Fast link ligase

To generate an expression plasmid for the ABUW_1645 open reading frame, a 697-bp DNA fragment, which began 60 bp upstream from the predicted ABUW_1645 start codon and ended 82 bp downstream from the predicted ABUW_1645 stop codon, was amplified by PCR using chromosomal DNA from A. baumannii strain AB5075 as the template (Phusion Hot Start Polymerase; Thermo Scientific, Waltham, MA). Oligonucleotide primers (1645 Exp. 1.1; 5′-GAGTGACGGCATGTCTATCT-3′ and 1645 Exp. 2.2; 5′-CTTATAGCCATAAGTGGTAATTGAG-3′) were treated with T4 Polynucleotide Kinase (New England Biolabs, Ipswich, MA) to add 5′-phosphates prior to PCR amplification. The fragment was purified from an agarose gel slice and ligated (Fast-Link Ligase; Epicentre, Madison, WI) into pWH1266^{36 (link)} that had been digested with ScaI (New England Biolabs) and subsequently treated with shrimp alkaline phosphatase (New England Biolabs) to dephosphorylate linearized vector. The ligation was transformed into E. coli Transformax EC100D competent cells (Epicentre) and plated on LB+Tet (10 μg/mL) plates, resulting in the expression vector pKT1645.