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.
Team:NCTU Formosa/Wet Lab/Expression
Cloning
All of the sequences contained T7 promoter, RBS, bacteriocin, intein and CBD, were shown in Figure 1.
Figure 1: Our BioBrick design
After amplification with PCR, all the PCR products have their length around 1200 b.p. Each directly length is in the Table 1.
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 |
Figure 2: The electrophoresis results of Leucocyclicin Q, Enteroicin B, Bovicin HJ50 and Enterocin 96.
Figure 3: The electrophoresis results of Lacticin Z, Enteroicin A, Durancin and Subtilosin.
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) |
|---|---|
| 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 4: 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 5: 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)
