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.
![](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/2018/0/08/T--NCTU_Formosa--Lu_SDS.png)
![](https://static.igem.org/mediawiki/2018/5/59/T--NCTU_Formosa--Dur_SDS.png)
Figure 4: SDS-PAGE result of the bacteriocin.
ER2566: E. coli ER2566 without plasmid; pTXB1: E. coli ER2566 with empty plasmid pTXB1; Leu: Leucocyclicin Q+intein+CBD(34.4kDa); Dur: Durancin +intein+CBD(35.3kDa)
![](https://static.igem.org/mediawiki/2018/c/c5/T--NCTU_Formosa--96_SDS.png)
![](https://static.igem.org/mediawiki/2018/a/ab/T--NCTU_Formosa--B_SDS.png)
Figure 5: SDS-PAGE result of the bacteriocin.
ER2566: E. coli ER2566 without plasmid; pTXB1: E. coli ER2566 with empty plasmid pTXB1; 96: Enterocin 96+intein+CBD(35.9kDa); B: Enteroicin B+intein+CBD(35.5kDa)