Computational protocol: Biodegradation of 17β-estradiol by Bacterial Co-culture Isolated from Manure

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[…] Cells of strain LM1 or LY1 were suspended in sterilized water and boiled at 100 °C for 5 min. One milliliter of the above lysate as a template was used to amplify the 16 S rDNA gene sequences of strain LM1 or LY1 via polymerase chain reaction (PCR). The universal eubacterial primers Pf (5′-agagtttgatcctggctcag-3′) and Pf (5′-acggctaccttgttacgact-3′), which represent bp 8–27 and 1495–1514 of the Escherichia coli 16 S rDNA (accession no. E05133), were used as forward and reverse primers, respectively. The PCR mixtures (50 μl) contained 5 μl of 10 × Ex Taq Buffer (containing Mg2+), 1 μl of Ex Taq enzyme (TaKaRa), 5 μl of dNTP mixture, 5 μl of each primer, and 5 μl of DNA template, adjusted to 50 μl with sterile ddH2O. PCR amplification was performed using a Gene Amp PCR system 9600 thermocycler (Perkin-Elmer, Norwalk, CT, USA) programmed as follows: 5 min of denaturation at 94 °C; 30 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 90 s; final extension at 72 °C for 10 min. The amplified PCR products were excised from 1.2% (w/v) agarose gel and ligated into the pGEM-T easy vector (Promega, Madison, WI, USA) according to the manufacturer’s instructions. The 16 S rDNA sequence was determined by Jilin Kumei Biotech Company (China). A BLAST similarity search of the nucleotide sequence retrieved sequences from species within the genus Acinetobacter and Pseudomonas. A multiple alignment of all the sequences included in the analysis was produced by ClustalX 2.1. The region of this alignment, in which all the sequences overlap (with intermittent gaps and uncertain bases omitted) was used to construct a phylogenetic tree using the neighbor-joining method in MEGA 6.0.In addition, morphological appearance, Gram-staining results, and other physiological and biochemical characteristics were used to identify the isolated bacteria. The two strains were deposited in the China General Microbiological Culture Collection Center in May 18, 2015. The CGMCC numbers of strains LM1 and LY1 are 10814 and 10815, respectively. […]

Pipeline specifications

Software tools Clustal W, MEGA
Application Phylogenetics
Organisms Bacteria, Escherichia coli
Chemicals Estrogens, Glucose, Sodium Acetate