Conjugation was carried out on LB agar plates overnight with a bacterial proportion of 4:1 of E. coli containing conjugative plasmid (donor) and V. cholerae as recipient strain. Bacterial cultures (mixed E. coli and V. cholerae) were plated on LB agar plate containing Carb (100 μg/ml) and Km (30 μg/ml) for selection of V. cholerae transconjugants carrying the plasmid. The removal of vector backbone from V. cholerae genome was achieved by favoring the homologous recombination Sapanisertib and use of lethal sacB gene while passaging the transconjugants in sodium chloride free LB medium supplemented with 10% sucrose. Attempts for construction of a kdpD knockout mutant using V. cholerae
strain NM06-058 The gene VC_A0531 encodes for the histidine kinase KdpD in V. cholerae and is flanked by the genes VC_A0530 encoding ��-Nicotinamide pyruvate-flavoredoxin oxidoreductase and VC_A0532 encoding response regulator KdpE homologue S3I-201 solubility dmso of E. coli. To generate a VC_A0531 deletion mutant, two fragments were amplified from the small chromosome of the wild type strain NM06-058 using two primer
pairs (i) kdpD_del_forw_1 / kdpD_del_rev_1 and (ii) kdpD_del_forw_2 / kdpD_del_rev_2. Using the first primer pair an approximately 600 pb fragment of gene VC_A0530 was amplified with a 24 bp homolog overhang to the start region of the VC_A0532 at the C-terminus. The second primer pair was used to amplify an approximately 400 bp fragment of the gene VC_A0532 with a 16 bp overhang homolog to the end region of the VC_A0530 at the N-terminus. Both amplicons were mixed together at equimolar ratio and a re-PCR was carried out with a combination of primers learn more kdpD_del_forw_1 and kdpD_del_rev_2 to generate an amplicon with a size of approximately 1,000 bp. The restriction of vector pEX18Ap and the insert was carried out with XbaI and PstI. After ligation and transformation into E. coli S17-1, a conjugation into the wild type V. cholerae strain NM06-058 was mediated according to the protocol described above. The cloning strategy was successful until transconjugation according selection on Carb / Km agar plates and sequencing, but homolog recombination attempts
with V. cholerae strain NM06-058 did not yield viable strains with deleted kdpD gene. Acknowledgments Authors thankfully acknowledge helpful technical assistance from Subhasis Barik. We thank the Indian Council of Medical Research (ICMR), Govt. of India and for funding support through the Indo-German Science Centre (Sanction No. TDR/491/2008-ECD-II). SR is recipient of a Junior Research Fellowship from the Council of Scientific and Industrial Research (CSIR), Govt. of India (Sanction No. 09/482(0054)/2010-EMR-I). References 1. WHO: Cholera. Fact sheet No 107 2011. http://www.who.int/mediacentre/factsheets/fs107/en/ 2. Kitaoka M, Miyata ST, Unterweger D, Pukatzki S: Antibiotic resistance mechanisms of Vibrio cholerae. J Med Microbiol 2011,60(4):397–407.PubMedCrossRef 3.