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

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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>
 
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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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       <img src="https://static.igem.org/mediawiki/2018/3/30/T--NCTU_Formosa--Experiment_design_title.png" class="title_title">
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      <div class="title_1"><p>Cloning</p></div>
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       <div class="text">
 
       <div class="text">
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         <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>&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.
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</p>
 
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      <div class="title"><p>BioBrick</p></div>
 
       <div class="text">
 
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         <p>All of the sequences contained T7 promoter, RBS, bacteriocin, intein and CBD, were shown in Figure 1.</p>
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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>
 
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       <img src="https://static.igem.org/mediawiki/2018/a/aa/T--NCTU_Formosa--biobrick.png" class="expression">
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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">
 
       <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: Our BioBrick design
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         Figure 1: BioBrick: T7 promoter + RBS + target peptide + intein-CBD
 
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       <div class="text">
 
       <div class="text">
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      <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>&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.
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      <div class="table">
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         <table>
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        <caption>
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          <p class="explanation">
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            <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>
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              Table 1: The DNA length of each Biobrick
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          </p>
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        <thead>
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          <tr>
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            <th><p>Bacteriocin</p></th>
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            <th style="text-align: center;"><p>Length</p></th>
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            <th style="text-align: center;"><p>Length of PCR product</p></th>
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          </tr>
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        <tbody>
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          <tr>
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            <td><p>Leucocyclicin Q</p></td>
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            <td style="text-align: center;"><p>186 b.p.</p></td>
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            <td style="text-align: center;"><p>1230 b.p.</p></td>
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          </tr>
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          <tr>
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            <td><p>Enterocin B</p></td>
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            <td style="text-align: center;"><p>210 b.p.</p></td>
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            <td style="text-align: center;"><p>1254 b.p.</p></td>
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          </tr>
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          <tr>
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            <td><p>Enterocin 96</p></td>
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            <td style="text-align: center;"><p>219 b.p.</p></td>
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            <td style="text-align: center;"><p>1263 b.p.</p></td>
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          </tr>
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          <tr>
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            <td><p>Lacticin Z</p></td>
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            <td style="text-align: center;"><p>153 b.p.</p></td>
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            <td style="text-align: center;"><p>1197 b.p.</p></td>
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          </tr>
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          <tr>
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            <td><p>Enteriocin A</p></td>
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            <td style="text-align: center;"><p>192 b.p.</p></td>
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            <td style="text-align: center;"><p>1236 b.p.</p></td>
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          </tr>
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          <tr>
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            <td><p>Bovicin HJ50</p></td>
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            <td style="text-align: center;"><p>171 b.p.</p></td>
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            <td style="text-align: center;"><p>1215 b.p.</p></td>
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          </tr>
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          <tr>
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            <td><p>Durancin TW-49M</p></td>
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            <td style="text-align: center;"><p>213 b.p.</p></td>
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            <td style="text-align: center;"><p>1257 b.p.</p></td>
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          </tr>
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          <tr>
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            <td><p>Subtilosin</p></td>
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            <td style="text-align: center;"><p>147 b.p.</p></td>
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            <td style="text-align: center;"><p>1291 b.p.</p></td>
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          </tr>
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        </tbody>
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      </table>
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      </div>
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      <div class="text">
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      <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>
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      <div class="cloning">
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      <img src="https://static.igem.org/mediawiki/2018/1/1d/T--NCTU_Formosa--Lu_comp.png" class="expression1">
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      <img src="https://static.igem.org/mediawiki/2018/4/46/T--NCTU_Formosa--B_comp.png" class="expression1">
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      <img src="https://static.igem.org/mediawiki/2018/1/17/T--NCTU_Formosa--Bov_comp.png" class="expression1">
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      <img src="https://static.igem.org/mediawiki/2018/5/5a/T--NCTU_Formosa--96_comp.png" class="expression1">
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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: The electrophoresis results of Leucocyclicin Q, Enteroicin B, Bovicin HJ50 and Enterocin 96.
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        Figure 2: Procedure of intein-mediated protein splicing
 
       </div>
 
       </div>
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       <div class="cloning">
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       <div class="title_1"><p>1. Protein expression</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="title_2"><p>Cloning:</p></div>
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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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      <div class="textnew">
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      <img src="https://static.igem.org/mediawiki/2018/d/d8/T--NCTU_Formosa--Dur_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>
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      <img src="https://static.igem.org/mediawiki/2018/0/08/T--NCTU_Formosa--Sub_comp.png" class="expression1">
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       <div class="explanation">
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       <div class="title_2"><p>Expression:</p></div>
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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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       <div class="textnew">
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      Figure 3: The electrophoresis results of Lacticin Z, Enteroicin A, Durancin and Subtilosin.
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        <p>We expressed the proteins by <i>E. coli</i> ER2566 and <i>E. coli</i> BL21 Rosetta-Gami.</p>
 
