Gene clean turbo

Standard molecular biology techniques were carried out as previously described (Sambrook & Russell, 2001 ). Plasmid DNA was prepared with a High Pure plasmid isolation kit (Roche Applied Science). DNA fragments were purified with Gene‐Clean Turbo (Q‐BIOgene). Oligonucleotides were supplied by Sigma Co. and their sequences are listed in Supporting Information Table S1. All cloned inserts and DNA fragments were confirmed by DNA sequencing through an ABI Prism 377 automated DNA sequencer (Applied Biosystems Inc.). Transformation of E. coli cells was carried out by using the RbCl method or by electroporation (Gene Pulser; Bio‐Rad) (Sambrook & Russell, 2001 ). Plasmids were transferred from E. coli S17–1λpir (donor strain) into Azoarcus and Paraburkholderia recipient strains by biparental filter mating as described previously (López‐Barragán et al., 2004 (link)). Plasmids were transferred to Acinetobacter and Pseudomonas strains by electroporation. The protein concentration in cell extracts was determined by the method of Bradford (1976 (link)) by using bovine serum albumin as the standard.

Recombinant DNA techniques were carried out according to published methods [26 ]. Plasmid DNA was prepared with a High Pure Plasmid Isolation Kit (Roche Applied Science, Penzberg, Germany). The DNA fragments were purified with Gene Clean Turbo (Q-BIOgene, Carlsbad, CA, USA). Oligonucleotides were supplied by Sigma (St. Louis, MO, USA). The sequence of inserts/DNA fragments was confirmed by DNA sequencing with an ABI Prism 377 automated DNA sequencer (Applied Biosystems, Foster City, CA, USA). Transformation of E. coli was carried out by using competent cells prepared by the RbCl method [34 ] or by electroporation (Gene Pulser, Bio-Rad, Cambridge, MA, USA) [34 ]. Plasmids were transferred from E. coli S17-1λpir (donor strain) to Azoarcus sp. CIB recipient strains via biparental filter mating as previously reported [23 (link)]. Proteins were analyzed by SDS-PAGE as described previously [26 ].

Standard molecular biology techniques were performed as described previously (31 ). Plasmid DNA was prepared with a High Pure plasmid isolation kit (Roche Applied Science). DNA fragments were purified with Gene-Clean Turbo (Q-biogene). Oligonucleotides were supplied by Sigma. The oligonucleotides employed for PCR amplification of the cloned fragments and other molecular biology techniques are summarized in Table 2. All cloned inserts and DNA fragments were confirmed by DNA sequencing with fluorescently labeled dideoxynucleotide terminators (41 (link)) and AmpliTaq FS DNA polymerase (Applied Biosystems) in an ABI Prism 377 automated DNA sequencer (Applied Biosystems). Transformation of E. coli cells was carried out by using the RbCl method or by electroporation (Gene Pulser; Bio-Rad) (31 ). The proteins were analyzed by SDS-PAGE and Coomassie-stained as described previously (31 ). The protein concentration was determined by the method of Bradford (42 (link)) using bovine serum albumin as the standard. Nucleotide sequence analyses were done at the National Center for Biotechnology Information (NCBI) server (www.ncbi.nlm.nih.gov). Pairwise and multiple protein sequence alignments were made with the ClustalW program (43 (link)) at the EMBL-EBI server.