Difference between revisions of "Team:Aalto-Helsinki/Practice project"
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<h2> Introduction </h2> | <h2> Introduction </h2> | ||
| − | <p>In synthetic biology it is | + | <p>In synthetic biology it is important to produce foreign compounds in a host. One aspect of this is constructing a synthetic genome and choosing the best compatible parts to have enough yield of a desired product. The most product is formed when host cells are big and physically stable.</p> |
<p>According to Ceroni <i>et al.</i> <a href="http://www.nature.com/nmeth/journal/v12/n5/full/nmeth.3339.html">[1]</a>, strong ribosomal binding sites (RBSs) affect cell growth and eventually lower the yield of a wanted protein compared to weaker RBSs. The reason behind this is that the strong RBSs reduce significantly the translation of endogenous mRNAs which are needed for cell growth. A ribosomal binding site is a location in a mRNA which a ribosome recognizes and binds to, thus initiating translation. The RBSs are defined by the efficiency with which they bind to ribosomes. Thus the strong RBSs bind more efficiently than the medium or the weak RBSs.</p> | <p>According to Ceroni <i>et al.</i> <a href="http://www.nature.com/nmeth/journal/v12/n5/full/nmeth.3339.html">[1]</a>, strong ribosomal binding sites (RBSs) affect cell growth and eventually lower the yield of a wanted protein compared to weaker RBSs. The reason behind this is that the strong RBSs reduce significantly the translation of endogenous mRNAs which are needed for cell growth. A ribosomal binding site is a location in a mRNA which a ribosome recognizes and binds to, thus initiating translation. The RBSs are defined by the efficiency with which they bind to ribosomes. Thus the strong RBSs bind more efficiently than the medium or the weak RBSs.</p> | ||
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<p>The constructs had a T7 promoter, so we had to use a BL21 (DE3) strain to induce transcription with IPTG. Then we measured the absorption of the blue chromoprotein via spectrophotometer with 588 nm. The results of the measurement would have specified what kind of RBS is the best one as the spectrophotometer would tell us which RBS resulted in the best production yield of the blue chromoprotein.</p> | <p>The constructs had a T7 promoter, so we had to use a BL21 (DE3) strain to induce transcription with IPTG. Then we measured the absorption of the blue chromoprotein via spectrophotometer with 588 nm. The results of the measurement would have specified what kind of RBS is the best one as the spectrophotometer would tell us which RBS resulted in the best production yield of the blue chromoprotein.</p> | ||
| − | <p>The detailed descriptions of our procedures and methods can be found | + | <p>The detailed descriptions of our procedures and methods can be found on <a href="https://2015.igem.org/Team:Aalto-Helsinki/Experiments">the Protocols page.</a></p> |
</section> | </section> | ||
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| − | <p>Our spectrophotometer results indicated that blue chromoprotein was not translated at all. After a while, we inspected our protocols and procedures thoroughly and finally had an answer to why it did not work: our 3A assembling was flawed, as shown in Figure 1. The main problem is that in the assembled plasmids the blue chromoprotein gene is before the T7 promoter and RBS | + | <p>Our spectrophotometer results indicated that blue chromoprotein was not translated at all. After a while, we inspected our protocols and procedures thoroughly and finally had an answer to why it did not work: our 3A assembling was flawed, as shown in Figure 1. The main problem is that in the assembled plasmids possible the blue chromoprotein gene is before the T7 promoter and RBS so the blue chromoprotein gene wont be transcripted. </p> |
<figure style="margin-bottom:2%;"> | <figure style="margin-bottom:2%;"> | ||
| − | <img src="https://static.igem.org/mediawiki/2015/b/b1/Aalto-Helsinki_RBS3A.png" style="max-width: | + | <img src="https://static.igem.org/mediawiki/2015/b/b1/Aalto-Helsinki_RBS3A.png" style="max-width:85%;" /> |
| − | <figcaption><b>Figure 1:</b> 3A | + | <figcaption><b>Figure 1:</b> BioBrick 3A Assembly. The left side shows how we could have got working plasmids following correctly the method. The right side shows the best case of a scenario of our attempt of the 3A assembly. For example, it is more propable that only terminator ligate with the KAN backbone without blue chromoprotein inserts. </figcaption> |
</figure> | </figure> | ||
<h3> What we have learned </h3> | <h3> What we have learned </h3> | ||
| − | <p style="margin-bottom:0; padding-bottom:10%;">In the end, we did not create constructs that we were expecting, but we have learned so much about lab procedures, for example different ligation methods, and the importance of thorough planning. If we had | + | <p style="margin-bottom:0; padding-bottom:10%;">In the end, we did not create constructs that we were expecting, but we have learned so much about lab procedures, for example different ligation methods, and the importance of thorough planning. If we had checked throughly our restriction plans with instructors, we believe that we would have got different results and also saved time. At least, we understand now what went wrong. To make sure that the same thing does not happen with the propane production and the creation of micelles, we asked for help or a confirmation even for the smallest details. |
</p> | </p> | ||
Revision as of 21:49, 15 September 2015
