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| </header> | | </header> |
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− | <h2>1. The expression of α-tubulin、β-tubulin、n-luciferase、c-luciferase</h2> | + | <h2>The expression of α-tubulin、β-tubulin、n-luciferase、c-luciferase</h2> |
− | <h4>1.1 vector construction</h4> | + | <h3>vector construction</h3> |
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| <figure class="text-center"> | | <figure class="text-center"> |
| <img src="https://static.igem.org/mediawiki/2016/3/3b/T--BNU-China--Results9.jpg" width="65%"> | | <img src="https://static.igem.org/mediawiki/2016/3/3b/T--BNU-China--Results9.jpg" width="65%"> |
| <figcaption> | | <figcaption> |
− | Fig.1.1.1 electrophoresis result of α-tubulin, β-tubulin, n-luciferase, c-luciferase gene fragments | + | Fig.1 electrophoresis result of α-tubulin, β-tubulin, n-luciferase, c-luciferase gene fragments |
| </figcaption> | | </figcaption> |
| </figure> | | </figure> |
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| <img src="https://static.igem.org/mediawiki/2016/c/c0/T--BNU-China--Results5.jpg" width="88%"> | | <img src="https://static.igem.org/mediawiki/2016/c/c0/T--BNU-China--Results5.jpg" width="88%"> |
| <figcaption> | | <figcaption> |
− | Fig.1.1.2 electrophoresis result of α-tubulin、β-tubulin、n-luciferase、c-luciferase expression vectors | + | Fig.2 electrophoresis result of α-tubulin、β-tubulin、n-luciferase、c-luciferase expression vectors |
| <br /> | | <br /> |
| <small style="font-size:66%">(A. electrophoresis result of colony PCR. The arrows show the correct size of target fragments B. We rebuilt and transformed the β-tubulin vector into Trans5α, then picked the single colony and extracted the plasmids. We also used PCR to check out the plasmids using primers of β-tubulin, and the band directed by arrows is correct. </small> | | <small style="font-size:66%">(A. electrophoresis result of colony PCR. The arrows show the correct size of target fragments B. We rebuilt and transformed the β-tubulin vector into Trans5α, then picked the single colony and extracted the plasmids. We also used PCR to check out the plasmids using primers of β-tubulin, and the band directed by arrows is correct. </small> |
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| <img src="https://static.igem.org/mediawiki/2016/7/7d/T--BNU-China--Results13.jpg" width="65%"> | | <img src="https://static.igem.org/mediawiki/2016/7/7d/T--BNU-China--Results13.jpg" width="65%"> |
| <figcaption> | | <figcaption> |
− | Fig.1.1.3 SDS-PAGE result of β-tubulin | + | Fig.3 SDS-PAGE result of β-tubulin |
| <br /> | | <br /> |
| <small style="font-size:66%">(left to right: non-induced group, control, induced group. arrow shows the correct molecular weight of target protein)</small> | | <small style="font-size:66%">(left to right: non-induced group, control, induced group. arrow shows the correct molecular weight of target protein)</small> |
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| <p>这里放发福的结果</p> | | <p>这里放发福的结果</p> |
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− | <h2>2. The expression of fusion protein | + | <h2>The expression of fusion protein |
− | <h4>2.1 PCR fusion PCR</h4> | + | <h3>PCR fusion PCR</h3> |
| <p>We constructed α-tubulin-YNE、YNE-α-tubulin、α-tubulin-YCE、YCE-α-tubulin、β-tubulin-YNE、YNE-β-tubulin、β-tubulin-YCE、YCE-β-tubulin、α-tubulin-nluc、nluc-α-tubulin、α-tubulin-cluc、cluc-α-tubulin respectively via fusion PCR. After ligating these fusion gene fragments to TransB(DE3), we transformed target plasmids to Trans5α. When colony PCR was done, we picked correct colony for plasmid amplification.</p> | | <p>We constructed α-tubulin-YNE、YNE-α-tubulin、α-tubulin-YCE、YCE-α-tubulin、β-tubulin-YNE、YNE-β-tubulin、β-tubulin-YCE、YCE-β-tubulin、α-tubulin-nluc、nluc-α-tubulin、α-tubulin-cluc、cluc-α-tubulin respectively via fusion PCR. After ligating these fusion gene fragments to TransB(DE3), we transformed target plasmids to Trans5α. When colony PCR was done, we picked correct colony for plasmid amplification.</p> |
