Figure 1

Strategy for obtaining a deletion of the pectinase gene cluster (Cbes1853-1856) and PCR analysis of the deletion in strain JWCB010. (A) The genome region containing the cluster with the deletion vector (pJFW54) containing about 2 kb flanking regions from up- and downstream of the cluster and the pyrF cassette [18]. Homologous recombination may occur at either the upstream or downstream flanking region, shown within the dotted-line box, integrating the plasmid into the genome and generating a strain that is a uracil prototroph. Counter-selection on 5-FOA selects for loss of the integrated plasmid and possible deletion of the pectinase gene cluster. Black arrows depict primers used for verification of the deletion. (B) Gel depicting PCR products of the pectinase gene cluster region in JWCB010 (2.1 kb), the deletion strain (lane 2), compared to its parent, JWCB005 (11 kb) (lane 1), using primers JF049 and JF204. JF204 anneals to a site outside of the homologous regions in the plasmid. (C) Gel depicting the 2.18 kb PCR product amplified using primer set (DC409/DC410) from the genome region that includes Cbes1854, pecB, JWCB005 (lane 1) or the deletion mutant (lane 2). (D) Gel depicting the 1.31 kb PCR product amplified using primer set (DC411/DC412) from the genome region that includes Cbes1855, pecA and Cbes1856, pecR from the JWCB005 (lane 1) or the deletion mutant (lane 2). M: 1 kb DNA ladder (NEB).