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' 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),
E_A: Leucocyclicin Q + intein + CBD (BBa_K2599014, 34.4 kDa) E_B: Enterocin B + intein + CBD (BBa_K2599011, 35.5 kDa) E_C: Bovicin HJ50 + intein + CBD (BBa_K2599009, 34.25 kDa) E_D: Enterocin 96 + intein + CBD (BBa_K2599012, 35.9 kDa) E_E: Lacticin Z + intein + CBD (BBa_K2599013, 33.9 kDa) E_F: 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).

Template

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+    }
+    .sds_page{
+      width: 60%;
+      margin-left: 20%;
+    }
+    .sds_entb{
+      width: 30%;
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        </div>
        <div class="text">
-       <p>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;After amplification with PCR, all the PCR products length around 1200 b.p. The exact lengths are listed in Table 1.</p>
+       <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">
@@ Line 305: / Line 315: @@
            <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
+               Table 1: The DNA length of each BioBrick
            </p>
          </caption>
@@ Line 350: / Line 360: @@
        </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. The lengths are labeled beside each band.</p>
+        <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">
@@ Line 357: / Line 367: @@
        <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 pattern of taq PCR products of six bacteriocin genes containing T7 promotor, RBS, intein, and CBD: (A) Leucocyclicin Q (BBa_K2599014, 1230 b.p.) (B) Enterocin B (BBa_K2599011, 1166 b.p.) (C) Bovicin HJ50 (BBa_K2599009, 1125 b.p.) (D) Enterocin 96 (BBa_K2599012, 1175 b.p.) (E) Lacticin Z (BBa_K2599013, 1112 b.p.) (F) Durancin TW-49M (BBa_K2599015, 1169b.p.)
+       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>
@@ Line 363: / Line 373: @@
        <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>
+       <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">
@@ Line 416: / Line 426: @@
        <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.
+           &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.
          </p>
        </div>
-       <img src="" class="sds_page">
+       <img src="https://static.igem.org/mediawiki/2018/f/f2/T--NCTU_Formosa--exp_SDS.png" class="sds_page">
        <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 3: SDS-PAGE electrophoresis of bacteriocin samples. M: protein Ladder 5–245 kDa, C: negative control (only intein+CBD ,28 kDa), EA: Leucocyclicin Q + intein + CBD (BBa_K2599014, 34.4kDa) 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 + intein + CBD(BBa_K2599015, 35.3kDa)
+           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)
            </p>
+    </div>
+    <div class="sec2" style="background-color:#ffffff;">
+      <div class="title_1"><p>Protein Purification</p></div>
+      <div class="text">
+        <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>
+      </div>
+      <img src="https://static.igem.org/mediawiki/2018/6/6f/T--NCTU_Formosa--entb11.png" class="sds_entb">
+      <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: The purification of Enterocin B.<br>
+        <p>M: Protein Ladder 5–245 kDa;<br>
+           C: Negative control. Enterocin B without purification(35.5 kDa);<br>
+           E: Experimental group. The purified Enterocin B(7.5 kDa).</p>
        </div>
      </div>
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