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

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   <div class="wrapper">
 
   <div class="wrapper">
 
     <div class="banner">
 
     <div class="banner">
−
       <img class="cover" src="https://static.igem.org/mediawiki/2018/b/b2/T--NCTU_Formosa--model_insite_cover.png">
+
       <img class="cover" src="https://static.igem.org/mediawiki/2018/c/c5/T--NCTU_Formosa--wetlab6.png">
 +
      <a href="https://2018.igem.org/Team:NCTU_Formosa/Wet_Lab/Expression"><img src="https://static.igem.org/mediawiki/2018/6/6e/T--NCTU_Formosa--protein_expression.png" class="protein"></a>
 +
      <a href="https://2018.igem.org/Team:NCTU_Formosa/Wet_Lab/Functional_Analysis"><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/Curcumin_Biosensor"><img src="https://static.igem.org/mediawiki/2018/2/20/T--NCTU_Formosa--biosensor.png" class="sensor"></a>
 
     </div>
 
     </div>
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     <div class="sec1">
+
     <div class="sec1" style="background-color:#fff;">
−
       <div class="title"><p>Protein Expression</p></div>
+
      <img src="https://static.igem.org/mediawiki/2018/a/ad/T--NCTU_Formosa--Expression_design_title.png" class="title_title">
 +
       <div class="title_1"><p>Cloning</p></div>
 +
      <div class="text">
 +
        <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>
 +
      </div>
 +
      <div class="text">
 +
        <p>All of the sequences contained T7 promoter, RBS, bacteriocin, intein and CBD, were shown in Figure 1.</p>
 +
      </div>
 +
      <img src="https://static.igem.org/mediawiki/2018/a/aa/T--NCTU_Formosa--biobrick.png" class="expression">
 +
      <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 1: Our BioBrick design
 +
      </div>
 +
      <div class="text">
 +
      <p>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;After amplification with PCR, all the PCR products' lengths are around 1200 b.p. The exact lengths are listed in Table 1.</p>
 +
      </div>
 
       <div class="table">
 
       <div class="table">
 
         <table>
 
         <table>
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           <p class="explanation">
 
           <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>
 
             <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 Bacteriocin we filtered.
+
               Table 1: The DNA length of each BioBrick
 
           </p>
 
           </p>
 
         </caption>
 
         </caption>
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           <tr>
 
           <tr>
 
             <th><p>Bacteriocin</p></th>
 
             <th><p>Bacteriocin</p></th>
−
             <th><p>Mass(kDa)</p></th>
+
             <th style="text-align: center;"><p>Length</p></th>
 +
            <th style="text-align: center;"><p>Length of PCR product</p></th>
 
           </tr>
 
           </tr>
 
         </thead>
 
         </thead>
 
         <tbody>
 
         <tbody>
 
           <tr>
 
           <tr>
−
             <td>Subtilocin</td>
+
             <td><p>Leucocyclicin Q</p></td>
−
             <td>5.8</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>
 
           <tr>
 
           <tr>
−
             <td>Enterocin A</td>
+
             <td><p>Enterocin B</p></td>
−
             <td>6.9</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>
 
           <tr>
 
           <tr>
−
             <td>Enterocin B</td>
+
             <td><p>Enterocin 96</p></td>
−
             <td>7.5</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>
 
           <tr>
 
           <tr>
−
             <td>Enterocin 96</td>
+
             <td><p>Lacticin Z</p></td>
−
             <td>7.9</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>
 
           <tr>
 
           <tr>
−
             <td>Bovicin HJ50</td>
+
             <td><p>Bovicin HJ50</p></td>
−
             <td>6.25</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>
 
