Figure 4

Schematic diagram of the overlap PCR. - 10, -35, TSS, SD, and Cre represent the -10 box, -35 box, transcription origin, SD box and catabolite repression element, respectively. Italic indicates the mutated nucleotides. Approximate positions of the primers are shown with arrows. Using primer pairs overlap-1-up/overlap-1-down and overlap-2-up/overlap-2-down, a 360 bp fragment upstream of the maltose promoter and a 300 bp fragment downstream of the maltose promoter were amplified from Bacillus subtilis 1A747 genome DNA, in which there are 30 bp overlap in the two amplified fragments and the two mutant sites were introduced by primers overlap-1-down and overlap-2-up. With overlap-1-up and overlap-2-down as primers, and the mixture of two obtained fragments as template, a 630 bp P _glv -M1 was overlap PCR amplified. Same mechanism was used to generate the other three mutants P _glv -M2, P _glv -M3 and P _glv -M4.