Standard PCR amplifications were https://www.selleckchem.com/products/ABT-263.html 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) . 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.