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.
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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> | ||
− | + | 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> | C: Negative control. Enterocin B without purification(35.5 kDa);<br> | ||
E: Experimental group. The purified Enterocin B(7.5 kDa).</p> | E: Experimental group. The purified Enterocin B(7.5 kDa).</p> |
Revision as of 09:29, 4 December 2018
![](https://static.igem.org/mediawiki/2018/f/ff/T--NCTU_Formosa--Navigation.png)
![](https://static.igem.org/mediawiki/2018/a/ad/T--NCTU_Formosa--Expression_design_title.png)
Cloning
All of the sequences contained T7 promoter, RBS, bacteriocin, intein and CBD, were shown in Figure 1.
![](https://static.igem.org/mediawiki/2018/a/aa/T--NCTU_Formosa--biobrick.png)
After amplification with PCR, all the PCR products' lengths are around 1200 b.p. The exact lengths are listed in Table 1.
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.
![](https://static.igem.org/mediawiki/2018/2/2b/T--NCTU_Formosa--exp_clone.png)
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 present in Table 2.
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.
![](https://static.igem.org/mediawiki/2018/f/f2/T--NCTU_Formosa--exp_SDS.png)
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 results were shown as below. We used Enterocin B as representative. The result showed that we could successfully purify bacteriocin.
![](https://static.igem.org/mediawiki/2018/6/6f/T--NCTU_Formosa--entb11.png)
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).
![](https://static.igem.org/mediawiki/2018/e/e8/T--NCTU_Formosa--top.png)