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.
Difference between revisions of "Team:NCTU Formosa/Wet Lab/Expression"
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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 1: The gel of Leucocyclicin Q, Enteroicin B, Bovicin HJ50 and Enterocin 96. | Figure 1: The gel of Leucocyclicin Q, Enteroicin B, Bovicin HJ50 and Enterocin 96. | ||
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<img src="https://static.igem.org/mediawiki/2018/d/d8/T--NCTU_Formosa--Dur_comp.png" class="expression1"> | <img src="https://static.igem.org/mediawiki/2018/d/d8/T--NCTU_Formosa--Dur_comp.png" class="expression1"> | ||
<img src="https://static.igem.org/mediawiki/2018/0/08/T--NCTU_Formosa--Sub_comp.png" class="expression1"> | <img src="https://static.igem.org/mediawiki/2018/0/08/T--NCTU_Formosa--Sub_comp.png" class="expression1"> | ||
| + | </div> | ||
| + | <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 1: The gel of Lacticin Z, Enteroicin A, Durancin and Subtilosin. | ||
</div> | </div> | ||
</div> | </div> | ||
Revision as of 13:18, 2 October 2018
Cloning
All of the sequences contained T7 promoter, RBS, bacteriocin, intein and CBD, were shown in Figure 1.
Figure 1: The picture of our BioBrick
Figure 1: The gel of Leucocyclicin Q, Enteroicin B, Bovicin HJ50 and Enterocin 96.
Figure 1: The gel of Lacticin Z, Enteroicin A, Durancin and Subtilosin.
Protein Expression
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 1: 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 2: 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)
