" id="sds_3">
 +
      <img src="https://static.igem.org/mediawiki/2018/a/ab/T--NCTU_Formosa--B_SDS.png" class="sds_2">
 +
      <div class="explanation">
 +
        <svg class="icon" aria-hidden="true" data-prefix="fas" data-icon="arrow-circle-up" class="svg-inline--fa fa-arrow-circle-up fa-w-16" role="img" xmlns="http://www.w3.org/2000/svg" viewBox="0 0 512 512"><path fill="currentColor" d="M8 256C8 119 119 8 256 8s248 111 248 248-111 248-248 248S8 393 8 256zm143.6 28.9l72.4-75.5V392c0 13.3 10.7 24 24 24h16c13.3 0 24-10.7 24-24V209.4l72.4 75.5c9.3 9.7 24.8 9.9 34.3.4l10.9-11c9.4-9.4 9.4-24.6 0-33.9L273 107.7c-9.4-9.4-24.6-9.4-33.9 0L106.3 240.4c-9.4 9.4-9.4 24.6 0 33.9l10.9 11c9.6 9.5 25.1 9.3 34.4-.4z"></path></svg>
 +
          Figure 5: SDS-PAGE result of the bacteriocin.<br>
 +
          <p style="width: 70%; text-align: center; margin-left: 15%;">
 +
            ER2566: E. coli ER2566 without plasmid; pTXB1: E. coli ER2566 with empty plasmid pTXB1;<br> 96: Enterocin 96+intein+CBD(35.9kDa); B: Enteroicin B+intein+CBD(35.5kDa)
 +
          </p>
 
       </div>
 
       </div>
−
      <div class="title_1"><p>2. MIC test</p></div>
 
−
      <div class="title_2"><p>Inhibition zone:</p></div>
 
−
      <div class="title_2"><p>Minimum Inhibitory Concentration Broth Microdilution:</p></div>
 
−
      <div class="title_1"><p>3. Growth curve experiment</p></div>
 
−
      <div class="title_1"><p>4. Soil experiment</p></div>
 
 
     </div>
 
     </div>
 +
  
 
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Revision as of 12:59, 6 October 2018

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Navigation Bar Protein Expression

Cloning

     To ensure our target genes were successfully cloned into the PSB1C3 backbone, we ran the electrophoresis of Taq PCR products to check the sizes of the insert genes.

All of the sequences contained T7 promoter, RBS, bacteriocin, intein and CBD, were shown in Figure 1.

Figure 1: Our BioBrick design

After amplification with PCR, all the PCR products have their length around 1200 b.p. Each directly length is in the Table 1.

Table 1: The DNA length of each Biobrick

Bacteriocin

Length

Length of PCR product

Leucocyclicin Q

186 b.p.

1230 b.p.

Enterocin B

210 b.p.

1254 b.p.

Enterocin 96

219 b.p.

1263 b.p.

Lacticin Z

153 b.p.

1197 b.p.

Enteriocin A

192 b.p.

1236 b.p.

Bovicin HJ50

171 b.p.

1215 b.p.

Durancin TW-49M

213 b.p.

1257 b.p.

Subtilosin

147 b.p.

1291 b.p.

     Electrophoresis results of the PCR products with marker on the left side and target gene on the right side.
The length are labeled beside each band.

Figure 2: The electrophoresis results of Leucocyclicin Q, Enteroicin B, Bovicin HJ50 and Enterocin 96.
Figure 3: The electrophoresis results of Lacticin Z, Enteroicin A, Durancin and Subtilosin.

Protein Expression

     After the expression from E. coli ER2566, we have to check whether the proteins are successfully expressed. We sonicate E. coli and run SDS-PAGE to make sure the correct sizes. The mass of each proteins are in Table 2.

Table 2. Mass of the bacteriocins

Bacteriocin

Mass (kDa)

Mass of peptides with intein

Leucocyclicin Q

6.4

34.4

Enterocin B

7.5

35.5

Bovicin HJ50

6.25

34.25

Enterocin 96

7.9

35.9

Lacticin Z

5.9

33.9

Durancin TW-49M

7.3

35.3

     The gel of SDS-Page result are shown below. The mass of intein-CBD tag is 28 kDa, therefore, all the result shows the initial mass of each bacteriocin plus the mass of intein-CBD tag. From each SDS-Page result, we can confirm the production of target peptides.

Figure 4: SDS-PAGE result of the bacteriocin.

ER2566: E. coli ER2566 without plasmid; pTXB1: E. coli ER2566 with empty plasmid pTXB1;
Leu: Leucocyclicin Q+intein+CBD(34.4kDa); Dur: Durancin +intein+CBD(35.3kDa)

Figure 5: SDS-PAGE result of the bacteriocin.

ER2566: E. coli ER2566 without plasmid; pTXB1: E. coli ER2566 with empty plasmid pTXB1;
96: Enterocin 96+intein+CBD(35.9kDa); B: Enteroicin B+intein+CBD(35.5kDa)