Team:Calgary/Notebook/Calendar/Binf
From 2011.igem.org
![](https://static.igem.org/mediawiki/2011/c/c1/UCalgary_CircleHome.png)
![](https://static.igem.org/mediawiki/2011/d/d4/UCalgary_CircleTeam.png)
![](https://static.igem.org/mediawiki/2011/7/7d/UCalgary_CircleProject.png)
![](https://static.igem.org/mediawiki/2011/8/8d/UCalgary_CircleParts.png)
![](https://static.igem.org/mediawiki/2011/0/03/UCalgary_CircleNotebook.png)
![](https://static.igem.org/mediawiki/2011/8/84/UCalgary_CircleOutreach.png)
![](https://static.igem.org/mediawiki/2011/3/3c/UCalgary_CircleSponsors.png)
![](https://static.igem.org/mediawiki/2011/8/85/UCalgary_CircleiGEM.png)
![](https://static.igem.org/mediawiki/2011/4/42/UCalgary_TitleBar.png)
Bioinformatic Survey
![](https://static.igem.org/mediawiki/2011/e/ed/UCalgary_BodyBoxTop.png)
Project Participants
Stephen Dixon, Patrick Wu
Author: Stephen
Rationale
Recently, David Lloyd, a TA, came across an interesting study which analyzed the capacity of bacteria to degrade naphthenic acids. According to the study, both Pseudomonas putida and Pseudomonas fluorescens are capable of degrading small amounts of naphthenic acids, but when put in co-cultures with each other, their capacity increases to 95% elimination. What's more is that this degradation was effective across a broad spectrum of naphthenic acids, including those with one, two, and even three rings. The inference we drew from this effect is that there are some unique genes within each bacteria that, when allowed to interact with each other, are responsible for the degradation of naphthenic acids. In a sense, one bacteria's garbage is another bacteria's treasure. This project's hypothesis is that it is possible to narrow down the candidates for this pathway by using a bioinformatic survey.
Author: Stephen
Original Strategy for the Bioinformatic Survey
The goal of the bioinformatic survey is to provide leads to the experimental side (or wet lab) of the project. Two assumptions were made at the beginning of the survey. The first is that the two genomes are homologous enough to eliminate a substantial portion of each genome from consideration. The other assumption is that the gene of interest is located within the non-homologous regions of the either genome. If both assumptions are correct, then it should be possible to create a short list of candidate genes involved in the degradation pathway, for investigation in the wet lab. Knowing what genes are involved in the degradation means that wet lab can simply look upstream for a naphthenic acid promoter.
Author: Stephen
June 27- July 1, 2011
My initial thinking is that both genomes consist of two parts: parts that have a homology to the other genome, and parts that don't. Eliminating the parts that do by definition would immediately reveal the parts that aren't. Since homology tends to be between similar sequences rather than exact matches, a statistical approach could be used that determines homology based on the significance of the finding.
Patrick Wu, my colleague, started off by looking for software that we could reuse for our application. DNA manipulation is sufficicently complicated enough that it makes no sense to reinvent the wheel; after some digging around, he found an open-source DNA alignment tool called MUMmer. MUMmer uses a suffix tree (a type of data structure) that works in O(n) time to rapidly align whole genomes; in the process, MUMmer provides information on single nucleotide changes, translocations, and homologous/similar genes.
Currently, we are looking into how MUMmer can be used to compare the homology of the genomes. If it turns out that the homology is not significant enough, then some other criteria must be used to narrow down the list of unique genes.
Author: Stephen
July 4-8, 2011
This week, Patrick went back to working on the Wiki, and I started to develop software for performing the big computation. My hope was to develop an original application based on several modules, which would process the entire genome. Module 1 would read and transcribe each of the genes in the entire genome. Module 2 would "compare the genes" and eliminate similar genes, and Module 3 would get the identity of each gene and sort them in some relevant order. However, my program for discarding junk DNA in Module 1 was not working as well as I had hoped; it identified over 150 genes in Mumps Virus, when there is only 7 according to BLAST. In the process of developing this software, I learned more about evolution and homology, horizontal gene transfer, the nitty-gritty details of transcription, and about codon tables.
Author: Stephen
July 11-15, 2011
This week, I realized that any organism with a sufficient number of genes that were horizontally transferred would, by definition, have genes that are non-homologous to other species within the same genus. Therefore, one way to find the naphthenic acid promoter is to look for genes which are horizontally transferred; horizontal transfer is a process by which bacteria share genetic information with dissimilar species.
