Standard PCR amplifications were

Standard PCR amplifications were performed with Biotools DNA polymerase (Biotools, Spain). All primers used

for PCR were synthesized by 1st Base Singapore and are listed in Additional file 1: Table S1. Electrocompetent cells were prepared from 6 ml overnight bacterial culture according to the procedure described by Choi et al (2005) [19]. Electroporation was carried out by placing 100 μl electrocompetent cells and 3 μl plasmid DNA in a sterile cuvette (0.1 cm electrode gap, Bio-Rad) and pulsed at 1.8 V using settings for bacteria in a Bio-Rad MicroPulser. The plasmid, pwFRT-TelR, was digested with XmaI and the 3.265 kb fragment carrying the tellurite-resistance cassette was isolated and ligated with XmaI-linearized pMo130 to produce the suicide plasmid, pMo130-TelR. The orientation of the tellurite-resistance cassette insert shown in Figure  1A was ascertained by digesting the plasmid with Xho1 and BamHI which gave a 4.161 kb and a 5.231 kb band. An insertion of the tellurite-resistance cassette into pMo130-TelR in the opposite orientation would have produced two bands of 1.150 kb and 8.242 kb. Conjugative transfer E. coli S17-1 donor

this website strain harboring the respective pMo130-TelR-(Up/Down) constructs and the A. baumannii recipient strains were cultured overnight at 37°C in 2 ml LB (supplemented with kanamycin for the donor E. coli strain). Aliquots of 0.2 ml each of donor and recipient EPZ5676 price cells were added to a microfuge

tube containing 1.2 ml of LB and washed twice with 2 ml LB each time. The cells were then suspended in 30 μl LB medium and added on to a sterile 0.45 μm cellulose nitrate filter paper (Sartorius Stedim, NY, U.S.A.) on LB agar and incubated at 30°C for 16 h. The cells were washed off from the filter by adding 0.4 ml Cobimetinib purchase of 0.9% NaCl. Aliquots of 0.1 ml were plated onto LB agar containing tellurite (30 mg/L) and gentamicin (25 mg/L) and incubated at 37°C for at least 16 h. Gentamicin was added for counter-selection against the donor cells. RNA analysis and quantitative real-time PCR (qRT-PCR) RNA was extracted from mid-log phase bacteria prepared by inoculating 10 ml Luria-Bertani (LB) broth Miller (1st BASE Pte Ltd, Singapore) with an overnight culture (1:50) and incubating at 37°C, with shaking at 120 rpm, until OD600 = 1.0. Triplicates of culture volumes containing two OD600 units (~ 2×109 cells) were centrifuged at 3,000 g for 10 min to harvest the cells. The cells were lysed by adding 1 mL of TRIzol® (Invitrogen, Carlsbad, CA) to the cell pellet and RNA was extracted according to the manufacturer’s protocol. Contaminating DNA was removed by treating the RNA sample with Ambion® TURBO™ DNase (Invitrogen) and cDNA was synthesized using random hexamer primers and TaqMan® Reverse Transcription Reagents (Invitrogen) according to the manufacturer’s protocol.

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