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| − | <p>An extra nucelotide has been added prior to this GFP brick's suffix to maintain the reading frame after fusion.</p>
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| − | <b>Fusable GFP / BBa_K1655001</b>
| + | An extra nucelotide has been added prior to this GFP brick's suffix to maintain the reading frame after fusion. |
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| − | <figure style="float:right">
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| − | <img src="https://static.igem.org/mediawiki/2015/1/1d/Aalto-Helsinki_gfp_plasmid_with_text.png" style="width:150px;"/>
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| − | <figcaption><center><b>Figure 4.</b> GFP Brick</center></figcaption>
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| − | </figure>
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| − | <p>We built a GFP biobrick which can be fused to any protein's aminoterminal end with the standard BioBrick assembly enzymes. We have added an extra nucleotide prior to the brick's suffix to maintain the reading frame after fusion, which is typically lost when the restriction enzyme assembly is used. </p>
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| − | <p><b>Validation:</b> Our GFP brick has been fully sequenced, and the sequencing results were as expected. See <span style="color:red">figure 5. </span>for the sequencing results. We have also been able to express the GFP after fusing it with an amphiphilic brick. This construct functioned under <a href="https://parts.igem.org/Part:BBa_K608003">BBa_K608003</a>, a strong constitutive promoter and a medium RBS. <a href="http://2015.igem.org/Team:Slovenia_HS">HS Slovenia Team</a> also helped us validate this brick. They gained positive results of their construct with the GFP through colony PCR and analytical restrictions, but were unable to detect the fluerescence under UV light or functionality of the fused protein. Figure 6 shows a positive result of colony PCR. The GFP has indeed been fused with another protein, CtfB, with the biobrick enzyme assembly. We were however able to show that the GFP is functional after fusion. See <span style="color:red">figure 7.</span> for microscopic pictures.</p>
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| − | <p> We restricted the GFP brick with XbaI & PstI to show that the insert in the brick was of the correct size. DNA from the colony which produced the band seen in Figure 8 in well 4 was sent to the registry under the name <b><a href="https://parts.igem.org/Part:BBa_K1655001">BBa_K1655001</a></b>.</p>
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| − | <p>Click <a href="https://static.igem.org/mediawiki/2015/5/51/Aalto-Helsinki_gfp_sequence_ah009.gb">here</a> to download the full sequence of our Fusable GFP in pSB1C3 backbone.</p>
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| − | <p>Click the images to enlarge them</p>
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| − | <figure style="float:left">
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| − | <a href="https://static.igem.org/mediawiki/2015/b/b3/Aalto-Helsinki_gfp_insert_validation_gel.png"><img src="https://static.igem.org/mediawiki/2015/b/b3/Aalto-Helsinki_gfp_insert_validation_gel.png" style="width:100px;"/></a>
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| − | <figcaption><b><center><span style="color:red">Figure 5.</b> sequencing.</span></center></figcaption>
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| − | </figure>
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| − | <figure style="float:left">
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| − | <a href="https://static.igem.org/mediawiki/2015/d/dc/Aalto-Helsinki_submittedparts_gfp_colonypcr_validation.png"><img src="https://static.igem.org/mediawiki/2015/d/dc/Aalto-Helsinki_submittedparts_gfp_colonypcr_validation.png" style="width:100px;"/></a>
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| − | <figcaption><b><center>Figure 6.</b> Slovenia's colony PCR showing that GFP is fused with CtfB</center></figcaption>
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| − | </figure>
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| − | <figure style="float:left">
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| − | <a href="https://static.igem.org/mediawiki/2015/d/dc/Aalto-Helsinki_submittedparts_gfp_colonypcr_validation.png"><img src="https://static.igem.org/mediawiki/2015/d/dc/Aalto-Helsinki_submittedparts_gfp_colonypcr_validation.png" style="width:100px;"/></a>
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| − | <figcaption><b><center><span style="color:red">Figure 7.</b> Microscope pics</span></center></figcaption>
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| − | </figure>
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| − | <figure style="float:left">
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| − | <a href="https://static.igem.org/mediawiki/2015/b/b3/Aalto-Helsinki_gfp_insert_validation_gel.png"><img src="https://static.igem.org/mediawiki/2015/b/b3/Aalto-Helsinki_gfp_insert_validation_gel.png" style="width:102px;"/></a>
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| − | <figcaption><b><center>Figure 8.</b> GFP brick restricted to show the right-sized insert.</center></figcaption>
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| − | </figure>
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| | <!-- Add more about the biology of this part here | | <!-- Add more about the biology of this part here |
An extra nucelotide has been added prior to this GFP brick's suffix to maintain the reading frame after fusion.
