The document summarizes Keith Sanders' research project analyzing genes capable of degrading the solvent 1,4-dioxane. Through literature reviews and database searches, Sanders identified the Monooxygenase MmoB/DmpM gene in Pseudonocardia Dioxanivorans as promising for degradation. Sequence analysis of this gene and related ones in other organisms supported Pseudonocardia sp. K1, Pseudonocardia sp. ENV478, and Rhodococcus sp. YYL as likely capable of true 1,4-dioxane degradation. While other genes may also degrade it, MmoB/DmpM appears most optimal.

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

12. Results: Monooxygenase MmoB/DmpM-DNA

13. Results: Monooxygenase MmoB/DmpM-DNA

14. Results: Monooxygenase MmoB/DmpM-DNA

15. Results: Monooxygenase MmoB/DmpM-DNA

16. Results: Monooxygenase MmoB/DmpM-Protein

17. Results: Monooxygenase MmoB/DmpM-Protein

18. Results: Monooxygenase MmoB/DmpM-Protein

19. Results: Monooxygenase MmoB/DmpM-Protein

20. Results Visuals: Gene Cluster

21. Results: Gene Cluster

22. Results: Gene Cluster

23. Results Visuals: Propane Monooxygenase

24. Results: Propane Monooxygenase-DNA

25. Results: Propane Monooxygenase-DNA

26. Results: Propane Monooxygenase-DNA

27. Results: Propane Monooxygenase-DNA

28. Results: Propane Monooxygenase-Protein

29. Results: Propane Monooxygenase-Protein

30. Results: Propane Monooxygenase-Protein

31. Results: Propane Monooxygenase-Protein

32. Results Visuals: Phenol-2 Monooxygenase

33. Results: Phenol-2 Monooxygenase-DNA

34. Results: Phenol-2 Monooxygenase-DNA

35. Results: Phenol-2 Monooxygenase-DNA

36. Results: Phenol-2 Monooxygenase-DNA

37. Results: Phenol-2 Monooxygenase-Protein

38. Results: Phenol-2 Monooxygenase-Protein

39. Results: Phenol-2 Monooxygenase-Protein

40. Results: Phenol-2 Monooxygenase-Protein

41. Results Visuals: Alcohol Dehydrogenase

42. Alcohol Dehydrogenase-Protein

43. Alcohol Dehydrogenase-Protein

44. Alcohol Dehydrogenase-Protein

45. Alcohol Dehydrogenase-Protein

46. Complete Phylogeny Tree

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.