Difference between revisions of "Team:Aalto-Helsinki/Future"
(sidenav working) |
(removed "and because mutations on domains change enzyme functions the most") |
||
(10 intermediate revisions by 3 users not shown) | |||
Line 52: | Line 52: | ||
<ul id="sidenav" class="nav nav-stacked"><!-- nav-pills if we want rounded corners --> | <ul id="sidenav" class="nav nav-stacked"><!-- nav-pills if we want rounded corners --> | ||
<li><a href="#" data-scroll="pathway"><h3>Propane pathway</h3></a></li> | <li><a href="#" data-scroll="pathway"><h3>Propane pathway</h3></a></li> | ||
+ | <li><a href="#" data-scroll="homolog"><h3>Searching homologs for CAR</h3></a></li> | ||
<li><a href="#" data-scroll="cyano"><h3>Propane out of sunlight, water and thin air</h3></a></li> | <li><a href="#" data-scroll="cyano"><h3>Propane out of sunlight, water and thin air</h3></a></li> | ||
<li><a href="#" data-scroll="safety"><h3>Improving safety</h3></a></li> | <li><a href="#" data-scroll="safety"><h3>Improving safety</h3></a></li> | ||
Line 72: | Line 73: | ||
<p>Even though it was not possible within our timeframe, one could try knocking out more endogenous aldehyde reductases and alcohol dehydrogenases that compete with ADO for butyraldehyde. This approach <a href="http://www.biotechnologyforbiofuels.com/content/8/1/61">has been tried</a> by Pauli Kallio and his associates with success: knocking out two endogenous aldehyde reductases Ahr and YqhD resulted in significant improvement in propane output.</p> | <p>Even though it was not possible within our timeframe, one could try knocking out more endogenous aldehyde reductases and alcohol dehydrogenases that compete with ADO for butyraldehyde. This approach <a href="http://www.biotechnologyforbiofuels.com/content/8/1/61">has been tried</a> by Pauli Kallio and his associates with success: knocking out two endogenous aldehyde reductases Ahr and YqhD resulted in significant improvement in propane output.</p> | ||
+ | |||
+ | <p>Our work focused on a pathway based on fatty acid synthesis, resulting in production of butyraldehyde, which is further turned to propane by ADO. This is not the only possible solution, as there are many other pathways that could be potentially used to generate butyraldehyde.</p> | ||
</section> | </section> | ||
+ | <section id="homolog" data-anchor="homolog"> | ||
+ | <h2> Searching homologs for CAR </h2> | ||
+ | |||
+ | <p>As <a href = https://2015.igem.org/Team:Aalto-Helsinki/Modeling_propane>our kinetic model</a> identified, CAR (Carboxylic acid reductase from <i>Mycobacterium marinum</i>) is the most significant bottleneck of the propane pathway after FadB2 and ADO. How can we solve this bottleneck? One method is to search for homologs, which have the same ancestor gene and possibly also have the same function as CAR, but a better performance on the kinetic level. We are focusing our efforts to find homologs, because enzyme analogs, <i>ie.</i> enzymes which have same functions but are evolutionarily from different species, are more difficult to identify as our methods cannot recognize different protein sequences that have the same functionality.</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 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 > | ||
+ | <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. 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 > | ||
+ | <a href="https://static.igem.org/mediawiki/2015/e/e4/Aalto-Helsinki_Bayesian.jpeg"><img src="https://static.igem.org/mediawiki/2015/e/e4/Aalto-Helsinki_Bayesian.jpeg" style="width:100%;margin-top:2%;" /></a> | ||
+ | <figcaption style="margin-bottom:2%;margin-top:2%;"><p><b>Figure 2:</b> The Bayesian MCMC phylogenetic tree. The node labels are posterior values, which describe how accurate a branch point is. Because almost all values are over 0.9, we can believe its accuracy, depending on initial values. Used Blosum62 as substitution model. ESS (effective sample size) was 6404.1033 for posterior. Click on the image to enlarge it.</p></figcaption> | ||
+ | </figure> | ||
+ | |||
+ | <p>Our phylogenetic trees are shown in Figure 1 and Figure 2. They are very similar to each other, even though they were calculated with different algorithms. One major difference is that the oxidoreductase from <i>Sciscionella marina</i> is considered to be evolutionarily closer to our CAR than the enzyme from <i>Rhodococcus wratislaviensis</i> in the Bayesian MCMC tree, but the enzymes closer to CAR are grouped alike in the both trees. In both figures the branch length to the thioester reductase-like protein of <i>Cryptosporangium arvum</i> is quite big. Therefore, we should consider the enzymes, which are closer to our CAR from the enzyme of <i>Cryptosporangium arvum</i>, as potential homologs which can replace CAR in the propane pathway as they have higher chance of having the same function. Little kinetic research has been done on the enzymes we found: BRENDA, an enzyme database, only had kinetic values for a CAR homolog from <i>Nocardia iowensis</i>, so we really cannot tell if their performance is better or worse. As CAR is one of the rate-limiting steps in the pathway, it would seem reasonable to screen its homologs for more efficient alternatives. If a homolog with better kinetic values than CAR is found, it can be used to improve the propane yield of our pathway.</p> | ||
+ | |||
+ | |||
+ | </section> | ||
<section id="cyano" data-anchor="cyano"> | <section id="cyano" data-anchor="cyano"> |
Latest revision as of 21:03, 18 September 2015