Fig. 2

Construction of integrative plasmid and conjugative donor strain for the Bacillus integrative plasmid (BIP) system. A Construction of the BIP system. The MICEaRep (a donor strain for modified integrative and conjugative element [MICE] system) was constructed by inserting the replication initiation gene (rep) of plasmid pAD123 into the amyE locus of the chromosome. The rep gene was under the control of the constitutive promoter P_ara which is modified from the Bacillus subtilis ara promoter. The BIP backbone, pSGC2iN, has a double-strand origin of replication (DSO) region and a pMB1 origin for E. coli but not the rep gene for Bacillus. B Quantitative real-time PCR to determine the plasmid copy number (PCN) for pAD123 backbone in B. subtilis 168 and pSGC2iN in MICEaRep donor. The dnaN and neo genes were selected as a single-copy reference gene on the chromosome and the target gene on the plasmid, respectively. The PCN was calculated via dividing the target plasmid absolute quantity by the chromosomal absolute quantity in the template total DNA. The bars display the means of three independent measurements, with the error bars indicating standard deviations