PCR‐based gene mutagenesis and plasmid multimerization.
A. Relevant features of the vector pNWP43N‐Bscel5. P43, SPnprB, Bscel5 and term represent the P43 promoter, the NprB signal peptide‐encoding sequence, gene of family 5 endoglucanse and terminator of Bscel5 from B. subtilis respectively. ColE1 ori, repB and cat represent the sequences coding for the ColE1 replication origin, replicase and chloramphenicol resistance marker respectively. The arrows show the transcription directions for these genes.
B. The flow scheme of the two‐step PCR procedure for the gene mutagenesis and plasmid multimerization. gh5, family 5 glycoside hydrolase‐encoding sequence; cbm3, family 3 carbohydrate‐binding module‐encoding sequence. P1, P2, P3 and P4 denote the positions of the primers for the PCR amplification. This figure was not drawn to scale.
C. Plasmid multimerization by PCR. Lanes: M, DNA markers; 1, PCR‐linearized pNWP43N‐Bscel5; 2, error‐prone PCR product of SPnprB‐Bscel5; 3, multimerized plasmid; 4, multimer digested with PstI/HindIII.