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| <img src="https://static.igem.org/mediawiki/2016/b/b9/T--BNU-China--Results6.jpg" width="75%"> | | <img src="https://static.igem.org/mediawiki/2016/b/b9/T--BNU-China--Results6.jpg" width="75%"> |
| <figcaption> | | <figcaption> |
− | figure 2.1.1 Result of colony PCR | + | Fig.4 Result of colony PCR |
| <br /> | | <br /> |
| <small style="font-size:66%">(arrows show the correct size of fusion gene fragments:α-tubulin-YNE is 1866 bp、α-tubulin-YCE is 1650bp、β-tubulin-YCE is 1629bp、α-tubulin-nluc is 2640bp、α-tubulin-cluc is1857bp)</small> | | <small style="font-size:66%">(arrows show the correct size of fusion gene fragments:α-tubulin-YNE is 1866 bp、α-tubulin-YCE is 1650bp、β-tubulin-YCE is 1629bp、α-tubulin-nluc is 2640bp、α-tubulin-cluc is1857bp)</small> |
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| <img src="https://static.igem.org/mediawiki/2016/a/a0/T--BNU-China--Results7.jpg" width="44%"> | | <img src="https://static.igem.org/mediawiki/2016/a/a0/T--BNU-China--Results7.jpg" width="44%"> |
| <figcaption> | | <figcaption> |
− | figure 2.1.2 SDS-PAGE of renatured bodies from α-tubulin-YNE、YNE-α-tubulin | + | Fig.5 SDS-PAGE of renatured bodies from α-tubulin-YNE、YNE-α-tubulin |
| <br /> | | <br /> |
| <small style="font-size:66%">(the molecular weight of target fusion protein is 74.6.arrows show the correct size)</small> | | <small style="font-size:66%">(the molecular weight of target fusion protein is 74.6.arrows show the correct size)</small> |
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| </figure> | | </figure> |
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− | <h4>2.2 Gateway</h4> | + | <h3>Gateway</h3> |
| <p>Apart from these, we also expressed our target protein through Rosetta(DE3).</p> | | <p>Apart from these, we also expressed our target protein through Rosetta(DE3).</p> |
| <p>In our experiment, we also try to construct fusion protein vectors with Gateway Large-scale Cloning technology. We used Invitrogen pENTR/D TOPO to clone β-tubulin into entry vector. Designing primer based on β-tubulin sequence and running PCR procedure. From this picture, the band of β-tubulin was correct.</p> | | <p>In our experiment, we also try to construct fusion protein vectors with Gateway Large-scale Cloning technology. We used Invitrogen pENTR/D TOPO to clone β-tubulin into entry vector. Designing primer based on β-tubulin sequence and running PCR procedure. From this picture, the band of β-tubulin was correct.</p> |
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| <img src="https://static.igem.org/mediawiki/2016/6/63/T--BNU-China--Results14.jpg" width="65%"> | | <img src="https://static.igem.org/mediawiki/2016/6/63/T--BNU-China--Results14.jpg" width="65%"> |
| <figcaption> | | <figcaption> |
− | figure 2.2.1 PCR result of entry vector | + | Fig.6 PCR result of entry vector |
| </figcaption> | | </figcaption> |
| </figure> | | </figure> |
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| <img src="https://static.igem.org/mediawiki/2016/2/2e/T--BNU-China--Results4.jpg" width="25%"> | | <img src="https://static.igem.org/mediawiki/2016/2/2e/T--BNU-China--Results4.jpg" width="25%"> |
| <figcaption> | | <figcaption> |
− | figure 2.2.2 single digestion result of entry vector | + | Fig.7 single digestion result of entry vector |
| </figcaption> | | </figcaption> |
| </figure> | | </figure> |
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| <img src="https://static.igem.org/mediawiki/2016/f/f0/T--BNU-China--Result15.jpg" width="65%"> | | <img src="https://static.igem.org/mediawiki/2016/f/f0/T--BNU-China--Result15.jpg" width="65%"> |
| <figcaption> | | <figcaption> |
− | figure 2.2.3 PCR result of target gene | + | Fig.8 PCR result of target gene |
| </figcaption> | | </figcaption> |
| </figure> | | </figure> |
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| </figure> | | </figure> |
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− | <h2>3. Microtubules extracted in vitro</h2> | + | <h2>Microtubules extracted in vitro</h2> |
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| </article> | | </article> |