We have also been able to inactivate specific loci on several other, globally successful plasmids including
those carrying the carbapenemases bla KPC and bla NDM-1, illustrating the utility of our approach and its broad applicability to the study of plasmid gene function (manuscripts in preparation). Recent advances in sequencing have identified AR-13324 various ‘successful’ plasmids such as those found associated with the globally disseminated strain E. coli ST131  or those carrying other prominent resistance genes such as bla CMY-2 or bla NDM-1. Investigating the factors key to their dissemination could also be examined using a similar approach [28, 29]. A better understanding of the biological relevance of plasmid ‘backbone’ genes in the successful survival and spread of antibiotic resistance plasmids will be of paramount importance if we are to prevent future persistence and further spread of both plasmid vectors and the antibiotic resistance genes that they carry. Methods Bacterial strains and plasmid extraction Wild-type plasmid pCT [Genbank: FN868832] was isolated from a veterinary E. coli strain C159/11 [15, 16]. Wild-type pCT and recombinant pCT DNA was extracted using a QIAprep Spin Miniprep Kit (Qiagen, Germany) and a QIAGEN Large selleck chemicals llc Construct Kit (Qiagen, Germany) according to the manufacturers’ instructions.
All plasmids were transformed into E. coli DH5α electro-competent cells (Bioline, UK) (1.25 kV, 25 μF, 200Ω, in chilled 2 mm electroporation cuvettes) and transformants selected by growing on agar containing 8 mg/L of cefotaxime (Sigma-Aldrich, USA) or 50 mg/L of kanamycin (Sigma-Aldrich, USA) when the aph cassette is used for gene inactivation. Inactivation
of the six selected pCT genes To inactivate the six selected pCT genes, pCT was transformed into the E. coli strain, SW102 which carried a chromosomal Lambda-Red Recombinase . Where transformation of the Atazanavir plasmid into this strain is difficult, conjugation by filter mating was done by selecting the transconjugants on media containing 50 mg/L of tetracycline and 8 mg/L of cefotaxime. The hybrid primers used to inactivate the selected pCT genes were designed to have 20 bp identity to the aph cassette on pKD4  and 40 bp sequence identity to the target genes (Table 2). Recombination of Selleck PF 2341066 amplimers encoding the aph gene with each pCT gene was carried out as previously described . Recombination was confirmed in each case by PCR and sequencing across the mutated DNA region (Table 2). The recombinant plasmid was then extracted and electroporated into DH5α or conjugated into another host strain to avoid further recombination from occurring and for further study.