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-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 DNA length of each | + | Table 1: The DNA length of each BioBrick |
</p> | </p> | ||
</caption> | </caption> |
Revision as of 17:33, 17 October 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' length is 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 pattern of Taq PCR products of six bacteriocin genes containing T7 promoter, 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.
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)
![](https://static.igem.org/mediawiki/2018/e/e8/T--NCTU_Formosa--top.png)