           <tr>
 
           <tr>
−
             <td>Durancin</td>
+
             <td><p>Durancin TW-49M</p></td>
−
             <td>7.3</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>
 +
        </tbody>
 +
      </table>
 +
      </div>
 +
      <div class="text">
 +
        <p>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;The figure 2 is electrophoresis results of the PCR products with marker on the left side and target gene on the right side. The lengths are labeled beside each band.</p>
 +
      </div>
 +
      <div class="cloning">
 +
        <img src="https://static.igem.org/mediawiki/2018/2/2b/T--NCTU_Formosa--exp_clone.png" class="clone">
 +
      </div>
 +
      <div class="explanation" style="width: 80%; margin-left: 10%; text-align: justify;"><p>
 +
      <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 2: Agarose gel electrophoretic patterns of Taq PCR products of six bacteriocin genes containing T7 promoter, RBS, gene segments of intein and CBD: (A) Leucocyclicin Q (BBa_K2599014, 1230 b.p.) (B) Enterocin B (BBa_K2599011, 1254 b.p.) (C) Bovicin HJ50 (BBa_K2599009, 1215 b.p.) (D) Lacticin Z (BBa_K2599013, 1197 b.p.) (E) Enterocin 96 (BBa_K2599012, 1263 b.p.) (F) Durancin TW-49M (BBa_K2599015, 1257 b.p.)
 +
      </p></div>
 +
    </div>
 +
    <div class="sec2" style="background-color:#ffffff;">
 +
      <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> BL21 Rosetta-gami, we had to check whether the proteins were successfully expressed. We broke <i>E. coli</i> with sonication and ran SDS-PAGE to check the correct sizes of the target proteins. The mass of each protein is presented 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 peptides with intein(kDa).
 +
          </p>
 +
        </caption>
 +
        <thead>
 
           <tr>
 
           <tr>
−
             <td>Leucocyclicin Q</td>
+
             <th><p>Bacteriocin</p></th>
−
             <td>6.4</td>
+
             <th><p style="white-space: nowrap;">Mass (kDa)</p></th>
 +
            <th><p style="white-space: nowrap;">Mass of peptides with intein</p></th>
 
           </tr>
 
           </tr>
 +
        </thead>
 +
        <tbody>
 
           <tr>
 
           <tr>
−
             <td>Lacticin Z</td>
+
             <td><p style="white-space: nowrap;">Leucocyclicin Q</p></td>
−
             <td>5.9</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>
 
           </tr>
 
         </tbody>
 
         </tbody>
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           We tried to get the bacteriocin from E. coli ER2566. To check whether the protein had been expressed, we used SDS-PAGE to confirm. Because our sequences content Intein and Chitin Binding Domain(CBD), which are 28 kDa, the result should be check as the bacteriocins’ initial mass plus 28 kDa. The figure showed our SDS-PAGE result. From here, we can know our target bacteriocin is produced.
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           &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;The gel of SDS-PAGE results were shown below. The mass of intein-CBD tag is 28 kDa. Therefore, all the results showed the initial mass of each bacteriocin plus the mass of intein-CBD tag. From SDS-PAGE results, we could confirm the production of target peptides.
 
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      <div class="explanation" style="width: 80%; margin-left: 10%; text-align: justify;"><p>
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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 3: SDS-PAGE analysis of bacteriocin samples. M: Protein Ladder 5–245 kDa, C: Negative control (only intein+CBD, 28 kDa),<br>
 +
E<sub>A</sub>: Leucocyclicin Q + intein + CBD (BBa_K2599014, 34.4 kDa) E<sub>B</sub>: Enterocin B + intein + CBD (BBa_K2599011, 35.5 kDa) E<sub>C</sub>: Bovicin HJ50 + intein + CBD (BBa_K2599009, 34.25 kDa) E<sub>D</sub>: Enterocin 96 + intein + CBD (BBa_K2599012, 35.9 kDa) E<sub>E</sub>: Lacticin Z + intein + CBD (BBa_K2599013, 33.9 kDa) E<sub>F</sub>: Durancin TW-49M + intein + CBD(BBa_K2599015, 35.3 kDa)
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      <div class="title_1"><p>Protein Purification</p></div>
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        <p>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Finally, we added 1,4-dithiothreitol(DTT) to purify bacteriocins from intein-CBD tag(28 kDa). The result is shown as below. We used Enterocin B as representative. The result showed that we could successfully purify bacteriocin.</p>
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       <img src="https://static.igem.org/mediawiki/2018/6/6f/T--NCTU_Formosa--entb11.png" class="sds_entb">
 
