Team:NCTU Formosa/Wet Lab/Expression

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.

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.

Table 1: The DNA length of each Biobrick
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.

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.

Table 2. Mass of the bacteriocins
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)