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.
Line 345: | Line 345: | ||
<tbody> | <tbody> | ||
<tr> | <tr> | ||
− | <td>Leucocyclicin Q</td> | + | <td><p>Leucocyclicin Q</p></td> |
− | <td>6.4</td> | + | <td><p>6.4</p></td> |
− | <td>34.4</td> | + | <td><p>34.4</p></td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td>Enterocin B</td> | + | <td><p>Enterocin B</p></td> |
− | <td>7.5</td> | + | <td><p>7.5</p></td> |
− | <td>35.5</td> | + | <td><p>35.5</p></td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td>Bovicin HJ50</td> | + | <td><p>Bovicin HJ50</p></td> |
− | <td>6.25</td> | + | <td><p>6.25</p></td> |
− | <td>34.25</td> | + | <td><p>34.25</p></td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td>Enterocin 96</td> | + | <td><p>Enterocin 96</p></td> |
− | <td>7.9</td> | + | <td><p>7.9</p></td> |
− | <td>35.9</td> | + | <td><p>35.9</p></td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td>Lacticin Z</td> | + | <td><p>Lacticin Z</p></td> |
− | <td>5.9</td> | + | <td><p>5.9</p></td> |
− | <td>33.9</td> | + | <td><p>33.9</p></td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td>Durancin TW-49M</td> | + | <td><p>Durancin TW-49M</p></td> |
− | <td>7.3</td> | + | <td><p>7.3</p></td> |
− | <td>35.3</td> | + | <td><p>35.3</p></td> |
</tr> | </tr> | ||
</tbody> | </tbody> |
Revision as of 15:13, 2 October 2018
![](https://static.igem.org/mediawiki/2018/f/ff/T--NCTU_Formosa--Navigation.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 have their length around 1200 b.p. Each directly length is in the 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. |
Enteriocin A |
192 b.p. |
1236 b.p. |
Bovicin HJ50 |
171 b.p. |
1215 b.p. |
Durancin TW-49M |
213 b.p. |
1257 b.p. |
Subtilosin |
147 b.p. |
1291 b.p. |
Electrophoresis results of the PCR products with marker on the left side and target gene on the right side.
The length are labeled beside each band.
![](https://static.igem.org/mediawiki/2018/1/1d/T--NCTU_Formosa--Lu_comp.png)
![](https://static.igem.org/mediawiki/2018/4/46/T--NCTU_Formosa--B_comp.png)
![](https://static.igem.org/mediawiki/2018/1/17/T--NCTU_Formosa--Bov_comp.png)
![](https://static.igem.org/mediawiki/2018/5/5a/T--NCTU_Formosa--96_comp.png)
![](https://static.igem.org/mediawiki/2018/2/24/T--NCTU_Formosa--Lac_comp.png)
![](https://static.igem.org/mediawiki/2018/2/23/T--NCTU_Formosa--A_comp.png)
![](https://static.igem.org/mediawiki/2018/d/d8/T--NCTU_Formosa--Dur_comp.png)
![](https://static.igem.org/mediawiki/2018/0/08/T--NCTU_Formosa--Sub_comp.png)
Protein Expression
After the expression from E. coli ER2566, we have to check whether the proteins are successfully expressed. We sonicate E. coli and run SDS-PAGE to make sure the correct sizes. The mass of each proteins are in Table 2.
Bacteriocin |
Mass (kDa) |
Mass of peptides with intein tag |
---|---|---|
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 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.
![](https://static.igem.org/mediawiki/2018/6/65/T--NCTU_Formosa--expression_96_b.png)
(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)
![](https://static.igem.org/mediawiki/2018/f/f7/T--NCTU_Formosa--expression_lu_dur.png)
(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)