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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>
 
         <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 : SDS-PAGE result of the bacteriocin. (N1) Negative control: E. coli ER2566 without plasmid (N2) Negative control: E. coli ER2566 with empty plasmid (A) Enterocin 96+intein+CBD(35.9kDa) (B) Enteroicin B+intein+CBD(35.5kDa) (C) Leucocyclicin Q+intein+CBD(34.4kDa) (D) Durancin +intein+CBD(35.3kDa) (E) Lacticin+intein+CBD(33.9kDa) (F)Bovicin HJ50+intein +CBD(34.25kDa)
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         Figure 4: The purification of Enterocin B.<br>
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        <p>M: Protein Ladder 5–245 kDa;<br>
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          C: Negative control. Enterocin B without purification(35.5 kDa);<br>
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          E: Experimental group. The purified Enterocin B(7.5 kDa).</p>
 
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Latest revision as of 15:20, 7 December 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' lengths are around 1200 b.p. The exact lengths are listed in 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.

Bovicin HJ50

171 b.p.

1215 b.p.

Durancin TW-49M

213 b.p.

1257 b.p.

     The figure 2 is electrophoresis results of the PCR products with marker on the left side and target gene on the right side. The lengths are labeled beside each band.

Figure 2: Agarose gel electrophoretic patterns of Taq PCR products of six bacteriocin genes containing T7 promoter, RBS, gene segments of intein and CBD: (A) Leucocyclicin Q (BBa_K2599014, 1230 b.p.) (B) Enterocin B (BBa_K2599011, 1254 b.p.) (C) Bovicin HJ50 (BBa_K2599009, 1215 b.p.) (D) Lacticin Z (BBa_K2599013, 1197 b.p.) (E) Enterocin 96 (BBa_K2599012, 1263 b.p.) (F) Durancin TW-49M (BBa_K2599015, 1257 b.p.)

Protein Expression

     After the expression from E. coli BL21 Rosetta-gami, we had to check whether the proteins were successfully expressed. We broke E. coli with sonication and ran SDS-PAGE to check the correct sizes of the target proteins. The mass of each protein is presented in Table 2.

Table 2. Mass of peptides with intein(kDa).

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 results were shown below. The mass of intein-CBD tag is 28 kDa. Therefore, all the results showed the initial mass of each bacteriocin plus the mass of intein-CBD tag. From SDS-PAGE results, we could confirm the production of target peptides.

Figure 3: SDS-PAGE analysis of bacteriocin samples. M: Protein Ladder 5–245 kDa, C: Negative control (only intein+CBD, 28 kDa),
EA: Leucocyclicin Q + intein + CBD (BBa_K2599014, 34.4 kDa) EB: Enterocin B + intein + CBD (BBa_K2599011, 35.5 kDa) EC: Bovicin HJ50 + intein + CBD (BBa_K2599009, 34.25 kDa) ED: Enterocin 96 + intein + CBD (BBa_K2599012, 35.9 kDa) EE: Lacticin Z + intein + CBD (BBa_K2599013, 33.9 kDa) EF: Durancin TW-49M + intein + CBD(BBa_K2599015, 35.3 kDa)

Protein Purification

     Finally, we added 1,4-dithiothreitol(DTT) to purify bacteriocins from intein-CBD tag(28 kDa). The result is shown as below. We used Enterocin B as representative. The result showed that we could successfully purify bacteriocin.

Figure 4: The purification of Enterocin B.

M: Protein Ladder 5–245 kDa;
C: Negative control. Enterocin B without purification(35.5 kDa);
E: Experimental group. The purified Enterocin B(7.5 kDa).

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