Thermoanaerobacter uzonensis strain AK85 ADH Lysate Activity Screen and Phylogenetic Data
Description
The dataset includes the raw lysate data of Thermoanaerobacter uzonensis strain AK85 (AK85) collected for a colorimetric Alcohol Dehydrogrenase activity screen with 29 different alcohol substrates. The substrates included primary and secondary alcohols with NAD+ and NADP+ respectively. Additionally, the data set includes all of the 16S rRNA sequences used to create a phylogenetic tree of AK85 in relation to the rest of the published species within the genus.
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Steps to reproduce
16S rRNA sequences were collected from NCBI with the correlated NCBI accession numbers listed in the excel file. 16S sequences were then aligned and the phylogenetic tree was created with https://ngphylogeny.fr/ using the FastMe selection. Lysate data was created by initially culturing Thermoanaerobacter uzonensis strain AK85 in BM media for 18 hours at 65°C. Cell pellets were collected and lysed by bead beating with 150-212 µm glass beads in a 1:1 ratio. 50 μL of lysate was then added a 122 μL colormetric assay mixture containing: 0.217 g NAD+ or 0.243 g NADP+, 0.135 g 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and 1.3 g gelatin in 900 mL 50 mM Tris-HCl, pH 8.5), 13.5 µL of 74 mM solution of alcohol of interest and 15 µL of phenazine methosulfate (PMS) solution (0.245 g/L PMS in 50 mM Tris-HCl, pH 8.5). The assay solution was incubated at room temperature for 10 minutes, followed by incubation at 59°C in a Bioscreen C microplate reader. Readings were collected at 590 nm every 10 minutes for a total of 100 or 110 minutes. Each alcohol substrate was analyzed in triplicate and a negative control included in the data set. A standard curve was prepared using NADH in the range of 0-50 nmol and used to calculate volumetric activity.
Institutions
- Foundation for Applied Molecular Evolution
- Haskolinn a Akureyri