Difference between revisions of "Team:NCTU Formosa/Wet Lab/Expression"

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         <p>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;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.</p>
 
         <p>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;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.</p>
 
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Revision as of 00:18, 18 October 2018

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' length is 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 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.

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 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

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