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' length is 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 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.)

Protein Expression

After the expression from E. coli ER2566, we had to check whether the proteins were successfully expressed. We sonicated E. coli and ran SDS-PAGE to make sure the correct sizes. The mass of each proteins present 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 result are shown below. The mass of intein-CBD tag is 28 kDa. therefore, all the result showed the initial mass of each bacteriocin plus the mass of intein-CBD tag. From each SDS-PAGE result, we could confirm the production of target peptides.

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 TW-49M + intein + CBD(BBa_K2599015, 35.3kDa)

Template

@@ Line 350: / Line 350: @@
        </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 357: @@
        <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 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.)
        </p></div>
      </div>
@@ Line 363: / Line 363: @@
        <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> ER2566, we had to check whether the proteins were successfully expressed. We sonicated <i>E. coli</i> and ran SDS-PAGE to make sure the correct sizes. The mass of each proteins present in Table 2.</p>
        </div>
        <div class="table">
@@ Line 416: / Line 416: @@
        <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 result are shown below. The mass of intein-CBD tag is 28 kDa. therefore, all the result showed the initial mass of each bacteriocin plus the mass of intein-CBD tag. From each SDS-PAGE result, we could confirm the production of target peptides.
          </p>
        </div>
@@ Line 422: / Line 422: @@
        <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 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 TW-49M + intein + CBD(BBa_K2599015, 35.3kDa)
            </p>
        </div>