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' lengths are around 1200 b.p. The exact lengths are listed in 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.
Bovicin HJ50	171 b.p.	1215 b.p.
Durancin TW-49M	213 b.p.	1257 b.p.

The figure 2 is electrophoresis results of the PCR products with marker on the left side and target gene on the right side. The lengths are labeled beside each band.

Figure 2: Agarose gel electrophoretic patterns of Taq PCR products of six bacteriocin genes containing T7 promoter, RBS, gene segments of intein and CBD: (A) Leucocyclicin Q (BBa_K2599014, 1230 b.p.) (B) Enterocin B (BBa_K2599011, 1254 b.p.) (C) Bovicin HJ50 (BBa_K2599009, 1215 b.p.) (D) Lacticin Z (BBa_K2599013, 1197 b.p.) (E) Enterocin 96 (BBa_K2599012, 1263 b.p.) (F) Durancin TW-49M (BBa_K2599015, 1257 b.p.)

Protein Expression

After the expression from E. coli BL21 Rosetta-gami, we had to check whether the proteins were successfully expressed. We broke E. coli with sonication and ran SDS-PAGE to check the correct sizes of the target proteins. The mass of each protein is present in Table 2.

Table 2. Mass of peptides with intein(kDa).
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 results were shown below. The mass of intein-CBD tag is 28 kDa. Therefore, all the results showed the initial mass of each bacteriocin plus the mass of intein-CBD tag. From SDS-PAGE results, we could confirm the production of target peptides.

Figure 3: SDS-PAGE analysis of bacteriocin samples. M: Protein Ladder 5–245 kDa, C: Negative control (only intein+CBD, 28 kDa), E_A: Leucocyclicin Q + intein + CBD (BBa_K2599014, 34.4 kDa) E_B: Enterocin B + intein + CBD (BBa_K2599011, 35.5 kDa) E_C: Bovicin HJ50 + intein + CBD (BBa_K2599009, 34.25 kDa) E_D: Enterocin 96 + intein + CBD (BBa_K2599012, 35.9 kDa) E_E: Lacticin Z + intein + CBD (BBa_K2599013, 33.9 kDa) E_F: Durancin TW-49M + intein + CBD(BBa_K2599015, 35.3 kDa)

Protein Purification

Finally, we added 1,4-dithiothreitol(DTT) to purify bacteriocins from intein+CBD tag. The results were shown as bellow. We used Enterocin B as representative. The result showed that we could successfully purify bacteriocin.

Figure 4: The purification of Enterocin B.

M: Protein Ladder 5–245 kDa; C: Negative control. Enterocin B without purification( + intein + CBD: 35.5kDa); E: Experimental group. The purified Enterocin B(7.5 kDa).

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