Figure 2

Cloning of complementation cassette into a recombinant protein expression plasmid. Above: EcoRI restriction and screening for positive recombinant clones. Numbers 1, 4, 8, and 9 refer to clones called pFC1, pFC4, pFC8 and pFC9. V refers to the restricted vector p582 as control. M refers to 1 kbp molecular size marker from Plasmid Factory GmbH. Below: Structure of plasmid constructs pFC1 (A) and pFC4 (B). The constructs are each 7 kb in size and contain the recombinant β-glucanase expression gene (bgl) and the gene for the bacteriocin release protein (kil). The origin of replication (ori) is based on pUC19. The plasmids carry the beta-lactamase gene for resistance against ampicillin and neomycin phosphotransferase gene for resistance against kanamycin (shown as Amp R and Kan R respectively). The auxotrophy-complementing tpiA gene along with its natural promoter (PtpiA) and terminator sequences has been cloned in the forward orientation in pFC1 and in reverse orientation in pFC4.