In the process of looking up how to identify such a gene, I encountered a wonderful program called Dark Horse, which uses an algorithm for finding phylogenetically atypical proteins among bacterial strains on a genome-wide basis - that is, proteins which are likely to have been horizontally transferred. So I looked up all strains of Pseudomonas putida (F1, GB1, KT2440, and W619) and fluorescens (Pf5 and Pf01) on the database, and instantly obtained a list of 200 such genes. Unfortunately, we know not what the exact sequence of the strains used in the Del Rio paper were, but we are hopeful that there is enough similarity to the species in the database that they will share the horizontal genes. We are now cataloguing the bacterial species in this list, in order to create a comprehensive list of genes to test in the lab. What is really interesting about this is that, assuming that Del Rio's bacteria are similar to the ones in the database, we may already be sitting on the answer.
Author: Stephen
July 16-22, 2011
David, our TA, went through the catalogue of species and added additional notes to some species. Patrick and Stephen spent most of the week preparing for the aGEM Conference.
Author: Stephen
July 23-29, 2011
This week we finally finished cataloguing the interesting results from the Dark Horse results. We also discovered ProteinWorld HB, an online web tool which searches for unique genes between two species. However, we found no genes were listed in both lists. On Friday, we discovered that Pseudomonas.com also offers a comparative genome search, and a list of annotated genes in each genome. We look forward to comparing the list of annotated genes to the Dark Horse and Protein World results.
We looked also at GenomeBlast, which claims to find unique genes between species; however, we were not successful in obtaining the program's output. The authors of GenomeBlast suggested by email that the problem may have arisen due to the size of the genomes we were trying to process.
Also, we emailed Dr Mayi about how our results can be verified in the wet lab.
Author: Stephen
July 29-August 4, 2011
This week was short, because Monday was a civic holiday.
Since Dr Mayi said that we could experimentally verify some of our results, the main focus this week has been on selecting around 5 or 6 genes that are responsive to naphthenic acids. We want genes which turn on as a response to NAs, and genes that are inactive when there are no NAs present. We continue to think about that the identity of the strains of LD1 (Pseudomonas putida) and LD2 (Pseudomonas fluorescens) remains unknown.
We also discovered that pseudomonas.com has a pre-computed gene comparison search, which allows you to look for genes in one group of species/strains which are not present in another list. We have the results from this list as well, but we are using it as a reference for what genes are actually present.
On Wednesday and Thursday, we BLASTed each of the interesting genes we found on our Dark Horse and ProteinWorld results, to learn more about them. We eliminated or "nominated" genes based on their supposed relevance to fatty acid degradation pathways, and how relevant they could be to naphthenic acid degradation. We also noted when a particular genes appeared to have conserved domains, but looked substantially different than other genes with a similar function. Using these standards, we short-listed our gene list to about six genes. Our final list includes a handful of transcription factors (such as GI 58003974), Enoyl-CoA hydratase, and LysR family proteins. Next week, we intend to experimentally verify whether the gene products of these sequences are affected by naphthenic acid presence.
Author: Patrick
July 29-August 4, 2011
This week I looked into results from the Dark Horse and ProteinWorldDB results in an attempt to find proteins of particular interest. A handful of transcription factors appear to be of interest. P. putida GB-1 offers a hypothetical protein that most closely aligns with a transcription factor (GI 58003974). As well, lysR family proteins appear to be promising because of their transcription regulation properties. Enoyl-CoA hydratases seem to be part of fatty acid degradation pathways, so it may be a good protein to pursue as well.
Author: Stephen
August 5-12, 2011
Having selected 5 genes for verification, we went about preparing ourselves for the verification step. Our plan is to perform a quantative RT-PCR on two samples of bacteria, one treated by naphthenic acid and one that is not. We plan to find out whether the selected genes are up-regulated in response to naphthenic acids. On Monday, I read about RT-PCR and found out how it worked. On Tuesday, I slept the entire day, which helped me recover from oral surgery. On Wednesday,
August 12-18
Author: Patrick
This week I was primarily working on primer design for the RT-PCR, and learning about the process. I also gathered names for the formation of an iGEM club for the upcoming Fall and Winter semesters and submitted the forms to the Students' Union. I have also been writing out thank-you letters to our sponsors and donators and filling out the SEEL application form for funding in the future.
![](https://static.igem.org/mediawiki/2011/c/cb/UCalgary_BodyBoxBottom.png)