       </div>
 
       </div>
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    </div>
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       <div class="title_2"><p>SDS-PAGE:</p></div>
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    <div class="sec2" style="background-color:#ffffff;">
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       <div class="textnew">
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       <div class="title_1"><p>Protein Expression</p></div>
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         <p>We checked the expression result through SDS-PAGE.</p>
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      <div class="text">
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      <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>
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      <div class="table">
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        <table>
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            <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>
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              Table 2. Mass of the bacteriocins
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          </p>
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        </caption>
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        <thead>
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          <tr>
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            <th><p>Bacteriocin</p></th>
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            <th><p style="white-space: nowrap;">Mass (kDa)</p></th>
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            <th><p style="white-space: nowrap;">Mass of peptides with intein</p></th>
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          </tr>
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        </thead>
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        <tbody>
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          <tr>
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            <td><p style="white-space: nowrap;">Leucocyclicin Q</p></td>
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            <td style="text-align: center;"><p>6.4</p></td>
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            <td style="text-align: center;"><p>34.4</p></td>
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          </tr>
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          <tr>
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            <td><p>Enterocin B</p></td>
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            <td style="text-align: center;"><p>7.5</p></td>
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            <td style="text-align: center;"><p>35.5</p></td>
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          </tr>
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−
          <tr>
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            <td><p>Bovicin HJ50</p></td>
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            <td style="text-align: center;"><p>6.25</p></td>
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            <td style="text-align: center;"><p>34.25</p></td>
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          </tr>
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          <tr>
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            <td><p>Enterocin 96</p></td>
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            <td style="text-align: center;"><p>7.9</p></td>
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            <td style="text-align: center;"><p>35.9</p></td>
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          </tr>
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          <tr>
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            <td><p>Lacticin Z</p></td>
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            <td style="text-align: center;"><p>5.9</p></td>
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            <td style="text-align: center;"><p>33.9</p></td>
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          </tr>
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          <tr>
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            <td><p style="white-space: nowrap;">Durancin TW-49M</p></td>
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            <td style="text-align: center;"><p>7.3</p></td>
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            <td style="text-align: center;"><p>35.3</p></td>
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          </tr>
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        </tbody>
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      </table>
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−
      </div>
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       <div class="text">
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         <p>
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          &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.
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        </p>
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      </div>
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      <img src="https://static.igem.org/mediawiki/2018/0/08/T--NCTU_Formosa--Lu_SDS.png" class="sds_1">
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      <img src="https://static.igem.org/mediawiki/2018/5/59/T--NCTU_Formosa--Dur_SDS.png" class="sds_2">
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      <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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          Figure 4: SDS-PAGE result of the bacteriocin.<br>
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−
          <p style="width: 70%; text-align: center; margin-left: 15%;">
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            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)
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−
          </p>
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      </div>
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−
      <img src="https://static.igem.org/mediawiki/2018/c/c5/T--NCTU_Formosa--96_SDS.png" class="sds_1" id="sds_3">
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−
      <img src="https://static.igem.org/mediawiki/2018/a/ab/T--NCTU_Formosa--B_SDS.png" class="sds_2">
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−
      <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>
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          Figure 5: SDS-PAGE result of the bacteriocin.<br>
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−
          <p style="width: 70%; text-align: center; margin-left: 15%;">
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−
            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)
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−
          </p>
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       </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 13:01, 6 October 2018

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Navigation Bar Experiment

     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.

BioBrick

     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.

Figure 1: BioBrick: T7 promoter + RBS + target peptide + intein-CBD

     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.
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.

Figure 2: Procedure of intein-mediated protein splicing

1. Protein expression

Cloning:

We cloned the insert gene into pSB1C3 backbone and transform into E. coli DH5α.
These have been submitted to iGEM.

Expression:

We expressed the proteins by E. coli ER2566 and E. coli BL21 Rosetta-Gami.

SDS-PAGE:

We checked the expression result through SDS-PAGE.

2. MIC test

Inhibition zone:

Minimum Inhibitory Concentration Broth Microdilution:

3. Growth curve experiment

4. Soil experiment