Team:NCTU Formosa/Wet Lab

Navigation Bar Protein Expression

Cloning

     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)

Mass of peptides with intein

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.

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)

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)