Louis, MO), and allowed to recover PCI-32765 cost for 18–24 h before plating in BSK-II containing kanamycin (340 μg ml-1) according to the protocol of Samuels et al [39]. Kanamycin resistant colonies, appearing approximately 10–14 days after plating, were screened for the presence

of the complementation plasmid by PCR using primers BB0771 F1 and BB0771 R1 2. A positive clone was chosen for further experiments and designated WC12. Construction of the rpoN mutant in B31-A A B. burgdorferi 297 rpoN mutant strain (donated by Michael Norgard) [19], in which rpoN was interrupted by the insertion of an erythromycin resistance gene, was maintained in BSK-II containing erythromycin (0.6 μg ml-1). Genomic DNA was extracted from the 297 rpoN mutant using the DNeasy Tissue Kit (Qiagen, Inc.) following the manufacturer’s instructions. Primers BB0450 mutF1 and BB0450 mutR1 (Table 2) were used to PCR amplify rpoN::ermC and flanking DNA

from 297 rpoN mutant genomic DNA. learn more The PCR product (~4.4 kb) was TA cloned into the pGEM T-Easy vector (Promega, Corp., Madison, WI) according to the manufacturer’s instructions, and the ligation reaction was transformed into competent E. coli DH5α. A

transformant containing 5-Fluoracil purchase the plasmid of interest was selected by blue-white screening on LB containing ampicillin (200 μg ml-1) and X-gal (40 μg ml-1), confirmed by PCR using the BB0450 mutF1 and BB0450 mutR1 primers, and designated pBB0450.1. See Table 2. The plasmid was extracted and concentrated to greater than 1 μg μl-1, and 10 μg were transformed into competent B31-A as described above. Transformants were selected by plating on BSK-II containing erythromycin (0.6 μg/ml) according to the protocol of Samuels et al [39]. The mutation in the rpoN gene of B31-A was confirmed by PCR using primers flanking the ermC insertion site (BB0450 mut confirm F1 and BB0450 mut confirm R1. See Table 2), and the mutant was designated RR22. In addition, DNA sequence analysis (ABI Prism® 3130XL Genetic Analyzer, Applied Biosystems, Forest City, CA) was performed to verify the rpoN::ermC junctions using primers 5′ ermC seq out and 3′ ermC seq out. See Table 2. The University of Rhode Island Genomics and Sequencing Center performed DNA sequencing.