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- 1. 1,4-Dioxane Degradation Gene Analysis Keith Sanders
- 2. Introduction • 1, 4-dioxane is a solvent used for organic and inorganic compounds. • In this project, I was tasked with finding genes capable of degrading 1, 4-dioxane and organisms which contained them. • After these genes were discovered many different analysis techniques were used to evaluate the these genes.
- 3. Purpose • Exposure to this chemical can lead to headaches, and irritation of the throat, lungs, and eyes. • 1, 4-dioxane can also be absorbed through the skin causing mild to severe skin irritation. • 1, 4-dioxane is also suspected to be a carcinogen. • Typically, exposure to this chemical is was limited to an occupational hazard, However substances of these chemicals have been found in ground and surface water.
- 4. Introduction • This project aims to find genes, and the organisms they belong to which can degrade 1, 4-dioxane. • True degradation means that the organism can use the chemical as a source of carbon. • Possible outcomes include finding either many different organisms which can degrade the compound, or finding a select group of highly related organisms with this gene.
- 5. Materials: Data Gathering Phase • During this phase of the project, data and sequences were gathered. • Databases: • NCBI Pubmed was used to gain articles for the literary review. • NCBI Databases like the gene, protein, and nucleotide were used to gather sequence data. • Programs like BLAST were used to search for multiple sequences using a particular sequence target.
- 6. Materials: Sequence Analysis • The program Clustal Omega was used create multiple sequence alignments (MSA). • From these MSA’s many different data elements could be obtained, all using Clustal Omega • The Phylips programs were used which contained multiple analysis programs. • Gene/Protein consensus logos were created using GENIO/logo • The phylogeny tree was created using TreeView
- 7. Methods: Gene Search • Using the results from my literary review I gained genes and organisms of interest. • Using BLAST, I was able to search databases for Protein(BLASTp) and DNA(BLASTn) sequences. • When using BLASTn, I set the search to exclude models and uncultured samples using the megablast algorithm. The database used to search was the nucleotide collection database. • When using BLASTp, I set the search to exclude models and uncultured samples. The algorithm set to DELTA-BLAST and was searched by the Uni/Swiss-prot database.
- 8. Methods: Multiple Sequence Alignment • The tool used to create the MSAs was Clustal Omega. • This program has the ability to use create sequences from DNA nucleotides and protein sequences. • All parameters kept at default. • The results of the MSA were saved for further examination.
- 9. Methods: Phylogeny Tree Creation • Clustal Omega created MSA and Phylips tree file which can be used in further programs. • The phylogeny tree was created using the TreeView Program. TreeView created the tree from sequences using an Average Distance % Identity.
- 10. Results • After performing the literary review, it was discovered that the Monooxygenase MmoB/DmpM found in Pseudonocardia Dioxanivorans. • My Research also pointed me to many genes to evaluate such as a Propane Monooxygenase, Phen-2 Monooxygenase, and an Alcohol Dehydrogenase
- 11. Result Visuals: Monooxygenase MmoB/DmpM
- 20. Results Visuals: Gene Cluster
- 23. Results Visuals: Propane Monooxygenase
- 32. Results Visuals: Phenol-2 Monooxygenase
- 41. Results Visuals: Alcohol Dehydrogenase
- 47. Conclusion • Thanks to the Literary review, I started this project knowing that the Monooxygenase MmoB/DmpM found in Pseudonocardia Dioxanivorans was the gene of interest. • The results showed that three organisms Pseudonocardia sp. K1, Pseudonocardia sp. ENV478, and Rhodococcus sp. YYL have genes most closely related to the gene of interest. • This supports the idea that these organisms are true 1, 4-dioxane degradation. • Furthermore, the genes associated with Rhodococcus sp. YYL are more closely related to Pseudonocardia Dioxanivorans.
- 48. Conclusion • Propane monooxygenase and Phen-2 monooxygenase have the ability to degrade 1, 4-dioxane, but only when their particular substrates are available. • This makes them less optimal than the Monooxygenase MmoB/DmpM. • Alcohol Dehydrogenase is the least related to any of the genes, which would suggest that it’s role in dioxane degradation is not as direct as the other genes located.