Difference between revisions of "Team:Aalto-Helsinki/Parts"
(actually GFP was fused with amph template protein's PCR product, which is different from PCR product which was sent as a brick (we never used brick in the lab work), so changed brick to protein. (Clarification: only brick PCR product was sequenced)) |
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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> | <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> | ||
− | <p><b>Validation:</b> Our GFP brick has been fully sequenced, and the sequencing results were as expected. See figure 5. for the sequencing results. We have also been able to express the GFP after fusing it with an amphiphilic | + | <p><b>Validation:</b> Our GFP brick has been fully sequenced, and the sequencing results were as expected. See figure 5. for the sequencing results. We have also been able to express the GFP after fusing it with an amphiphilic protein. This construct functioned under <a href="http://parts.igem.org/Part:BBa_K608003" target="_blank">BBa_K608003</a>, a strong constitutive promoter and a medium RBS. <a href="https://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 fluorescence 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 figure 7. for microscopic pictures.</p> |
<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="http://parts.igem.org/Part:BBa_K1655001">BBa_K1655001</a></b>.</p> | <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="http://parts.igem.org/Part:BBa_K1655001">BBa_K1655001</a></b>.</p> | ||
<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> | <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>We submitted a sole amphiphilic brick, containing only the coding region of the ampihphilic protein. This brick can be used to produce intracellular micelles or vesicles under any chosen expression system. Our amphiphilic protein is constructed so that the hydrophilic part is translated first. According to <a href="http://www.nature.com/nmat/journal/v14/n1/abs/nmat4118.html">Huber <i>et al.</i></a> this allows for the formation on of intracellular micelles and vesicles. If the hydrophobic domain was to be translated first, the amphiphils would only form micelles.<p> | <p>We submitted a sole amphiphilic brick, containing only the coding region of the ampihphilic protein. This brick can be used to produce intracellular micelles or vesicles under any chosen expression system. Our amphiphilic protein is constructed so that the hydrophilic part is translated first. According to <a href="http://www.nature.com/nmat/journal/v14/n1/abs/nmat4118.html">Huber <i>et al.</i></a> this allows for the formation on of intracellular micelles and vesicles. If the hydrophobic domain was to be translated first, the amphiphils would only form micelles.<p> | ||
− | <p><b>Validation:</b> Our amphiphilic brick's sequencing results were unclear, as the brick is mainly built of short repeats. We have however been able to show that the size on this insert in the pSB1C3 backbone is correct. See figure 10 for a gel electrophoresis image. The DNA from the colony which produced the product seen in well 2 was sent to the registry. We were also able to show, that once | + | <p><b>Validation:</b> Our amphiphilic brick's sequencing results were unclear, as the brick is mainly built of short repeats. We have however been able to show that the size on this insert in the pSB1C3 backbone is correct. See figure 10 for a gel electrophoresis image. The DNA from the colony which produced the product seen in well 2 was sent to the registry. We were also able to show, that once amphiphilic protein is fused to a GFP, the GFP is expressed and again, the construct size is correct. See figure 11 where the last two wells before the ladder show a correct plasmid size. Although the sequencing results are unclear, the correct sized mRNA seemed to be produced. This lead us to believe, that the Amphiphilic protein was indeed transcribed and possibly translated as well. We thought that we weren't able to detect the micelles and vesicles because of low accuracy of the microscopic images or because our expression level was too high. Our promoter was 10 times more efficient than the one that has been previously used to produce micelles and vesicles with this amphiphilic protein. The high concentration of amphiphils is likely to interfere with the micelle and vesicle formation.<br/><br/></p> |
<p><span style="color:red"><b>Note!</b></span> The submitted BioBrick does most likely not contain the correct sequence. Restriction analysis lead us to initially believe we had the correct insert, as the insert size in pSB1C3 backbone matched that of the correct sequence (figure 10). However, as we proceeded to the sequencing results of the then already submitted brick a day before the wiki freeze, we found out that the sequence was not that of the amphiphilic protein, but instead that of the <a href="http://parts.igem.org/Part:BBa_K592009">blue chromoprotein</a>.</p> | <p><span style="color:red"><b>Note!</b></span> The submitted BioBrick does most likely not contain the correct sequence. Restriction analysis lead us to initially believe we had the correct insert, as the insert size in pSB1C3 backbone matched that of the correct sequence (figure 10). However, as we proceeded to the sequencing results of the then already submitted brick a day before the wiki freeze, we found out that the sequence was not that of the amphiphilic protein, but instead that of the <a href="http://parts.igem.org/Part:BBa_K592009">blue chromoprotein</a>.</p> |
Latest revision as of 04:59, 29 October 2015