Computational protocol: Isolation and Characterization of Hydrocarbon-Degrading Yeast Strains from Petroleum Contaminated Industrial Wastewater

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Protocol publication

[…] Genomic DNA of the strains HC1 and HC4 was isolated using a protocol described by Masneuf-Pomarède et al. (2007) []. The primers ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) described by White et al. (1990) [] were used to amplify the 5.8S ITS region of strain HC4. The D1/D2 domain of 26S rDNA gene of strain HC1 was amplified under the same conditions with primers NL1 (5′-GCATATCAATAAGCGGAGGAAAAG-3′) and NL4 (5′-GGTCCGTGTTTCAAGACGG-3′) []. The PCR reaction was performed on a Thermocycler GeneAmp PCR System 9700 (Applied Biosystems) in a 50 µl reaction mixture containing 5x GoTaq reaction buffer (Promega), 0.25 mM each dNTP, 0.2 µM each primer, 50 ng DNA template, and 2.5 U GoTaq DNA polymerase (Promega). The PCR program was as follows: denatured by heating for 2 min at 94°C and subjected to 30 cycles for 30 s at 94°C, 45 s at 58°C, and 1 min 45 s at 72°C, this was followed by a final elongation step for 10 min at 72°C. PCR products were purified with illustra GFX PCR DNA and Gel Band Purification kit (Amersham Biosciences, GE Healthcare) according to the manufacturer's protocol. DNA sequencing of PCR products was performed using a BigDye Terminator v3.1 Cycle Sequencing Kit on the ABI PRISM 3100-Avant Genetic Analyser (Applied Biosystems). Target sequences were analyzed using BLAST online (https://blast.ncbi.nlm.nih.gov/) []. The related sequences were collected and aligned using the MUSCLE software [, ]. A phylogenetic tree was constructed using the neighbor-joining method in MEGA version 4.1. Yeast species were identified based on the phylogenetic analysis results []. The topology of the distance tree was tested by resampling data with 1000 bootstraps to provide confidence estimates. Nucleotide sequences obtained in this work were deposited in the NCBI GenBank data library. […]

Pipeline specifications

Software tools MUSCLE, MEGA
Applications Phylogenetics, Nucleotide sequence alignment
Organisms Saccharomyces cerevisiae
Chemicals Carbon, Hydrocarbons, Hydrocarbons, Aromatic