Computational protocol: Biodegradation of 3-methyldiphenylether (MDE) by Hydrogenophaga atypical strain QY7-2 and cloning of the methy-oxidation gene mdeABCD

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Protocol publication

[…] The isolation and manipulation of the recombinant DNA were performed as described by Sambrook and Russel. The restriction enzymes, the PrimeSTAR HS DNA polymerase, and the TA cloning vector used in this work were commercial preparations and were used as specified by the supplier, TaKaRa Biotechnology Co., Ltd. (Dalian, China). The standard PCR mixture contained 10 ng of template DNA, 0.2 μM of each primer, 200 μM of each deoxynucleoside triphosphate (dNTP), 10 μL of 5 X PrimeSTAR buffer (Mg2+ Plus), and 1.25 U of PrimeSTAR HS DNA polymerase in a total volume of 50 μl. The standard PCR conditions were as follows: 98 °C for 30 s; 30 cycles of 98 °C for 10 s, 58 °C for 10 s, and 72 °C for 1 min kb−1; and 72 °C for 5 min. Oligonucleotide synthesis and DNA sequencing were performed by Invitrogen Technology Co., Ltd. (Shanghai, China).BLASTN and BLASTP were used for the nucleotide sequence and deduced amino acid identity searches, respectively. For phylogenetic analysis, Clustal W was used to align all of the protein sequences. The multiple sequence alignment was then imported into MEGA version 5.0 software. A phylogenetic tree was then constructed using the neighbour-joining method. Distances were calculated using the Kimura two-parameter distance model, and a bootstrap consensus tree, inferred from 1,000 replicates, was taken to represent the evolutionary history of the sequences analysed. Predictions of the open reading frames (ORFs) were performed with ORF Finder (NCBI) and GeneMark. […]

Pipeline specifications

Software tools BLASTN, BLASTP, Clustal W, MEGA, Open Reading Frame Finder, GeneMark
Applications Genome annotation, Phylogenetics
Organisms Escherichia coli BL21(DE3)
Chemicals Carbon