Expression profiling by high throughput sequencing
Summary
Paenarthrobacter nicotinovorans pAO1 is a nicotine degrading microorganism that shows promising applications in converting nicotine-containing waste into useful green chemicals. Its biotechnological applications are nevertheless hampered by the lack of knowledge and tools to perform genetic and metabolic engineering. The objective of the work is to provide the first transcriptome of the strain and is a second step in our envisioned complete omics characterization of nicotine metabolism in P. nicotinovorans ATCC 49919. Acknowledgements. This work was supported by a grant of the Romanian Ministry of Education and Research, CNCS - UEFISCDI, project number PN-III-P4-ID-PCE-2020-0656, within PNCDI III.
Overall design
The strain was grown on citrate medium with and without nicotine. Cells were harvested at three different timepoints correlated with cellular growth and nicotine catabolism. The first timepoint (T1) was set at ~5 hours after inoculation (and nicotine-supplementation), representing the start of the exponential phase (and of nicotine catabolism). The second timepoint (T2), at ~10 HAI, captures the transition from the exponential to stationary phase: the on-going degradation of nicotine and its depletion from the growth medium. The third timepoint (T3), at 24 HAI, represents the stationary phase, when nicotine has been depleted from the growth medium. Total RNA was isolated, poly-adenylated, and directly sequenced using the Oxford Nanopore Technology (ONT) MinION Mk1B device coupled to a Flongle adapter. Basecalling was performed using Guppy_6.3.2 and high accuracy. Basecalled data was analysed using the nf-core/nanoseq pipeline for quality assessment, mapping to the reference genome, transcriptome assembly, transcript quantification and differential gene expression.
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