Computational protocol: Isolation and Molecular Characterization of Novel Chlorpyrifos and 3,5,6-trichloro-2-pyridinol-degrading Bacteria from Sugarcane Farm Soils

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Protocol publication

[…] Purified PCR products were sequenced using the BigDye Terminator v3.1 kit (Applied Biosystems). About 15 ng of DNA template and 3 pmol of both forward (27f) and reverse primer (1492r) were used per 20 μL reaction mixture in separate reactions. 16S rRNA gene was amplified with the above primer set resulting in product length of ∼1400 bp. The amplified PCR products were collected, purified and sequenced. The quality of the sequence was checked using Sequencher 4.10.1 (Gene Codes Corporation) where both forward and reverse strands were edited, assembled and aligned together to generate a contig for further analyzes.All 16S rRNA partial sequences (>1200 bp) isolates were compared with those available on a BLAST search of the GenBank database. Sequences with the greatest similarity were extracted and aligned with MUSCLE (MEGA 6). Those aligned DNA sequences were then used to construct a phylogenetic tree using MEGA 6 (Molecular Evolutionary Genetics Analysis; ) by performing the maximum likelihood tree analysis with 1000 bootstrap replicates using Kimura-2-parameter model with Gamma distribution (K2 + G). All sequences have been submitted to National Centre for Biotechnology Information (NCBI) database (accession number: KY646471–KY646476). [...] To investigate the differences in the pesticide degradation between different isolates, an independent samples t-test was carried out, with the significance level (p value) set at 0.05. The degradation of the pesticides was ascribed by the first-order function (Ct = Co × e-kt). The rate constant, k, was estimated by plotting lnC over time with –k being equal to the slope of the linear regression representing this relationship; hence, k is an estimate of the average rate constant across all sampling points for each sample. The half-lives of the pesticides were obtained by function t1/2 = ln2/k. Statistical analyses were carried out with the software package IBM SPSS Statistics 17 (SPSS, Inc.). […]

Pipeline specifications

Software tools Sequencher, MUSCLE, MEGA, SPSS
Applications Miscellaneous, Phylogenetics, Nucleotide sequence alignment
Chemicals Carbon, Chlorpyrifos, Nitrogen, Organophosphates