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

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	Mass (kDa)
Enterocin B	7.5
Enterocin 96	7.9
Bovicin HJ50	6.25
Durancin	7.3
Leucocyclicin Q	6.4
Lacticin Z	5.9

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)
Enterocin B	7.5
Enterocin 96	7.9
Bovicin HJ50	6.25
Durancin	7.3
Leucocyclicin Q	6.4
Lacticin Z	5.9

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.

Figure 4: SDS-PAGE result of Enterocin 96 and Enteroicin B.

(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)

Figure 5: SDS-PAGE result of Leucocyclicin Q and Durancin.

(N1) Negative control: E. coli ER2566 without plasmid (N2) Negative control: E. coli ER2566 with empty plasmid
(C) Leucocyclicin Q+intein+CBD (34.4kDa) (D) Durancin +intein+CBD (35.3kDa)

@@ Line 236: / Line 236: @@
          <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>After amplification with PCR, all the PCR products have their length around 1200 b.p. Each directly length is in the Table 1.</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 1: The DNA length of each Biobrick
+          </p>
+        </caption>
+        <thead>
+          <tr>
+            <th><p>Bacteriocin</p></th>
+            <th><p>Mass (kDa)</p></th>
+          </tr>
+        </thead>
+        <tbody>
+          <tr>
+            <td>Enterocin B</td>
+            <td>7.5</td>
+          </tr>
+          <tr>
+            <td>Enterocin 96</td>
+            <td>7.9</td>
+          </tr>
+          <tr>
+            <td>Bovicin HJ50</td>
+            <td>6.25</td>
+          </tr>
+          <tr>
+            <td>Durancin</td>
+            <td>7.3</td>
+          </tr>
+          <tr>
+            <td>Leucocyclicin Q</td>
+            <td>6.4</td>
+          </tr>
+          <tr>
+            <td>Lacticin Z</td>
+            <td>5.9</td>
+          </tr>
+        </tbody>
+      </table>
        </div>
        <div class="cloning">
@@ Line 260: / Line 305: @@
      <div class="sec2" style="background-color:#ffffff;">
        <div class="title"><p>Protein Expression</p></div>
+      <div class="text">
+      <p>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>
@@ Line 265: / Line 313: @@
            <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 Bacteriocins we selected.
+               Table 2. Mass of the bacteriocins
            </p>
          </caption>