Overview
In this year's project, Team Michigan is experimenting with transforming Pseudomonas putida S16 to break down 1,4-dioxane. We selected P. putida for its ability to thrive in aquatic environments while maintaining a short doubling time of 1.8 hours1. Unlike other bacteria such as E. coli, the specific strain of P. putida we utilized, S16, already possesses most of the machinery necessary to degrade 1,4-dioxane efficiently. The transformation involves utilizing a plasmid containing the gene for the enzyme tetrahydrofuran monooxygenase (THFMO), a crucial enzyme for the breakdown pathway1.
To engineer the plasmid, we first had to remove the original nicotine degradation gene, NicA2, using the XbaI and EcoRI-HF restriction sites. Due to the instability of gene fragments over 1.5kb, we decided to split the THFMO gene into three fragments, which were subsequently ligated into the pJN105 plasmid backbone via Gibson Assembly. To assess the degradation efficacy of the bacteria, we employed gas chromatography with mass spectroscopy (GC-MS). To enhance the degradation efficacy, we plan to incorporate another plasmid into the host genome containing aldehyde dehydrogenase (ALDH), which co-regulates the breakdown of 1,4-dioxane with THFMO. To ensure the bacteria primarily broke down dioxane rather than other nutrients in the media, we utilized R2A, a minimal growth media.
