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.
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Figure 2: Agarose gel electrophoretic pattern of Taq PCR products of six bacteriocin genes containing T7 promoter, RBS, intein, and CBD: (A) Leucocyclicin Q (BBa_K2599014, 1230 b.p.) (B) Enterocin B (BBa_K2599011, 1166 b.p.) (C) Bovicin HJ50 (BBa_K2599009, 1125 b.p.) (D) Enterocin 96 (BBa_K2599012, 1175 b.p.) (E) Lacticin Z (BBa_K2599013, 1112 b.p.) (F) Durancin TW-49M (BBa_K2599015, 1169 b.p.) | Figure 2: Agarose gel electrophoretic pattern of Taq PCR products of six bacteriocin genes containing T7 promoter, RBS, intein, and CBD: (A) Leucocyclicin Q (BBa_K2599014, 1230 b.p.) (B) Enterocin B (BBa_K2599011, 1166 b.p.) (C) Bovicin HJ50 (BBa_K2599009, 1125 b.p.) (D) Enterocin 96 (BBa_K2599012, 1175 b.p.) (E) Lacticin Z (BBa_K2599013, 1112 b.p.) (F) Durancin TW-49M (BBa_K2599015, 1169 b.p.) | ||
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<div class="title_1"><p>Protein Expression</p></div> | <div class="title_1"><p>Protein Expression</p></div> | ||
Revision as of 00:04, 18 October 2018
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' length is around 1200 b.p. The exact lengths are listed in 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. |
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 pattern of Taq PCR products of six bacteriocin genes containing T7 promoter, RBS, intein, and CBD: (A) Leucocyclicin Q (BBa_K2599014, 1230 b.p.) (B) Enterocin B (BBa_K2599011, 1166 b.p.) (C) Bovicin HJ50 (BBa_K2599009, 1125 b.p.) (D) Enterocin 96 (BBa_K2599012, 1175 b.p.) (E) Lacticin Z (BBa_K2599013, 1112 b.p.) (F) Durancin TW-49M (BBa_K2599015, 1169 b.p.)
Protein Expression
After the expression from E. coli BL21 Rosetta-gami, we had to check whether the proteins were successfully expressed. We sonicated E. coli and ran SDS-PAGE to make sure the correct sizes. The mass of each proteins present in Table 2.
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 showed the initial mass of each bacteriocin plus the mass of intein-CBD tag. From each SDS-PAGE result, 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), EA: Leucocyclicin Q + intein + CBD (BBa_K2599014, 34.4 kDa) EB: Enterocin B + intein + CBD (BBa_K2599011, 35.5 kDa) EC: Bovicin HJ50 + intein + CBD (BBa_K2599009, 34.25 kDa) ED: Enterocin 96 + intein + CBD (BBa_K2599012, 35.9 kDa) EE: Lacticin Z + intein + CBD (BBa_K2599013, 33.9 kDa) EF: 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 result is as bellow. We use Enterocin B as representative. The result shows that we can successfully purify bacteriocin.
Figure 4: The purification of Enterocin B.
M: Protein Ladder 5–245 kDa; C: Negative control. Enterocin B without purified; E: Experimental group. The pure Enterocin B(7.5kDa
