Difference between revisions of "Team:Aalto-Helsinki/Future"
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<p>To search for CAR homologs, we created phylogenetic trees using protein sequences, because we are searching for proteins with a slightly altered structure (and therefore possibly better kinetic properties) and amino acid mutations can affect this. Conversely, nucleic acid mutations might have no effect on the amino acid sequence and therefore the protein structure, as most amino acids have multiple codons encoding them. We created two phylogenetic trees using two different methods: UPGMA (Unweighted Pair Group Method with Arithmetic Mean) and Bayesian MCMC (Markov chain Monte Carlo). UPGMA is faster but more crude than Bayesian MCMC. An UPGMA tree was done with Geneious v8.1.7 and for a Bayesian MCMC tree used <a href = http://beast.bio.ed.ac.uk/beast>BEAST v1.8.2</a>, BEAUti v1.8.2 <a href = http://mbe.oxfordjournals.org/content/29/8/1969>[1]</a>, <a href = http://beast.bio.ed.ac.uk/tracer>Tracer v1.6</a>, TreeAnnotator v1.8.2 ja FigTree v1.4.2. Needed multiple protein alignment was done with MUSCLE (with 16 iterations) in Geneious.</p> | <p>To search for CAR homologs, we created phylogenetic trees using protein sequences, because we are searching for proteins with a slightly altered structure (and therefore possibly better kinetic properties) and amino acid mutations can affect this. Conversely, nucleic acid mutations might have no effect on the amino acid sequence and therefore the protein structure, as most amino acids have multiple codons encoding them. We created two phylogenetic trees using two different methods: UPGMA (Unweighted Pair Group Method with Arithmetic Mean) and Bayesian MCMC (Markov chain Monte Carlo). UPGMA is faster but more crude than Bayesian MCMC. An UPGMA tree was done with Geneious v8.1.7 and for a Bayesian MCMC tree used <a href = http://beast.bio.ed.ac.uk/beast>BEAST v1.8.2</a>, BEAUti v1.8.2 <a href = http://mbe.oxfordjournals.org/content/29/8/1969>[1]</a>, <a href = http://beast.bio.ed.ac.uk/tracer>Tracer v1.6</a>, TreeAnnotator v1.8.2 ja FigTree v1.4.2. Needed multiple protein alignment was done with MUSCLE (with 16 iterations) in Geneious.</p> | ||
− | <p>We used diverse resources to find potential CAR homologs, which have the same function. InterPro had <a href = http://www.ebi.ac.uk/interpro/protein/B2HN69>an entry</a> for our CAR and we used the site’s “Similar protein” -link to look for similar proteins, which are likely to be homologs. The proteins | + | <p>We used diverse resources to find potential CAR homologs, which have the same function. InterPro had <a href = http://www.ebi.ac.uk/interpro/protein/B2HN69>an entry</a> for our CAR and we used the site’s “Similar protein” -link to look for similar proteins, which are likely to be homologs. The proteins which have AMP-dependent synthetase/ligase, acyl carrier protein-like and thioester reductase-like domains were chosen, because CAR has them. We searched through UniProt database using keywords, such as “short fatty acid coa ligase” and “Carboxylic acid reductase”. The proteins, which have similar GO-classes and a comparable description as CAR, were picked. Many similar proteins were found by protein BLAST (Blosum62). BLAST results with E value of 0 and over 80 % identity with our CAR sequence were chosen. From the protein sequence of CAR, Blastp recognized several related superfamilies, <i>ie.</i> protein families which are similar on the sequence level. They are adenylate forming domain Class I, phosphopantetheine attachment site and Rossmann-fold NAD(P)(+)-binding proteins, the descriptions of which match with the domain descriptions of the CAR InterPro entry.</p> |
<figure > | <figure > | ||
<a href="https://static.igem.org/mediawiki/2015/e/ee/Aalto-Helsinki_upgma.png"><img src="https://static.igem.org/mediawiki/2015/e/ee/Aalto-Helsinki_upgma.png" style="width:100%;margin-top:2%;" /></a> | <a href="https://static.igem.org/mediawiki/2015/e/ee/Aalto-Helsinki_upgma.png"><img src="https://static.igem.org/mediawiki/2015/e/ee/Aalto-Helsinki_upgma.png" style="width:100%;margin-top:2%;" /></a> | ||
− | <figcaption style="margin-bottom:2%;margin-top:2%;"><p><b>Figure 1:</b> The UPGMA phylogenetic tree and sources for the proteins. The node labels describe how evolutionarily distant the proteins are: the higher a value is the more distant they are. The search method tells how we found the proteins and the ID its respective identification code. The enzyme from the human is picked to be an outsider group for calculations. Underlined enzyme is our CAR. The used substitution model for UPGMA was Jukes-Cantor. Click on the image to enlarge it.</p></figcaption> | + | <figcaption style="margin-bottom:2%;margin-top:2%;"><p><b>Figure 1:</b> The UPGMA phylogenetic tree and sources for the proteins. The node labels describe how evolutionarily distant the proteins are: the higher a value is the more distant they are. The search method tells how we found the proteins and the ID its respective identification code. The enzyme from the human is picked to be an outsider group for calculations. The human enzyme catalyzes the opposite reaction as our CAR, oxidation of butyraldehyde to butyric acid. Underlined enzyme is our CAR. The used substitution model for UPGMA was Jukes-Cantor. Click on the image to enlarge it.</p></figcaption> |
</figure> | </figure> | ||
Latest revision as of 21:03, 18 September 2015