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

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       <p>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;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.</p>
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       <p>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;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.</p>
 
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         <svg class="icon" aria-hidden="true" data-prefix="fas" data-icon="arrow-circle-up" class="svg-inline--fa fa-arrow-circle-up fa-w-16" role="img" xmlns="http://www.w3.org/2000/svg" viewBox="0 0 512 512"><path fill="currentColor" d="M8 256C8 119 119 8 256 8s248 111 248 248-111 248-248 248S8 393 8 256zm143.6 28.9l72.4-75.5V392c0 13.3 10.7 24 24 24h16c13.3 0 24-10.7 24-24V209.4l72.4 75.5c9.3 9.7 24.8 9.9 34.3.4l10.9-11c9.4-9.4 9.4-24.6 0-33.9L273 107.7c-9.4-9.4-24.6-9.4-33.9 0L106.3 240.4c-9.4 9.4-9.4 24.6 0 33.9l10.9 11c9.6 9.5 25.1 9.3 34.4-.4z"></path></svg>
 
         <svg class="icon" aria-hidden="true" data-prefix="fas" data-icon="arrow-circle-up" class="svg-inline--fa fa-arrow-circle-up fa-w-16" role="img" xmlns="http://www.w3.org/2000/svg" viewBox="0 0 512 512"><path fill="currentColor" d="M8 256C8 119 119 8 256 8s248 111 248 248-111 248-248 248S8 393 8 256zm143.6 28.9l72.4-75.5V392c0 13.3 10.7 24 24 24h16c13.3 0 24-10.7 24-24V209.4l72.4 75.5c9.3 9.7 24.8 9.9 34.3.4l10.9-11c9.4-9.4 9.4-24.6 0-33.9L273 107.7c-9.4-9.4-24.6-9.4-33.9 0L106.3 240.4c-9.4 9.4-9.4 24.6 0 33.9l10.9 11c9.6 9.5 25.1 9.3 34.4-.4z"></path></svg>
           Figure 3: SDS-PAGE electrophoresis of bacteriocin samples. M: protein Ladder 5–245 kDa, C: negative control (only intein+CBD ,28 kDa), EA: Leucocyclicin Q+ intein+ CBD (BBa_K2599014, 34.4kDa) 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+ intein+ CBD(BBa_K2599015, 35.3kDa)
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           Figure 3: SDS-PAGE electrophoresis of bacteriocin samples. M: protein Ladder 5–245 kDa, C: negative control (only intein+CBD ,28 kDa), EA: Leucocyclicin Q + intein + CBD (BBa_K2599014, 34.4kDa) 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 + intein + CBD(BBa_K2599015, 35.3kDa)
 
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Revision as of 14:44, 17 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 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.

     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 promotor, 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, 1169b.p.)

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 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 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 3: SDS-PAGE electrophoresis of bacteriocin samples. M: protein Ladder 5–245 kDa, C: negative control (only intein+CBD ,28 kDa), EA: Leucocyclicin Q + intein + CBD (BBa_K2599014, 34.4kDa) 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 + intein + CBD(BBa_K2599015, 35.3kDa)

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