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.
Difference between revisions of "Team:NCTU Formosa/Wet Lab/Expression"
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| − | <p> After the expression from <i>E. coli</i> BL21 Rosetta-gami, we had to check whether the proteins were successfully expressed. We sonicated <i>E. coli</i> and ran SDS-PAGE to make sure the correct sizes. The mass of each | + | <p> After the expression from <i>E. coli</i> BL21 Rosetta-gami, we had to check whether the proteins were successfully expressed. We sonicated <i>E. coli</i> and ran SDS-PAGE to make sure the correct sizes. The mass of each protein is present in Table 2.</p> |
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<div class="table"> | <div class="table"> | ||
Revision as of 02:16, 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' lengths are 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 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 sonicated E. coli and ran SDS-PAGE to make sure the correct sizes. The mass of each protein is 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 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), 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 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; E: Experimental group. The purified Enterocin B(7.5kDa).
